a method for selective concentrating of dna targets by ......a method for the selective...
TRANSCRIPT
A Method for Selective Concentrating of DNA Targets by Capillary Affinity Gel Electrophoresis
by
Andrew Chan
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Department of Chemistry University of Toronto
© Copyright by Andrew Chan, 2013
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A Method for Selective Concentrating of DNA Targets by Capillary
Affinity Gel Electrophoresis
Andrew Chan
Doctor of Philosophy
Department of Chemistry University of Toronto
2013
Abstract
A method for the selective concentrating of DNA targets using capillary affinity gel
electrophoresis is presented. Complementary ssDNA targets are retained through hybridization
with oligonucleotide probes immobilized within polyacrylamide gels while non-complementary
targets are removed. The captured DNA targets were concentrated by step elution, where a
localized thermal zone was applied in small steps along the capillary.
Evaluation of the selective capture of a 150 nt DNA target in a complicated mixture was carried
out by factorial analysis. Gels with a smaller average pore size were found to retain a higher
amount of complementary targets. This was thought to be due to the ssDNA target migrating
through the gel by reptation, eliminating hairpin structures, making the complementary region of
the target available for hybridization.
This method was applied to a series of DNA targets of different lengths, 19 nt, 150 nt, 250 nt and
400 nt. The recovery of the method ranged from 0.5 to 4% for the PCR targets, and 13 to 18%
for the 19 nt oligonucleotide target. The purity was calculated to be up to 44% for the PCR
targets and up to 86% for the 19 nt target. This was an improvement in purity of up to 15 times
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and 1100 times in comparison to the original samples for the PCR targets and 19 nt
oligonucleotide, respectively.
The 19 nt targets were selective concentrated and delivered into a microfluidic based DNA
biosensing platform. The purity of the sample improved from 0.01% to 50% while recovery
decreased from 100% to 20% for a sample with 0.5 nM complementary and 1 µM non-
complementary targets. An improvement in the response of the sensing platform was
demonstrated on 19 nt oligonucleotide targets delivered by selective concentration versus
concentration alone into the microfluidic biosensing system.
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Acknowledgments
Firstly, I would like to thank my supervisor, Professor Ulrich Krull for his guidance, support and
patience throughout this journey. I would not have been able to achieve this without his help. I
would also like to thank the members of my supervisory committee, Professors Aaron Wheeler
and Julie Audet for their feedback over the years.
I would also like to thank Dr. Lu Chen, Uvaraj Uddayasankar and Omair Noor for their
assistance in the work with interfacing the capillary and microfluidic biosensing platform. I
would also like to thank past and present members of the Chemical Sensors Group, especially
Drs. Russ Algar, Melissa Massey, Ying Lim and April Wong for their friendship, support and
laughs.
Finally, I would like to thank my parents and family for bearing with me throughout this pursuit.
This is dedicated to you.
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Table of Contents
Acknowledgments.......................................................................................................................... iv
Table of Contents ............................................................................................................................ v
Abbreviations...................................................................................................................................x
Publications....................................................................................................................................xii
List of Tables ............................................................................................................................... xiii
List of Figures .............................................................................................................................. xix
List of Appendices ................................................................................................................... xxxiv
Chapter 1 Introduction .................................................................................................................... 1
1.1 DNA Biosensors for Detection of Real-World Targets ....................................................... 1
1.2 Goals of Pre-treatment ......................................................................................................... 5
1.2.1 Extraction................................................................................................................... 5
1.2.2 Purification................................................................................................................. 5
1.2.3 Sample Loss and Quantification ................................................................................ 6
1.3 Methods for DNA Purification............................................................................................. 8
1.3.1 Purification Methods based on Solid Phase Extraction (SPE)................................... 8
1.3.2 Purification of DNA in Conjunction with Selective Hybridization......................... 11
1.4 Enrichment and Amplification of Sample.......................................................................... 14
1.4.1 DNA Amplification by Polymerase Chain Reaction ............................................... 15
1.4.2 Concentrating by Volume Reduction....................................................................... 16
1.5 Fragmentation and Denaturation of DNA.......................................................................... 17
1.5.1 Fragmentation .......................................................................................................... 17
1.5.2 Preparation of Single-Stranded DNA Target ........................................................... 18
1.6 Integrated Microfluidic Devices ........................................................................................ 20
1.7 Methods for Purification and Concentrating in Microfluidic Devices............................... 22
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1.7.1 Filtration................................................................................................................... 22
1.7.2 Solid-Phase Extraction............................................................................................. 23
1.8 Sample Concentrating by Electrokinetic Methods............................................................. 26
1.8.1 Field Amplified Stacking......................................................................................... 26
1.8.2 Isotachophoresis....................................................................................................... 27
1.9 Contributions of this Thesis ............................................................................................... 28
Chapter 2 Materials and Method................................................................................................... 33
2.1 Reagents ............................................................................................................................. 33
2.2 DNA Targets ...................................................................................................................... 33
2.3 Instrumentation .................................................................................................................. 35
2.3.1 Capillary Electrophoresis......................................................................................... 35
2.3.2 Instrumentation for on-line capillary electrophoresis/step elution experiments...... 36
2.3.3 Confocal Fluorescence Microscope Images ............................................................ 36
2.3.3.1 Confocal fluorescence microscope slide reader for 532nm/635nm excitation (Chipreader) ........................................................................... 37
2.3.3.2 Epifluorescence microscope for 635 nm excitation (Alpha) .................... 38
2.3.3.3 Confocal Fluorescence Microscope for 534 nm excitation (Confocal) .... 38
2.3.4 UV-VIS .................................................................................................................... 38
2.3.5 Steady-State Solution phase Fluorescence Measurements ...................................... 39
2.3.6 Other Equipment ...................................................................................................... 39
2.4 Generation of Longer lengths of DNA Targets.................................................................. 39
2.4.1 150 bp targets .......................................................................................................... 39
2.4.2 250 bp targets .......................................................................................................... 40
2.4.3 400 bp targets .......................................................................................................... 41
2.4.4 Validation of DNA targets ...................................................................................... 42
2.5 Preparation of Capillary Affinity Capture Gel................................................................... 42
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2.6 Capture and elution experiments....................................................................................... 43
2.6.1 Pre-Conditioning of Affinity Capture Gel in Capillaries......................................... 43
2.6.2 Capture and Elution Experiments ........................................................................... 43
2.6.3 Factorial Design Experiments.................................................................................. 43
2.6.4 Step Elution of Captured DNA targets .................................................................... 45
2.7 Delivery of concentrated targets into microfluidic based DNA biosensing platform........ 45
2.7.1 Construction of DNA Microfluidic Biosensing Platform........................................ 45
Chapter 3 Results and Discussion................................................................................................. 49
3.0 Capture of Oligonucleotides of 20 nt Length..................................................................... 49
3.0.1 Considerations for Imaging Fused Silica Capillaries by Confocal Fluorescence Microscopy ........................................................................................................... 49
3.0.2 Capture and Elution Experiment for a 20 nt Target................................................. 50
3.0.3 Autofluorescence and Non-Selective Adsorption.................................................... 52
3.0.4 Variation of Polymer Density .................................................................................. 55
3.0.5 Quantity of Probe that was Immobilized in the Polyacrylamide Matrix ................. 57
3.0.6 Influence of Concentration of Target on the Efficiency of Capture ........................ 59
3.0.7 Capture and Elution using a Non-Complementary Target ...................................... 60
3.0.8 Examination of Selectivity Using a Five Base Pair Mismatch Target..................... 62
3.0.9 Separations of Mixtures of Complementary and Non-complementary Targets ...... 63
3.0.10 Five Base Pair Mismatch in Mixture with Fully Complementary Target ............. 65
3.0.11 Capture of 40 nt Length Targets ............................................................................ 67
3.1 Capture of Targets of Greater Lengths than 40 nt – Moving Towards Handling of NAs From Real Samples ........................................................................................................... 68
3.1.1 DNA Targets Selected for Experiments .................................................................. 69
3.2 Compositions of Affinity Capture Gels ............................................................................ 70
3.3 Selective Capture of 150 nt Target.................................................................................... 71
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3.3.1 Comparison of Capture of 150 nt DNA Targets Using Complementary and Non-complementary Probe ................................................................................... 79
3.4 Performance of the Affinity Gel for the Capture DNA Targets......................................... 82
3.5 Examination of the Effects of Varying Gel Formulation on Performance ........................ 90
3.5.1 Affect of Gel Formulation on the Quantity of Probe that was Incorporated ........... 91
3.5.2 Effect of Radical Initiators on Oligonucleotide Sequence....................................... 95
3.5.3 Cleavage of Oligonucleotide Probe by Radicals .................................................... 98
3.5.4 Examination of Damage to Nucleobases by Radical ............................................ 103
3.5.5 Examination of Conditions that Affect Capture of Complementary Targets ........ 107
3.5.6 Affinity Capture of Complementary Targets with Probes that are Immobilized in 3D Gel Supports.................................................................................................. 109
3.5.7 Effects of Gel Formulation on the Concentration of Target Injected .................... 113
3.5.8 Effects of Gel Formulation on the Quantity of Target Captured ........................... 115
3.5.9 Effects of Polymerization of Polyacrylamide ........................................................ 117
3.5.10 Effect of Probe Availability As a Function of Gel Formulation.......................... 120
3.5.11 Effect of Gel Formulation on Migration of DNA Targets ................................... 121
3.5.12 Effect of Stringency Conditions on Percent Recovery and Purity during Washing Step ...................................................................................................... 125
3.6 Selective Concentrating of Oligonucleotide Targets by Step Elution From Affinity Gels ................................................................................................................................. 133
3.6.1 Concentrating the 150 nt, 250 nt and 400 nt Targets by Step Elution ................... 139
3.7 Delivery of Concentrated Targets into Microfluidic DNA Biosensing Platform ............ 141
3.7.1 Design Aspects for Sample Transfer from the Capillary to the Microfluidic Biosensing Platform............................................................................................ 141
3.7.2 Delivery of Oligonucleotide Targets to the Microfluidic Biosensing Platform by Direct Injection and by Selective Concentration ................................................ 146
3.7.3 Response of Microfluidic Biosensing Platform to Mixtures of Targets Delivered by Direct Injection and Following Selective Concentration............................... 150
3.7.4 Response of Microfluidic Biosensing Platform to Delivery of Concentrated Oligonucleotide Targets With and Without Purification .................................... 155
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Chapter 4 Future Directions........................................................................................................ 159
4.1 Determination of Oligonucleotide Probe Incorporated into Affinity Capture Gel .......... 159
4.2 Further Factorial Experiments on Gel Formulations ....................................................... 159
4.3 Improvements to Capillary-Microfluidic Platform .......................................................... 160
4.4 Moving Towards DNA Targets in Complex Matrices..................................................... 161
Chapter 5 Conclusions ................................................................................................................ 163
References................................................................................................................................... 165
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Abbreviations
APS - Ammonium Persulfate
ATP - Adenonsine Triphosphate
AU - Arbitrary Units
BSA - Bovine Serum Albumin
CD - Compact Disc
CE - Capillary Electrophoresis
CE-MS - Capillary Electrophoresis-Mass Spectrometry
CFU - Colony Forming Units
CGE - Capillary Gel Electrophoresis
DEAE - Diethylaminoethanol
DMF - Digital Microfluidics
DNA - Deoxyribonucleic Acid
dsDNA - Double Stranded Deoxyribonucleic Acid
DTT - Dithiothreitol
EDTA - Ethylenediaminetetraacetic Acid
EKS - Electrokinetic Supercharging
ELWD - Extra Long Working Distance
EOF - Electroosmotic Flow
EWOD - Electrowetting on Dielectric
FAS - Field Amplified Stacking
HCV - Hepatitis C Virus
HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HPV - Human Papillomavirus
ID - Inner Diameter
ITP - Isotachophoresis
LATE-PCR - Linear-After-the-Exponential Polymerase Chain Reaction
LOD - Limit of Detection
MAGIChip - Microarray of Gel-immobilized Compounds on a Chip
MPS - 3-methacryloxypropyltrimethoxysilane
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NA - Nucleic Acid
OD - Outer Diameter
PCR - Polymerase Chain Reaction
PDMS - Polydimethoxysilane
PMT - Photomultiplier Tube
PNA - Peptide Nucleic Acid
POC - Point of Care
PVA - Polyvinyl Alcohol
PVP - Polyvinylpyrrolidone
RNA - Ribonucleic Acid
rRNA - Ribosomal Ribonucleic Acid
RT - Room Temperature
SMN - Survival Motor Neuron
SPE - Solid Phase Extraction
SPR - Surface Plasmon Resonance
ssDNA - Single Stranded Deoxyribnucleic Acid
TBE - Tris-Borate-Ethylenediaminetetraacetic Acid
TEMED - N,N,N′,N′-tetramethylethane-1,2-diamine
TMOS - Tetramethyl Orthosilicate
TMSPM - 3-(trimethoxysilyl)propyl methacrylate
UV - Ultraviolet
UV-VIS - Ultraviolet-Visible
WD - Working Distance
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Publications
A. Chan and U.J. Krull, "Capillary electrophoresis for capture and concentrating of target nucleic
acids by affinity gels modified to contain single-stranded nucleic acid probes", Analytica
Chimica Acta, 578(2006), pg 31-42.
A. Chan, T. Artuso, U.J. Krull, "Sample Handling Protocols for Biosensor Applications" in
Handbook of Sample Preparation. Hoboken, N.J.:John Wiley & Sons, 2010, Chapter 21, pg 385-
418.
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List of Tables
Table 1.1: Examples of biosensors used for detection of NA targets from various different
sample matrices. Steps for pre-treatment protocols prior to detection are identified, as well as the
total time needed for pre-treatment and detection. When available, detection time is also
provided in parenthesis. .................................................................................................................. 4
Table 2.1: Oligonucleotide targets that were used in the experiments. Longer PCR targets are
described in a subsequent section. Melt temperature was provided by the supplier.
*oligonucleotide probes used in capillary affinity capture gels contained the Acrydite
modification at the 5' end, while probes used for immobilization onto epoxy-modified glass
slides in the microfluidic device contained a primary amino group with a C12 spacer on the 5'
end. Cy3 fluorescent label was attached to the 3' end when used.**fluorophores on these
oligonucleotide sequences were attached at 5' end when used. .................................................... 34
Table 2.2: Design matrix for the quarter 2-level fractional factorial analysis for the examination
of gel formulation on the performance of the capillary affinity capture gels. .............................. 44
Table 2.3: Experimental conditions for the two levels used for the fractional factorial design
matrix. ........................................................................................................................................... 44
Table 2.4: Design matrix for three level factorial experiment to explore capture efficiency and
selectivity of the affinity capture gel. Factors A and B are defined in Table 2.5. ....................... 45
Table 2.5: Experimental conditions for each level tested in the design matrix of Table 2.4...... 45
Table 3.1: Tabulated results for extent of probe incorporation and performance in capture for
three different probe concentrations. Affinity gel: Varying concentrations of Cy3-dT20 probe
(182 nM, 454 nM and 727 nM) in a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL
of a sample containing 1 µM Cy5-dT20 at 267 Vcm-1 for 60 s. Electrophoresis condition: 133
Vcm-1 with 1x TBE/0.1% PVP for 20 minutes. Elution condition: 267 Vcm-1 with 1x TBE/0.5 M
NaSCN/0.1%PVP for 15 minutes at 60 °C. Error bars are propagated error following correlation
of average fluorescence intensity to concentration using a calibration curve. ............................. 58
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Table 3.2: Tabulated results for effect of concentration of sample on loading of affinity gel.
Amount of target in loading of a sample was calculated based on a 10 µL of the target
concentration solution used for loading. Amount of probe and target were calculated using the
geometric volume of 0.59 µL for the capillary. Affinity gel: 0.45 µM Cy3-dT20 probe in a
12.5%T linear polyacrylamide gel. Injection condition: 10 µL sample containing Cy5-dA20 at
533 Vcm-1 for 60 seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP for 10
minutes. Elution condition: 267 Vcm-1 with 1xTBE/0.5 M NaSCN/0.1%PVP for 5 minutes at
60°C. Error bars are propagated error following correlation of average fluorescence intensity to
concentration using a calibration curve. ....................................................................................... 59
Table 3.3: Summary results for Recovery and Purity for mixture containing 150 nt and 1.5 pmol
non-complementary targets by affinity capture gel. Recovery and purity of the original solution
and by selective capture are presented. The recovery and purity were calculated based on
removal of material from the affinity capture gel by elution of the entire capillary (no
concentrating). Experimental conditions shown in Figure 3.29. .................................................. 88
Table 3.4: Summary results for Recovery and Purity for mixture containing 250 nt and 1.5 pmol
non-complementary targets by affinity capture gel. Recovery and purity of the original solution
and by selective capture are presented. The recovery and purity were calculated based on
removal of material from the affinity capture gel by elution of the entire capillary (no
concentrating). Experimental conditions shown in Figure 3.30. .................................................. 89
Table 3.5: Summary results for Recovery and Purity for mixture containing 400 nt and 1.5 pmol
non-complementary targets by affinity capture gel. Recovery and purity of the original solution
and by selective capture are presented. The recovery and purity were calculated based on
removal of material from the affinity capture gel by elution of the entire capillary (no
concentrating). Experimental conditions shown in Figure 3.31. .................................................. 89
Table 3.6: Summary of the migration times of the peaks observed from the CGE experiment.
The sequence of the oligonucleotide target used in these experiments: 5’ Cy5 - ACA GGG TTT
CAG ACA AAA T 3’. Error represents 1 standard from three trials. ........................................ 102
Table 3.7: Summary of melt temperatures of the oligonucleotide duplex following reaction with
the different radical initiator ratio. Error represent 1 standard deviation of three trials. ............ 107
xv
Table 3.8: Summary factors which were identified as significant from factorial analysis (95%
confidence). The (+) and (-) after each factor denotes whether the effect was positive or
negative. ...................................................................................................................................... 109
Table 3.9: Determination of pore size from the Ogston plot presented in Figure 3.46. The range
of DNA fragments where log(µ/µo) deviates from linearity is assumed to be the size range where
DNA transitions from Ogston to reptation. The radius of gyration of the dsDNA fragments was
calculated by Eq (11), where persistence length for dsDNA was 50 nm, and contour length of
DNA was 0.34 nm/base. ............................................................................................................. 123
Table 3.10: Probe density based on 0.5 µM probe in the original monomer solution. Values
were calculated based on the number of pores that could fit in a 1000x1000x1000 nm cube at the
lowest gel formulation (7.5 %T/1 %C). The calculation assumes that the pore volume for the
remaining gel formulation was the same as the lowest monomer concentration and number of
pores for the remaining gel formulations were calculated as such. The amount of probe was
determined based on previous experiments examining the percentage of probe incorporated, and
the accessibility of the probe from experiments performed with 19 nt targets in Figure 3.45. .. 125
Table 3.11: Summary of results of the concentrating effect of step elution of complementary
target as a function of elution length. Volume of the eluting targets was calculated based on the
peak width and mobility of the oligonucleotide, which was 86 µms-1 at 96 Vcm-1. Error
represent 1 standard deviation of three trials. Affinity capture gel: 50 nM SMN probe, 10%
LAAm affinity capture gel. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150
Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer. Step
elution for concentrating effect: coverage length: 12.5, 25 and 37.5 mm; step size: 250 µm; step
rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain 400
mV. Sampling rate: 10 Hz ......................................................................................................... 136
Table 3.12: Summary of results from step elution of complementary target as determined from
data obtained from the experiment depicted in Figure 3.51b. Results for integrated Peak Area,
Width, Height were calculated using Origin Pro 8.0 Volume of the eluting targets was calculated
based on the peak width and mobility of the oligonucleotide, which was 86 µms-1 at 96 Vcm-1.
Error represent 1 standard deviation of three trials. Affinity capture gel: 50 nM SMN probe,
10% LAAm affinity capture gel. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at
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150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer.
Step elution for concentrating effect: coverage length: 12.5, 25 and 37.5 mm; step size: 250 µm;
step rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain
400 mV. Sampling rate: 10 Hz .................................................................................................. 138
Table 3.13: Summary results for Recovery and Purity from affinity capture gel for mixtures
containing varying amounts of 150 nt complementary and 1.5 pmol non-complementary targets.
The Recovery and Purity were calculated from quantitative concentration data for the eluting
peak by use of calibration curves. Errors represent propagated error following correlation of
average fluorescence intensity to concentration using a calibration curve. Affinity capture gel: 3
µM β-actin probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic injection for 20 min at
133 Vcm-1. Incubation time 5 min. Wash step: electrophoresis for 25 min at 133 Vcm-1, 45 °C,
with 25% v/v formamide/1X TBE/PVP. Concentrating step: coverage length: 12.5 mm; step
size: 250 µm; step rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition settings (Cy5): PMT gain 500
mV, translation speed: 50 µms-1, scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110
mV, Pinhole: 60 µm, 1 FPS ........................................................................................................ 139
Table 3.14: Summary results for Recovery and Purity for mixture containing varying amounts of
250 nt of complementary and 1.5 pmol non-complementary targets by affinity capture gel. Errors
represent propagated error following correlation of average fluorescence intensity to
concentration using a calibration curve. Affinity capture gel: 3 µM β-actin probe, 12.5 %T, 1
%C. Capture conditions: electrokinetic injection for 30 min at 133 Vcm-1. Incubation time 5
min. Wash step: electrophoresis for 40 min at 133 Vcm-1, 45 °C, with 25% v/v formamide/1X
TBE/PVP. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 66 µms-1;
Voltage: 96 Vcm-1; Acquisition settings (Cy5): PMT gain 500 mV, translation speed: 50 µms-1,
scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS . 140
Table 3.15: Summary results for Recovery and Purity for mixture containing 400 nt and 1.5
pmol non-complementary targets by affinity capture gel. Errors represent propagated error
following correlation of average fluorescence intensity to concentration using a calibration curve.
Affinity capture gel: 3 µM uidA probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic
injection for 40 min at 133 Vcm-1. Incubation time 5 min. Wash step: electrophoresis for 50 min
at 133 Vcm-1, 45 °C, with 25% v/v formamide/1X TBE/PVP. Concentrating step: coverage
xvii
length: 12.5 mm; step size: 250 µm; step rate: 52 µms-1; Voltage: 96 Vcm-1; Acquisition settings
(Cy5): PMT gain 500 mV, translation speed: 50 µms-1, scan rate, 50 Hz. (Cy3): Image
resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS...................................................... 140
Table 3.16: Summary of data for the response of the microfluidic DNA biosensing platform to
delivery of complementary targets by selective concentrating using the affinity capture gel. The
amount of target injected into the affinity capture gel by electrokinetic injection from the original
target solution is shown in parenthesis. The equivalent quantity was determined based on
correlation to the concentration-response curve of Figure 3.55. The enhancement factor was
calculated based on the ratio of the equivalent quantity and quantity of material injected. Errors
represent 1 standard of three trials expect for Equivalent Quantity Determined from Calibration
Curve, which is propagated error from correlation with calibration curve. Affinity capture gel:
100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL A647 SMN target, electrokinetic
injection for 1 min at 150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x
TBE/PVP running buffer. Concentrating Step: coverage length: 12.5 mm; step size: 250 µm; step
rate: 86 µms-1; Voltage: 96 Vcm-1; Delivery of purified and concentrated targets into
microfluidic biosensing platform: 500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition
settings: PMT gain 700 mV, translation speed: 50 µms-1 scan rate: 50 Hz. .............................. 149
Table 3.17: Summary of the performance of the two delivery methods. Percent recovery is
based on the proportion of the amount of target delivered to the microfluidic biosensing platform
from of the original starting sample. The values for delivery by direct injection were calculated
based on the concentration of the targets in the original sample. The values used for the delivery
selective concentrating were calculated based on the response of the biosensing platform. The
enrichment factor is the ratio of the percent complementary target with and without selective
concentrating. Errors represent propagated error resulting from calculating derived values. ... 154
Table 3.18: Position and peak width of the eluted targets from the concentrating of the two
captured targets along the capillary (from the injection end). Error represent 1 standard deviation
of three trials. Affinity capture gel: 10% LAAm, 100 nM SMN and 5 µM β-actin probes. Target
injection: 10 µL of 1 nM A647-SMN and 1 µM Cy3-β-actin targets, electrokinetic injection for 1
min at 150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running
buffer. Concentration step: coverage length: 12.5 mm; Step rate: 86 µms-1; Voltage: 96 Vcm-1;
xviii
Acquisition settings (Alexa647): PMT gain 700 mV, translation speed: 50 µms-1, scan rate, 50
Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS ...................... 156
Table 3.19: Quantitative information of the eluted A647-SMN target during stacking from gels
which containing only SMN probe (selective concentrating) and containing both β-actin and
SMN probe (concentrating only). Errors represent 1 standard deviation of three trials. Affinity
capture gel: Selective concentrating: 10% LAAm with 100 nM SMN probe. Concentrating only:
10% LAAm with 100 nM SMN and 5 µM β-actin probes. Target injection: 10 µL of 1 nM
A647-SMN and 1 µM Cy3-β-actin targets, electrokinetic injection for 1 min at 150 Vcm-1.
Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer. Concentrating
Step: coverage length: 12.5 mm; Step rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition settings
(Alexa647): PMT gain 700 mV, translation speed: 50 µms-1, scan rate, 50 Hz. ....................... 156
xix
List of Figures
Figure 1.1: Radical polymerization reaction of acrylamide in the presence of an Acrydite™
modified oligonucleotide. ............................................................................................................. 13
Figure 1.2: Schematic of differences between (a) conventional denaturation by heat and (b)
denaturation by heat with ancillary blocking oligonucleotides. With permission from Analytica
Chimica Acta. Copyright 2004, Elsevier [131]. .......................................................................... 19
Figure 1.3: (a) Schematic diagram of the microchip layout for pre-concentration, (b) image of
the pre-concentrator channel, and (c) schematic of how the filtration membrane is placed in
between the microchip and the coverplate. With permission from Analytical Chemistry.
Copyright 2004, American Chemical Society [153]..................................................................... 23
Figure 1.4: Fluorescence images of fluorescein-labeled ricin injected a) without pre-
concentration, and b) with pre-concentration for 1 minute. With permission from Analytical
Chemistry. Copyright 2004, American Chemical Society [153]. ................................................ 23
Figure 1.5: Schematic and SEM images of the microfabricated silica pillars for SPE of DNA.
With permission from Biosensors and Bioelectronics. Copyright 2003, Elsevier [158]............. 25
Figure 1.6: Schematic representation of selective concentrating as done in the work of this
thesis. First, the target was captured onto the affinity capture gel column. Elution took place in a
localized area of the capillary by means of application of heating to a narrow zone such that only
targets captured in that region were denatured. This process took place during electrophoresis,
and the denatured targets moved along in the electric field. The heated zone was then physically
moved along the column. This allowed for the continual release of targets into a stacked zone of
significantly smaller volume than the original sample volume. ................................................... 30
Figure 2.1: Schematic of the capillary electrophoresis set-up .................................................... 35
Figure 2.2: Set-up for online capillary electrophoresis and step elution experiments................ 36
Figure 2.3: Schematic of the instrumental setup for how confocal fluorescence microscope
images were obtained.................................................................................................................... 37
xx
Figure 2.4: Schematic for the construction of the microfluidic DNA sensing platform with a
capillary interface. Left: original microfluidics template and position of the template capillary.
The capillary (100 µm I.D., 375 µm O.D.) was positioned over the microfluidic channel such that
the inner diameter was within the width of the channel. PDMS was poured over the template and
cured on a hotplate. The template capillary and microfluidic template were removed and the
PDMS chip had the channel structure and capillary port. Right: schematic of the microfluidic
DNA sensor platform. The PDMS template was trimmed such that only the straight channel
remained, and this was positioned over the two oligonucleotide probe spots on the epoxy
modified slides. ............................................................................................................................. 46
Figure 2.5: Schematic of the electrophoresis setup for the capillary to microfluidic DNA
biosensor. ...................................................................................................................................... 47
Figure 3.1: Confocal fluorescence microscope images of affinity capture capillaries from a
capture and elution run using a complementary target showing: (a) the affinity gel material
inside the capillary prior to loading of target oligonucleotide sequence, (b) running for 35
minutes following electrokinetic injection of target Cy5 – dA20 and (c) after elution for 25
minutes at 60 °C. Affinity gel: 0.45 µM Cy3-dT20 probe in a 12.5%T linear polyacrylamide
gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dA20 at 267 Vcm-1 for 60
seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP for 35 minutes. Elution
condition: 267 Vcm-1 with 1x TBE/0.5 M NaSCN/0.1% PVP for 25 minutes at 60 °C. Images
were obtained using the Chipreader.............................................................................................. 51
Figure 3.2: Confocal fluorescence microscope images of affinity capture capillaries showing the
Cy3 (left) and Cy5 (right) channels from a portion of the capillary shown in Figure 3.1(a), which
shows the autofluorescence signal of the system before the loading of any fluorescently labelled
materials. Images were enhanced in ImageJ using the Window/Level function for better clarity.
....................................................................................................................................................... 52
Figure 3.3: Confocal fluorescence microscope images of affinity capture capillaries showing the
Cy5 channel (a) before loading of complementary target and (b) following elution. A
fluorescence signal was apparent following elution, indicating retention of 13.6 nM or 8 fmol of
target. Injection condition: 5 µL of a sample containing 1 µM Cy5-dT20 at 267 Vcm-1 for 60
seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1 %PVP for 35 minutes. Elution
xxi
condition: 267 Vcm-1 with 1x TBE/0.5 M NaSCN/0.1 %PVP for 25 minutes at 60 °C. Images
were enhanced in ImageJ using the Window/Level function for better clarity. ........................... 54
Figure 3.4: (a) Change in the average Cy5 fluorescence intensity over time, measuring the loss
of any adsorbed materials used in the pre-treatment of the columns. Pre-treatment protocol: a 5
µL sample of 2 µM Cy5-dC20 at 267 Vcm-1 for 2 minutes, followed by electrophoresis at 133
Vcm-1 in 1xTBE/0.1% PVP for 5 minutes, and a second injection of a 5 µL 2 µM Cy5-dC20 at
267 Vcm-1 for 2 minutes and electrophoresis at 133 Vcm-1 in 1xTBE/0.1% PVP for 15 minutes.
(b) The original fluorescence intensity of the capillary channel prior to the loading of any
material (baseline). Error bars represent 1 standard deviation of three trials. ............................. 54
Figure 3.5: Confocal fluorescence microscopy images of capillary affinity capture gel of the
Cy3 channel showing the elution of 0.45 µM Cy3-dT20 probe immobilized in a 7.5%T linear
polyacrylamide gel. The image was taken of the capillary in (a), following pre-conditioning and
pre-treatment and (b), following loading 1 µM Cy5-dA20 and running for 35 minutes. Injection
condition: 5 µL of a sample containing 1 µM Cy5-dA20 at 267 Vcm-1 for 60 seconds.
Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP for 35 minutes. Images were
acquired using the Chipreader. ..................................................................................................... 56
Figure 3.6: Effect of amount of target on the efficiency of capture using a 454 nM Cy3-dT20
probe affinity gel. The ratio of captured target versus available probe was plotted against
varying concentrations of Cy5-dA20 targets. Error bars are propagated error following
correlation of average fluorescence intensity to concentration using a calibration curve. ........... 60
Figure 3.7: Confocal microscope images of capillaries examining the use of a non-
complementary target in the affinity gel from (a) prior to loading the non-complementary target,
(b) after loading and running for 25 minutes and (c) after the elution step was applied. Affinity
gel: 0.45 µM Cy3-dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection condition:
5 µL of a sample containing 1 µM Cy5-dC20 at 267 Vcm-1 for 60 seconds. Electrophoresis
condition: 133 Vcm-1 with 1x TBE/0.1% PVP for 35 minutes. Elution condition: 267 Vcm-1
with 1xTBE/0.5 M NaSCN/0.1%PVP for 25 minutes at 60°C. Images were obtained using the
Chipreader..................................................................................................................................... 61
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Figure 3.8: Confocal microscope images of capillaries showing non-complementary target
(Cy5-dC20) as it moved electrophoretically through the capillary after (a) 60 seconds, (b) 120
seconds, (c) 240 seconds and (d) 420 seconds. Only the images of the Cy5 channel are shown.
Affinity gel: 0.45 µM Cy3-dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection
condition: 5 µL of a sample containing 1 µM Cy5-dC20 at 267 Vcm-1 for 60 seconds.
Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP. Images were obtained using the
Chipreader..................................................................................................................................... 61
Figure 3.9: Confocal microscope images of capillaries examining the loading of Cy3-dA8C5A8
(5 base pair mismatch) target through an affinity capture gel. Images were taken after (a) 5
minutes and (b) 30 minutes. Affinity gel: 1.8 µM dT20 probe immobilized in a 12.5%T
polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy3-dA8C5A8 at
267 Vcm-1 for 60 seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP.
Images were obtained using the Chipreader. The images in (b) was enhanced in ImageJ using
the Window/Level function for better clarity. .............................................................................. 62
Figure 3.10: Confocal microscope images of capillaries tracking a time course experiment for
loading a mixture of dT20-Cy3 and dC20-Cy5. Images shown were taken after (a) 120 seconds,
(b) 240 seconds, (c) 540 seconds and (d) 840 seconds. Affinity gel: 1.8 µM dA20 probe
immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition: 5 µL of a sample
containing 0.5 µM Cy3-dT20, 0.5 µM Cy5-dC20 at 267 Vcm-1 for 60 seconds. Electrophoresis
condition: 133 Vcm-1 with 1x TBE/0.1% PVP. Images were obtained using the Chipreader. ... 63
Figure 3.11: Profile plot taken from the inlet to outlet end of the capillary from confocal
microscope images of the Cy5 channel after a 4 minute run of the (a) fully complementary
(dT20-Cy3) target with (b) non-complementary (dC20-Cy5) targets in a dA20-probe affinity gel.
Images were acquired using the Chipreader. ................................................................................ 64
Figure 3.12: Profile plot taken from the inlet to outlet end of the capillary from confocal
microscope images of the Cy5 channel. Profile plots following migration of (a) Cy3-dT20 and
(b) Cy5-dC20 through an unmodified polyacrylamide gel after five minutes. The profile of the
entire capillary length is not shown. The distance is shown from the injection end to the elution
end. Images acquired using the Chipreader. ................................................................................. 65
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Figure 3.13: Confocal microscope images taken from an affinity gel column containing
immobilized dT20-probe after loading a 1:1 mixture of Cy3-dA8C5A8 and Cy5-dA20. Images
taken after (a) 120 seconds, (b) 240 seconds, (c) 360 seconds, (d) 660 seconds and (e) 960
seconds. Affinity gel: 1.8 µM dT20 probe immobilized 12.5%T linear polyacrylamide affinity
gel. Injection condition: 5 µL of a sample containing 0.5 µM Cy3-dA8C5A8 and 0.5 µM Cy5-
dA20 at 267 Vcm-1 for 60 seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1%
PVP. Images acquired using the Chipreader. The Cy3 channel image in (e) was enhanced using
the Window/Level function in ImageJ.......................................................................................... 66
Figure 3.14: Confocal microscope images taken from an affinity gel column containing
immobilized dT20-probe after loading a 9:1 mixture of Cy3-dA8C5A8 Cy5-dA20 target. Images
were taken after (a) 240 s, (b) 540 s and (c) 840s. Affinity gel: 0.9 µM dT20 probe immobilized
12.5%T linear polyacrylamide affinity gel. Injection condition: 5 µL of a sample containing 1.8
µM Cy3-dA8C5A8 and 0.1 µM Cy5-dA20 at 267 Vcm-1 for 60 seconds. Electrophoresis
condition: 133 Vcm-1 with 1x TBE/0.1% PVP. Images acquired using the Chipreader. ............ 67
Figure 3.15: Confocal microscope images of capillaries for the Cy5 channel demonstrating the
loading and capture of a 1 µM 40 nt target sequence, Cy5-dC10T20C10 through the affinity gel.
Images were taken after electrophoresis following electrokinetic injection for (a) 300 s and (b) 25
min. Affinity gel: 1.8 µM dA20 probe immobilized 12.5%T linear polyacrylamide affinity gel.
Injection condition: 5 µL of a sample containing 1 µM Cy5-dC10T20C10at 267 Vcm-1 for 60
seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP. Images were acquired
using the Chipreader. .................................................................................................................... 68
Figure 3.16: Fluorescence intensity values from the Cy5 channel as measured from confocal
microscope images of the capillary taken at various times during the capture and elution
experiment. Values were obtained by taking the average of the values generated from the profile
plot of the confocal image. The capillary containing the affinity capture matrix was first imaged
to establish the background fluorescence signal (‘before’). A 10 µL, 1.67 µM solution of the
Cy5 labeled 150 nt complementary target was then loaded into the affinity capture gel
(7.5%T/6%C, 1.8 µM β-actin probe) electrokinetically for 20 minutes at 167 Vcm-1 (‘load’)
(The fluorescence intensity following this step saturated the detector and the actual value is not
shown). The fluorescence intensity for the (‘wash’) step was taken after the entire capillary was
xxiv
heated to 95 °C for 5 minutes, allowed to sit for 20 minutes at 20 °C, and following the
application of a voltage of 167 Vcm-1 for 20 minutes at 20 °C. Finally the captured targets were
eluted by the application of a voltage of 167 Vcm-1 for 15 minutes at 65 °C. Images were
acquired using the Chipreader. ..................................................................................................... 72
Figure 3.17: Profile plots from the outlet end to inlet end of the capillary from confocal
microscope images obtained for the Cy5 channel of the capillary. a) Fluorescence intensity
profile of the Cy5 channel of the affinity gel following the capture of the (0.14 µM) Cy5 labeled
150 nt target. Affinity capture gel: 7.5%T, 6 %C, 3 µM β-actin probe. b) Profile of the Cy5
channel of the affinity gel following the capture of the (1 µM) Cy5 labeled 19 nt target (SMN).
Affinity capture gel: 10%T, 5%C, 0.5 µM affinity capture probe (SMN). Images were acquired
using the Chipreader. .................................................................................................................... 73
Figure 3.18: Profile plots from the outlet end to inlet end of the capillary from confocal
microscope images obtained for the Cy5 channel of the capillary. Profile plots for the capture of
a (0.14 µM) 150 nt target using a 20 nt length probe and a 10 nt length probe. Affinity capture
gel: 7.5%T, 6 %C, 3 µM affinity capture probe (β-actin). Images acquired using the Chipreader.
....................................................................................................................................................... 76
Figure 3.19: Examples of hairpin structures as calculated by OligoAnalyzer software. Settings
used for calculations were 25 °C, 50 mM Na+ concentration, suboptimality 50% and maximum
foldings 20. Probe region is highlighted in the drawn box. ......................................................... 77
Figure 3.20: A histogram of the number of partial interactions possible between the target, its
complementary strand and the probe as calculated by OligoAnalyzer software. The number of
interactions was binned by the number of base pairs forming the interactions. Calculation
conditions were oligonucleotide concentration = 0.25 µM, [Na+] = 50 mM................................ 78
Figure 3.21: Data for comparison of the amount of 150 nt target (100 nM) captured by the
affinity capture gel after the capillary was heated to 95 °C for 5 minutes following the injection
relative to the amount captured by an unheated column. Affinity capture gel: 7.5%T, 6 %C, 3
µM β-actin probe. Error bars are propagated error following correlation of average fluorescence
intensity to concentration using a calibration curve. .................................................................... 79
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Figure 3.22: Difference in the amount of target retained after the ‘wash’ step from capture and
elution experiments between affinity capture gels that were complementary (3 µM β-actin) and
non-complementary (3 µM non-β-Actin) to a 150 nt length DNA target (20 nM). The data was
obtained from confocal fluorescence images (Chipreader) of the capillaries and values were
obtained from the profile plot function. Error bars are propagated error following correlation of
average fluorescence intensity to concentration using a calibration curve. .................................. 80
Figure 3.23: Difference in the amount of target retained after the ‘wash’ step from capture and
elution experiments between affinity capture gels that were complementary (3 µM β-actin) and
non-complementary probe (3 µM non-β-Actin) to a Cy5 labelled 250 nt DNA target (10 nM).
The experimental conditions are the same as in Figure 3.17 except the last step in the ‘wash’ step
was applied for 40 minutes rather than 20 minutes. The data was obtained from confocal
fluorescence images (Chipreader) of the capillaries and values were obtained from the profile
plot function. Error bars are propagated error following correlation of average fluorescence
intensity to concentration using a calibration curve. .................................................................... 81
Figure 3.24: Difference in the amount of target retained after the ‘wash’ step from capture and
elution experiments between affinity capture gels that contained complementary (3 µM uidA)
and non-complementary (3 µM SMN) to a Cy5 labelled 400 nt DNA target (130 nM). The
experimental conditions are the same as in Figure 3.17 except the last step in the ‘wash’ step was
applied for 50 minutes. The data was obtained from confocal fluorescence images (Chipreader)
of the capillaries and values were obtained from the profile plot function. Error bars are
propagated error following correlation of average fluorescence intensity to concentration using a
calibration curve............................................................................................................................ 81
Figure 3.25: Amount of 150 nt target captured onto affinity capture gel as a function of
concentration. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of
150 nt target in 1xTBE/PVP, 20 minute electrokinetic injection at 133 Vcm-1. Incubation time: 5
min. Wash Step: electrophoresis at 133 Vcm-1 for 25 min at 25 °C with 1xTBE/PVP buffer. The
data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values
were obtained from the profile plot function. Error bars are propagated error following
correlation of average fluorescence intensity to concentration using a calibration curve. ........... 82
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Figure 3.26: Amount of 150 nt target captured from samples in mixtures of complementary and
non-complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection
step: 10 µL of 150 nt target and non-complementary target in 1XTBE/PVP, 20 minute
electrokinetic injection at 133 Vcm-1. Incubation time: 5 min. Wash Step: electrophoresis at 133
Vcm-1 for 25 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP buffer. The data was
obtained from confocal fluorescence images (Chipreader) of the capillaries and values were
obtained from the profile plot function. Error bars are propagated error following correlation of
average fluorescence intensity to concentration using a calibration curve. .................................. 83
Figure 3.27: Comparison of amount of material retained by the 150 nt complementary target
when treating with 100 nt non-complementary target. Affinity capture gel: 12.5%T/1%C, 3 µM
β-actin probe. Injection step: 10 µL of 40 nM complementary (150 nt Cy5-β-actin) and 45 nM
non-complementary targets (100 nt Cy3-LAMA3) in 1xTBE/PVP, 20 minute electrokinetic
injection at 133 Vcm-1. Incubation time 5 min. Wash Step: electrophoresis at 133 Vcm-1 for 25
minutes at 40 °C with 1xTBE/PVP with different concentrations of formamide. Data was derived
from images obtained from epifluorescence images of Cy3 channel (Alpha). Error bars represent
1 standard deviation of three trials................................................................................................ 84
Figure 3.28: 1% Agarose gel electrophoresis for non-complementary target used in efficiency
experiments. Lane 1: DNA Ladder. Lanes 2 and 3: non-complementary target used in factorial
analysis. Run conditions: 100 V, 1 hour, 1x TBE buffer............................................................ 85
Figure 3.29: Amount of material captured for 150 nt target in mixture of constant concentration
of non-complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe.
Injection step: 10 µL of 150 nt target and 1.5 pmol of non-complementary target in 1XTBE/PVP,
20 minute electrokinetic injection at 133 Vcm-1. Incubation time: 5 min. Wash Step:
electrophoresis at 133 Vcm-1 for 25 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP
buffer. The data was obtained from epifluorescence microscope images (Alpha) of the capillaries
and values were obtained from the profile plot function. Error bars are propagated error
following correlation of average fluorescence intensity to concentration using a calibration curve.
....................................................................................................................................................... 86
Figure 3.30: Amount of material captured for 250 nt target in mixture of constant concentration
of non-complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe.
xxvii
Injection step: 10 µL of 250 nt target and 1.5 pmol of non-complementary target in 1XTBE/PVP,
30 minutes electrokinetic injection at 133 Vcm-1. Incubation time: 5 min. Wash Step:
electrophoresis at 133 Vcm-1 for 40 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP
buffer. The data was obtained from epifluorescence microscope images (Alpha) of the capillaries
and values were obtained from the profile plot function. Error bars are propagated error
following correlation of average fluorescence intensity to concentration using a calibration curve.
....................................................................................................................................................... 87
Figure 3.31: Amount of 400 nt target captured in mixture of non-complementary targets onto
affinity capture gel as a function of concentration. Affinity Capture Gel: 12.5%T/1%C, 3 µM
uidA probe. Injection step: 10 µL of 400 nt target and 1.5 pmol of non-complementary target in
1XTBE/PVP, 34 minutes electrokinetic injection at 133 Vcm-1. Incubation time: 5 min. Wash
Step: electrophoresis at 133 Vcm-1 for 50 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP
buffer. The data was obtained from epifluorescence microscope images (Alpha) of the capillaries
and values were obtained from the profile plot function. Error bars are propagated error
following correlation of average fluorescence intensity to concentration using a calibration curve.
....................................................................................................................................................... 87
Figure 3.32: The relative fluorescence intensity following reaction between a 1 µM Cy3 labeled
oligonucleotide with different ratios of TEMED/APS radical initiator system after a period of 20
minutes. The loss was calculated relative to the in initial fluorescence intensity measured of the
solution immediately following the addition of the radical initiator. Error bars represent 1
standard deviation of three trials................................................................................................... 92
Figure 3.33: UV-VIS spectra of bulk polyacrylamide gels before and after polymerization for
20 minutes as a function of different gel formulations. a) 12.5%T/5%C, b) 12.5%T/1%C, c)
7.5%T/5%C, d) 7.5%T/1%C ........................................................................................................ 94
Figure 3.34: Reaction products between the sulfate radical anion and (a) adenine, (b) guanine,
(c) cytosine and (d) thymine. Adapted from [207]. ..................................................................... 97
Figure 3.35: Scheme of the reaction between the radical and the nucleotide base that can lead to
either removal of the nucleoside base or strand cleavage. Adapted from [213]........................... 98
xxviii
Figure 3.36: Electrophoretogram for a solution containing 2 µM 12nt, 0.5 µM 19 nt and 1.0 µM
20 nt oligonucleotides. Cy5 labeled targets. Injection: 10 µL sample volume, 142 Vcm-1, 4 s.
Run condition: 142 Vcm-1 in 1xTBE/PVP buffer........................................................................ 99
Figure 3.37: Electrophoretograms of the reaction products between a 19 nt Cy5 labelled target
and different amounts of TEMED/APS. Injection: 10 µL sample volume, 142 Vcm-1, 4 s. Run
condition: 142 Vcm-1 in 1xTBE/PVP buffer. a) Control b) TEMED/APS in 10%/10%. c)
TEMED/APS 10%/4%, d) TEMED/APS 4%/10%, e) TEMED/APS 4%/4% ........................... 102
Figure 3.38: Representative melt curve for a sample of 0.3 µM 19 bp duplex (SMN) in 1 x TBE.
Error bars represent 1 standard deviation of three trials. ............................................................ 104
Figure 3.39: Melt curves for a sample of 0.3 µM 19 bp duplex in 1 x TBE where the probe was
reacted with different amounts of TEMED and APS for 20 minutes prior to addition of the
complementary target. a) TEMED/APS 10%/10%, b) TEMED/APS 10%/4%, c) TEMED/APS,
4%/10%, d) TEMED/APS 4%/4%. Error bars represent 1 standard deviation of three trials. ... 106
Figure 3.40: Decrease in average fluorescence intensity as labelled DNA targets wash out of the
capillary following injection of the DNA target into affinity capture gels containing
complementary (�) and non-complementary (x) probe. Fluorescence intensity is normalized
against the initial fluorescence intensity. Experimental conditions: Affinity capture gel: 10%T,
5%C, 2 µM β-actin probe, 2 µM non-β-actin probe. Injection conditions: 10 µL, 250 nM Cy5-
50 nt target, electrokinetic injection at 133 Vcm-1 for 20 minutes. Incubation Time: 5 mins.
Wash conditions: 133 Vcm-1 at 25 °C with 1xTBE/PVP. The data was obtained from confocal
fluorescence images (Chipreader) of the capillaries and values were obtained from the profile
plot function. Error bars represent 1 standard deviation of three trials....................................... 111
Figure 3.41: Concentration of Target Injected. Values corrected for differences in fluorescence
intensity as a function of different gel formulations. Affinity capture gel: Conditions as
prescribed in factorial design. Injection: 10 µL of 170 nM (low probe) 500 nM (high probe)
target. Electrokinetic injection at 133 Vcm-1 for 20 minutes. The data was obtained from confocal
fluorescence images (Chipreader) of the capillaries and values were obtained from the profile
plot function. Error bars are propagated error following correlation of average fluorescence
intensity to concentration using a calibration curve. .................................................................. 113
xxix
Figure 3.42: Electrophoretic mobility of 150 bp target using different gel filled capillaries with
different gel formulations. Gel formulations used were as previously prescribed, with radical
initiator concentrations of TEMED/APS of 10%/10%. Mobility calculated based on the time
required to travel 2.6 cm along the capillary. Injection conditions, 10 µL, 0.5 µM Cy5-150 bp
target, 86 Vcm-1, 15 s. Run conditions, 143 Vcm-1, 1x TBE/PVP buffer. PMT gain 400 mV.
Error bars represent 1 standard deviation of three trials. ............................................................ 114
Figure 3.43: Amount of material captured by affinity capture gel. Values were corrected for
differences in fluorescence intensity as a function of different gel formulations. Affinity capture
gel: Conditions as prescribed in factorial experiment. Injection: 10 µL of 170 nM (low probe)
or 500 nM (high probe) Cy5-150 nt target. Electrokinetic injection at 133 Vcm-1 for 20 minutes.
Incubation time: 5 mins at 10 °C. Wash Step: electrophoresis at 133 Vcm-1 for 25 mins at 10 °C
with 1xTBE/PVP. The data was obtained from confocal fluorescence images (Chipreader) of the
capillaries and values were obtained from the profile plot function. Error bars are propagated
error following correlation of average fluorescence intensity to concentration using a calibration
curve............................................................................................................................................ 116
Figure 3.44: Schematic representation of polyacrylamide gel and how oligonucleotide probes
are incorporated into the gel. a) indicates how gels with large pores are formed and b) indicates
how gels with small pores are formed. ssDNA with hairpin structures are presented in the center
and illustrates the difference between how DNA might move through the different gel structures;
a) unhindered via Ogston, and b) stretched by reptation. ........................................................... 118
Figure 3.45: Amount of target captured for a 19 nt probe/target pair at different monomer and
crosslinker levels as examined in the factorial analysis. Affinity capture gel: 0.5 µM SMN
probe. TEMED and APS used were at 10% w/v and v/v, respectively. Injection: 10 µL, 0.5 µM
Cy5 complementary target. Electrokinetic injection at 133 Vcm-1 for 10 min. Incubation time: 5
mins at 10 °C. Wash step: electrophoresis at 133 Vcm-1 for 15 min at 10 °C with 1xTBE/PVP.
The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and
values were obtained from the profile plot function. Error bars are propagated error following
correlation of average fluorescence intensity to concentration using a calibration curve. ......... 120
Figure 3.46: log(µ/µo) versus number of bases for DNA fragments from a Low Range DNA Gel
ladder (Fast Ladder) using TOPRO3 in different gel formulations. Capillary Gel: Different gel
xxx
formulations as described in factorial analysis with 1 µM TOPRO-3. Injection: 10 µL of ladder
incubated with 10 µM TOPRO3 (1:1), 5 seconds, 570 Vcm-1. Run: 93 Vcm-1 in
1xTBE/PVP/1µM TOPRO3. Acquisition settings: PMT Gain 400 mV, sampling rate 1Hz. .. 122
Figure 3.47: Percent recovery and percent purity of sample following washing of the affinity
capture gel. Data represents the average from the duplicates defined in the factorial design.
Affinity capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt
target and 12 nM of non-complementary target. Electrokinetic injection at 181 Vcm-1 for 20
minutes. Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm-1 for 25
minutes at 10 °C, 25 °C, 40 °C using 0%, 10% and 25% v/v formamide of 1xTBE/PVP. The data
was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were
obtained from the profile plot function. Error bars are propagated error following correlation of
average fluorescence intensity to concentration using a calibration curve. ................................ 128
Figure 3.48: Fluorescence profile plots of capillaries taken from the outlet to inlet end generated
from confocal microscope (Chipreader) images tracking the non-complementary target following
affinity capture and a subsequent wash step using two different stringency conditions. Affinity
capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and 12
nM of non-complementary target. Electrokinetic injection at 181 Vcm-1 for 20 minutes.
Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm-1 for 25
minutes, at (a) 10 °C with 1xTBE/PVP and (b) 40 °C with 25%v/v formamide/1x TBE/PVP. 131
Figure 3.49: Profile plots of capillaries from the outlet to inlet end from confocal microscope
(Chipreader) images of the complementary target following affinity capture the wash step in the
affinity capture and a subsequent wash step using two different stringency conditions. Affinity
capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and 12
nM of non-complementary target. Electrokinetic injection at 181 Vcm-1 for 20 minutes.
Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm-1 for 25
minutes, at (a) 10 °C with 1xTBE/PVP and (b) 40 °C with 25%v/v formamide/1x TBE/PVP. 132
Figure 3.50: Electrophoretograms comparing the relative fluorescence intensities
(concentrations) of short oligonucleotide target by step elution from complementary and non-
complementary probes. a) Affinity capture gel: 50 nM SMN probe, 10% LAAm. Target
injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150 Vcm-1. b) Affinity capture gel: 50 nM
xxxi
β-actin probe, 10% LAAm. Target injection: 10 µL 5 µM Cy5-SMN target for 1 min at 150
Vcm-1. Capture step: 10 min, 1xTBE/PVP running buffer, 150 Vcm-1. Concentrating step:
coverage length: 25 mm; step size: 250 µm; step rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition
settings: ND 4, 8 and 16 filters, PMT gain 400 mV. Sampling rate: 10 Hz. ............................ 135
Figure 3.51: Schematic diagram of different permutations of the step elution sweeps where a)
the resistive heating element was started at the same point at the injection end of the capillary,
and the terminal position was varied. In b), the step elution again swept through different
distances, but stopped at the same position along the capillary.................................................. 137
Figure 3.52: Schematic diagram illustrating two possible orientations for creating an
interconnect between the capillary column and the microfluidic channel. The interconnect can
be created by orienting the capillary (a) orthogonal to the microfluidic channel and (b) in-plane
with the microfluidic channel. The area that is shaded in blue represents the filled area of the
capillary and microfluidic channel.............................................................................................. 143
Figure 3.53: Line scans of the microfluidic channel of the DNA biosensing platform following
delivery of fluorescently-labelled complementary target by selective concentration. Affinity
capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL 5 nM A647 SMN
target, electrokinetic injection for 1 min at 150 Vcm-1. Capture: electrophoresis for 10 min at
150 Vcm-1 with 1x TBE/PVP running buffer. Concentration step: coverage length: 12.5 mm;
step size: 250 µm; step rate: 86 µms-1; Voltage: 96 Vcm-1; Delivery of concentrated targets into
microfluidic biosensing platform: 500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition
settings: PMT gain 700 mV, translation speed: 50 µms-1 scan rate: 50 Hz. .............................. 144
Figure 3.54: Line scans of the fluorescence intensity along a microfluidic channel of the DNA
biosensing platform following delivery of complementary target by selective concentration. a)
both pad probe spots are complementary to the SMN target sequence, and b) one probe pad is
complementary (SMN) and the second is non-complementary (β-actin probe). Affinity capture
gel: 100 nM SMN probe, 10% LAAm gel. Target injection: a) 10 µL 1 nM A647 SMN target,
b) 10 µL 1nM A647 SMN target, 1 µM Cy3 β-actin target, electrokinetic injection for 1 min at
150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer.
Concentration step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms-1; Voltage:
96 Vcm-1; Delivery of purified and concentrated targets into microfluidic biosensing platform:
xxxii
500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV,
translation speed: 50 µms-1 scan rate: 50 Hz............................................................................... 147
Figure 3.55: Response of the microfluidic based DNA biosensing platform to quantities of
complementary target a) average fluorescence intensity signal level and b) integrated
fluorescence intensity. Different concentrations of DNA were mixed in 10% LAAm gel and
injected into an empty fused silica capillary using a syringe to CE adapter. Delivery of
complementary oligonucleotide into microfluidic biosensor: 500 V, 10 minutes, 1xTB/PVP/20
mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms-1, scan rate: 50
Hz. Error bars represent 1 standard deviation of three trials. ..................................................... 148
Figure 3.56: The response of the microfluidic biosensing platform for samples containing
complementary and non-complementary target comparing delivery with and without selective
concentrating. Direct Injection: mixture of A647 SMN and Cy3 β-actin target in 10% LAAm
gel. Selective concentrating: Affinity capture gel: 100 nM SMN probe, 10% LAAm gel.
Target injection: 10 µL of A647 SMN and Cy3 β-actin target, electrokinetic injection for 1 min
at 150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer.
Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms-1; Voltage:
96 Vcm-1; Delivery of oligonucleotide targets into microfluidic biosensing platform: 500 V, 10
minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed:
50 µms-1 scan rate: 50 Hz. Error bars represent 1 standard deviation of three trials. ................. 151
Figure 3.57: Effect of the probe concentration on the amount of target introduced into the
affinity gel by electrokinetic injection. Affinity gel: Varying concentrations of dA20 probe (1.8
µM, 0.45 µM, no probe) in a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL
sample containing 0.5 µM Cy3-dT20 at 267 Vcm-1 for 60 seconds. The amount of target in the
original sample was 2.5 pmol. Error bars represent 1 standard deviation of three trials............ 154
Figure 3.58: Comparison of the response of the microfluidic biosensing platform (containing
probe for SMN) for a sample containing A647-SMN and Cy3-β-actin targets as prepared by
selective concentrating and concentrating of the all oligonucleotide targets. Delivery of
oligonucleotide targets by selective concentrating was done as previously described. Delivery of
oligonucleotide targets by non-selective pre-concentration was done using 10% LAAm affinity
xxxiii
capture gels which contained 100 nM SMN probe and 5 µM β-actin probe. Error bars represent 1
standard deviation of three trials................................................................................................. 158
xxxiv
List of Appendices
A. Factorial Design Experiment ................................................................................................. 182
A1. Fractional Factorial Designs............................................................................................ 183
A2. Design Matrix for Quarter Fractional Factorial Design .................................................. 184
A3. Choice of Factors and Levels .......................................................................................... 185
B. Synthesis of DNA Targets...................................................................................................... 186
B1. Construction of 250 bp Target......................................................................................... 186
B2. Construction of 400 bp Target......................................................................................... 189
B3. Confirmation of DNA Targets......................................................................................... 190
B4. Examination of Sequence for the 150 nt Target .............................................................. 195
C. Generation of Single Stranded DNA Targets......................................................................... 199
D. Suppression of Electroosmotic Flow (EOF) in Capillary Affinity Capture Gels ................. 203
E. Factorial Analysis for Probe Incorporated into Affinity Capture Gel .................................... 207
E1. Table of Results ............................................................................................................... 207
E2. Factorial Analysis for Percentage of Probe Incorporated................................................ 207
E2.1 Analysis of Results ................................................................................................. 207
E2.2 Pareto Effects Plot .................................................................................................. 208
E2.3 Magnitude of Effects .............................................................................................. 208
E2.4 ANOVA Table........................................................................................................ 210
E2.5 Normal Probability Plot.......................................................................................... 211
E2.6 Examination of Model Adequacy........................................................................... 212
E3. Amount of Probe Incorporated ........................................................................................ 213
F. Amount of Target Captured.................................................................................................... 216
F1. Data Reflecting the Quantity of Target Captured ............................................................ 216
F2. Concentration of Target Injected ..................................................................................... 216
xxxv
F3. Amount of Target Captured ............................................................................................. 219
G. Evaluation of Hybridization and Stringency Conditions ...................................................... 222
G1. Effectiveness of Gels for Purification of Target ............................................................. 222
G2. Hybridization Time and Wash Voltage for Samples Containing Only Complementary Targets............................................................................................................................. 222
G3. Stringency Conditions for Samples Containing Complementary and Non-complementary Targets................................................................................................... 223
H. Effect of Step Size in the Step Elution Process ..................................................................... 226
1
Chapter 1 Introduction
Nucleic acid (NA) sequences can be used for species identification, to identify the presence of
pathogens, and to detect genes and gene mutations that are associated with risk of various
dysfunctions. NAs typically represent a relatively small component of the biological make up of
cells, and are in mixture with a wide variety of other molecules when real samples from
biological sources are considered. The intention of the work in this thesis is the investigation and
development of a method for the selective concentration of one or more targeted NAs within a
mixture, for delivery to a microfluidic based NA biosensor. The DNA targets of interest were
captured selectively onto an affinity gel inside a capillary column. This affinity capture gel
consisted of oligonucleotide probes incorporated into a polyacrylamide gel matrix.
Concentrating of the captured targets was accomplished by eluting the targets into a smaller
volume than the original sample solution. This was achieved by using a localized thermal elution
zone that was swept across the capillary to stack the targets as they dehybridized from the
capture gel. Selective concentrating of the DNA target prior to detection was intended to
improve the limit of detection of the biosensor by increasing the concentration of the target DNA
while removing non-complementary NA sequences from the original sample solution. An
overview of the importance of sample pre-treatment protocols that are used prior to the detection
of DNA from real world samples, as well as some of the most common methods for DNA
purification will first be considered.
1.1 DNA Biosensors for Detection of Real-World Targets
A DNA biosensor is a self-contained device that is designed to provide quantitative information
about the presence of a specific target. It typically uses single stranded oligonucleotide probe
sequences to interact with a specific region of the target. Typically these probes are immobilized
onto a surface near the transduction element to integrate the biological recognition element with
the transducer [1, 2]. When the target DNA hybridizes with the immobilized probe sequences,
the extent of binding is interrogated to produce a signal by the transduction strategy, ultimately
resulting in production of a quantifiable electrical signal [3, 4].
2
Biosensors are intended to operate as highly sensitive and selective devices with the ability to
achieve detection of low quantities of targets, and excellent discrimination within mixtures of
closely related compounds [3, 5]. Biosensors are defined as reusable devices, and it is desirable
that the sensor can quickly be regenerated to be capable of analysis of a series of samples [1, 3,
5, 6]. DNA based biosensors have been of interest for the rapid and sensitive detection of targets
such as pathogens in environmental samples, meat, water and soil; the presence of an infectious
disease in various clinical samples such as blood, urine or saliva; and for the identification of
genetic markers which differ by as little as one base pair [1, 2, 5, 7, 8]. Such applications require
rapid detection of very low concentrations of nucleic acids in complex sample matrices [9].
Early detection of diseases may allow for early treatment which may provide a better outcomes
[10–12]. Small amounts of foodborne pathogens can result in illness. For pathogens such as
Listeria monocytogenes and Campylobactor spp, infectious doses starting from 400 Colony
Forming Units (CFU) have been reported [3]. Ten bacterial cells of E. coli O157:H7 can result in
infection [13]. Inhalation of more than 104 spores of Bacillus anthracis requires medical
attention within 24-48 hours. However, symptoms can take up to 60 days to appear in humans,
delaying necessary treatment [14].
In addition to the need to detect low concentrations of target, it must be recognized that the target
is also present in a large amount of background that can interfere with analysis. For example, E.
coli O15:H7 in stool samples require detection of below 105 CFU/mL, and are contained in a
complex mixture of non-pathogenic strains [15]. For blood samples, the amount of genomic
DNA present from the blood may be 1014 times higher than that DNA from pathogenic species
[9]. Cells in whole blood are predominantly erythrocytes and account for 99% of total cells,
while the DNA containing leukocytes make up less than 1% [16]. Detection of B. anthracis from
soil samples requires differentiation with other closely related non-pathogenic Bacillus species
such as B. cereus, B. mycoides and B. thuringiensis [17]. The presence of a high background in a
sample matrix may hinder detection and can compromise selectivity [9, 15]. If sample analyte is
in very low concentration, detection may become an issue if the signal generated by background
is sizeable [18]. Components in the sample matrix may also affect the long-term stability of the
recognition element and transducer [9]. These can include nucleases which can degrade the DNA
target and immobilized probes, compounds which can inhibit target amplification by Polymerase
3
Chain Reaction (PCR), and aggregation factors or other cells or cell fragments that can clog
downstream processing [13, 15].
Currently, tests for the identification of cell-based pathogens are usually performed with
inclusion of a culturing step. The cells are allowed to grow and multiply in selective media to
enrich the concentration, and this is followed by biochemical tests selective towards that strain's
phenotypes. This can take 20 hours to several days to complete [8, 9, 19, 20]. There is a need for
technology that can offer more rapid detection of targets with high sensitivity in complex
matrices at the point of care level [9, 15, 21, 22]. DNA biosensors have demonstrated
quantitative function within several hours, depending on the type of sample preparation required
[9, 20, 23, 24].
Table 1.1 provides some examples of biosensors that have been applied for the detection of DNA
targets in real samples. Listed are the transduction method, type of sample pre-treatment
protocol as well as the limit of detection that was reported. Additionally, the total analysis time
from the point of obtaining the sample to the actual quantitative detection step is provided. This
information was compiled based on the time for each pre-treatment step as listed in the
experimental method, as well as time that was indicated for detection. The time required for
only the detection step is also shown when available. Some of the pre-treatment protocols listed
will be discussed further.
As can be seen in Table 1.1, detection of targets from real samples often require multiple pre-
treatment steps for purification and to also concentrate the targets prior to detection. Some of
these pre-treatment steps can become quite involved, and in some cases can take more time than
the actual analysis.
Sample purification and concentrating are typically necessary for detection of NA targets from
real samples. In addition to removal of background matrix and amplification of the target, the
NA targets must be released from inside cells, and must be in a form in terms of length and
folding that it is amenable for detection using hybridization [18]. Concentrating the NA targets
can occur by reduction of volume in which the analyte is contained and can alleviate sensitivity
demands placed on a detector [18, 25]. For NA analysis, a more common strategy for
improvement of detection sensitivity is amplification of NAs during sample pre-treatment by
Polymerase Chain Reaction (PCR).
4
Table 1.1: Examples of biosensors used for detection of NA targets from various different sample matrices. Steps for pre-treatment protocols prior to detection are identified, as well as the total time
needed for pre-treatment and detection. When available, detection time is also provided in parenthesis.
Transduction Mode
Target Sample Pre-treatment
Protocol LOD
Total Analysis
Time Ref
Spectroscopic E. coli In buffer
Culture enrichment,
centrifugation, lysis, chloroform-
phenol extraction,
ultrasonication
1 pg/mL
> 12 hours (20
second detection)
[26]
Visual Vibrio cholerae Clinical
Samples
PCR, concentration by
lateral flow
5 ng in 10 µL
~1 hour (10 min
detection) [27]
SPR Tumor necrosis
factor (TNF-alpha)
In buffer PCR,
denaturation by heat
0.677 pM
~120 min [23]
Piezoelectric HPV Clinical cervical
scrapings
Centrifugation, DNA Extraction
(Commercial Kit), PCR
50 nM ~10 hrs (20 min
detection) [28]
Piezoelectric E. coli Water
Culture enrichment, lysis,
phenol-chloroform extraction,
centrifugation, PCR,
denaturation
1 µg/mL >12 hrs (10 min
detection) [29]
Piezoelectric E. coli O157:H7 In buffer
Culture enrichment,
centrifugation, extraction,
Asymmetric PCR
2.67x102
CFU/mL 22 hrs [30]
Piezoelectric Aspergillus
flavus and A. parasiticus
Flour/feed Extraction, PCR 0.03 µM 2.5 hours (20 min
detection) [31]
Piezoelectric HPV Clinical samples
Plasmid extraction
1.21 pg/L
3 hours [32]
Piezoelectric CaMV 35S GMO -
Tabacco Plants
Extraction, ultrasonication
0.25 ng/µL
30 minutes
[33]
Electrochemical Phosphinothricin acetyltransferase
GMO soybean
PCR 2.7x
10-14
M
2 hours (40 min
detection) [34]
Electrochemical E. coli Water Magnetic beads,
heat lysis/denaturation
0.5ng/µL
40 minutes
(30 minute
detection)
[35]
Electrochemical E. coli O157:H7 Cultured samples
Centrifugation, cell lysis,
concentration using magnetic
2.5 aM , 0.01
CFU/mL 6 hours [8, 20]
5
beads, asymmetric PCR
Electrochemical Human
Interleukin 2
From cultured samples
Centrifugation, Liquid N2 lysis,
genomic extraction,
purification, PCR
69 pM 3 hours (15 min
detection) [24]
1.2 Goals of Pre-treatment
The major goals of the pre-treatment of a sample for detection by a biosensor are: extraction of
the target material from the sample matrix, the purification of the target, sample enrichment
through amplification or volume reduction and conversion of the NA targets into short, single
stranded fragments.
1.2.1 Extraction
Extraction may be required to isolate the analyte of interest from the background matrix. For the
extraction of NA, the cells of interest must be lysed and the NA separated from the intracellular
material. Common methods for isolation of NAs from the sample include filtration and
centrifugation. Cell lysis can be accomplished by chemical or mechanical means. Such
techniques have been reported and extensively reviewed [36–43].
1.2.2 Purification
The removal of background components may be necessary to avoid inhibition or competition
with the bio-recognition between the probe and its target [44, 45], or if contaminants can also
generate a signal [37, 46]. Since biological materials are generally used as the selective reagents
to develop biosensors, issues related to the stability of the probes in the background matrix may
arise [37].
Purification may also be required in order to differentiate between viable and dead cells prior to
the extraction of DNA. This is important where only viable cells are of interest; for example in
the detection of a pathogen in consumable products where only live cells will adversely affect
human health. One challenge is that extraction methods will extract NAs from both viable and
dead cells [44], making it very difficult to differentiate whether there is any actual danger
associated with biologicals in a consumable product [44, 47, 48].
6
Purification of samples can also be important when considering the quantitative determination of
a target. Variations in the components of the matrix between samples may pose a problem for
quantitative analysis [1]. Calibration curves are often constructed using solutions of known
concentrations of the target in water or buffer. Unless steps are taken to ensure that the
background matrix from a “real” sample would not interfere with the quantitative determination,
purification is necessary to ensure that the background matrix of the sample was compatible with
that used with the calibration curve [1]. Alternatively, a standard addition approach might be
considered, but this is of little interest if the matrix interferes with signal development.
The majority of purification protocols done to extract DNA are intended to remove compounds
that might inhibit amplification of NA targets by PCR. PCR is a common step for amplification
of NAs prior to detection by a biosensor to improve sensitivity, and can also serve in itself as a
detection strategy, confirming the presence or absence of the analyte [8]. As can be seen from
the examples listed in Table 1.1, PCR amplification is commonly used to amplify the target
sequence.
Environmental samples, such as those from water, soil and food materials are known to contain
compounds which can inhibit PCR or that can interfere with detection [49–51]. Such
compounds include humic acids, polyphenolic compounds, polysaccharides, urea, soot, dust and
pollen, silt, clay, metal ions, chelators, milk products, fat in foods, hemoglobin, iron, heparin,
acidic polysaccharides, as well as NAs from non-target microorganisms [38, 45, 49, 50, 52–54].
Humic acids and polysaccharides can bind to DNA polymerase, and to chelating agents which
may be co-factors for the enzyme. It has been shown that as little as 1 ng of humic acid can
inhibit PCR [45]. Haemachrom in erythrocytes is also known to be an inhibitor of PCR.
Removal of red blood cells from white blood cells is required for NA purification [16].
1.2.3 Sample Loss and Quantification
The sample pre-treatment protocols outlined in examples in Table 1.1 often involve multiple
steps before the target molecules can be detected by a biosensor. Therefore, consideration must
be given that with each purification step taken, there is the possibility of loss of the analyte by
partial transfer and non-selective adsorption. An extensive multi-step protocol may lead to
significant loss of target, which can be disproportionately detrimental in cases where the quantity
7
of target is low [55]. Oftentimes each sample pre-treatment protocol is optimized to suit each
application and sample type, resulting in variations in the pre-treatment protocols [44, 56, 57].
The loss induced by the purification protocol must be accounted for when quantitative
information provided by a biosensor is to be meaningful. However, achievement of reproducible
efficiency of a purification protocol is not always a simple matter. For example, differences in
cell wall structures may also result in differences of adhesion behaviour of cells with particulate
matter in the sample, resulting in variation of loss due to adsorption [36, 58]. Differences
between bacterial cells will also alter the effectiveness of typical lysis protocols when attempting
to release intracellular materials for assay [36, 56, 58]. While gram-negative organisms were
successfully lysed using an alkaline agent, gram-positive organisms, having a thicker cell walls,
required the action of a surfactant (TritonX-100) and an enzyme (lysozyme) in order to
efficiently lyse the cells [59, 60]. It was reported by Honoré-Bouakline et al. and Kotlowski et
al. that some commercially available kits do not allow for the complete lysis of mycobacterial
cells such as Mycobacterium tuberculosis [59, 61].
The recovery (quantity recovered) and purity (relative amount of target within sample) of
extracted NA often varies depending on the protocol that is being used, as well as the
composition and type of sample [36, 44, 54, 62, 63]. The recovery of DNA from standard
phenol-chloroform extraction was demonstrated to be lower when using a sample such as canned
tuna in comparison to DNA extracted from raw, fresh tuna [64]. Purification methods such as
gradient centrifugation, glass bead extraction, chromatography and spin columns may also result
in a significant loss of extracted NA [50, 54, 65]. As a general rule, the more extensive the
purification protocol, the lower will be the recovery of NA [54, 66]. Additionally, since many
biological molecules are charged, loss due to adsorption by electrostatic interaction with the
materials used for purification is common.
Many of the established purification protocols can only provide semi-quantitative assays unless
steps are taken to account for the loss of material during purification [67]. Quantification at each
stage of a multi-step extraction procedure is considered important to account for loss during
purification [68].
8
1.3 Methods for DNA Purification
The following presents an overview of some of the most common methods used for DNA
purification. Most are based on the capture of the DNA onto a solid support surface, removal of
any non-DNA material by washing, and elution from the support surface. Many of the methods
are not selective towards specific DNA sequences, while a few methods exist where the target of
interest is purified selectively.
1.3.1 Purification Methods based on Solid Phase Extraction (SPE)
Solid phase extraction is a commonly used technique for the purification of DNA from a
complex matrix following cell lysis [69, 70]. DNA will bind to silica or glass particles by
hydrogen bonding or electrostatic interactions in the presence of a chaotropic agents such as NaI,
NaClO4, guanidine hydrochloride or guanidine thiocyanate [59, 71–73]. The captured DNA can
be eluted by application of a low salt buffer [72].
The use of solid phase extraction eliminates the need for organic solvents associated with
conventional extraction and purification techniques such as phenol-chloroform extraction [70].
Many commercially available kits for extraction and purification of DNA also use a solid phase
extraction step [69, 70]. Solid phase extraction has been used for the purification of DNA from a
wide variety of different types of samples including plants, fungi, micro-organisms as well as
human hair, teeth, bone and blood [70]. Concentrating the sample can be accomplished by
eluting the DNA into a smaller volume than the original sample [72, 74].
Common solid phase extraction materials include hydrophobic surfaces such as alkyl-bonded
silicas (C18, C20), and copolymers such as cross-linked polystyrene and divinylbenzene [2, 75].
Normal phase materials such as those containing diol, aminopropyl or cyanopropyl functional
groups have also been used [75].
Proteins that are present in a biological sample can also bind to solid phase extraction material,
limiting the binding capacity of DNA on the columns [76]. Capture is non-selective, and any
compounds with similar properties to NAs may also be retained by the resin, decreasing the
purity of the extracted material [77].
9
Landers’ group has demonstrated a SPE purification system for DNA by using monolithic
extraction columns made from 3-(trimethoxysilyl)propyl methacrylate (TMSPM) modified with
85% v/v tetramethyl orthosilicate (TMOS). The addition of TMOS has been shown to increase
DNA binding by the monolith. Elution of the captured targets into a final volume of 1 µL was
done by using a low ionic strength Tris buffer at pH 8. Solid phase capture of PCR amplified
380 bp human genomic β-globin fragments as well as human genomic DNA purified from blood
was demonstrated. Both samples still required the addition of guanidine hydrochloride prior to
binding. Extraction efficiency as quantified by a PicoGreen assay was 86% for the PCR
amplified genomic DNA and 60% for DNA from whole blood. These efficiencies were greater
than DNA purified using a commercially available solid phase extraction kit [78].
Chromatographic techniques such as size exclusion, ion-exchange and affinity chromatography
has also been demonstrated for SPE purification of DNA [36, 79]. Gel filtration is a common
method for size exclusion that can purify DNA with minimal loss of the target [36, 80].
Ion exchange columns can selectively bind or elute DNA based on the pH and ionic strength of
the buffers used. Anion-exchange has been the most prominent technique. Positively charged
diethylaminoethyl (DEAE) tertiary or quaternary amine anion exchange resins will bind to
negatively charged DNA [79]. Elution of NA from the resin can be done by adjusting the pH of
the mobile phase [77]. Recovery of 80% of NA bound on the resin has been reported [36, 79].
Polymeric monoliths can be used as an alternative to resins. Monoliths are single pieces of
porous material where the pores are interconnected, forming channels with diameters ranging
from 13 nm to 4000 nm and 60-80% porosity within the monolith material. The pore size can be
selected to increase the surface area available for binding of NAs. Monoliths tend to offer good
mass transfer between mobile and stationary phases within channels and low back pressures at
high flow rates [36, 81, 82]. Methacrylate-based monoliths have been demonstrated for the
separation and purification of varying sizes of DNA molecules. For example, DNA binding was
calculated to be 9 mg/mL for a number of different DNA targets, including plasmid DNA from
E. coli as well as 200 kbp and 50 kbp genomic DNA. These results are notable when compared
to binding of less than 200 µg/mL on anionic Sepharose resins. Recovery of the DNA was
achieved by adjusting pH and ionic strength of the elution buffer. Under optimal conditions,
recoveries of up to 80% were obtained within 10 minutes [79].
10
A DEAE monolithic anion exchange column has been used to purify genomic DNA [82].
Samples of bacterial and eukaryotic genomic DNA purified by a commercially available kit were
tested on the monolithic column. Elution of the DNA was performed by increasing the
concentration of salt in the elution buffer. The 3 mm x 12 mm monolithic column could also be
used to process large volumes of DNA samples. Binding of ~4 mg of DNA from a 30 mL
sample was demonstrated with no DNA being detected in the wash fractions. It was also shown
that RNA present in the purified samples could also be removed by the column [82].
Magnetic beads can also be used for the capture of DNA similar to solid phase extraction [77,
71, 83–85]. Modifications of the magnetic beads can include a silica surface [86], polymer
surfaces [87], polyvinyl alcohol (PVA) or hyperbranched polyamidoamine dendrimers [69]. The
latter two provide for an additional electrostatic component for the capture of charged material
[69]. Carboxy-coated magnetic beads have also been used for bioaffinity adsorption of DNA
[88].
Anion-exchange resins that contain a paramagnetic core are available commercially (Whatman
DEAE-Magarose). These beads have been used to isolate plasmid DNA from bacterial cell
lysate and genomic DNA from bacterial cells and blood samples by means of electrostatic
attraction [77]. Magnetic beads can be functionalized with a dense coating of amino groups on
the particle surface by an aminosilane reagent, 3-[2-2-aminoethy(amino)-ethylamino-
propyetrimethoxysilane]. DNA can then be captured by electrostatic interaction between the
positively charged amino-modified beads and the negatively charged DNA [84]. An automated
sample pre-treatment using magnetic beads has been introduced for the extraction of DNA from
solid biomaterials [71].
However, the different methods for non-selective purification of NA material presented may not
have the same efficacy in purifying a sample. For example, a study conducted by Trochimchuk
et al. compared the efficacy of purification of DNA from E. coli present in cattle manure by
phenol-chloroform extraction, gel filtration and SPE. The efficacy was measured by its removal
of compounds which inhibit PCR amplification. It was noted that NA samples purified by SPE
purified were successfully amplified by PCR, while samples purified by phenol/chloroform and
gel filtration did not yield successful PCR amplicons unless the original sample was diluted [80].
11
Bencina et al. also compared the efficacy of NA purification using ion-exchange columns and by
SPE. It was observed that both methods recovered comparable quantities of NA content and
removed inhibitor compounds to PCR. However, optimization of anion-exchange columns
required adjusting a number of factors such as flow rate, ionic strength and pH. Additionally,
fragments of 50kbp and larger resulted in poor recovery with the ion-exchange columns, which
might be due to the large number of charge interaction between the DNA and the anion exchange
resin [79]. The efficacy of SPE to purify NA samples for PCR amplification, its relatively ease of
use as well as its widespread and commercial availability makes SPE one of the most attractive
and commonly used methods for non-selective NA purification.
1.3.2 Purification of DNA in Conjunction with Selective Hybridization
The purification methods outlined in the previous section can be considered non-selective
methods that collect any NA material present in the sample. An issue that arises by such non-
selective concentration methods is that a large amount of non-target NA is collected concurrent
with the target NA, which may hinder detection downstream.
Methods where only the NA target of interest is retained and purified from other NA in the
sample have also been reported. These methods use oligonucleotide probes selective towards a
particular target incorporated into support materials for the selective capture and purification of
DNA from a sample. Following the selective capture of the DNA targets, the column can be
washed, and the captured DNA can be eluted as a relatively pure sample [77]. Through selective
capture of complementary material followed, it may be possible to reduce or eliminate issues that
are associated with non-selective adsorption of non-complementary target on detection elements.
Magnetic beads have been modified with oligonucleotide probes to capture a specific DNA
target [69]. DNA that is captured on the magnetic beads can be used for direct detection without
eluting the DNA [85, 89, 90]. For example, an electrochemical biosensor using magnetic beads
and PCR for E. coli. analysis demonstrated that 2.5 aM of PCR products could be detected [20].
Fuentes et al. demonstrated the detection of 10-18 g/mL (two molecules) of HCV cDNA from a 1
mL sample by PCR amplification of DNA targets that were selectively captured on magnetic
beads. The detection was not impaired by the addition to the sample solution of 2.5 million fold
excess of non-complementary DNA. Non-selective adsorption of DNA was decreased by
blocking active sites with aldehyde-aspartic-dextran. The use of the negatively charged polymer
12
reduced the positive charge of the support surface and also allowed for a strong secondary amine
bond to be formed between the oligonucleotide probe and the polymer coated magnetic beads.
However, some non-specific adsorption was still observed [85].
Even though purification using magnetic beads may offer a much simpler and faster extraction
method than conventional phenol-chloroform extraction, the recovery and purity of the extracted
DNA may not be as high as conventional methods [83]. A study by Faggi et al. comparing
purification of DNA from yeast using magnetic beads and conventional phenol-chloroform
extraction found that phenol-chloroform resulted in higher recovery of extracted DNA with
better purity than magnetic beads. The amount of DNA extracted from three different types of
fungi by phenol-chloroform was 6684, 2022 and 945 µg/mL. Purity was indicated as the ratio of
optical densities of UV absorbance at 260 nm and 280 nm, which was reported to be 1.8, 1.4 and
1.4, respectively. By comparison, the amount of DNA extracted from the same three types fungi
by magnetic beads were 443, 223 and 329 µg/mL with purities of 1.3, 1.2 and 1.4, respectively.
Additionally, the cost per test using magnetic beads was higher than phenol-chloroform
extraction [83].
Peptide nucleic acids (PNAs) have also been demonstrated for affinity capture of DNA targets.
PNAs are synthetic mimics of oligonucleotides that are stable against the action of peptidases
and nucleases [91]. Duplexes of PNA-DNA and PNA-RNA show higher thermal stability and
faster hybridization kinetics versus their DNA-DNA and DNA-RNA counterparts [92, 93]. The
improvement in hybridization kinetics is believed to be due to the neutrality of the PNA
backbone, eliminating the electrostatic repulsion observed for the hybridization of two DNA
strands [92]. PNA immobilized onto beads has been reported to offer enhanced recovery and
detection of rRNA. rRNA was captured and detected on PNA-coated Lumavidin beads from 0.1
ng of RNA within 15 minutes. By comparison, the use of DNA immobilized beads required an
overnight hybridization before a detectable signal was obtained from the same amount of RNA
[92]. Unfortunately, PNAs are not highly soluble in water and have a tendency to self-aggregate
and sediment in low salt concentrations [91].
Oligonucleotide probes can also be incorporated into chromatography support materials for the
selective capture and purification of DNA from a sample. Various research groups, including
13
those of Mathies, Olsen and ours have utilized a commercially available oligonucleotide
modification sold under the name of Acrydite™.
The acrylamide modified oliogonucleotide probe was first introduced by Rehman et al., and
allows for the oligonucleotide probes to be incorporated into polyacrylamide gels during radical
polymerization [94]. Acrydite™ represents a modification that adds an acrylamide monomer
unit to the 5’ end of an oligonucleotide sequence. When mixed with a solution of acrylamide
monomer and polymerized, the resulting polyacrylamide gel has oligonucleotide probes
incorporated into the gel matrix [95–97]. DNA targets are then driven through the modified gels
by electrophoresis [98]. Any debris or unwanted salts can be washed away, and the
complementary targets are subsequently released by denaturation [95–97]. This chemistry has
also been used for incorporation of the NA probes into a 3D gel matrix, and has been used to
clean up of PCR amplicons for CE sequencing, pyroseqeuncing and as a biosensor platform [96,
97, 99–101].
Figure 1.1: Radical polymerization reaction of acrylamide in the presence of an Acrydite™ modified
oligonucleotide.
14
Whitney et al. manufactured polyacrylamide gels that were modified with oligonucleotides.
These affinity gels were cut into 1 cm diameter disks and were used to capture DNA from a
sample volume of 2.4 mL by electrophoresis. The captured target was subsequently eluted into a
volume of 50 µL by the addition of NaOH. A 5 fold increase in the quantity of DNA was
recovered from stool samples when compared to the use of immobilized probes on magnetic
beads [98].
Polyacrylamide gels modified with capture probe have also been used inside microfluidic
channels by Olsen and Mathies [96, 97]. After the running conditions were optimized, this
method was capable of desalting and concentrating a sample of PCR amplification product in
120 seconds with a 100 fold increase in concentration based on volume reduction [96].
We have previously reported the quantitative determination of the efficiency of capture by
probe-modified gels loaded inside fused silica capillaries (100 µm I.D. X 7.5 cm length). By
measuring the fluorescence intensity of labelled oligonucleotide targets, the capture efficiency
was determined to be approximately 90% of the amount of probe molecules that were
incorporated into the gel. Elution by heating and chemical denaturation released approximately
95% of the captured target. A constant loss of 8 femtomoles of the target DNA by adsorption
onto the gels was observed after elution, and was independent of probe and target concentration.
This loss of DNA was still observed even after efforts to block active sites by pre-conditioning
the capture gels with a solution of non-complementary oligonucleotides [95]. One objective of
the work presented in this thesis will be to examine the response of a biosensing platform to
samples processed by selective versus non-selective methods of NA purification.
1.4 Enrichment and Amplification of Sample
Enrichment and amplification of the target DNA is often necessary in order to detect low target
concentrations that are present in clinical, food and environmental samples. The most common
methods increase the total amount of target present, and make use of methods such as cell
culturing prior to DNA extraction, or by PCR.
15
1.4.1 DNA Amplification by Polymerase Chain Reaction
PCR allows for a small number of copies of DNA to be amplified exponentially. This process is
often used for amplification of DNA targets for applications in trace analysis, and is still a
necessary step for detection of low concentrations of DNA by biosensors [20, 32, 102].
PCR uses the target DNA as a template that will be copied. DNA primers are used to flank the
target sequence to mark the initiation and termination locations for copying. Nucleotide
triphosphates are used to build the new DNA copies, and are stitched together by DNA
polymerase. Heat stable DNA polymerase is used. Amplification starts by first annealing the
primers to the target DNA, followed by an increase in temperature to 72°C for polymerase
activity and 94°C to denature the newly formed double-stranded DNA (dsDNA). The
temperature of the reaction vial is lowered again to anneal the primers to the target DNA and the
cycle is repeated, and the quantity of DNA is amplified exponentially [103]. Theoretically, more
than 109 copies of target DNA can be produced after 25 to 30 cycles [104]. The length of the
PCR amplicons can be controlled by designing the DNA primers to bind to different areas of the
DNA target. Fluorescently labelled-DNA targets can be generated by adding specific
fluorescence modification to the monomeric nucleotides prior to assembly by polymerase..
On average, a typical 30 – 40 cycle PCR amplification in bulk solution can take upwards of 2-3
hours to complete, but is wholly dependent on the reaction times for each step [23, 28, 29].
Amplification yields are rarely truly exponential and will plateau over the course of the PCR
amplification [105]. This is thought to be the result of the consumption of reagent at higher
cycle numbers, poor template to primer hybridization, contamination in the sample, inefficient
thermal cycling and poor temperature control [103]. Efficient amplification will depend on rapid
heat cycling so to allow for rapid temperature transitions. More rapid and efficient heat cycling
can be achieved by reducing the volume of the PCR mixture to less than 1 µL [103]. Such small
volume PCR reactions has been demonstrated by construction of small microreactors on
microfluidic devices [106–108].
Another major contributor to amplification efficiency is the purity of the template DNA.
Inhibiting compounds can deactivate the thermostable DNA polymerase or degrade the target
nucleic acids [22, 109]. For any sample pre-treatment protocol that includes amplification by
PCR, stringent purification protocols are usually implemented in order to remove most if not all
16
of the inhibitor compounds. Some recent relevant developments to overcome such limitations
include work by Kermekchiev et al., where they recently identified mutants of thermostable Taq
DNA polymerase that were found to be more resistant to inhibitor compounds in whole blood
and crude soil samples [110]. Also, the Collins group has examined a method for performing
PCR directly from lysed cells without requiring purification of the DNA from the cell lysates
[111, 112]. A sufficient dilution factor was found where the concentration of inhibitors in the
solution no longer inhibited PCR. PCR was performed successfully on a 10 fold dilution of
E. coli O157:H7 cell lysates (original concentration of 200 cells/mL) [112].
1.4.2 Concentrating by Volume Reduction
Biosensors typically require small sample volumes for detection. Volumes usually range from
nano- to microlitres. However, for trace detection of analytes in “real” samples, collection of
microlitres may not be statistically representative of the sample.
Bacteria or viruses may not be homogenously distributed within samples, necessitating that large
volumes be collected for representative sampling [113]. Additionally, the target pathogens may
be present in low numbers, again requiring large volumes to be collected so as to have
confidence in quantification [13, 47, 114, 115].
For example, Mycobacterium ulcerans, which is a human pathogen that causes chronic necrotic
skin disease, has a threshold “load of concern” that is estimated as approximately 0.5 cells per
100 mL of water [59]. The infectious dose for many foodborne pathogenic bacteria is often only
a few cells [116, 117]. For E. coli O157:H7 in ground beef, contamination levels are usually less
than 100 CFU/g of beef [52, 118].
In the detection of cancerous colon cells from stool samples, the amount of DNA obtained from
colon cells represents only 0.01 to 0.1 % of the total DNA that is recovered from the samples. Of
that small percentage, only about 1% of those cells may be cancerous [98]; the balance being
primarily from DNA associated with bacterial cells.
The problem of low abundance of the target for these cases results in the need for large sampling
volumes. The challenge is to ensure that a sufficient quantity of pathogen has been collected to
meet the limit of detection for the sensor, as well as to provide replicates for statistical
confidence measures of results. For example, assume that a RNA-based biosensor had a limit of
17
detection of 16 ng/µL of RNA for a 2 µL sample. Assuming a homogeneous sample, and that
the extraction and purification protocol recovery an average of 0.02 ng of RNA per cell, then the
sampling volume would be about 6.4 L if the solution contained 250 cells/L (i.e. 800 cells in
total) to achieve a detectable amount of rRNA [119]. Volumes taken for environmental samples
are often several orders of magnitude larger than the volumes actually required by a biosensor in
the measurement step. This requires some form of volume reduction so that the volume is
reduced without significant loss of the target. Volume reduction is usually done by capture or
sequestering of the target, purification, and subsequent elution of the target into a smaller
volume. This selective concentrating of the target results in enhancement in signal-to-noise,
improving detection sensitivity and the reliability of analysis [120, 121].
1.5 Fragmentation and Denaturation of DNA
The length of the DNA targets can impact hybridization kinetics, and this is particularly
important for hybridization with immobilized probes. DNA fragments need to be sufficiently
short to allow for fast hybridization. Lengths of DNA target commonly used are on the order of
25 to 300 base pairs. By comparison, the lengths of DNA from cells are orders of magnitude
longer. For example, the genomic DNA for most E. coli strains is on the order of 4 million base
pairs in length [122]. Short target sequences within long fragments of DNA may be sterically
hindered from interacting with immobilized probes [122, 123]. Furthermore, if the target
fragments are too long then intramolecular structures may be present that prevent hybridization.
1.5.1 Fragmentation
Various mechanical cell lysis methods used to release intercellular material have been shown to
fragment DNA to differing degrees [36, 38]. Bead beating often results in the most fragmented
DNA [52, 124, 125] while ultrasonication can be used to shear as well as denature DNA [122–
124].
Fragmentation of DNA using ultrasound occurs by mechanical damage through heating of the
solution and by chemical damage by free radicals attack on the DNA. Our group has previously
demonstrated that ultrasonication (85 W power, 20 kHz) can fragment genomic DNA extracted
from E. coli cells to produce products in the 100-400 bp range within 30 seconds. Application of
18
ultrasound will also heat the sample solution, which will also cause denaturation of the DNA
fragments [122].
Enzyme catalyzed scission of DNA involves use of restriction enzymes, and this approach has
also been used to generate shorter DNA fragments than typically obtained from short periods of
application of high power ultrasound [122, 126].
1.5.2 Preparation of Single-Stranded DNA Target
The most common method for denaturation of DNA targets is by heating the DNA solution at
95 °C for 5 minutes followed immediately by cooling to ice temperature [126–128]. A
modification to thermal denaturation has been to introduce blocking oligonucleotides directly to
the denaturation mixture. These blocking oligonucleotides are 10-30 nt length single-stranded
oligonucleotides that are complementary to a region on the DNA target. Following thermal
denaturation at 95 °C, the solution is cooled to allow for annealing of the blocking
oligonucleotides to the DNA targets. This prevents the re-annealing of the denatured strands
while still leaving the target region available for binding to the oligonucleotide probes on the
biosensor [126–128].
As one example, a SPR-based DNA biosensor was used to examine samples of bovine DNA
processed by thermal denaturation with blocking oligonucleotides. These experiments showed a
43% increase in signal in comparison to the same samples that were only thermally denatured
[126]. Similar results were also observed for samples of apolipoprotein E gene in humans and
the 35S promoter sequence that is present in most genetically modified organisms [126].
Short, single stranded DNA targets can also be produced by asymmetric PCR. In conventional
symmetric PCR, equal concentrations of forward and reverse primers are used in the reaction
mixture and the amplicons are in double stranded form. Denaturation by heat followed by rapid
cooling has been used to generate single stranded products. In asymmetric PCR, a higher
concentration of one primer is added over the other [129]. Over the course of the amplification,
the amplicons will be predominantly in a single stranded form, extended from the excess primer
[130].
19
Figure 1.2: Schematic of differences between (a) conventional denaturation by heat and (b) denaturation by heat with ancillary blocking oligonucleotides. With permission from Analytica Chimica Acta. Copyright
2004, Elsevier [131].
The use of asymmetric PCR provides an amplification yield that is significantly less than that
from conventional PCR. For asymmetric PCR, amplification occurs exponentially until the
primer in lower abundance is exhausted. Subsequently, the available number of templates
remains constant, and amplification occurs in a linear fashion as cycling continues. Asymmetric
PCR often requires extensive optimization to determine of the ratio between the primers,
amounts of starting materials and number of cycles to provide sufficient amplification of a
particular target [130].
Wangh’s group has introduced a modification to asymmetric PCR called Linear-After-The-
Exponential PCR (LATE-PCR). In LATE-PCR, the length and nucleotide base composition of
the two primers are adjusted so that the difference in melt temperatures is zero or the limiting
primer has a higher melt temperature than the excess primer.
The amplification efficiency of LATE-PCR over asymmetric PCR was demonstrated for an
application involving the CD∆508 cystic fibrosis allele and the TSD 1278 Tay-Sachs disease
allele, where more products were detected by fluorescence from LATE-PCR after 80 cycles
[130].
20
1.6 Integrated Microfluidic Devices
There is an interest to integrate the extraction, purification and concentration steps with detection
into a single package. A miniaturized device can reduce reagent use and assay cost, often
increases speed of the relevant processes, and allows a lab to become “portable”. A system that is
free of human interaction with the sample improves reproducibility and ameliorates opportunity
for contamination or sample loss [15, 102].
A fully integrated microfluidic device was recently developed by Liu et al., where cell capture
by immunomagnetic beads, purification, concentration and thermal lysis were all done within the
same device. The capture efficiency using the magnetic beads was determined to be at 40%.
Discrimination of single-nucleotide polymorphism targets was demonstrated following on-chip
PCR amplification. The detection of 103-106 E. coli K12 cells from a 1 mL whole blood sample
was shown and required 2.7 hours. It was observed that the presence of the magnetic beads in
the PCR mixture reduced the amplification efficiency by 50% [132].
Landers’ group has demonstrated a fully integrated microfluidic device based on purification by
SPE that could generate a genetic profile from whole blood. Upon loading the sample into a
glass-based microfluidic device, a chemical lysis agent was mixed with the blood. This was
followed by loading onto a silica bed for purification of genomic DNA. The eluted DNA was
amplified by PCR. The entire process took less than 30 minutes, and 1500 to 2000 CFU of
Bacillus anthrax was detected in 750 nL of whole blood [25].
Integrated microfluidic systems for DNA detection have also been demonstrated using digital
microfluidic (DMF) platforms. In contrast to conventional microfluidic where solution is
continuously moved through the microchannels in the chip, DMF moves independently
addressed droplets of solution with typical volumes of micro- to pico-litres discretely on an
electrode array by a phenomenon termed electrowetting on dielectric (EWOD) [133, 134].
Using DMF, droplets of different chemical reagents can be dispensed from a reservoir and mixed
together by moving the droplets onto the same location on the array. This allows for bench-top
protocols to be reduced in scale for implementation in DMF. DMF may provide a modular
platform for the miniaturization and integration of multiple conventional bench-top scale pre-
treatment techniques and assays into a microfluidic scale [133–135]. Sample pre-treatment
techniques such as immunomagnetic capture of cells onto magnetic beads followed by
21
concentrating [133, 136], liquid-liquid extraction of oligonucleotides from histones [137] and
PCR [134–136, 138] have been demonstrated using DMF.
Besides electrokinetics, fluids can also be manipulated in a microfluidic platform by centrifugal
forces. Centrifugal or Compact Disc (CD) based microfluidic platforms use disc-shaped devices
where a centrifugal force is generated by rotation of the disc [139–143]. Centrifugal-
microfluidic platforms have an advantage over electrokinetic based microfluidic devices in that
they can be operated using inexpensive CD drives and do not require the use of specialized
equipment such as high voltage power supplies [144, 145]. Additionally, commercially available
CDs or DVDs can be used as substrate materials, which are low cost and offer a number of
surface attachment chemistries for probe immobilization [144, 146]. Other polymer materials
such as PDMS can be made into the CD form factor for use in centrifugal microfluidics [142].
Multiple channels can also be designed into the discs, allowing for high throughput screening or
parallel processing of the sample in one device [139–142].
Fluid flow rate in the disc can be determined by the rotation speed of the motor [145]. The
system can handle a variety of fluids such as organic solvents or other aqueous buffers;
movement of sample matrices such as blood, milk and urine have been demonstrated [145].
Fluid velocity is not influenced as strongly by factors such as pH, ionic strength as compared to
electrophoretic methods, and flow rates from 10 nL/s to 100 µL/s can be achieved [139–141].
The main driving force for fluid transport is centrifugal force, but can be further controlled by
coriolis forces [139–141, 143, 145], valves [139–141] or external magnets [143]. A number of
pre-treatment techniques for biomolecules have been demonstrated in the centrifugal
microfluidic platform, including: cell culturing [139]; separation of red blood cells from plasma
in a whole blood sample by sedimentation [143]; immunomagnetic separation [147]; cell lysis
using glass beads [142]; separation by affinity [145], ion exchange [145] and size exclusion
chromatography [139–141]; DNA extraction based on solid phase extraction [145, 147]; PCR
[147]. Detection strategies in centrifugal microfluidic devices include: DNA hybridization [144,
145, 147]; immunoassays [143, 147–149] and colorimetric analysis [143].
For development of fully integrated microfluidic-based DNA biosensors, there is a need to
develop sample pre-treatment and purification methods that are amenable to channel based and
digital microfluidic devices. Sample pre-treatment for microfluidic applications is still a
22
challenge and a limiting factor in many chip designs [150]. Many of the pre-treatment and
purification technologies have aimed to miniaturize macro-scale pre-treatment techniques for use
inside microfluidic channels. Conventional techniques such as centrifugation or precipitation are
not readily adaptable into a microfluidic device [16, 69, 88, 151]. Other techniques such as
filtration, magnetic particle based separation and solid-phase extraction have been miniaturized
to operate on chips [103, 151]. Section 1.7 will review some of the purification methods as well
as concentrating methods that are available for channel based microfluidic devices, with the
focus of the work in this thesis aligning with channel-based microfluidic systems.
1.7 Methods for Purification and Concentrating in Microfluidic Devices
1.7.1 Filtration
Filtration in a microfluidic device can be accomplished either by incorporating a piece of
membrane filter material, most commonly by sandwiching it in between two pieces of the
microfluidic device, or by construction of microstructures such as micropillar arrays or
microweirs [16, 152]. Sample filtration and concentrating in a microchip was demonstrated with
porous membranes. Long et al. sandwiched a 10 nm pore diameter membrane between two
pieces of PDMS which had microchannels etched inside. Molecules small enough to pass
through the nanopores were moved across the membrane by the application of a voltage. This
allowed the collection of larger molecules at the membrane by size exclusion, resulting in
concentrating of the sample [152]. The filter was used to concentrate a sample of PCR amplified
DNA target. An 80 fold enhancement in the signal was observed after 40 seconds of filtration.
This enhancement factor could be further increased by using longer sampling times as well as an
increased applied voltage [152].
Foote et al. have demonstrated a nanoporous silica membrane that is capable of a 100 fold
enrichment of DNA after 5 minutes. As shown in Figure 1.3(c), the silica filter membrane was
placed between the silicon-based microchip and a glass cover plate, which trapped the DNA
molecules while still allowing current to flow. Figure 1.4 shows the concentration of
fluorescently labeled ovalbumin using this chip. When coupled with enrichment by field
amplified stacking, a 600 fold enhancement in the signal was observed [153].
23
Figure 1.3: (a) Schematic diagram of the microchip layout for pre-concentration, (b) image of the pre-
concentrator channel, and (c) schematic of how the filtration membrane is placed in between the microchip and the coverplate. With permission from Analytical Chemistry. Copyright 2004, American
Chemical Society [153].
Figure 1.4: Fluorescence images of fluorescein-labeled ricin injected a) without pre-concentration, and
b) with pre-concentration for 1 minute. With permission from Analytical Chemistry. Copyright 2004, American Chemical Society [153].
1.7.2 Solid-Phase Extraction
The incorporation of solid-phase extraction in microfluidic devices has been demonstrated for
purification and concentration of biological targets such as DNA [72, 73, 154]. Capture
efficiency can be improved by repetitively cycling the sample through the SPE matrix in the
microchannel [88]. Following capture, the target can be released into a smaller volume,
concentrating the sample for further downstream processing and detection [155, 156]. A method
to increase concentration has been achieved using an inline solid phase extraction using amino-
silica monoliths. Concentrating factors of 100 fold were achieved for DNA from crude E. coli
lysates. [157]
24
Methods of incorporating SPE into microfluidic devices include filling the microfluidic channels
with silica resins or beads [72, 88, 155, 156, 158, 159], as well as constructing monoliths or
microstructures inside the channels [158]. Wolfe et al. demonstrated a silica material that was
able to extract 500 bp DNA from a 25 µL sample of HindIII digested λ-phage DNA. The
extraction efficiency was reported at 80% and the material was eluted into a final volume of 5 µL
[160]. Other examples of work using SPE for purification of DNA samples in microfluidic
devices have been reported by Yu et al. [161] and Oleschuk et al. [162].
Binding capacity of DNA can be improved by use of beads of smaller diameter to increase the
surface area of capture. However, this results in an increase in back pressure and requires the
application of higher pressure to achieve reasonable flow rates [73, 74, 76]. In addition, the use
of microbeads in the channel requires consideration of a mechanism to retain microbeads inside
the microfluidic channel. This is most commonly done by placing a frit in the channels [28,
163].
Landers’ group has introduced an alternative approach based on immobilization of the silica
beads with sol-gels. The sol-gel is thought to act as an “interparticle glue” to hold the particles
in place. Extraction and purification of genomic DNA from samples of human whole blood in 25
minutes was demonstrated using a solid-phase extraction material that was based on silica gel
immobilized using a sol-gel within a microchannel [160].
Monoliths can be used for the isolation and concentrating of DNA. Monoliths offer larger
surface areas, controllable pore size, and higher mass transfer from porous structures than silica
beads [74, 76, 164] and can be cast in situ by photopolymerization. Pore size and porosity can
be adjusted by altering the concentration and type of porogenic solvent that is selected [72, 164].
Examples of the use of porous monoliths for the purification of DNA have been reported by
Bhattacharyya et al. [72] and Satterfield et al. [164]. A sol-gel based monolith has been
demonstrated for the isolation of DNA from clinical samples. The monolith was a tetramethyl
ortho-silicate based sol-gel loaded inside a microchannel. The addition of ethylene glycol as a
porogen resulted in a monolith with pores in the micrometer scale, providing a large surface area
for adsorption of DNA with little back pressure. Extraction efficiencies of 85% for λ-phage
DNA and 70% for the extraction of human genomic DNA from human blood were observed.
Using this system, a 200 µL solution containing DNA can be concentrated by eluting into a final
25
volume of 12 – 18 µL. Extraction of viral DNA from human spinal fluid using this monolithic
material has also been demonstrated. Repeated extraction using a single device showed blockage
of the pores by components in the lysed cells [165].
DNA can be captured onto silica-based microstructures such as pillars inside the microchannels
[72, 88]. The use of these pillars increases the surface area, but requires complex fabrication
procedures, increasing the cost of each chip [73]. Cady et al. demonstrated an increase in
surface area available for the capture of DNA by using silica-coated pillars (Figure 1.5).
Depending on the etch depth (20 to 50 µm), an increase in surface area from 300% to 600% was
observed. Selective binding of genomic DNA was observed in the presence of a chaotropic salt
followed by washing with ethanol and elution in a low-ionic strength buffer. Samples were
moved through the extraction device by application of positive pressure, eliminating the need for
centrifugation. The binding capacity for DNA in the device was calculated to be approximately
82 ng/cm2 [158]. Polycarbonate based microchips with microposts inside the polycarbonate
channels have also been used for the capture of DNA [88].
Figure 1.5: Schematic and SEM images of the microfabricated silica pillars for SPE of DNA. With
permission from Biosensors and Bioelectronics. Copyright 2003, Elsevier [158].
Yeung et al. developed a gel-based method for concentrating DNA following PCR that was
accomplished on a microfluidic chip. One of the primers used in the PCR process was modified
with a biotin label while the other primer was fluorescently labeled. The capture gel consisted of
immobilized strepavidin moeities. The biotin labeled PCR amplicons interacted with the
immobilized streptavidin to be captured as the PCR products were moved through the gel. Since
the PCR products are double stranded, application of heat to melt the target was used to release
26
the second strand that carried the fluorescent label. This allowed for the capture of all targets in
the PCR amplicon, including those that had mismatches, which would otherwise not have been
captured if specific capture strands for hybridization had been immobilized into the gel. The
method required a total of 40 minutes; half the time of conventional methods, with provision of a
10 fold increase in concentration [166].
1.8 Sample Concentrating by Electrokinetic Methods
The manipulation of solutions and targets by an electric field in microfluidic devices has
demonstrated enrichment of low concentrations of targets by manipulation of their
electrophoretic mobility inside a channel so that they are confined in a smaller volume. Such
techniques include field amplified stacking and isotachophoresis [163, 167, 168].
1.8.1 Field Amplified Stacking
Field amplified stacking (FAS) involves a concentrating effect that is achieved when the target is
introduced electrokinetically into the separation channel. The targets are first dissolved in a
buffer that has conductivity lower than that of the buffer in the channel. Upon electrokinetic
injection, the targets move at a higher velocity in the sample buffer until they reach the interface
between the sample buffer and the buffer in the separation channel. At this point the targets
experience a sharp decrease in velocity. Stacking into a smaller volume occurs, concentrating
the targets. Concentration increases of 3-100 fold have been observed, depending on the charge
of the targets and the conductivities of the buffer [163, 167].
The difference in conductivity between the two buffers should be as large as possible to achieve
the largest stacking effect. A large difference in conductivity allows the targets to stack much
more quickly while limiting the velocity of the stacked band in the high conductivity buffer.
Ideally, the use of water as the low conductivity buffer would yield the greatest enhancement.
However, real sample matrices often contain substances which could increase the conductivity of
the buffer [169–171]. A solution to this is to dilute the sample with water prior to injection to
lower the conductivity of the sample [169, 172]. Alternatively, a short plug of water can be
introduced into the injection end of a channel for sample stacking. The target is injected by
electrokinetic injection into the water and is stacked against the separation buffer. The increase
in concentration is improved since stacking occurs between the water and the separation buffer
27
rather than within the buffer of the original sample. Concentrating factors of 1000 fold have
been reported using such an approach [163].
As an example, Shim’s group has introduced a method to concentrate DNA to improve
sensitivity of downstream electrochemical detection. The system first introduces a short plug of
water into the channel where DNA is loaded by electrokinetic injection. Stacking occurs against
a buffer that contains hydroxypropyl cellulose modified with gold nanoparticles and sodium
citrate. Enhanced stacking occurs due to an increase in conductivity of the stacking buffer, as
well as a decrease of the electrophoretic mobility of the DNA by adsorption onto the large gold
nanoparticles. A concentrating factor of 25000 was observed using this method, which is 2 to 3
orders of magnitude higher than other stacking methods. The stacking, separation and detection
of a sample of 100 bp DNA ladder was completed within 435 seconds [167].
1.8.2 Isotachophoresis
In isotachophoresis (ITP), the target is concentrated and separated from other components in a
discontinuous buffer system. The target plug is positioned between two different buffers. A
leading buffer contains a large concentration of higher mobility ions (higher conductivity) than
those of the targets, and a terminating buffer contains ions with lower mobility at the injection
side (lower conductivity) [171–173]. When a potential is applied across the channel, the local
field strength across the leading buffer will be less than that of the terminating buffer to maintain
a constant velocity of all charged species across the entire channel [172]. Therefore, an ion
traveling through the low conductivity buffer will experience a higher field strength than one in a
high conductivity buffer. The velocity of the ions will decrease upon moving from the low
conductivity buffer to the high conductivity buffer [172].
During isotachophoresis the charged targets in the sample plug having faster electrophoretic
mobilities move ahead into the leading buffer. As the targets begin to separate from the other
ions in the sample plug, they create a local region of high conductivity, reducing the local field
strength. The velocity of the fast moving targets decrease until they reach a constant velocity
[172, 173].
Other targets with different mobilities that are present in the sample will also separate into
different zones until a constant velocity is achieved [172]. At equilibrium, the sample
28
components are contained in sharp zones with the same concentration as the leading electrolyte.
Therefore, the degree of concentration is determined by the composition of the leading buffer
[163]. ITP requires loading and maintaining zones of the different buffers, and knowledge of the
conductivity of the targets is needed for an efficient separation [120]. This method can be
cumbersome due to the need to use discontinuous buffer systems [174]. Selection of proper
buffers to use in ITP for sample stacking can also become complex [168].
As one example, the concentration of digested HaeIII dsDNA fragments has been demonstrated
by ITP. The ITP device was constructed from poly(methyl methacrylate). The leading edge
buffer consisted of 15 mM HCl, 36 mM imidazole (pH 7) and the terminating edge buffer was a
solution of 20 mM HEPES, 36 mM imidazole (pH 7.2). The high mobility ion in the leading
buffer was chloride and the low mobility ion in the terminating buffer was HEPES. Injection
and stacking was completed in 70 seconds. By measurement of fluorescence from an
intercalating dye, stacking was shown to increase the fluorescence intensity by 40 fold. It is
noteworthy that the high salt content used in ITP may interfere with PCR of the stacked DNA
[173–175].
Recently, electrokinetic supercharging (EKS) has been reported as a concentration technique that
uses field amplified stacking followed by transient ITP [175, 176]. It has been demonstrated to
be capable of handling very dilute samples. Here, the sample is first injected into a channel
filled with the leading electrolyte. The trailing electrolyte is then injected and stacking by ITP
occurs. Concentration of targets inside capillaries was observed to be improved by up to 100 000
fold. Inside microchannels, concentration factors from 10 to 100 fold were observed [175].
1.9 Contributions of this Thesis
The work presented in this thesis examines a method for the selective concentrating of DNA
targets for delivery into channel-based microfluidic. These studies were combined with work
done by other researchers in the Krull group who are developing biosensor technology for use in
a microfluidic system. It can be observed in the review of other purification methods that many
of the methods for extraction, purification and concentrating of DNA from cellular samples have
been based on the non-selective collection of DNA targets onto a solid support. The materials
captured on the support are subsequently washed, and then eluted into a volume smaller than the
original sample volume. As previously noted, a significant issue that arises by implementation
29
of such non-selective concentration methods is that a large amount of non-target DNA is
collected with the target DNA, which may hinder selective detection downstream. Through the
selective concentrating of the complementary material, it may be possible to reduce or eliminate
issues that are associated with non-selective adsorption of non-complementary target on
detection elements. The Acrydite capillary affinity gel electrophoresis chemistry that I have
previously reported to capture short oligonucleotide targets was further examined and developed
herein for the selective purification and concentration of longer base pair DNA targets [95, 177].
Although a number of pre-treatment techniques have been adapted and miniaturized to operate in
the microfluidic regime, an issue still present is the processing of large volume samples with
these devices. As eluded to in the introduction, large sample volumes (mL to L) are often
required where the target analyte is present in very small concentration; for example, for the
detection of pathogenic bacteria from consumable products. Such samples must be "scaled-
down" prior to detection using a microfluidic device.
Additionally, since it would be difficult to account for all the components present in the sample
matrix for any given sample, it is likely that an unforseen component in the matrix could
irreversible damage the integrated microfluidic biosensor and limit its reusability. For example,
clogging of the channels can be a possibility if the solution was not adequately filtered. A
modular approach to developing pre-treatment protocols, such as the one used in this thesis
where the selective purification chemistry is inside a capillary that can be inserted into the main
biosensing, be more practical. The chemistry inside the capillary can be replaced if damaged or
used without having to replace the biosensing platform.
As already indicated, the Mathies group has used this affinity capture gel chemistry as a method
for the purification of DNA targets [96, 178–180]. Mathies has used this chemistry for the
removal of buffers, excess primers and template PCR products for downstream sequencing
experiments in a channel-based microfluidic device. They have also reported the optimization of
binding efficiency for PCR products in the affinity gel as a function of electric field strength and
temperature [96].
The work presented in this thesis further examines the ability of Acrydite chemistry to be used
for purification of a target that is in a mixture containing non-complementary DNA. A thorough
evaluation of the selective capture of one oligonucleotide target in a complicated mixture has not
30
yet been reported. The investigation considered the effects of gel formulation, and different
loading and elution conditions on recovery and purity. This was addressed systematically and
guided by a factorial analysis (see Appendix A for background of factorial analysis). It was
identified that a higher amount of complementary target was retained in the affinity capture gels
made with higher concentrations of monomer. This was thought to be a result of such gels
having a smaller average pore size than the radius of gyration of the ssDNA target examined,
causing the ssDNA target to migrate by reptation. It was proposed that migration by reptation
stretches out the ssDNA targets, eliminating any hairpin structures present in the ssDNA. This
made the complementary region on the target available for hybridization with the gel
immobilized probe, resulting in a higher amount of capture of the complementary target by the
affinity capture gel.
The release and delivery of the DNA targets was performed by step elution for concentrating the
DNA targets. Use of a localized elution zone that was applied in small steps along the length of
a capillary column allowed for the targets to stack as they were denatured from the capture gel.
This is shown schematically in Figure 1.6. The elution was accomplished by denaturation using
a resistive heating element.
Figure 1.6: Schematic representation of selective concentrating as done in the work of this thesis. First, the target was captured onto the affinity capture gel column. Elution took place in a localized area of the
capillary by means of application of heating to a narrow zone such that only targets captured in that region were denatured. This process took place during electrophoresis, and the denatured targets moved along in the electric field. The heated zone was then physically moved along the column. This allowed for
the continual release of targets into a stacked zone of significantly smaller volume than the original sample volume.
31
Since only the target of interest was retained by the affinity capture gel, the target was
concentrated and eluted at high purity. The concentrated target was delivered into a microfluidic
channel that had been modified with immobilized probe oligonucleotides at various spots on one
channel wall. The capillary interfaced to the microfluidic device acted to bridge the disparity in
volumes between the macro-environment to the microfluidic device.
A capillary-to-microfluidic interconnect was used to deliver target analyte into the microfluidic
device. The volume of the capillary (350 nL) was larger than the microfluidic channel (19 nL),
and this offered the opportunity to use stacking to reduce the volume of the material delivered
into the microfluidic device. The use of affinity capture gel inside the capillary as a component
separate from the microfluidic channel allowed for sequential processing of samples, and the
opportunity from replacement of the gel in a manner that was independent of the sensor
component.
This method of selective concentrating of DNA targets was intended as an adjunct protocol for
the purification of samples following PCR amplification or ultrasonication, where DNA targets
would be available as shorter, single-stranded sequences. This method is not meant to replace
any particular purification method, but provides an additional method to further purify and
concentrate a sample.
The targets examined in this thesis were selected to cover a range of lengths as might be
encountered in real samples, and included 19 nt, 150 nt, 250 nt and 400 nt oligonucleotides. The
19 nt oligonucleotide was a synthetic oligonucleotide target while the 150, 250 and 400 nt targets
was obtained by PCR. Selective capture of these targets in the presence of non-complementary
material was achieved by the affinity capture gel, and this was followed by step elution to
achieve target concentration. The recovery of the method ranged from 0.5 to 4% for the PCR
targets, while it was 13 to 18% for the 20 bp oligonucleotide target. The purity was calculated to
be up to 44% for the PCR target and up to 86% for the 19 nt target. This was an improvement in
purity of 15 times and 1100 times in comparison to the original samples for the PCR targets and
19 nt oligonucleotide, respectively. The lowest concentration of the 150, 250 and 400 nt targets
that saw an advantage by selective concentration was 1 nM of complementary in 150 nM non-
complementary target. The lowest concentration of the 19 nt oligonucleotide target that could be
32
processed to see advantage in selective purification and concentration was 0.5 nM
complementary target in 1 µM non-complementary target.
Finally, the 19 nt oliogonucleotide targets were delivered by selective and non-selective
concentration into a channel based microfluidic DNA biosensing platform, and the response of
the was clearly improved when selective concentration was invoked for sample processing.
33
Chapter 2 Materials and Method
2.1 Reagents
Acrylamide, bis-acrylamide, agarose gel, APS and TEMED were all electrophoresis grade from
Sigma-Aldrich (Mississauga, ON, Canada). DNA modifying enzymes, DNA ladders, gel
loading dyes and all other molecular biology reagents were from Fermentas Inc. (Burlington,
ON, Canada). The QIAquick PCR purification kit and Genomic DNA extraction kit were from
Qiagen Inc (Mississauga, ON, Canada). Fluorescent DNA intercalating dyes were from
Invitrogen Inc. (Burlington, ON, Canada). Fused silica capillary (100 µm I.D., 375 µm O.D.)
was from Molex Corporation (Phoenix, AZ, USA). PDMS microfluidic chips were cast using
Sylgard 184 Silicone Elastomer kit (Ellsworth Adhesives, Stoney Creek, ON, Canada). Epoxy
modified glass substrates (Super Epoxy II) were from Array-it (Sunnyvale, CA, USA). All other
reagents were from Sigma-Aldrich (Mississauga, ON, Canada) unless stated otherwise.
2.2 DNA Targets
Table 2.1 lists all of the oligonucleotide sequences (probes, primers, targets) that are used in the
experimental work. Oligonucleotide sequences were from Integrated DNA Technologies
(Coralville, IW, USA), and were used as received.
34
Table 2.1: Oligonucleotide targets that were used in the experiments. Longer PCR targets are described in a subsequent section. Melt temperature was provided by the supplier. *oligonucleotide probes used in
capillary affinity capture gels contained the Acrydite modification at the 5' end, while probes used for immobilization onto epoxy-modified glass slides in the microfluidic device contained a primary amino group with a C12 spacer on the 5' end. Cy3 fluorescent label was attached to the 3' end when used.
**fluorophores on these oligonucleotide sequences were attached at 5' end when used. Oligonucleotide
Probes* Length
(bp) Sequence Tm*
(°C)
SMN 19 5' ATT TTG TCT GAA ACC CTG T 3' 49.3
β-actin 19 5’ CCC TCC CCC ATG CCA TCC T 3’ 62.3
β-actin (1 bpm) 19 5’ CCC TCC CCC ATG CCA CCC T 3’ 65.2
Non-complementary
β-actin
19 5' ACG CGG TCT GAT GCC CTG T 3' 62.3
β-actin (short) 14 5’ CCC TCC CTC ATG CC 3’ 51.8
uidA 22 5’ AGT CTT ACT TCC ATG ATT TCT T 3’ 49.3
DNA Targets**
SMN 19 5’ ACA GGG TTT CAG ACA AAA T 3’ 49.3
Β-actin 19 5' AGG GTG GCA TGG GGG AGG G 3' 62.3
uidA 22 5' AAG AAA TCA TGG AAG TAA GAC T 3' 49.3
Non-complementary
20 5' CCG CGA CGG ATT GAT TGT TT 3' 56.2
PCR Primers**
β-actin forward 20 5’ TCA CCC ACA CTG TGC CCA TC 3’ 60.0
β-actin reverse 20 5’ GTG GTG GTG AAG CTG TAG CC 3’ 58.4
LAMA3 forward 20 5’ CTG GGC TAC AGT TCA CAG CA 3’ 57.3
LAMA3 reverse 20 5’ TCC ACA TAA CTC GCT TGC AG 3’ 55.2
400 forward 24 5’ CTT GTC CAG TTG CAA CCA CCT GTT 3’ 60.0
400 reverse 24 5’ ATG CGG TCA CTC ATT ACG GCA AAG 3’ 59.8
35
2.3 Instrumentation
2.3.1 Capillary Electrophoresis
Figure 2.1: Schematic of the capillary electrophoresis set-up.
Figure 2.1 shows the capillary electrophoresis setup used for the experiments. Microcentrifuge
tubes of 0.65 mL volume were used to hold the running buffer (0.1 % PVP in 1x TBE, pH 8.0).
The capillary was inserted into the microcentrifuge tubes via holes drilled into the top of the
tubes. A Spellman CZE1000R (Hauppauge, New York, USA) high voltage power source was
used as the power supply. Electrodes were inserted through a second hole made in the side of
the microcentrifuge tube. Temperature was controlled by pumping water heated to the desired
temperature into a syringe connected to a water jacket system surrounding the capillary.
36
2.3.2 Instrumentation for on-line capillary electrophoresis/step elution experiments
Figure 2.2: Set-up for online capillary electrophoresis and step elution experiments.
Figure 2.2 shows the set-up used for online electrophoresis experiments and step elution
experiments. A Labsmith HVS448 3000V (Livermore, CA, USA) was used as the high voltage
power supply. Microcentrifuge tubes of 250 µL volume were used to hold running buffer (1x
TBE with 0.1% PVP) and were mounted onto a flat, plastic surface using epoxy glue. The level
of tubes were adjusted to ensure that the capillary was level when placed inside the setup.
Localized step elution of the captured targets on the capillary was accomplished by heating,
using a 45W soldering iron with approximately 0.8 mm metal wire coiled around the soldering
tip. The resistive heating element covered approximately 0.8 mm of the capillary at a time. The
temperature of the resistive heating element was measured to be around 85 °C. The heating
element was moved across the capillary in discrete steps at a defined rate using a Sigma-Koki
SGSP20-85 motorized Stage attached to a Shot-602 two-Axis Stage controller (Tokyo, Japan)
and controlled using custom software written in National Instruments LabVIEW 8.0 (Austin,
TX, USA).
2.3.3 Confocal Fluorescence Microscope Images
In addition to obtaining data from electrophoretograms, images of the capillaries were acquired
by confocal fluorescence microscope. Such images were obtained by placing the capillary on the
stage of the fluorescence microscope and measuring the fluorescence emission intensity as the
stage was rastered in the x and y directions. This produces a fluorescence emission map of the
37
capillary. In order to measure fluorescence emission from the inside of the capillary, a large
portion of the polyimide coating was burned off the capillary. Figure 2.3 shows a schematic of
how fluorescence images of the capillaries were acquired. Offline images of the entire fused
silica capillary were also obtained for data processing.
Figure 2.3: Schematic of the instrumental setup for how confocal fluorescence microscope images were
obtained.
The images were then processed further as detailed in the results and discussion section. The
affinity gel capillaries were imaged using the following instruments.
2.3.3.1 Confocal fluorescence microscope slide reader for 532nm/635nm excitation (Chipreader)
Affinity gel-filled capillaries were imaged off-line using a Bio-Rad VersArray ChipReader
confocal fluorescence microscope (Hercules, California, USA). The ChipReader was designed
to excite and detect fluorescence of Cy3 and Cy5 dyes. Laser excitation of the Cy3 and Cy5
dyes occurred at 532 nm and 635 nm, and the fluorescence emission was detected at 570 nm and
670 nm, respectively. The focal plane was first determined by scanning a capillary filled with a
solution of dye-labelled oligonucleotide and focusing until the maximum signal intensity of the
dye was obtained. Scanning parameters were as follows: laser power 10% (100% = 8 mW
power), detector sensitivity 800mV, 1x detector gain, image resolution 15 µm, and an image scan
speed of 25 lines per second.
38
2.3.3.2 Epifluorescence microscope for 635 nm excitation (Alpha)
Real-time monitoring of Cy5 fluorescence during electrophoresis and step elution pre-
concentration experiments was performed using a custom a epifluorescent microscope based on a
Nikon Eclipse L150 microscope platform (Melville, NY, USA). The excitation source was a
10 mW 635 nm solid-state laser (Coherent, Santa Clara, CA, USA) directed through a 40x
microscope objective (Plan Fluor, ELWD, Numerical Aperture 0.60) using a 630-650 nm band
pass excitation filter. Emission was collected through the same objective using a 660 nm long
pass dichroic filter and a 665-695 nm band pass emission filter (Chroma Technology Corp,
Bellows Falls, VT, USA) into a Hamamatsu H574-20 PMT detector. The microscope stage was
controlled by a Conix Research Inc. XYZ 4000ML Stage Manipulator (Springfield, OR, USA).
Image acquisition software for the custom microscope was written in LabVIEW.
2.3.3.3 Confocal Fluorescence Microscope for 534 nm excitation (Confocal)
A Nikon C2 confocal system mounted onto a Nikon L150 microscope setup was used for real-
time monitoring and some off-line images of Cy3 fluorescence. The excitation source was a
10 mW 534 nm laser (Research Electro-Optics, Boulder, CO, USA). Excitation radiation was
delivered through a 4x microscope objective (Plan Fluor , WD 17.2 mm, Numerical Aperture
0.13) by the Nikon C2 system. Emission was collected through the same objective and passed
into the 3-PMT detector. Filters were set up such that emission intensity collected from 560 to
610 nm were assigned to the Cy3 channel. Data obtained from off-line images from this
instrument will be noted.
All fluorescence intensity images were analyzed and processed using ImageJ (NIH).
Enhancements were made to some images by adjusting the Window/Level parameters to aid in
visualization. Such enhanced images were identified in the figure caption.
2.3.4 UV-VIS
UV-VIS absorbance measurements were collected using a HP8425A. Melt temperature
experiments were done using a HP89090A Peltier temperature controller.
39
2.3.5 Steady-State Solution phase Fluorescence Measurements
All steady state fluorescence measurements were done using a QuantaMaster PTI
Spectrofluorimeter (Photon Technology International, Lawrenceville, New Jersey, USA). A 45
µL volume ultramicro fluorescence cell was used to hold samples (path length 3 mm, Hellma
Ltd, Concord, Ontario, Canada). The excitation and emission settings were as follows: Cy3 –
Excitation 513 nm, Emission 550 – 600 nm, integration time 1 second, Cy5 – Excitation 625 nm,
Emission 645 – 680 nm, integration time 1 second. Each sample was scanned three times to
provide an average signal.
2.3.6 Other Equipment
Ultrasonication of DNA was done using a Vibra-cell Ultrasonic Processor VC-250 equipped
with a 5 mm diameter tapered microtip (Sonics & Materials, Danbury, CT, USA). PCR was
performed on an iCycler Thermal Cycler (Biorad, Hercules, CA, USA). Aqueous solutions of
PCR amplified DNA were dried using a centrifugal vacuum evaporator (DNA Plus, Jencons,
Leighton Buzzard, UK). Agarose gel electrophoresis made use of a BioRAD powerPAC power
supply and gel chamber, and the Mini-Sub Cell GT Agarose Gel Electrophoresis System. Gels
were imaged using a Biorad Gel-Doc 1000 system. PDMS microfluidic chips were plasma
oxidized using a Herrick PDC-32G Plasma Cleaner/Sterilizer.
2.4 Generation of Longer lengths of DNA Targets
2.4.1 150 bp targets
Longer DNA target sequences were synthesized from samples provided by Dr. Paul Piunno from
the Department of Chemical and Physical Sciences, UTM (Mississauga, ON, Canada). The
150 bp target provided is a target from the β-actin gene, which codes a cytoskeletal protein found
in networks of microfilaments and stress fibers that influence the cytoarchitecture of a cell [181].
It is a highly conserved housekeeping gene, and typically is used to normalize molecular
expression studies [182]. The 150 bp length DNA target was generated by PCR. The PCR used
a combination of: 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.08% Nonidet P40, 1.5 mM MgCl2,
0.5 µM of each primer, 25 nM dNTPs, 0.02 µg/mL template, 2.5 units of Taq. PCR conditions
were as follows: an initial denaturation step at 95 °C for 5 minutes, 45 cycles of denaturation: 1
min at 95 °C, annealing: 30 seconds at 61 °C, extension: 30 seconds at 72 °C, and a final
40
extension step at 72 °C for 10 minutes. Attachment of a Cy5 fluorescent label to the target
strand of the interest was accomplished by using a Cy5 labeled reverse primer in the PCR mix.
2.4.2 250 bp targets
The 250 bp target was constructed by ligation of the 150 bp target prepared as described in
Section 2.4.1 with a 100 bp target supplied by Paul Piunno. The 100 bp fragment is a target for
the LAMA3 gene. This gene codes the α3A part of the Laminin-5 heterotrimer filament protein,
which is an important structural component in basement membranes. Laminins contribute to cell
proliferation, migration, differentiation and promotion of tissue survival [183].
The enzymatic reactions were carried out using the protocols provided by the supplier. The 5’
end of the 150 bp target was first phosphorylated with T4 Polynucleotide Kinase so that the
subsequent reaction with T4 DNA Ligase would occur. The reaction conditions were as follows:
20 pmol of DNA target, 1x supplied reaction buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl2,
6 mM DTT, 0.1 mM spermidine, 0.1 mM EDTA), 20 pmol of ATP, 10 units of T4
Polynucleotide Kinase. The reaction was allowed to proceed for 30 minutes at 37 °C, following
which the enzyme was inactivated by increasing the temperature to 75 °C for 10 minutes. The
reaction mixture was used in the ligation step without further purification.
The 150 and 100 bp target was then incubated with T4 DNA Ligase for 7 hours at 22 °C to allow
for ligation to occur. The reaction used equal volumes of 150 and 100 bp length DNA targets, 1x
ligation buffer as provided (40 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM DTT, 0.5 mM
ATP), 5%v/v PEG 4000 and 2 units of T4 DNA Ligase. Following ligation, the enzyme was
inactivated by heating at 65 °C for 10 minutes, as suggested by the supplier. Following the
reaction, the reaction mixture was run on a 1% agarose gel with 1x SYBR Gold intercalating dye
in 1x TBE (100 V, 1 hour) along with a 100 bp DNA Ladder. DNA bands were visualized on a
UV trans-illuminator and the appropriate gel bands were excised and DNA recovered using the
QIAquick Gel extraction kit. Cy5 labeled targets were generated using fluorescently tagged
primer in the PCR mixture [40 cycles, denaturing at 95 °C for 1 min, annealing at 53 °C for 30 s,
extension at 72 °C, 30s].
41
2.4.3 400 bp targets
400 bp length targets were obtained from genomic DNA extracted from E. coli K12 cell cultures
in a number of steps. The E. coli K12 strain was provided by Fong Ly from the Department of
Biology, UTM (Mississauga, ON, Canada).
First, genomic DNA was extracted from the cell cultures using a Qiagen genomic DNA
extraction kit (Qiagen). The protocol was followed without modification. Three volumes of a
proprietary solution which contained a chaotropic agent was added to 1 volume of the excised
agarose gel and incubated at 50 °C until the gel was dissolved. The same volume of isopropanol
was added to the solution as the volume of gel. The resulting solution was added to a silica gel
based spin column and centrifuged for 1 minute at 13 000 x g. A volume of 500 µL of the initial
binding solution was added to the spin column and centrifuged again. A volume of 750 µL of a
wash buffer that contained absolute ethanol was added to the spin column, and this was allowed
to sit for 2 minutes before centrifugation. The eluent was discarded and the spin column was
centrifuged for an additional minute. 50 µL of elution buffer (10 mM Tris-Cl, pH 8.5) was
added to the spin column and allowed to stand for 1 minute before the spin column was
centrifuged for one minute to collect the purified DNA.
Next, a 1000 bp length target was generated from the genomic DNA by a double restriction
enzyme digest. Two restriction enzymes, BfmI and Bsp119I, were used in a restriction enzyme
digest of the extracted genomic DNA to generate a 1000 bp length DNA target. Reaction
conditions were as follows: 0.5 µg of extracted genomic DNA , 1x Tango™ buffer (33 mM
Tris-acetate (pH 7.9), 10 mM magnesium acetate, 66 mM potassium acetate, 0.1 mg/mL BSA), 2
units of Bsp119I and BfmI each. The double digest was allowed to proceed at 37 °C overnight,
after which the enzymes were inactivated by heating to 80 °C for 20 min.
The 400 bp length target was generated from the 1000 bp length target by PCR. Primers were
designed using the web-based PrimerQuest tool available at www.idtdna.com. The sequence of
the 1000 bp length target was obtained from geneBLAST. The design criteria were that the
primers were 24 bp in length, with a Tm of 60 °C and a target length of 400 bp containing the
target region, which was in the middle of the sequence. PCR was used to generate Cy5 labeled
DNA targets. PCR conditions (40 cycles of 95 °C denaturation for 1 min, 55 °C annealing for 30
seconds and extension at 72 °C for 30 seconds)
42
After the PCR reaction was completed, all DNA targets were purified using a QIAquick PCR
purification kit. The protocol was as follows: Binding buffer (which is a proprietary solution
that contains guanidine HCl and isopropanol) was added in 5:1 volume of the PCR reaction
solution. The resulting mixture was added to the silica gel based spin column and centrifuged at
13 000 x g for 1 minute. A volume of 750 µL of 30% w/v Guanidine HCl was added to the spin
column following the initial binding of the DNA to the silica column, and the spin column was
then centrifuged at 13 000 x g for 1 minute. A volume of 750 µL of the wash buffer
(proprietary) with absolute ethanol was added to the spin column, followed by two rounds of
centrifugation at 13 000 x g for 1 minute. The bound DNA target was eluted by adding 50 µL of
the buffer in the kit (Tris-HCl) to the spin column, which was then centrifuged at 13 000 x g for
1 min.
2.4.4 Validation of DNA targets
The targets as synthesized by PCR were run in an agarose gel electrophoresis system (1%
agarose, 1x TBE, 25 Vcm-1) and compared with standard DNA ladders. The gels contained 0.8x
SYBR Gold to allow for visualization by use of a standard UV trans-illuminator. DNA targets
were sequenced at The Centre for Applied Genomics (Toronto, ON, Canada).
2.5 Preparation of Capillary Affinity Capture Gel
Fused silica capillaries were first cut into 5.5 cm long pieces and the polyimide coating was
removed by burning with a flame to create a 4.5 cm window in the center of the capillary to
allow for imaging by fluorescence microscopy.
The capillary was filled with a solution of 2% w/v polyvinylpyrrolidone (average molecular
weight 360,000, PVP) for 5 minutes. The affinity capture gel material, which consisted of
varying amounts of 50% w/v acrylamide, 2% bis-acrylamide, 0.1% PVP in 1X TBE solution and
acrylamide modified probe, was initiated using 0.5 µL of 4% w/v ammonium persulfate (APS)
and 0.5 µL of 4% v/v N,N,N,N-tetramethyl-ethylenediamine (TEMED). The initiated affinity
gel mixture was injected into the capillary and allowed to polymerize for one hour, and then was
stored at room temperature overnight prior to use. The capillary was filled using a vacuum
injection system similar to that described by Baba et al. [184]. A rubber septum was used to cap
a small, empty glass vial, where a capillary was inserted into the septum using a needle. A
43
vacuum line was connected to the vial through the septum, and solution was injected into the
capillary by placing the end of the capillary not inside the vial in the solution while a vacuum
was applied.
2.6 Capture and elution experiments
2.6.1 Pre-Conditioning of Affinity Capture Gel in Capillaries
The capillaries were pre-conditioned prior to use by operating the electrophoretic system at 67
Vcm-1 for 30 minutes. A 10 µL, 2 µM sample of the non-complementary oligonucleotide
sequence was electrokinetically loaded at 267 Vcm-1 for 2 minutes into the capillary to block
non-selective adsorption sites. After injection of the non-complementary oligonucleotide, the
sample was run for 5 minutes at 133 Vcm-1. The non-complementary target was loaded a second
time in the same manner, and the process was repeated for 15 minutes at 133 Vcm-1.
2.6.2 Capture and Elution Experiments
A general protocol for the loading and capture of the longer length DNA targets was followed.
Target solutions contained varying concentrations of the DNA targets in1x TBE and 0.1% PVP
in deionized water. DNA target solutions were first boiled at 95 °C for 5 minutes and
immediately put on ice for 10 minutes. A 10 µL sample of the denatured DNA target was loaded
into the affinity capture gel by electrokinetic injection (167 Vcm-1 for 15 minutes).
Electrophoresis was stopped following electrokinetic injection and the temperature surrounding
the capillary was then maintained at 20 °C for 20 minutes using a water jacket system that
surrounded the outside of the capillary. A voltage was then applied across the capillary at 167
Vcm-1 for 20 minutes while the temperature around the capillary was maintained at 20 °C for
removal of any excess, uncaptured target. Elution of the captured targets was achieved by
application of the same voltage while the temperature of the capillary was increased to 65 °C
using the water jacket.
2.6.3 Factorial Design Experiments
Fractional factorial design experiments were used to identify optimal gel formulation and capture
conditions for the DNA targets. A quarter fractional 2-level factorial design was used for the
examination of gel formulation on effect of probe incorporation and target loading. Table 2.2
and 2.3 shows the design matrix used and experimental conditions, respectively. The factors
44
examined were the monomer concentration (%T), crosslinker content (%C), the concentration of
oligonucleotide probes, and the concentrations of TEMED and APS used for initiation of the
polymerization reaction.
Table 2.2: Design matrix for the quarter 2-level fractional factorial analysis for the examination of gel formulation on the performance of the capillary affinity capture gels.
Standard Order
Run Order %T (A)
%C (B)
Probe (C)
TEMED (D)
APS (E)
1 9 3 2 - - - + +
2 10 5 8 - - + - -
3 11 6 3 - + - + -
4 12 2 1 - + + - +
5 13 8 4 + - - - +
6 14 4 6 + - + + -
7 15 1 7 + + - - -
8 16 7 5 + + + + +
Table 2.3: Experimental conditions for the two levels used for the fractional factorial design matrix.
Factor - +
A (%T) 7.5 % 12.5 % B (%C) 1 % 5 % C (DNA probe)
0.5 µM 3.0 µM
D (TEMED) 4 %v/v 10 %v/v E (APS) 4 %w/v 10 %w/v
The total monomer concentration (%T) and percentage of crosslinker (%C) is given by:
%100100
% xsolutionmL
ercrosslinkgmonomergT
+= (1)
%100% xrecrosslinkgmonomerg
rekcrosslingC
+= (2)
Examination of conditions for capture efficiency and selectivity of the affinity capture gel were
explored using a 3 level 2 factor factorial design. The amount of target captured was examined
based on the incubation time for hybridization and the wash voltage. Response of the affinity
capture gel to mixtures of targets was examined by changing the elution temperature and
formamide content as a denaturant. Tables 2.4 and 2.5 show the design matrix and experimental
conditions that were used. Statistical analysis was done using Minitab R14 (Minitab Inc, State
College, PA, USA).
45
Table 2.4: Design matrix for three level factorial experiment to explore capture efficiency and selectivity of the affinity capture gel. Factors A and B are defined in Table 2.5.
Standard Order
Run Order
A B
1 10 5 13 1 1
2 11 3 12 1 2
3 12 7 10 1 3
4 13 6 14 2 1
5 14 1 17 2 2
6 15 8 18 2 3
7 16 4 11 3 1
8 17 2 16 3 2
9 18 9 15 3 3
Table 2.5: Experimental conditions for each level tested in the design matrix of Table 2.4.
Capture Efficiency Selectivity
Level Sit Time (A)
Wash Voltage (B)
Temperature
(°C) (A)
%v/v formamide (B)
1 5 min 300 V 10 0
2 20 min 600 V 25 10
3 40 min 900 V 40 25
2.6.4 Step Elution of Captured DNA targets
Localized elution of the captured DNA targets was done using the set-up shown in Figure 2. For
these experiments, 7 cm long capillary pieces were cut and filled with the affinity capture gel. A
1.5 cm detection window was made at the elution end of the capillary. Following the capture of
the DNA targets, the resistive heating element was moved across the capillary using a step size
of 250 µm, at a step rate that was determined based on the electrophoretic velocity of the DNA
target of interest. The elution of the stacked oligonucleotide target as it moved through the
detection windows was monitored in real time by an epifluorescence microscope setup.
2.7 Delivery of concentrated targets into microfluidic based DNA biosensing platform
The stacked, eluted targets were delivered from the capillary into a microfluidic DNA biosensing
platform based on a design by Erickson et al. via a capillary-microfluidic interconnect [185].
2.7.1 Construction of DNA Microfluidic Biosensing Platform
Amine terminated oligonucleotide probes at 25 µM were covalently immobilized onto epoxy
modified glass slides by spotting them in borate buffer followed by storage in a humid chamber
46
for 48 hours. Excess probes were removed by washing the slide in 70 °C water for 1 minute.
The location of the probe spots on the glass slide is shown in Figure 2.4.
The design of the microfluidic DNA biosensor is shown in Figure 2.4. The master template was
provided courtesy of Uvaraj Uddayasankar and Omair Noor. The design was based on an H-
shaped channel. The dimension of the main microfluidic channel was 185 µm (W) x 8 µm (H).
Figure 2.4: Schematic for the construction of the microfluidic DNA sensing platform with a capillary
interface. Left: original microfluidics template and position of the template capillary. The capillary (100 µm I.D., 375 µm O.D.) was positioned over the microfluidic channel such that the inner diameter was within the width of the channel. PDMS was poured over the template and cured on a hotplate. The
template capillary and microfluidic template were removed and the PDMS chip had the channel structure and capillary port. Right: schematic of the microfluidic DNA sensor platform. The PDMS template was
trimmed such that only the straight channel remained, and this was positioned over the two oligonucleotide probe spots on the epoxy modified slides.
The capillary-to-microfluidic interconnect was made by positioning a piece of empty capillary
using an XYZ translation stage (Coherent Inc., Santa Clara, CA, USA) orthogonally over the
microfluidic channel on the template. The microfluidic channel was made by casting a 10:1
mixture of the silicone elastomer base and curing agent over the template. Curing was
accomplished by heating at 120 °C on a hotplate for 20 minutes. Removal of the capillary
47
following casting left a hole where the affinity capture capillary could be inserted. The PDMS
mold was trimmed and then plasma oxidized for 30 seconds. The side-arm channels were
removed from the mold so that only the straight channel was present in the final PDMS chip.
The length of the final channel was 1.5 cm from the interconnect to the reservoir well. The mold
was then placed over the epoxy modified glass slides so that the channel was positioned over the
immobilized probe spots. The open ends of the channel were sealed with a drop of PDMS. A
schematic of the final microfluidic system is shown in Figure 2.4.
The microfluidic chip was first pre-conditioned by running electrophoresis in buffer on a 65°C
block heater for 5 minutes, followed by injection of a 5 µM non-labelled non-complementary
sequence to block non-selective absorption sites.
Step elution of the oligonucleotide targets was done prior to interfacing with the microfluidic
chip. The affinity capture capillary was 4.5 cm in length, and was filled with 10% LAAm gel
containing 100 nM complementary (SMN) probe. Step elution of the targets was done as
outlined previously. Immediately following the elution process, the leading 2.5 cm portion of
the capillary was cut and removed, and the remaining 2 cm portion of the affinity capture
capillary was inserted into the microfluidic chip via the interconnect port. Voltage (500V) was
applied across the capillary and microfluidic channel as shown in Figure 2.5.
Figure 2.5: Schematic of the electrophoresis setup for the capillary to microfluidic DNA biosensor.
48
Delivery of material without any pre-treatment (direct injection) was done using the same
capillary-to-microfluidic interface. Solutions of 10% LAAm were made and mixed with the dye-
labeled DNA targets. The complementary target used for these experiments was Alexa647-SMN
and the non-complementary target was Cy3-β-actin. The solutions were then injected into pre-
cut capillaries using a syringe-to-CE coupler, and these were used to deliver sample into the
microfluidic channel.
Experiments examining samples where all DNA targets were concentrated (without purification)
were performed using affinity capture gels that contained a mixture of 100 nM (SMN) and 5 µM
(β-actin) probes.
49
Chapter 3 Results and Discussion
3.0 Capture of Oligonucleotides of 20 nt Length
I have previously published results which demonstrated the successful attachment of the
acrylamide modified oligonucleotide probe into polyacrylamide gel in Analytica Chimica Acta,
578(2006), pg 31-42, titled "Capillary Electrophoresis for Capture and Concentrating of Target
Nucleic Acids by Affinity Gels Modified to Contain Single-Stranded Nucleic Acid Probes".
This system was used to capture a 20 nt length oligonucleotide target, and demonstrated
selectivity of the capture gel when a mixture containing both fully complementary target and a 5
base pair mismatch was used. This work reported the quantity of probe that could be
incorporated into a polyacrylamide gel, as well as the capability of the affinity gel to capture
targets at different concentrations. A summary of the methods and highlights from this
published work is now presented.
3.0.1 Considerations for Imaging Fused Silica Capillaries by Confocal Fluorescence Microscopy
Data for the affinity capture gel experiments were obtained from images of the capillary acquired
by confocal fluorescence microscopy. A number of instruments were used to obtain
fluorescence microscope images and described in Section 2.3.3.1 to 2.3.3.3. At various steps of
the experiment (i.e., following pre-conditioning of the capillaries, following injection of the
DNA targets, following elution of the targets), the capillary was removed from the CE setup,
placed on a microscope slide and the entire capillary was imaged using the fluorescence
microscope. Data which were obtained from images taken by confocal microscopy will be
indicated as such in the caption. Quantitative analysis of signal intensity of fluorescence images
was done using ImageJ. A straight line with a width of 20 pixels was drawn along the capillary
and a profile plot was generated using the Plot Profile function. This plots the fluorescence
intensity (integrating the 20 pixel width) along the 4 cm window of capillary length. Since the
capillary may not be completely straight when being imaged, a 20 pixel wide line was used so
that the fluorescence information from the entire capillary was captured in a single straight line
during analysis. The average fluorescence intensity was then calculated from the profile plot.
50
In order to image the fluorescently labelled NAs were used in the experiments, and these were
detected by the confocal fluorescence microscope (Chipreader). Fused silica capillaries are
coated with a protective layer of polyimide. This material is fluorescent, and the intensity of
emission was sufficient to saturate the detector of the microscope. This polyimide layer was first
removed so that the affinity gel inside the capillary could be imaged. However, removal of the
polyimide coating resulted in mechanical brittleness when handling the bare fused silica.
Therefore, only 4 cm of the polyimide coating was removed from the center of the capillary to
allow for imaging while retaining some durability for physical manipulation of the capillaries.
The confocal fluorescence microscopy images represent a 4 cm window, rather than the entire
length of the capillary. It was assumed that the affinity gel was homogeneous throughout the
capillary. Confocal fluorescence microscope images of the capillaries were stored as two
separate files, one containing the fluorescence emission data from the Cy3 channel and the other
from the Cy5 channel. For experiments where both fluorophores were present, the images are
shown as a pair. The left image represents the fluorescence emission data from the Cy3 channel
and the right image the emission from the Cy5 channel. For experiments where only one
fluorophore was used, only a single image of that dye channel is presented. Some images shown
were processed using ImageJ by adjusting the Window/Level parameters to aid in visualization.
Such enhancement of images has been identified in the figure captions.
3.0.2 Capture and Elution Experiment for a 20 nt Target
Figure 3.1 shows an example of confocal fluorescence microscopy images of capillaries obtained
during a capture and elution experiment for a 20 nt length complementary target using the
affinity gel. The affinity gel consisted of a 0.45 µM dT20-Cy3 oligonucleotide probe in a 12.5
%T linear polyacrylamide gel. Briefly, the left channel represents the fluorescence image of the
Cy3 channel, which corresponds to the Cy3 labelled oligonucleotide probe (dT20-Cy3) used in
the affinity capture gel while the right channel represents the fluorescence image of the Cy5
channel, corresponding to the Cy5 labelled oligonucleotide target (dA20-Cy5). The
complementary target, a 5 µL sample of 1 µM dA20-Cy5, was introduced electrokinetically at
267 Vcm-1 for 1 minute. Figure 3.1(a) shows the image of the capillary prior to the loading of
the complementary target. Successful capture of the complementary Cy5 labelled target can be
seen by the images in Figure 3.1(b) after injection and electrophoresis for 35 minutes. Elution of
the captured target was achieved by the addition of 0.5 M NaSCN to the running buffer and
51
application of a 60 °C water jacket around the capillary for 25 minutes, as shown in Figure
3.1(c). The use of sodium thiocyanate salt has been previously demonstrated to be a potent
denaturant of double stranded DNA [186].
Figure 3.1: Confocal fluorescence microscope images of affinity capture capillaries from a capture and
elution run using a complementary target showing: (a) the affinity gel material inside the capillary prior to loading of target oligonucleotide sequence, (b) running for 35 minutes following electrokinetic injection of
target Cy5 – dA20 and (c) after elution for 25 minutes at 60 °C. Affinity gel: 0.45 µM Cy3-dT20 probe in a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dA20 at 267 Vcm
-1 for 60 seconds. Electrophoresis condition: 133 Vcm
-1
with 1x TBE/0.1% PVP for 35 minutes. Elution condition: 267 Vcm-1
with 1x TBE/0.5 M NaSCN/0.1% PVP for 25 minutes at 60 °C. Images were obtained using the Chipreader.
The probe was labelled with a Cy3 fluorophore. The presence of a Cy3 fluorescence signal inside
the capillary indicated that the Acrydite™ modified probes were successfully incorporated into
the polyacrylamide matrix. The Cy3 signal was observed throughout the entire capture and
elution experiment, showing that the EOF was adequately suppressed by the dynamic coating
with PVP; otherwise, a loss in Cy3 fluorescence as the gel was physically ejected from the
capillary would have been observed.
52
3.0.3 Autofluorescence and Non-Selective Adsorption
A low level of fluorescence intensity was observed in the Cy5 channel following the elution of
the captured target in the experiments described in Figure 3.1. An effort was made to identify
the source of this residual fluorescence signal.
Prior to the loading of any materials into the gel filled capillary, a fluorescence signal was
observed from the capillary in both the Cy3 and Cy5 channels, as shown in Figure 3.2. The
average fluorescence signals along the length of the capillary column in the Cy3 and Cy5
channels were 379 AU and 699 AU, respectively. This background fluorescence was attributed
to autofluorescence from the fused silica capillary and polyacrylamide gel. It was also important
to note that while the fluorescence emission observed in the Cy3 channel was only from the gel
region inside the capillary channel, the fluorescence emission in the Cy5 channel was from the
entire capillary.
Figure 3.2: Confocal fluorescence microscope images of affinity capture capillaries showing the Cy3 (left) and Cy5 (right) channels from a portion of the capillary shown in Figure 3.1(a), which shows the
autofluorescence signal of the system before the loading of any fluorescently labelled materials. Images were enhanced in ImageJ using the Window/Level function for better clarity.
A control experiment included introduction of both Cy3 and Cy5 labelled targets to a
polyacrylamide gel that contained no immobilized probe. There was an increase in the intensity
relative to the background fluorescence following the loading and migration of a sample through
the capillary. The increase represented retention of 5 ± 5 fmol and 10 ± 5 fmol of Cy3 and Cy5
labelled oligonucleotides, respectively (as determined using a concentration-response calibration
curve). In these experiments the affinity gels had not been pre-treated using non-labelled non-
complementary targets and the labelled targets were being non-selectively adsorbed onto the gel.
53
For applications that involve the selective capture of low concentrations of material, any non-
selective adsorption would be unfavourable. Therefore, as a pre-treatment step, non-labelled non-
complementary oligonucleotide target was pre-loaded into the capillary.
The effectiveness of the pre-treatment protocol was tested using a 5 µL sample of 1 µM dC20-
Cy5 non-complementary target that was introduced to an affinity gel containing 0.45 µM Cy3-
dT20 probe in 12.5 %T linear polyacrylamide. The amount of material left in the gel as a result
of non-selective adsorption corresponded to 10 ± 9 fmol for columns without any pre-treatment,
and 4 ± 4 fmol for columns with pre-treatment (data corrected for autofluorescence).
The effectiveness of the pre-treatment protocol was examined using complementary targets. The
affinity gels consisted of 0.45 µM Cy3-dT20 probe in a 12.5 %T linear polyacrylamide. Samples
containing 1 µM dA20-Cy5 were used as the complementary target. Following sample elution
with denaturation of hybrids, affinity gels that were not pre-treated retained approximately 19 ±
8 fmol of dye-labelled oligonucleotide while the affinity gels that were pre-treated indicated
retention of 8 ± 8 fmol of oligonucleotide target. Pre-treatment helped in reducing but did not
completely eliminate non-selective adsorption.
Figure 3.3 shows Cy5 fluorescence emission following elution of the captured target. Since
retention of material was observed with both complementary and non-complementary targets
following elution, it was concluded that the retention of material was based on non-selective
adsorption, rather than incomplete elution of the captured targets.
The pre-treatment likely resulted in a condition where adsorbed material was being leached from
the gel during the capture and release experiment. This is analogous to the process encountered
when using dynamic coatings to suppress electroosmotic flow on capillary walls, where a small
amount of the surface active agent must be included in the running buffers to replenish the
coating.
The possibility of loss of adsorbed material used in pre-treatment over time due to leaching was
examined. Here, Cy5-labelled dC20 was used in place of the non-labelled oligonucleotide
during the pre-treatment protocol. Following pre-treatment, the capillary was subjected to an
electric field in 1xTBE / 0.1% PVP running buffer and scanned for fluorescence at various times.
54
The resulting plot shows the average fluorescence intensity of the Cy5-labelled dC20 inside the
capillary over time.
Figure 3.3: Confocal fluorescence microscope images of affinity capture capillaries showing the Cy5
channel (a) before loading of complementary target and (b) following elution. A fluorescence signal was apparent following elution, indicating retention of 13.6 nM or 8 fmol of target.
Injection condition: 5 µL of a sample containing 1 µM Cy5-dT20 at 267 Vcm-1
for 60 seconds. Electrophoresis condition: 133 Vcm
-1 with 1x TBE/0.1 %PVP for 35 minutes. Elution condition: 267 Vcm
-1
with 1x TBE/0.5 M NaSCN/0.1 %PVP for 25 minutes at 60 °C. Images were enhanced in ImageJ using the Window/Level function for better clarity.
600
700
800
900
1000
1100
1200
1300
1400
15 35 55 75 95
Time (minutes)
Avera
ge F
luo
rescen
ce I
nte
nsit
y (
AU
)
(a)
(b)
Figure 3.4: (a) Change in the average Cy5 fluorescence intensity over time, measuring the loss of any
adsorbed materials used in the pre-treatment of the columns. Pre-treatment protocol: a 5 µL sample of 2 µM Cy5-dC20 at 267 Vcm
-1 for 2 minutes, followed by electrophoresis at 133 Vcm
-1 in 1xTBE/0.1% PVP
for 5 minutes, and a second injection of a 5 µL 2 µM Cy5-dC20 at 267 Vcm-1
for 2 minutes and electrophoresis at 133 Vcm
-1 in 1xTBE/0.1% PVP for 15 minutes. (b) The original fluorescence intensity
of the capillary channel prior to the loading of any material (baseline). Error bars represent 1 standard deviation of three trials.
55
The data of Figure 3.4 shows a decrease in the average fluorescence intensity over time,
indicative of loss of the non-complementary material used to pre-treat the capillary. It can be
observed that the decrease in average fluorescence intensity was greatest during the initial 40
minutes, which coincides with the time frame for the capture of complementary target. The loss
of the non-complementary pre-treatment material may have allowed the complementary targets
to adsorb onto the gel.
The addition of non-complementary dC20 (10% of the total DNA sample) to the running buffers
did not reduce the amount of non-selective adsorption of target. The average fluorescence
intensity following elution of captured targets was similar with and without the addition of the
dC20 in the running buffer. This suggested the need for a more rigorous pre-treatment protocol
if low concentrations of target were to be handled using DNA-modified polyacrylamide gels.
Work by other groups using the Acrydite™ immobilization chemistry in polyacrylamide gels has
made no mention of non-selective adsorption [96, 97, 187]. Those reports have used a lower
concentration of acrylamide monomer in the affinity gel, but it is not clear whether this should be
related to the trend in adsorption that was noted.
Some loss due to non-specific adsorption could be expected because the blocking agent was of
the same chemistry as the sample. The goal of this work was not to obtain 100% efficiency in
the release of the captured targets, but rather to achieve a reproducible efficiency of capture and
release. Even with an efficiency less than 100%, a correction factor may be applied to the data
for correction to achieve quantitative results, but reproducibility must be achieved for this
correction to be applicable.
Using the pre-treatment scheme, the loss due to adsorption was observed to be constant at 8 fmol
for complementary targets regardless of factors such as probe concentration, or the initial target
concentration within the range that was examined. This suggested a constant error of 8 ± 8 fmol
using the affinity gel, which provided for confidence in reproducibility of capture and elution,
and for determination of relative loss.
3.0.4 Variation of Polymer Density
The mobility of a target through a polyacrylamide gel is dependent on the density of the gel.
Therefore, it was of interest to examine the capture and elution process of affinity gels
56
manufactured using different polymer densities. However, when the density was below 12%T of
linear polyacrylamide gel, the polyacrylamide appeared to be physically unstable; elution of the
probe-modified polyacrylamide from the capillary was observed.
Figure 3.5 show images of the Cy3 channel of the capillary for a 0.45 µM Cy3-dT20 probe in a
7.5 %T linear polyacrylamide affinity gel (a) before and (b) after the introduction of
complementary target. Elution of the probe-modified polymer chains from the column was
observed following the capture of complementary targets.
Figure 3.5: Confocal fluorescence microscopy images of capillary affinity capture gel of the Cy3 channel showing the elution of 0.45 µM Cy3-dT20 probe immobilized in a 7.5%T linear polyacrylamide gel. The image was taken of the capillary in (a), following pre-conditioning and pre-treatment and (b), following
loading 1 µM Cy5-dA20 and running for 35 minutes. Injection condition: 5 µL of a sample containing 1 µM Cy5-dA20 at 267 Vcm
-1 for 60 seconds.
Electrophoresis condition: 133 Vcm-1
with 1x TBE/0.1% PVP for 35 minutes. Images were acquired using the Chipreader.
The incorporation of oligonucleotide probes into the polyacrylamide imparts a net negative
charge on the polymer chains. When subjected to an electric field, the charged polymer chains
would exhibit an electrophoretic mobility if not anchored, and would elute from the column.
The fact that this was not observed for the 12.5 %T polyacrylamide gels is attributed to the
higher degree of entanglement for higher polymer density.
57
The polyacrylamide used to manufacture the affinity gels did not contain any N,N’-methylene-
bis-acrylamide cross-linker in the monomeric solution, meaning that the gel was not chemically
cross-linked. Therefore, the polymer chains must entangle with one another in order to form
‘pores’ within the matrix for size sieving to occur. The concentration where entanglement takes
place is referred to as the entanglement concentration c*. At concentrations ten times greater
than c* (usually greater than 10% w/w [188]) the polymers will form a strongly entangled
network and are considered to behave similarly to cross-linked counterparts [189]. The 12.5 %T
linear polyacrylamide gels used may be considered a concentrated gel.
In the work reported by Mathies and Olsen, the affinity capture probes were incorporated into
lower density polymers, 5 %T and 10 %T polyacrylamide gels, respectively. However, elution
of the polymer chains was not reported. This could be due to the shorter times used for capture
and elution in these experiments. Since smaller volumes of the affinity gel were used inside the
microfluidics channels versus in the capillary, the processing time was significantly reduced.
From the experiment shown in Figure 3.5, the elution of the polymer was not observed until after
having subjected the gel to the electric field for approximately 90 minutes (30 minutes for pre-
conditioning, 25 minutes during the pre-treatment, and 35 minutes following the loading of
complementary target). By contrast, the total capture and elution process was completed in 120
seconds and 15 minutes, respectively, by Mathies and Olsen.
3.0.5 Quantity of Probe that was Immobilized in the Polyacrylamide Matrix
The amount of probe (dT20) that was incorporated into the affinity gels was examined by
confocal fluorescence microscopy. Three different concentrations of Cy3-labelled dT20 probe,
182 nM, 454 nM, and 727 nM in 12.5 %T linear polyacrylamide gels were prepared. Once the
gels were cast, the capillaries were pre-conditioned by running the capillaries at 67 Vcm-1 for 30
minutes in 1x TBE /0.1% PVP buffer. This was followed by pre-treatment with non-labelled
non-complementary target as previously described. Subsequently, a 5 µL sample of 1 µM dA20-
Cy5 was injected and was allowed to run through the capillary.
It was expected that any probe molecules that were not incorporated into the polyacrylamide gel
matrix would be eluted from the column during the pre-conditioning step. The Cy3 fluorescence
intensity measured inside the capillary channel would correspond to the amount of probe that
was successfully incorporated. Concentrations were determined using calibration data and the
58
amount of dye-labelled probe inside the gel was calculated based on a geometric volume of the
capillary of 0.59 µL. Calculation of the percentage of probe that was incorporated into the gel
was done by considering the quantity of probe in the gel in comparison to the amount of probe in
the original monomeric solution.
As shown in Table 3.1, an average of about 40% of probe molecules was incorporated into the
gels, and this result did not appear to be affected by the initial concentration of probe in the
reaction solution (in the concentration range examined).
Table 3.1: Tabulated results for extent of probe incorporation and performance in capture for three different probe concentrations.
Affinity gel: Varying concentrations of Cy3-dT20 probe (182 nM, 454 nM and 727 nM) in a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dT20 at 267 Vcm
-1 for
60 s. Electrophoresis condition: 133 Vcm-1
with 1x TBE/0.1% PVP for 20 minutes. Elution condition: 267 Vcm
-1 with 1x TBE/0.5 M NaSCN/0.1%PVP for 15 minutes at 60 °C. Error bars are propagated error
following correlation of average fluorescence intensity to concentration using a calibration curve. Original
amount of Probe (fmol)
Amount of probe (fmol)
% probe incorporated
Amount of target captured
(fmol)
% target captured versus probe
Amount of target
remaining (fmol)
% elution based on amount of
capture
107 46±6 43±6 42±9 93 8 ± 8 81
268 107±7 40±3 86±9 80 8 ± 8 91
429 179±8 42±2 159±11 89 8 ± 8 95
The efficiency of capture of target was based on the ratio of concentration of fully
complementary target that was captured versus the concentration of probe that had been
immobilized in the gel. Binding efficiencies less than 100% would suggest some binding sites
were not available for binding. Since the polymer chains were entangled, some entanglement
points might be situated between two probe sites, thereby preventing hybridization with
complementary targets. Conversely, binding efficiencies greater than 100% would suggest non-
selective adsorption or incomplete elution of any excess, unbound targets off the column.
No obvious correlation in efficiency was observed between the amount of target captured versus
the amount of probe that was available for the three different probe concentrations that were
examined. The capture efficiency appeared to be fairly high, and was in the range of 80% to
93% for the three probe concentrations that were used. A sample of 1 µM target was used for all
three cases to examine the effect of saturation of the probe sites (amount of target available to
load into the capillary was 5000 fmol).
Approximately 80 to 95% of the captured target was released after elution. The relative quantity
of target eluted was somewhat dependent on the quantity of probe that was in the gel, with higher
59
elution efficiencies being apparent for gels that contained more probe molecules. Following
elution, a constant residual retention of 8 fmol was noted. Non-selective adsorption was not
directly related to the concentration of the immobilized probe molecules in the gel.
3.0.6 Influence of Concentration of Target on the Efficiency of Capture
The ratio of amount of target-to-probe was determined and was compared to the amount of target
in the original sample. The intention was to assess the working range of the affinity gel. All
experiments were based on an electrokinetic injection for 1 minute at 533 Vcm-1 of a 10 µL
sample into a DNA-modified gel (454 nM Cy3-dT20 probe affinity gel). The target was allowed
to migrate for 10 minutes and the gel was then imaged. This was followed by elution for 5
minutes, after which the gel was imaged again. Note that not all of the available target was
actually loaded onto the column in the process of electrokinetic injection. This is the cause for
the differences observed in data for experiments related to Table 3.1 and Table 3.2. Different
loading conditions were used to collect each data set.
Table 3.2: Tabulated results for effect of concentration of sample on loading of affinity gel. Amount of target in loading of a sample was calculated based on a 10 µL of the target concentration solution used for loading. Amount of probe and target were calculated using the geometric volume of 0.59 µL for the
capillary. Affinity gel: 0.45 µM Cy3-dT20 probe in a 12.5%T linear polyacrylamide gel. Injection condition: 10 µL sample containing Cy5-dA20 at 533 Vcm
-1 for 60 seconds. Electrophoresis condition: 133 Vcm
-1 with 1x
TBE/0.1% PVP for 10 minutes. Elution condition: 267 Vcm-1
with 1xTBE/0.5 M NaSCN/0.1%PVP for 5 minutes at 60°C. Error bars are propagated error following correlation of average fluorescence intensity
to concentration using a calibration curve. Amount of Target in
sample (fmol)
Amount of Probe incorporated
(fmol)
Amount of Target
Captured (fmol)
Target / Probe
Amount of Target after
Elution (fmol)
5 71 ± 6 10 ± 9 0.1 ± 0.1 8 ± 8
10 54 ± 6 14 ± 9 0.3 ± 0.2 8 ± 8
50 71 ± 6 29 ± 9 0.4 ± 0.1 8 ± 8
100 82 ± 6 16 ± 2 0.19 ± 0.02 8 ± 8
200 84 ± 6 54 ± 3 0.64 ± 0.06 8 ± 8
300 85 ± 7 41 ± 2 0.48 ± 0.05 8 ± 8
400 74 ± 6 99 ± 4 1.3 ± 0.1 8 ± 8
500 76 ± 6 78 ± 9 1.0 ± 0.1 8 ± 8
1000 75 ± 6 90 ± 9 1.2 ± 0.2 8 ± 8
2500 60 ± 6 83 ± 9 1.4 ± 0.2 8 ± 8
60
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 500 1000 1500 2000 2500
Amount of Target (fmol)
Am
ou
nt
of
Targ
et
Cap
ture
d (
fmo
l) /
Am
ou
nt
of
Pro
be (
fmo
l)
Figure 3.6: Effect of amount of target on the efficiency of capture using a 454 nM Cy3-dT20 probe
affinity gel. The ratio of captured target versus available probe was plotted against varying concentrations of Cy5-dA20 targets. Error bars are propagated error following correlation of average
fluorescence intensity to concentration using a calibration curve.
This analysis provided an indication of the working range of the affinity gel and also indicated a
non-linearity between the amount of target that was processed and the amount captured. This
was expected since a saturation point would be reached for amounts of target higher than the
amount of probe available to capture the targets. This was observed for samples containing
amounts of target greater than 400 fmoles in the original sample. The fluorescence intensity in
the affinity gel after loading a 5 fmol sample was not significantly different from the
background. The experimental data indicates that the lower limit of target that can be reliably
captured and detected by confocal fluorescence microscopy is 50 fmol.
For amounts of target greater than 400 fmol, it was observed that the ratio of target-to-probe was
greater than one. These results suggested that the column was overloaded and insufficient time
was allowed for the removal of excess target, resulting in capture efficiencies greater than unity.
3.0.7 Capture and Elution using a Non-Complementary Target
Figure 3.7 shows that non-complementary target (Cy5-dC20) was not captured by the affinity
probe gel. The average fluorescence intensity following elution corresponded to retention of
35 ± 15 fmol of material. In Figure 3.8, the plug of non-complementary target migrated through
61
the affinity gel without any significant tailing, indicating very little interaction between the
affinity gel and the target.
Figure 3.7: Confocal microscope images of capillaries examining the use of a non-complementary target in the affinity gel from (a) prior to loading the non-complementary target, (b) after loading and running for
25 minutes and (c) after the elution step was applied. Affinity gel: 0.45 µM Cy3-dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dC20 at 267 Vcm
-1 for 60 seconds. Electrophoresis condition:
133 Vcm-1
with 1x TBE/0.1% PVP for 35 minutes. Elution condition: 267 Vcm-1
with 1xTBE/0.5 M NaSCN/0.1%PVP for 25 minutes at 60°C. Images were obtained using the Chipreader.
Figure 3.8: Confocal microscope images of capillaries showing non-complementary target (Cy5-dC20)
as it moved electrophoretically through the capillary after (a) 60 seconds, (b) 120 seconds, (c) 240 seconds and (d) 420 seconds. Only the images of the Cy5 channel are shown.
Affinity gel: 0.45 µM Cy3-dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dC20 at 267 Vcm
-1 for 60 seconds. Electrophoresis condition:
133 Vcm-1
with 1x TBE/0.1% PVP. Images were obtained using the Chipreader.
62
3.0.8 Examination of Selectivity Using a Five Base Pair Mismatch Target
A Cy3 labelled oligonucleotide target with a five base pair mismatch in the center of the 20 nt
sequence (Cy3-dA8C5A8) was run through the affinity gel. From Figure 3.9 it can be seen that
the five base pair mismatch target traveled through the affinity gel with very little interaction
with the affinity probe. No statistically significant increase in the average fluorescence intensity
was observed in the Cy3 channel following the migration of the 5 base pair mismatch targets.
Fluorescence images indicated that there was no trapping of the target, and the absence of tailing
suggested that the five base pair mismatch target traveled through the gel with very little
interaction with the affinity probe.
Figure 3.9: Confocal microscope images of capillaries examining the loading of Cy3-dA8C5A8 (5 base pair mismatch) target through an affinity capture gel. Images were taken after (a) 5 minutes and (b) 30
minutes. Affinity gel: 1.8 µM dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection condition: 5 µL of a
sample containing 1 µM Cy3-dA8C5A8 at 267 Vcm-1
for 60 seconds. Electrophoresis condition: 133 Vcm
-1 with 1x TBE/0.1% PVP. Images were obtained using the Chipreader. The images in (b) was
enhanced in ImageJ using the Window/Level function for better clarity.
The five base pair mismatch was not expected to be captured at room temperature conditions.
The melt temperature of the probe and fully complementary target hybrid that was examined was
37.3 °C [190]. The melt temperature for the mismatch target was calculated to be 17.4 °C (1.8
63
µM probe, 1.0 µM target, 50 mM [Na+]) using the online DNA Thermodynamics &
Hybridization Tool (biophysics.idtdna.com). This set of experiments was done at room
temperature (23 °C) and therefore was not expected to lead to formation of stable hybrids.
3.0.9 Separations of Mixtures of Complementary and Non-complementary Targets
Figure 3.10 shows the results of the passage of a mixture of 0.5 µM dT20-Cy3 (complementary)
and 0.5 µM dC20-Cy5 (non-complementary) targets through a 12.5%T linear polyacrylamide gel
containing 1.8 µM dA20 probe.
Figure 3.10: Confocal microscope images of capillaries tracking a time course experiment for loading a mixture of dT20-Cy3 and dC20-Cy5. Images shown were taken after (a) 120 seconds, (b) 240 seconds,
(c) 540 seconds and (d) 840 seconds. Affinity gel: 1.8 µM dA20 probe immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition:
5 µL of a sample containing 0.5 µM Cy3-dT20, 0.5 µM Cy5-dC20 at 267 Vcm
-1 for 60 seconds. Electrophoresis condition: 133 Vcm
-1 with 1x TBE/0.1% PVP. Images were
obtained using the Chipreader.
As the sample containing the mixture of complementary and non-complementary targets
migrated through the affinity gel, selective capture of the complementary targets by the
immobilized probes was observed. This was indicated by a retardation of the electrophoretic
mobility as well as tailing of the complementary target (Figure 3.11).
64
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Figure 3.11: Profile plot taken from the inlet to outlet end of the capillary from confocal microscope
images of the Cy5 channel after a 4 minute run of the (a) fully complementary (dT20-Cy3) target with (b) non-complementary (dC20-Cy5) targets in a dA20-probe affinity gel. Images were acquired using the
Chipreader.
Selectivity in a capillary gel electrophoresis experiment is expressed as the ratio of the corrected
migration time between two species [191].
)(
)(
1
2
o
o
tt
tt
−
−=α (3)
where t2 and t1 are the migration time of the first and second eluted analyte being compared, and
t0 is the void time. The migration time of a target inside the capillary is directly related to
electrophoretic mobility. In the affinity gel experiment, the electrophoretic mobility is
determined by the concentration of free affinity ligands as well as the association constant for
complex formation [192]:
][1 LKt
tA+==
ο
ο
µ
µ (4)
where µo and to are the electrophoretic mobility and migration time of the target in the absence of
the affinity ligand, µ and t are the electrophoretic mobility and migration time of the target in the
presence of the affinity ligand, respectively. The concentration of the free ligand is [L], and KA is
the association constant.
It can be seen in Figure 3.12 that both targets exhibited similar electrophoretic mobility
migrating through an unmodified polyacrylamide gel. In Figure 3.11, the electrophoretic
65
mobility of the complementary target was retarded by the presence of the affinity probe in the
polyacrylamide gel matrix, demonstrating selectivity of the affinity probe for the complementary
target.
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Figure 3.12: Profile plot taken from the inlet to outlet end of the capillary from confocal microscope
images of the Cy5 channel. Profile plots following migration of (a) Cy3-dT20 and (b) Cy5-dC20 through an unmodified polyacrylamide gel after five minutes. The profile of the entire capillary length is not shown. The distance is shown from the injection end to the elution end. Images acquired using the Chipreader.
3.0.10 Five Base Pair Mismatch in Mixture with Fully Complementary Target
Selectivity for the fully complementary target was shown using mixtures containing 1:1 (Figure
3.13) and 1:9 mixture (Figure 3.14) of the fully complementary target (dA20 – Cy5) to five base
pair mismatch target (Cy3 – dA8C5A8).
The competitive experiments demonstrated selectivity of the affinity probe towards the
complementary target in the presence of non-complementary and also 5 base-pair mismatched
targets.
66
Figure 3.13: Confocal microscope images taken from an affinity gel column containing immobilized dT20-probe after loading a 1:1 mixture of Cy3-dA8C5A8 and Cy5-dA20. Images taken after (a) 120
seconds, (b) 240 seconds, (c) 360 seconds, (d) 660 seconds and (e) 960 seconds. Affinity gel: 1.8 µM dT20 probe immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition: 5 µL of a sample containing 0.5 µM Cy3-dA8C5A8 and 0.5 µM Cy5-dA20 at 267 Vcm
-1 for 60 seconds.
Electrophoresis condition: 133 Vcm-1
with 1x TBE/0.1% PVP. Images acquired using the Chipreader. The Cy3 channel image in (e) was enhanced using the Window/Level function in ImageJ.
67
Figure 3.14: Confocal microscope images taken from an affinity gel column containing immobilized
dT20-probe after loading a 9:1 mixture of Cy3-dA8C5A8 Cy5-dA20 target. Images were taken after (a) 240 s, (b) 540 s and (c) 840s.
Affinity gel: 0.9 µM dT20 probe immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition: 5 µL of a sample containing 1.8 µM Cy3-dA8C5A8 and 0.1 µM Cy5-dA20 at 267 Vcm
-1 for 60 seconds.
Electrophoresis condition: 133 Vcm-1
with 1x TBE/0.1% PVP. Images acquired using the Chipreader.
3.0.11 Capture of 40 nt Length Targets
Figure 3.15 demonstrated the successful capture of a 40 nt length target, Cy5-dC10T20C10 using
the affinity gel.
68
Figure 3.15: Confocal microscope images of capillaries for the Cy5 channel demonstrating the loading and capture of a 1 µM 40 nt target sequence, Cy5-dC10T20C10 through the affinity gel. Images were
taken after electrophoresis following electrokinetic injection for (a) 300 s and (b) 25 min. Affinity gel: 1.8 µM dA20 probe immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition:
5 µL of a sample containing 1 µM Cy5-dC10T20C10at 267 Vcm-1
for 60 seconds. Electrophoresis condition: 133 Vcm
-1 with 1x TBE/0.1% PVP. Images were acquired using the Chipreader.
Following elution, the average fluorescence intensity change indicated a capture of 184 ± 12
fmol of target and adsorption of 8 ± 8 fmol with a relative error of -5%. Complete removal of the
target from the gel had again not occurred for the elution conditions reported herein, and the
marginal loss of material was similar to that observed for all the other experiments that used
complementary targets.
3.1 Capture of Targets of Greater Lengths than 40 nt – Moving Towards Handling of NAs From Real Samples
Building on the preliminary findings using 20 and 40 nt length of NAs, my interest was to
capture longer DNA targets such as would be available in real samples that were prepared by
PCR, or by use of ultrasonication to shear genomic DNA into smaller fragments [122, 193–195].
It was necessary to identify the most important factors that affect the performance of the affinity
gel towards the capture and subsequent release of longer DNA targets.
69
Various gel formulations were examined to better understand the incorporation of probes for
capture of longer DNA sequences. A systematic factorial approach was implemented to assist in
the identification of the primary factors that determined optimal loading of the targets, and
concurrent reduction of non-selective adsorption for target solutions that contained mixtures of
complementary and non-complementary sequences. These studies allowed for optimization of
affinity gels for evaluation of the practical potential for handling longer oligonucleotides to
increase target concentration prior to delivery into an analytical biosensing device.
3.1.1 DNA Targets Selected for Experiments
Typical methods for the processing of DNA to generate single stranded targets after extraction
from cells have included PCR and ultrasonication [26, 28, 23, 196, 30, 29]. Examples of lengths
of DNA targets generated by PCR from real samples that have been detected with DNA
biosensors are commonly in the range of 150 - 600 nt [197–200]. The majority of the reported
targets were generated by symmetric PCR, and single stranded DNA targets were generated by
heating at 90-95 °C for 5 to 10 minutes, followed by fast cooling and storage on ice [198–200].
An example of a report that used asymmetric PCR generated single stranded targets of 168 and
340 nt lengths [197].
Larguinho et al. examined the fragmentation of genomic and plasmid DNA using a number of
different ultrasonication devices. They were able to generate fragment distributions between 100
and 5000 base for plasmid DNA, and fragments between 100 and 800 base distribution for
genomic DNA [193].
Work previously published by our group has demonstrated the ultrasonication of genomic E. coli
DNA to generate fragments of 100-400 nt in length. Additionally, experiments done by Mann
have shown that the length of DNA target generated by ultrasonication can be adjusted by
changing various factors such as ionic strength, ultrasound power, temperature of the solution
and exposure time [122].
The lengths of DNA targets chosen in this study were lengths that could be generated using
either PCR or ultrasound. Three representative lengths of DNA targets were chosen; 150 nt,
250 nt and 400 nt. The 150 nt target was the provided by Dr. Paul Piunno and is a target from
the β-actin gene. As previously mentioned in the Material and Methods section, the β-actin gene
70
is a highly conserved housekeeping gene, and typically is used to normalize molecular
expression studies. Some studies have shown that the level of expression of β-actin can change
in the presence of a disease such as Alzheimer’s disease, as well as in cancer samples [182].
The 250 nt target was generated by combining the 150 nt β-actin fragment with the 100 nt
fragment LAMA3 target also provided by Dr. Paul Piunno. The 100 nt fragment is a target for
the LAMA3 gene. This gene codes the α3A part of the Laminin-5 heterotrimer filament protein,
which is an important structural component in basement membranes. There is evidence that
Laminin-5 is expressed in invading tumor cells and can be strongly active in promoting the
migration of some tumor cells [183].
The 400 nt target was generated by the genomic E. coli DNA that was extracted from E. coli K12
strain. The target of interest was a region in the uidA gene, which is present in all E. coli strains.
This was selected because this gene was previously used by our group as a control [201].
Supplemental information regarding the synthesis of the 250 nt and 400 nt targets, as well as gel
electrophoresis and sequencing data for confirmation of the correct DNA targets can be found in
Appendix B. Experiments examining the generation of single stranded targets by heat
denaturation are included in Appendix C.
3.2 Compositions of Affinity Capture Gels
Most of the work using capillary electrophoresis in the literature has been with linear
polyacrylamide solutions instead of crosslinked polyacrylamide gels due to the advantage of
relatively facile replacement of the gels, as well as problems that arise with bubble or void
formation when casting crosslinked polyacrylamide gels in situ inside capillary columns. During
the polymerization process that creates crosslinked polyacrylamide, shrinkage of the polymer can
often result in the formation of voids inside the capillary [189, 202, 203].
It was previously shown in Figure 3.5 that the use of lower density gels resulted in the elution of
the polyacrylamide affinity gel from the capillary. The polyacrylamide affinity gel matrix was
based on the covalent attachment of negatively charged oligonucleotide probes. This imparted a
negative charge to the polyacrylamide strands. It was observed that the gel could elute from the
column under the influence of an electric field. For the longer DNA targets of interest in this
thesis, the time required to move the DNA sample through the capillary would increase, meaning
71
a significantly longer total analysis time and greater probability of loss of gel due to the applied
electric field. The elution of polyacrylamide affinity gels limits the use of linear gels at low
concentrations. Therefore, the decision was made to switch to crosslinked polyacrylamide gels
that would offer physical stability as well as an advantage in speeding the elution of longer DNA
sequences. One technical problem was that previous experiments already had indicated bubble
and void formation in the gel was apparent for recipes where the amount of crosslinker used was
2.5% w/v (approximately 20-33 %C given a %T range used in these experiments of 7.5-12.5%).
However, further investigations revealed that this was minimized when the amount of crosslinker
was decreased to 0.5% w/v (approximately 4-7 %C for the same %T range). Electroosmotic
Flow (EOF) in the capillaries was suppressed using dynamic coating with PVP. Experiments
comparing the efficacy between a dynamic and a covalent coating for suppression of EOF can be
found in Appendix D.
3.3 Selective Capture of 150 nt Target
The sequence for the 150 nt target is shown in Appendix B. The region on the 150 nt target that
is complementary to the oligonucleotide probe lies between base number 54-72 on the DNA
strand. Initial experimental work associated with this thesis began with use the of 20 nt targets.
The targets were introduced into the affinity capture column by elution chromatography; a small
amount of the sample was introduced into the capillary column and was transported by the
mobile phase (running buffer). The target interacted with the stationary phase, and could be
captured by the affinity gel. Initially, capture experiments for the longer DNA targets made use
of this method of sample introduction. However, reproducible target capture was not achieved.
When the 150 nt target was introduced in a similar fashion as the 20 nt target, offline images
scanned of the capillary showed successful introduction of the target materia, but hybridization
of the complementary target as the injected DNA targets moved through the capillary was not
observed.
It was believed that either; 1) not enough material was injected into the capillary during the
injection step since the mobility of the longer DNA targets was less than the 20 nt
oligonucleotides, or 2) that the kinetics of reassociation was slower for the longer DNA targets
and the longer targets were not efficiently captured as the sample plug moved through the
affinity gel.
72
To allow more material to be injected into the capillary, the sample solution was continuously
loaded into the column, filling the capillary with the DNA target. A period of incubation
following the injection step was introduced to allow the DNA targets time to hybridize with the
affinity probes. Any unbound material was removed from the column by electrophoresis.
Figure 3.16 demonstrates an example of selective capture and elution of a 150 nt DNA target.
The average fluorescence intensities shown correspond to: the affinity capture gel prior to the
introduction of target; following injection of the target; removal of any unbound target by
electrophoresis of the affinity gel; and elution of the captured target by heating the affinity gel to
65 °C via a water jacket surrounding the capillary.
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Figure 3.16: Fluorescence intensity values from the Cy5 channel as measured from confocal microscope
images of the capillary taken at various times during the capture and elution experiment. Values were obtained by taking the average of the values generated from the profile plot of the confocal image. The
capillary containing the affinity capture matrix was first imaged to establish the background fluorescence signal (‘before’). A 10 µL, 1.67 µM solution of the Cy5 labeled 150 nt complementary target was then
loaded into the affinity capture gel (7.5%T/6%C, 1.8 µM β-actin probe) electrokinetically for 20 minutes at 167 Vcm
-1 (‘load’) (The fluorescence intensity following this step saturated the detector and the actual
value is not shown). The fluorescence intensity for the (‘wash’) step was taken after the entire capillary was heated to 95 °C for 5 minutes, allowed to sit for 20 minutes at 20 °C, and following the application of
a voltage of 167 Vcm-1
for 20 minutes at 20 °C. Finally the captured targets were eluted by the application of a voltage of 167 Vcm
-1 for 15 minutes at 65 °C. Images were acquired using the
Chipreader.
Figure 3.17 shows an example of a profile plots of images of the capillary obtained by confocal
microscopy following capture of the longer DNA targets (3.17(a)) versus that for the 20 nt target
(3.17(b)).
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Figure 3.17: Profile plots from the outlet end to inlet end of the capillary from confocal microscope
images obtained for the Cy5 channel of the capillary. a) Fluorescence intensity profile of the Cy5 channel of the affinity gel following the capture of the (0.14 µM) Cy5 labeled 150 nt target. Affinity capture gel:
7.5%T, 6 %C, 3 µM β-actin probe. b) Profile of the Cy5 channel of the affinity gel following the capture of the (1 µM) Cy5 labeled 19 nt target (SMN). Affinity capture gel: 10%T, 5%C, 0.5 µM affinity capture
probe (SMN). Images were acquired using the Chipreader.
74
The shape of the fluorescence intensity profile observed for the capture of the 150 nt DNA target
(3.17(a)) is different than that observed for the 19 nt oligonucleotide target (3.17(b)), with the
latter showing a relatively homogeneous distribution profile along the length of the channel. The
experimental conditions between these two experiments were conducted differently. The
experiment shown in Figure 3.17 (b) was performed with the Cy5-labelled 20 nt target using a
sample introduction mode similar to elution chromatography. Here, the 20 nt target was injected
for a short amount of time (1 minute) from the original sample solution and then the target was
moved through the affinity capture gel by electrophoresis and is seen to be captured more
homogeneously throughout the affinity capture gel . By contrast, the experiment using the 150
nt target was run by frontal chromatography. Here, the sample solution containing the Cy5-150
nt target was used as the feed solution and was continually injected into the capillary under the
moved to the outlet end of the capillary. The sample solution was replaced with the run buffer
and electrophoresis was conducted.
Retarded diffusion results in the shape observed of the profile plots in Figure 3.17(a). The
electrophoretic mobility of the complementary target as it moves through the affinity capture gel
is decreased due to interaction the target has with the immobilized probe. Livshits et. al. have
proposed that DNA moves through such affinity gels via a mechanism called "retarded
diffusion". As the DNA targets move through the gels, they will continuously associate and
dissociate with the immobilized capture probes. A higher probe concentration or the formation
of fully complementary hybrids would result in the material taking a longer time to move
through the gel. This model applies for a system where the DNA is moved through the gel by
diffusion and will be discussed further in Section 3.5.6 [204].
In the system examined in this thesis, the DNA is being moved through the capillary gel by
electrophoresis, but will still association and disassociation of the target with immobilized
probes. After the complementary target is loaded onto the column, those strands that form partial
duplexes with the probe will be retarded to a lesser degree versus those that form full duplexes
with the immobilized probe. Over time, targets that form partial duplexes with the probe as well
as targets that are near the outlet end of the capillary will elute out of the capillary column first,
resulting in very little fluorescence intensity observed near the outlet end of the capillary in the
fluorescence image in Figure 3.17(a). Those targets which form full duplexes with the
immobilized probe and closer to the inlet end of the capillary will have traveled the shortest
75
distance in the capillary during the wash step, and is seen as higher fluorescence intensity
observed near the inlet end of the capillary in Figure 3.17(a). What is observed in Figure 3.17(b)
is the fluorescence intensity observed is the material that formed perfect duplexes remaining in
the capillary column following the wash step. Here, the material has remained in the inlet end of
the capillary, indicating that its mobility has been reduced to the largest degree and that
population is expected to be purely composed of the complementary material. Fluorescence
images taken of the Cy3 labeled probes showed a homogeneous fluorescence, confirming that
the probes remained inside the capillary column.
Figure 3.18 shows profile plots of confocal images taken of the affinity capture gels following
the washing step for capture experiments that used a probe length that had been reduced to 10 nt.
The shorter probe sequence would result in weaker association and lower thermodynamic
stability, and would affect the ability of the capture probe to retard the movement of the DNA
targets. From Figure 3.18, the use of a 10 nt probe caused a shift in the position profile of the
captured material that was consistent with a reduction in affinity. The position of the
complementary material appears to be shifted forward along the capillary when the shorter probe
was used versus the 19 nt probe again due to the decrease in mobility expected between the
different probes.
From Equation 4, the degree of retardation of electrophoretic mobility is dependent on
association constant. Since the shorter probe is expected to have a smaller affinity binding
constant than the 19 nt probe, the overall result is a smaller effect on electrophoretic mobility of
the complementary target with the shorter probe. For the same amount of electrophoresis time
during the wash step, the population of DNA targets that form a full duplex with the immobilized
probe will have a smaller decrease in electrophoretic mobility and appears to have moved further
along the capillary.
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Figure 3.18: Profile plots from the outlet end to inlet end of the capillary from confocal microscope
images obtained for the Cy5 channel of the capillary. Profile plots for the capture of a (0.14 µM) 150 nt target using a 20 nt length probe and a 10 nt length probe. Affinity capture gel: 7.5%T, 6 %C, 3 µM
affinity capture probe (β-actin). Images acquired using the Chipreader.
The use of the longer DNA targets in the affinity capture gels may raise issues not observed
previously with the 20 base oliogonucleotide targets. A reduction of the amount of target that
could be captured might arise from:
1) The reassociation of target with complementary sequence in the sample solution. Additionally,
the target and its complementary strand could decrease the availability of the probe through non-
specific interactions such as adsorption.
2) A longer target could possess secondary structures such as hairpins through intra-strand
interactions.
Figure 3.19 shows 3 of 15 possible hairpin structures of the 150 nt target, as calculated using the
OligoAnalyzer 3.1 software provided by Integrated DNA Technologies. The melt temperature of
these hairpin structures ranged from 32.6 to 40.1 °C.
77
Figure 3.19: Examples of hairpin structures as calculated by OligoAnalyzer software. Settings used for calculations were 25 °C, 50 mM Na
+ concentration, suboptimality 50% and maximum foldings 20. Probe
region is highlighted in the drawn box.
The target region that is complementary to the probe lies between base number 54-72 on the
DNA strand, and is highlighted in the drawn box in Figure 3.19. Therefore, it is possible that the
target adopted hairpin structures during the injection step, ultimately limiting the availability of
conformations of target that were suitable for capture by hybridization.
Figure 3.20 shows a histogram of all the partial interactions possible between the target, probe
and complementary strand as calculated using the OligoAnalyzer software (www.idtdna.com),
sorted by the number of base pairs interacting between the two sequences.
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Target-Target Target-Complement Target-Probe Complement-Probe
Figure 3.20: A histogram of the number of partial interactions possible between the target, its complementary strand and the probe as calculated by OligoAnalyzer software. The number of
interactions was binned by the number of base pairs forming the interactions. Calculation conditions were oligonucleotide concentration = 0.25 µM, [Na
+] = 50 mM.
As can be seen in Figure 3.20, a large number of possible partial interactions exist between the
target strands and probes, ranging from 2 base pairs to 50 base pairs. This is reasonable
considering that the target and its complementary strand are relatively long, increasing the
likelihood of partial interactions. It is important to note that the interacting bases may not
necessarily be in a conserved sequence. Some interactions are between the ends of the two
targets, while others are between bases where the alignment of the two targets are shifted from
one another. Based on this, it is reasonable to assume that the target will participate in
interactions other than with the probe. Therefore, not all of the target that is injected into the
affinity capture gel may actually be available for hybridization, resulting in a reduction of the
amount of material that can be captured.
In one set of experiments the capillary column was heated to 95 °C following injection of sample
onto the column. This was done to drive hybridization to completion by first disrupting all
79
interactions between the target and other sequences present, and to denature any strands that may
have reannealed after the initial heat denaturation step. It is likely that heating the capillary to
95 °C denatured the target-probe hybrids as well, and upon cooling the targets were still able to
form partial interactions. A difference in the capture profile from that shown in Figure 3.17
would indicate that heating could free target for preferential hybridization with probes, resulting
in a more homogenous fluorescence intensity profile. However, this was not seen in the data of
Figure 3.21. Even if targets were captured along the capillary column, dissociation and
reassociation would occur during electrophoresis as described by Livshits, and targets captured
near the elution end of the capillary would still be eluted from the column during the time frame
of the washing step.
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Figure 3.21: Data for comparison of the amount of 150 nt target (100 nM) captured by the affinity capture
gel after the capillary was heated to 95 °C for 5 minutes following the injection relative to the amount captured by an unheated column. Affinity capture gel: 7.5%T, 6 %C, 3 µM β-actin probe. Error bars are
propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.
3.3.1 Comparison of Capture of 150 nt DNA Targets Using Complementary and Non-complementary Probe
Figure 3.22 compares the amount of material captured based on correlation of the average
fluorescence intensity to a calibration curve. The data is collected following the ‘wash’ step, and
represents the difference between affinity gels that are complementary and non-complementary
80
to the 150 nt target. It is clear from this data that much more target was captured with the
complementary probe than the non-complementary probe, supporting the conclusion that the
capture was selective.
Figures 3.23 and 3.24 demonstrate the selective capture of the 250 nt and 400 nt length targets by
affinity capture gels containing complementary and non-complementary probes. The
complementary region on the 250 nt target is between base number 25 to 43; the complementary
region on the 400 nt target is between base number 162 to 184. The sequences for the target can
be found in Appendix B. From these sets of data, it can be observed that there is a difference
between the amount of material remaining in the capillary when a complementary and non-
complementary target bound. The amount of material remaining for uidA and SMN may
represent some interaction of the uidA with the SMN probe. The uidA probe is selective towards
the uidA gene in E. coli K12, while SMN is a probe for the spinal muscle neuron gene. The SMN
probe was meant to be a non-complementary probe to the 400 nt target. The two probes were
used for complementary and non-complementary probe due to similar GC content as well as melt
temperatures.
-20
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Figure 3.22: Difference in the amount of target retained after the ‘wash’ step from capture and elution
experiments between affinity capture gels that were complementary (3 µM β-actin) and non-complementary (3 µM non-β-Actin) to a 150 nt length DNA target (20 nM). The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to
concentration using a calibration curve.
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Figure 3.23: Difference in the amount of target retained after the ‘wash’ step from capture and elution
experiments between affinity capture gels that were complementary (3 µM β-actin) and non-complementary probe (3 µM non-β-Actin) to a Cy5 labelled 250 nt DNA target (10 nM). The experimental
conditions are the same as in Figure 3.17 except the last step in the ‘wash’ step was applied for 40 minutes rather than 20 minutes. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error
following correlation of average fluorescence intensity to concentration using a calibration curve.
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uidA SMN
Am
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fmol)
Figure 3.24: Difference in the amount of target retained after the ‘wash’ step from capture and elution
experiments between affinity capture gels that contained complementary (3 µM uidA) and non-complementary (3 µM SMN) to a Cy5 labelled 400 nt DNA target (130 nM). The experimental conditions are the same as in Figure 3.17 except the last step in the ‘wash’ step was applied for 50 minutes. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average
fluorescence intensity to concentration using a calibration curve.
82
3.4 Performance of the Affinity Gel for the Capture DNA Targets
Figure 3.25 shows the amount of complementary 150 nt target captured by the affinity capture
gel to different concentrations of the 150 nt target in the original sample solution and Figure 3.26
shows the response to a mixture of the 150 nt and non-complementary target. The conditions
that were selected for the gel formulation and capture conditions were based on criteria for
optimization that were identified from a quarter-fractional factorial for gel formulation and
analysis of stringency conditions using a three-level factorial analysis: Affinity capture gel:
12.5%T, 1%C, 3 µM probe. Capture conditions: Incubation time: 5 min, injection voltage: 222
Vcm-1, wash voltage: 222 Vcm-1, wash temperature 40 °C, wash buffer: 1xTBE/PVP/25% v/v
formamide.
0
20
40
60
80
100
120
140
0 0.1 0.2 0.3 0.4 0.5 0.6
Original Amount of Target in Sample (pmol)
Am
ount
Captu
red (
fmol)
Figure 3.25: Amount of 150 nt target captured onto affinity capture gel as a function of concentration.
Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 150 nt target in 1XTBE/PVP, 20 minute electrokinetic injection at 133 Vcm
-1. Incubation time: 5 min. Wash Step:
electrophoresis at 133 Vcm-1
for 25 minutes at 25 °C with 1x TBE/PVP buffer. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to
concentration using a calibration curve.
83
0
5
10
15
20
25
30
35
1.31/0.71 0.48/0.71 0.32/0.71 0.16/0.71
Amount non-complementary/complementary in original target solution
(pmol)
Am
ount
of
Mate
rial C
aptu
red (
fmol)
non-complementary complementary
Figure 3.26: Amount of 150 nt target captured from samples in mixtures of complementary and non-
complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 150 nt target and non-complementary target in 1XTBE/PVP, 20 minute electrokinetic injection at 133 Vcm
-1. Incubation time:
5 min. Wash Step: electrophoresis at 133 Vcm-1
for 25 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP buffer. The data was obtained from confocal fluorescence images (Chipreader) of the
capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.
From Figure 3.25, the amount of complementary target retained by affinity capture gel did not
show a linear response, and may indicate saturation of the immobilized probe. In Figure 3.26
examines the affect of different amounts of non-complementary target in a constant amount of
complementary target. The results indicate non-selective adsorption of the non-complementary
target on the affinity capture gel as well as a saturation effect for the non-complementary target
at approximately 0.48 pmol, which might indicate a maximum level of the non-complementary
target being non-selectively adsorbed. There might also be a suppression of the amount of non-
complementary target at the highest non-complementary content used (at 1.31 pmol non-
complementary).
Originally, a Cy3-labelled 100 nt sequence (LAMA3) was selected for use as the non-
complementary target in these sets of experiments. The interaction of the 100 nt sequence that
was non-complementary to the probe was examined. Figure 3.27 shows the level of retention of
84
the non-complementary target was not influenced at different formamide concentrations in the
buffer during the wash step.
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.01
0 10 25
%formamide (v/v)
Flu
ore
scen
ce In
ten
sit
y (
AU
)
Figure 3.27: Comparison of amount of material retained by the 150 nt complementary target when
treating with 100 nt non-complementary target. Affinity capture gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 40 nM complementary
(150 nt Cy5-β-actin) and 45 nM non-complementary targets (100 nt Cy3-LAMA3) in 1xTBE/PVP, 20 minute electrokinetic injection at 133 Vcm
-1. Incubation time 5 min. Wash Step: electrophoresis at
133 Vcm-1
for 25 minutes at 40 °C with 1xTBE/PVP with different concentrations of formamide. Data was derived from images obtained from epifluorescence images of Cy3 channel (Alpha). Error bars represent
1 standard deviation of three trials.
Based on the results, it was observed that the fluorescence intensity resulting from the 100 nt
non-complementary target used originally was retained to a small degree and did not vary at
different stringency conditions examined. This indicates a small amount of non-selective
adsorption. Since the objective of this experiment was to examine the change in non-
complementary level with increasing formamide to increase stringency conditions, and the use of
the 100 nt target was not influenced by stringency conditions, the 100 nt non-complementary
was not used for these experiments.
Therefore a mixture of longer non-complementary PCR target containing targets of 500 nt and
greater was used to examine the affect of different stringency conditions in the factorial
experiment. Figure 3.28 shows the agarose gel electrophoresis of the targets. This target was
obtained by circumstance rather than by design, and originated from a contaminated DNA
template used for PCR. Therefore, the actual sequence was not known. The sequence was
85
assumed to be non-complementary to the probe sequence used. Results summarized below in
Table 3.5 shows a similar quantity of non-complementary target captured using a different probe
(uidA), suggesting that the non-complementary target was non-complementary to the probe
sequences used in these experiments.
Figure 3.28: 1% Agarose gel electrophoresis for non-complementary target used in efficiency
experiments. Lane 1: DNA Ladder. Lanes 2 and 3: non-complementary target used in factorial analysis. Run conditions: 100 V, 1 hour, 1x TBE buffer.
Figures 3.29 - 3.31 indicate the quantity of complementary material captured by the affinity
capture gel for the 150, 250 and 400 nt targets in a mixture of 150 nM (1.5 pmol) non-
complementary targets shown in Figure 3.28. This concentration of non-complementary target
was chosen as the maximum amount of material tested that previously had demonstrated the
highest level of retention in the capture gel. Again, a non-linear response was observed, which
may indicate saturation of the immobilized probe.
The results between Figures 3.25 and 3.29 represent data for samples containing complementary
150 nt target and samples containing 150 nt complementary target and non-complementary,
respectively. It was observed that samples containing the same quantity of complementary
86
material, a higher amount of complementary target was captured when the sample contained the
complementary target alone versus when the non-complementary target was present. This could
be due to a number of effects. Since experiments conducted containing mixtures was washed at
high stringency conditions (25%v/v formamide at 40 °C), loss of some complementary target
would be expected. Additionally, the presence of the non-complementary target can also add
additional sources of interactions between the complementary target strand and immobilized
probe, occupying these targets for hybridization. It would be expected the same trend for the
other two targets examined.
0
20
40
60
80
100
120
0 0.1 0.2 0.3 0.4 0.5
Original Amount of Target in Sample (pmol)
Am
ount
Captu
red (
fmol)
Figure 3.29: Amount of material captured for 150 nt target in mixture of constant concentration of non-
complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 150 nt target and
1.5 pmol of non-complementary target in 1XTBE/PVP, 20 minute electrokinetic injection at 133 Vcm-1
. Incubation time: 5 min. Wash Step: electrophoresis at 133 Vcm
-1 for 25 minutes at 40 °C with 25%v/v
formamide/1x TBE/PVP buffer. The data was obtained from epifluorescence microscope images (Alpha) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error
following correlation of average fluorescence intensity to concentration using a calibration curve.
87
0
50
100
150
200
250
0 0.2 0.4 0.6 0.8 1 1.2
Original Amount of Target in Sample (pmol)
Am
ount
Captu
red (
fmol)
Figure 3.30: Amount of material captured for 250 nt target in mixture of constant concentration of non-
complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 250 nt target and
1.5 pmol of non-complementary target in 1XTBE/PVP, 30 minutes electrokinetic injection at 133 Vcm-1
. Incubation time: 5 min. Wash Step: electrophoresis at 133 Vcm
-1 for 40 minutes at 40 °C with 25%v/v
formamide/1x TBE/PVP buffer. The data was obtained from epifluorescence microscope images (Alpha) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error
following correlation of average fluorescence intensity to concentration using a calibration curve.
0
50
100
150
200
250
300
350
400
450
0 0.2 0.4 0.6 0.8 1 1.2
Original Amount of Target in Sample (pmol)
Am
ount
Captu
red (
fmol)
Figure 3.31: Amount of 400 nt target captured in mixture of non-complementary targets onto affinity
capture gel as a function of concentration. Affinity Capture Gel: 12.5%T/1%C, 3 µM uidA probe. Injection step: 10 µL of 400 nt target and 1.5 pmol of non-complementary target in 1XTBE/PVP, 34 minutes electrokinetic injection at 133 Vcm
-1. Incubation
time: 5 min. Wash Step: electrophoresis at 133 Vcm-1
for 50 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP buffer. The data was obtained from epifluorescence microscope images (Alpha) of the
capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.
88
Tables 3.3-3.5 presents a summary of the performance of the affinity capture gel in terms of
recovery (amount recovered relative to the amount of complementary target in the original
sample solution) and purity (relative amount of complementary target versus the total amount of
DNA target (complementary and non-complementary) in the original sample solution). It is
important to note that these results are based on elution of the entire capillary without
concentration as achieved by heating of the entire capillary at once. Therefore, the volume of
material eluting through was assumed to be the volume of the capillary (350 nL). For
comparison, Section 3.6 will describe the performance of the affinity capture gel for the same
targets are concentrated by step elution.
Table 3.3: Summary results for Recovery and Purity for mixture containing 150 nt and 1.5 pmol non-
complementary targets by affinity capture gel. Recovery and purity of the original solution and by selective capture are presented. The recovery and purity were calculated based on removal of material from the affinity capture gel by elution of the entire capillary (no concentrating). Experimental conditions
shown in Figure 3.29.
150 nt Without selective
purification By selective purification (no concentrating)
Amount of
comple-mentary target in original sample solution
Recovery (%)
Purity (%)
Amount of Complemen-
tary target captured
Amount of Non-
complemen-tary material
captured
Recovery (%)
Purity (%)
Enhance-ment
400 fmol 100 23.12 5.52 ± 0.7
fmol 12.4 ± 2 fmol 1.4 ± 0.2 31 ± 6 1.5 ± 0.3
10 fmol 100 0.75 470 ± 160
amol 12.4 ± 4 fmol 4.7 ± 1.6
3.6 ± 1.5
5 ± 2
5 fmol 100 0.37 86 ± 50 amol 12.4 ± 4 fmol 1.7 ± 1.0 0.77 ±
0.4 2.0 ± 1.3
1 fmol 100 0.08 0 0 0 0 0
89
Table 3.4: Summary results for Recovery and Purity for mixture containing 250 nt and 1.5 pmol non-complementary targets by affinity capture gel. Recovery and purity of the original solution and by
selective capture are presented. The recovery and purity were calculated based on removal of material from the affinity capture gel by elution of the entire capillary (no concentrating). Experimental conditions
shown in Figure 3.30.
250 nt Without selective
purification By selective purification (no concentrating)
Amount of
comple-mentary target in original sample solution
Recovery (%)
Purity (%)
Amount of Complemen-
tary target captured
Amount of Non-
complemen-tary material
captured
Recovery (%)
Purity (%)
Enhance-ment
1 pmol 100 42.92 10 ± 4 fmol 13 ± 4 fmol 1.0 ± 0.5 44 ± 22
1.1 ± 0.5
500 fmol 100 27.32 6.0 ± 0.9 fmol 16 ± 3 fmol 1.1 ± 0.2 27 ± 6 1.1 ± 0.2
250 fmol 100 15.82 6 ± 2 fmol 14 ± 2 fmol 2.6 ± 0.8 31 ± 11
2.2 ± 0.7
100 fmol 100 6.99 1.8 ± 0.7 fmol 14 ± 2 fmol 1.8 ± 0.7 11 ± 5 1.8 ± 0.8
50 fmol 100 3.62 0.7 ± 0.2 fmol 14 ± 2 fmol 1.4 ± 0.5 4.8 ± 1.7
1.5 ± 0.5
10 fmol 100 0.75 0.32 ± 0.1
fmol 14 ± 2 fmol 3.2 ± 1.3
2.2 ± 0.9
3.3 ± 1.4
Table 3.5: Summary results for Recovery and Purity for mixture containing 400 nt and 1.5 pmol non-
complementary targets by affinity capture gel. Recovery and purity of the original solution and by selective capture are presented. The recovery and purity were calculated based on removal of material from the affinity capture gel by elution of the entire capillary (no concentrating). Experimental conditions
shown in Figure 3.31.
400 nt Without selective
purification By selective purification (no concentrating)
Amount of
comple-mentary target in original sample solution
Recovery (%)
Purity (%)
Amount of Complemen-
tary target captured
Amount of Non-
complemen-tary material
captured
Recovery (%)
Purity (%)
Enhance-ment
1 pmol 100 42.92 22 ± 4 fmol 14 ± 2 fmol 2.2 ± 0.4 61 ± 13
1.5 ± 0.3
500 fmol 100 27.32 11 ± 3 fmol 14 ± 2 fmol 2.3 ± 0.5 44 ± 12
1.8 ± 0.5
250 fmol 100 15.82 10 ± 2 fmol 14 ± 2 fmol 4.4 ± 0.9 44 ± 10
3.1 ± 0.7
100 fmol 100 6.99 9.2 ± 0.4 fmol 14 ± 2 fmol 9.2 ± 0.4 40 ± 5 6.3 ± 0.7
50 fmol 100 3.62 2.8 ± 0.7 fmol 14 ± 2 fmol 5.7 ± 1.3 17 ± 5 5.2 ± 1.5
10 fmol 100 0.75 0.69 ± 0.20
fmol 19 ± 2 fmol 7 ± 2
3.5 ± 1.1
5.3 ± 1.6
1 fmol 100 0.08 35 ± 30 amol 12 ± 2 fmol 3.5 ± 2.9 0.28 ± 0.24
4.3 ± 3.6
90
Based on the results presented, the lowest quantity of material that could be processed and
purified with the affinity capture gel was 5 fmol, 10 fmol and 1 fmol for the 150 nt, 250 nt and
400 nt targets, respectively. The improvement in purity through delivery of the material by
affinity capture and elution ranged from a factor of 1.1 to 6.3. However, the recovery of the
method was low, and ranged from 2-10%.
3.5 Examination of the Effects of Varying Gel Formulation on Performance
The conditions used to assess the performance of the affinity capture gel were determined based
on optimization as was identified by factorial analysis. Factorial analysis allows for an efficient
systematic multiplexed approach to identify important factors that influence analytical
performance, and furthermore allows identification of interactions between different factors that
have impact on the analytical response.
Two independent factorial experiments were performed to assess and improve the recovery and
purity of the affinity capture gel. A quarter fractional 2-level 5 factor factorial experiment was
carried out to determine the effects of differences in gel formulation on the performance of the
affinity capture gel. Different gel formulations included variation of the quantities of the
following components: the total monomer concentration (%T), the amount of crosslinker (%C),
the oligonucleotide probe concentration, and the concentrations of TEMED and APS radical
initiator. Performance considerations included the amount of probe that was incorporated
(Appendix E) and the amount of target that was captured (Appendix F). The amount of material
eluted from the capillary column following the elution step (electrophoresis at 167 Vcm-1 for
15 minutes at 65 °C) was determined by measuring the fluorescence intensity remaining in the
capillary. Correlation of the measured fluorescence intensity with the calibration curve resulted
in negative concentration values. Therefore, the amount of material remaining was set as zero,
and no differences were observed for the gel formulations examined.
A three level factorial analysis was then completed to determine optimal conditions for capture
of targets and selectivity. Conditions that were examined included incubation time, wash
voltage, wash temperature and formamide content in the elution buffer (Appendix G).
91
The factorial experiments screened for the impact of a large number of factors to identify
dominant trends. The affinity capture gel was meant to be used on a series of targets of different
lengths. Therefore, the intent was to identify general trends rather than optimization of
conditions for a single target length. An exhaustive optimization and exploration of the response
surface was not performed.
The next sections will provide an overview of some of the primary factors that were identified by
the factorial experiments. Primary or significant factors are identified at the 95% confidence
interval. Detailed reports and calculations generated from the factorial experiment are included
in the appendix.
3.5.1 Affect of Gel Formulation on the Quantity of Probe that was Incorporated
The amount of oligonucleotide probe immobilized into the affinity capture gel was determined
on a relative basis by measuring the change in fluorescence intensity following polymerization of
the affinity capture gel, and also following pre-conditioning of the affinity capture gel. This
latter step was considered significant because materials such as unreacted monomer,
oligonucleotide probes and other compounds were removed by electrophoresis. The change in
fluorescence intensity measured following the preconditioning step more accurately indicates the
amount of probe immobilized into the polyacrylamide gel matrix.
A factorial analysis was carried out to examine the influence of factors changing gel formulation.
The factors examined were the monomer and crosslinker content, the amount of oligonucleotide
probe, and the concentrations of the radical initiator system (TEMED and APS). Processing of
the factorial data showed no significant influence (at 95% confidence) (Appendix E) of the
factors on the amount of probe incorporated. The average quantity of oligonucleotide probe
incorporated into the affinity capture gel was 88% ± 9% of the amount initially in the mixture.
This result suggested that the conversion efficiency of the oligonucleotide modified monomers
into the polyacrylamide gel was not affected by differences in the gel formulation for the factors
and concentration ranges examined at the 95% confidence limit. This also implies that the
polymerization efficiency was similar across the different gel formulations
92
The change in fluorescence intensity was reported rather than the absolute fluorescence intensity
due to a possible reaction between the fluorophore and the radicals as well as possible
differences in the degree of light scatter. Initially, the factorial analysis on the absolute
fluorescence intensity of the capillary following polymerization showed a negative result on the
amount of probe by the radical initiator (APS) used (Appendix E2). This result was attributed to
a possible reaction between the fluorophore and radical initiator and also was possibly due to
changes in the physical structure of the gel, leading to changes in the extent of light scatter.
Figure 3.32 shows the percentage of fluorescence intensity lost of the Cy3 labelled
oligonucleotide probe following polymerization after 20 minutes with different concentrations of
TEMED and APS as compared with the fluorescence intensity of the solution before the addition
of the radical initiator. The fluorescence intensity lost was greatest when the TEMED/APS ratio
used was at 4%/10%. At this ratio, the concentration of TEMED and APS were 0.22 M and 0.44
M. For all the other ratios tested, the concentration of TEMED was in excess of the
concentration of APS. A larger APS concentration than TEMED may result in excess APS
available to react with the oligonucleotide probe directly.
0
10
20
30
40
50
60
70
80
90
10% / 10% 10% / 4% 4% / 10% 4% / 4%
%v/v TEMED/ %w/w APS Ratio
Perc
enta
ge F
luore
scence I
nte
nsity
Rem
ain
ing (
%)
Figure 3.32: The relative fluorescence intensity following reaction between a 1 µM Cy3 labeled
oligonucleotide with different ratios of TEMED/APS radical initiator system after a period of 20 minutes. The loss was calculated relative to the in initial fluorescence intensity measured of the solution
immediately following the addition of the radical initiator. Error bars represent 1 standard deviation of three trials.
93
Additionally, UV-VIS spectra collected before and after polymerization (Figure 3.33) show
differences in light scattering of the gel. An apparent increase in absorbance above 280 nm is
due to increase in light scattering as the polymer gel is formed [205]. As can be observed, all gel
formulations exhibit some scattering, but scattering was most pronounced when the
concentration for 7.5% and 5%C polyacrylamide gels. The disappearance of the signal at 275
nm has been previously used to track the disappearance of the C=C double bond as the
acrylamide and bis-acrylamide monomers are incorporated into the growing polymer chains
during polymerization [206].
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
200 300 400 500 600 700 800
wavelength (nm)
ab
so
rba
nc
e (
AU
)
before polymerization after 20 min polymerizationa)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
200 300 400 500 600 700 800
wavelength (nm)
ab
so
rba
nc
e (
AU
)
before polymerization after 20 min polymerizationb)
94
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
200 300 400 500 600 700 800
wavelength (nm)
ab
so
rba
nc
e (
AU
)
before polymerization after 20 min polymerizationc)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
200 300 400 500 600 700 800
wavelength (nm)
ab
so
rba
nc
e (
AU
)
before polymerization after 20 min polymerizationd)
Figure 3.33: UV-VIS spectra of bulk polyacrylamide gels before and after polymerization for 20 minutes
as a function of different gel formulations. a) 12.5%T/5%C, b) 12.5%T/1%C, c) 7.5%T/5%C, d) 7.5%T/1%C
The results from Figure 3.32 and 3.33 demonstrate that absolute fluorescence intensity data from
the factorial analysis might be influenced by reaction with radicals and light scattering and may
not necessarily be representative of the amount of probe present in the gel. Therefore, the
relative difference in fluorescence intensity before and after pre-conditioning was used to
determine the amount of probe incorporation. The fluorescence intensity inside the capillary
following the polymerization of the affinity capture gels was obtained. Since nothing had been
done to the capillaries at this point, the amount of probe inside should still be equal to the
amount added in the original solution. Differences in the fluorescence intensity between
different capillaries would be the result of undesired reactions by the radical initiator as well as
differences in local environment, but not due to difference in the amount of oligonucleotide
95
probes incorporated into the capillary. Therefore, the factorial analysis was carried out to
examine if differences in gel formulation would result in a difference in the percentage of probes
incorporated into the gel. Since none of the factors showed a significant result, the average
percentage of incorporation was determined to be 88%.
It is important to note that the total quantity of probe incorporated into the gel is determined on
the concentration of oligonucleotide probe in the pre-polymer solution. The results highlighted
in the previous paragraph indicate that for the concentration range examined, the percentage of
probe incorporated into the gel was not influenced by the gel formulation. This suggests that for
an a initial probe concentration of 3 µM and 0.5 µM, the final probe concentration inside the
affinity capture gel after polymerization pre-conditioning would be 2.64 µM and 0.44 µM.
3.5.2 Effect of Radical Initiators on Oligonucleotide Sequence
The results from the previous section indicate that the radicals may react with the fluorophore
attached to the oligonucleotide target. This raises the issue of possible effects of the radical
initiator on the oligonucleotide sequence, with potential cleavage of the probe or structural
changes of the nucleotides.
Persulfate radical anion is a strong oxidizing agent and can attack organic species aside from the
acrylamide monomer [207–209]. Sulfate radical anions are generated from thermal homolysis of
the sulfur-sulfur bond. Sulfate radical anion may also be converted to hydroxyl radical in
aqueous solution, as shown in the reactions below.
⋅→ −−4
282 2SOOS
⋅+→+⋅ −− OHHSOOHSO 424
⋅++⋅→ −−−OHHSOSOOS 44
282
The rate of hydrolysis of sulfate radical anion at neutral pH is slow (k=107 M-1s-1) compared to
rate of reaction with most monomers (k=108-109 M-1s-1) and becomes significant when the
monomer concentration is very low. The persulfate radical anion initiates polymerization by
attacking the vinyl group of the monomer either through direct addition to the double bond or an
96
electron transfer to generate a radical anion in the monomer species. The formation of a radical
anion can lead to chain propagation through either a radical or anionic mechanism [210].
⋅−−−→=+⋅ −−CXYCHOSOCXYCHSO 2324
⋅++→=+⋅ +−− CXYCHSOCXYCHSO 22424
Since the sulfate radical anions initiate polymerization by attacking vinyl bonds, the sulfate
radical anion can also attack the double bonds present in all the nucleotide bases [209, 211, 212].
Again, this can proceed either through an electron transfer to the double bond or the addition of
the sulfate radical to the nucleobase. Elimination of SO42- produces a one-electron oxidized
species [212].
It has been reported that guanine is the most easily oxidized of the nucleic acid bases [213]. The
dominant reaction of the sulfate radical anion is the one-electron oxidization of guanine, with a
minor contribution from adenine [213]. The reaction is proposed to first produce an addition-
adduct, followed by a rapid loss of SO42- to give the one electron oxidized radical species [207].
The one electron oxidation produces a positive charge, and guanine is the major site for this
positive hole localization. The cationic radical of guanine is often deprotonated to become a
neutral radical species at pH 4.5 to 9.5 [207, 209, 213].
Figure 3.34 shows possible reaction product(s) between the nucleobases and SO4- radical. It is
also possible for the other components besides the nucleobase to be damaged. Figure 3.35
demonstrates the transfer of the radical from the nucleobase to the sugar moiety for ribonucleic
acid. The lone electron can cleave the nucleobase from the sugar, releasing it from the
oligonucleotide strand, or can cleave the phosphate backbone, breaking the oligonucleotide
strand [211, 214, 215]. Attack of the anion radical directly on the negatively charged phosphate
backbone is unlikely as there is electrostatic repulsion [213]. It has been shown that the SO4-
radical preferentially attacks the nucleobase rather than the deoxyribose or phosphate [211–213].
Studies done on polyribonucleotide polyA and single stranded DNA of mixed base sequence
with sulfate radical anions showed a strand breakage efficiency (yield of strand breakage for total
radical induced) of less than 5% strand breakage [213]. This was due to inefficient transfer of
the radical from the guanine base to the sugar moiety as shown in Figure 3.35 when the radical
97
of attack was the sulfate radical anion [213]. Radiation induced radicals interacting with guanine
have also demonstrated inefficient strand breakage [213].
Figure 3.34: Reaction products between the sulfate radical anion and (a) adenine, (b) guanine, (c)
cytosine and (d) thymine. Adapted from [207].
98
Figure 3.35: Scheme of the reaction between the radical and the nucleotide base that can lead to either
removal of the nucleoside base or strand cleavage. Adapted from [213].
The inclusion of TEMED in the polymerization of acrylamide serves as a catalyst for the
generation of radicals with APS [216–218]. The TEMED/APS combination acts as a redox pair,
where a one electron reduction of TEMED by persulfate generates one TEMED radical and one
sulfate radical anion [219, 220]. This enhances the polymerization rate by a factor of 3 [216].
Although there have been a lot of studies on the reaction between sulfate radical anion and DNA,
the reaction of the TEMED radical with DNA does not appear to be as well studied. Possible
reaction of the radical initiator system with the oligonucleotide probes used in our experiment
will be explored in the next section.
3.5.3 Cleavage of Oligonucleotide Probe by Radicals
The following experiment examined the possibility of cleavage of the oligonucleotide probes due
to reaction with the radical initiators. A 5 µM sample of Cy5 labeled oligonucleotide was mixed
with different ratios of TEMED and APS for five minutes, followed by the addition of 12 mM of
99
hydroquinone to the solution. Hydroquinone has been reported as a free radical scavenger [221,
222]. The reaction mixtures were then injected into a 12.5%w/v linear polyacrylamide gel
capillary and the products were tracked by capillary gel electrophoresis. Figure 3.36 shows the
electrophoretogram of a mixture of 12 nt (2 µM Cy5 - dT4A3T5), 19 nt (0.5 µM Cy5 - SMN
Target) and 20 nt (1 µM Cy5-dC20) oligonucleotides.
0
0.5
1
1.5
2
2.5
300 320 340 360 380 400 420 440
Time (s)
Flu
ore
scen
ce In
ten
sit
y (
AU
)
12nt
19 nt
20 nt
Figure 3.36: Electrophoretogram for a solution containing 2 µM 12nt, 0.5 µM 19 nt and 1.0 µM 20 nt
oligonucleotides. Cy5 labeled targets. Injection: 10 µL sample volume, 142 Vcm-1
, 4 s. Run condition: 142 Vcm
-1 in 1xTBE/PVP buffer.
Figure 3.37 shows the electrophoretograms for the solutions of 19 nt oligonucleotide targets
(Cy5-SMN) after reacting with different amounts of TEMED and APS for five minutes and
quenched with hydroquinone. Figure 3.37(a) shows the electrophoretogram of the 19 nt
oligonucleotide mixed with hydroquinone (no radical initiator).
100
0
0.2
0.4
0.6
0.8
1
1.2
400 420 440 460 480 500
Time (s)
Flu
ore
scence I
nte
nsity (
AU
)
a)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
400 420 440 460 480 500
Time (s)
Flu
ore
scence I
nte
nsity (
AU
)
b)
101
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
400 420 440 460 480 500
Time (s)
Flu
ore
scence I
nte
nsity (
AU
)
c)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
400 420 440 460 480 500
Time (s)
Flu
ore
scence I
nte
nsity (
AU
)
d)
102
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
400 420 440 460 480 500
Time (s)
Flu
ore
scence I
nte
nsity (
AU
)
e)
Figure 3.37: Electrophoretograms of the reaction products between a 19 nt Cy5 labelled target and different amounts of TEMED/APS. Injection: 10 µL sample volume, 142 Vcm
-1, 4 s. Run condition:
142 Vcm-1
in 1xTBE/PVP buffer. a) Control b) TEMED/APS in 10%/10%. c) TEMED/APS 10%/4%, d) TEMED/APS 4%/10%, e) TEMED/APS 4%/4%
A summary of the migration times of the various peaks is presented in Table 3.6. The results
show, based on migration time, a peak corresponding to the original 19 nt oligonucleotide target
as well as the appearance of a second, slower moving peak after reaction with the radical
initiator for 5 minutes. The intensity of this second peak is higher for cases where a higher
concentration of persulfate was used, suggesting that this second peak is a reaction product
between the oligonucleotide and the sulfate radical anion.
Table 3.6: Summary of the migration times of the peaks observed from the CGE experiment. The sequence of the oligonucleotide target used in these experiments: 5’ Cy5 - ACA GGG TTT CAG ACA
AAA T 3’. Error represents 1 standard from three trials. Reaction mixture Migration
Time (s)
19 nt and hydoquinone only (no radical initiator)
457 ± 11
457 ± 18 19 nt + TEMED/APS 10% v/v/10%w/v 471 ± 13
443 ± 11 19 nt + TEMED/APS 10% v/v/4%w/v 461 ± 13
437 ± 11 19 nt + TEMED/APS 4% v/v/10%w/v 455 ± 14
440 ± 14 19 nt + TEMED/APS 4% v/v/4%w/v 458 ± 17
103
It is possible that this second peak is the one electron oxidation of the guanine base present on
the oligonucleotide strand. The one electron oxidation of guanine can add a positive charge to
the oligonucleotide sequence, assuming the base is not deprotonated. The resulting
oligonucleotide product would migrate slower (smaller charge density) and observed as the
secondary peak. Since the purpose of this the examination was to examine possible cleavage
reaction products, further analysis of the second peak was not performed.
Guanine is the base most susceptible to radical attack, cleavage would be expected to have the
highest probability to at these sites. Fragments that are of at least 3 bases and 12 bases in length
would be observed in the electrophoretograms if cleavage had occurred. The absence of peaks
migrating faster than the original 19 nt peak from the electrophoretograms suggests that cleavage
did not occur, or occurred in amounts too small to be detected by fluorescence. This agrees with
experiments performed on single stranded DNA where only low levels of cleavage was observed
[209].
3.5.4 Examination of Damage to Nucleobases by Radical
Damage to the nucleobases by the radical initiator was also considered. If the fidelity of the
probe sequence was compromised by reaction with the radical initiator, then this would
negatively affect the selectivity as well as the capture efficiency of the oligonucleotide probes.
Previous experiments have shown the ability of the affinity capture gels to selectively capture
complementary oligonucleotide targets. Selective capture by the affinity gel was also observed
for 150 nt and as well as other targets complementary to the probe. Additionally, displacement
chromatography experiments demonstrated an ability to discriminate a system where a 1 bp
mismatch was present, and an increase in displacement was observed (Appendix B4).
Experiments performed using a probe that was 10 nt in length also demonstrated the successful
capture of the 150 nt target. The use of the TEMED/APS radical initiator system has also been
reported by other groups for the immobilization of oligonucleotide sequences onto gel matrices
[97, 151, 100]. Possible negative effects of the sulfate radical anion on the oligonucleotide
targets were not reported to be an issue. These results and observations already strongly suggest
that the probe sequence was not significantly damaged by the radical initiators.
Melt curves were obtained using oligonucleotide probes that had been subjected to reactions with
the radical initiator system to further examine whether there was evidence of damage of the
104
nucleobases. A shift in the melt temperature would be observed if damage to a significant
proportion of nucleobases had occurred.
The radical initiator was allowed to react with the oligonucleotide probes for 20 minutes, and the
target strand was introduced and allowed to hybridize for an additional 20 minutes prior to
determining the melt temperature. Hydroquinone was not added to stop the reaction since it
showed a large UV absorbance in the region of interest, obscuring the absorbance from the
DNA. Additionally, APS and TEMED exhibited an absorbance peak in the UV region, which
changed as a function of temperature. Therefore melt curves of solutions containing just APS
and TEMED were first run as controls and this background signal was subtracted from the
spectra obtained for the melt curve experiments. Figure 3.38 shows a representative melt curve
where the oligonucleotide probe was not reacted with the radical initiator (control). Figure 3.39
presents melt curves where the oligonucleotide probe was reacted with the different
concentrations of TEMED and APS.
1
1.1
1.2
1.3
1.4
1.5
1.6
45 50 55 60 65 70
Temperature (C)
norm
aliz
ed a
bsorb
ance (
AU
)
Figure 3.38: Representative melt curve for a sample of 0.3 µM 19 bp duplex (SMN) in 1 x TBE. Error
bars represent 1 standard deviation of three trials.
105
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
40 45 50 55 60 65 70
Temperature (C)
Norm
aliz
ed A
bsorb
ance (
AU
)
a)
1
1.2
1.4
1.6
1.8
2
2.2
2.4
45 50 55 60 65 70 75
Temperature (C)
Norm
aliz
ed A
bsorb
ance (
AU
)
b)
106
0.9
1.1
1.3
1.5
1.7
1.9
2.1
50 55 60 65 70 75
Temperature (C)
Norm
aliz
ed A
bsorb
ance (
AU
)
c)
0.9
0.95
1
1.05
1.1
1.15
1.2
1.25
1.3
1.35
1.4
45 50 55 60 65 70
Temperature (C)
Norm
aliz
ed A
bsorb
ance (
AU
)
d)
Figure 3.39: Melt curves for a sample of 0.3 µM 19 bp duplex in 1 x TBE where the probe was reacted with different amounts of TEMED and APS for 20 minutes prior to addition of the complementary target. a) TEMED/APS 10%/10%, b) TEMED/APS 10%/4%, c) TEMED/APS, 4%/10%, d) TEMED/APS 4%/4%.
Error bars represent 1 standard deviation of three trials.
The melt temperature was determined by taking the first derivative of the melt curve and taking
the temperature point at the inflection point between 50 and 70 °C. The results are presented in
Table 3.7.
107
Table 3.7: Summary of melt temperatures of the oligonucleotide duplex following reaction with the different radical initiator ratio. Error represent 1 standard deviation of three trials.
Concentration of
TEMED/APS added
Melt Temperature
(°C)
Control 58.5 ± 3
10%/10% 62.5 ± 1.7
10%/4% 59.5 ± 3.5
4%/10% 62.5 ± 2.3
4%/4% 57.5 ± 1.7
The sequence of the oligonucleotide target examined was: 5' ATT TTG TCT GAA ACC CTG T
3'. Guanine is predominantly the base attacked by sulfate radical anion, and 3 such nucleotides
were available. The expected change in melt temperature for 3 base pair mismatches would be a
reduction of around 5 to 10 °C, assuming a 1-3 °C shift per 1% mismatch [223]. Such shifts of
melt temperature were not observed. However, it does not preclude the possibility that the any
possible damage only resulted in partial loss of hydrogen bonding ability (i.e., 1 or 2 hydrogen
bonds between GC versus 3 hydrogen bonds normally).
3.5.5 Examination of Conditions that Affect Capture of Complementary Targets
This section examines various factors that affect the amount of material captured by affinity
capture gels, and that influence the discrimination between complementary and non-
complementary target. This work focused on:
1) A fractional factorial design to determine effects of different gel formulations on the amount
of target captured. The design matrix for this analysis was the same as used previously to
examine the amount of probe incorporated into the polyacrylamide gel matrix. The effects were
measured as the relative difference between the concentration of target injected and
concentration of target retained.
2) The discrimination between complementary and non-complementary target on the affinity
capture gel. This was examined at different stringency conditions (temperature, and increasing
formamide content in buffer) using a 3 level 2 factor factorial experiment. Two responses were
measured: the percent recovery and purity.
108
After a year of work it became obvious that some polyacrylamide gels failed either right after
polymerization or during the pre-conditioning step. Failure was observed in the form of voids
inside the polyacrylamide gel (gel did not fill continuously inside the capillary), which prevented
a current from being carried across the capillary. It was also noted that even if gels did not fail
immediately, discontinuities in the gel developed following 1-2 hours of use. These mechanical
failures of gels were observed to be more frequent for gels made with the higher monomer
concentrations used in the factorial experiment (about 50% of the time) than in lower monomer
concentration gels (less than 10%). The amide group of polyacrylamide chains is hydrolytically
unstable above pH 7; hydrolysis of the amine group forms charged carboxylic acid groups along
the polyacrylamide, transforming it to polyacrylate. EOF can occur along the charged surface,
causing matrix swelling, distortion and collapse of the gel matrix [224, 225]. Additionally,
volume changes during the gelation phase can also lead to shrinkage of the gel versus the
original volume of the pre-polymerization mixture, resulting in voids in the gel matrix [226,
227].
Mechanical failure was not encountered for gels made with linear polyacrylamide (no
crosslinker). Therefore, the factorial experiment to examine stringency conditions was
conducted using 12.5% T linear polyacrylamide gels. Factors identified as significant in
adjustment of stringency conditions were expected to be largely independent of gel formulation,
with the results being indicative of other gel formulations.
Table 3.8 summarizes the factors identified as significant (at 95% confidence) and whether a
positive or negative effect was observed. Increases in monomer and crosslinker content
increased the concentration of the target injected. Increasing monomer level increased the
quantity of target captured by the gel. A negative effect was observed for increasing the amount
of crosslinker and concentration of TEMED. Negative interaction effects were also observed
between the monomer and crosslinker content and monomer content and concentration of APS.
The use of higher temperature and increasing formamide content increased the stringency and
improved the purity of the sample that was recovered from the capillary affinity capture gel.
109
Table 3.8: Summary factors which were identified as significant from factorial analysis (95% confidence). The (+) and (-) after each factor denotes whether the effect was positive or negative. Response Measured:
Concentration of target Injected
(Appendix F2)
Amount of Target
Captured (Appendix F3)
Percent Recovery (Appendix
G2)
Percent Purity (Appendix G3)
Total Monomer (+)
Total Monomer (+)
None Formamide (+)
Crosslinker content (+)
Crosslinker content (-)
Temperature x Formamide (+)
Probe (+) Probe (+)
TEMED (-)
Total Monomer x APS (-)
Significant Factors:
Total Monomer x Crosslinker
content (-)
3.5.6 Affinity Capture of Complementary Targets with Probes that are Immobilized in 3D Gel Supports
The immobilization of probes into a 3D gel matrix for DNA diagnostics has been reported and
studied extensively by Mirzabekov et al., and termed as MAGIChip (Microarrays of Gel-
immobilized Compounds on a chip). In the MAGIChip technology, pads of polyacrylamide gels
containing oligonucleotide probes that have been covalently incorporated are spotted onto a solid
substrate surface with dimensions of approximately 100 µm x 100 µm x 20 µm (0.2 nL) [228].
The MAGIChip has been used for discrimination of duplexes and mismatched 126 nt targets
using immobilized oligonucleotide probes of 17-26 nt length [204, 229–233].
The suspension of oligonucleotide probes inside the 3D gel matrix allows the probes to be
spaced out from one another. This may alleviate issues of steric hindrance associated with
immobilization of oligonucleotide probes on a 2D interface. Hybridization kinetics inside 3D
gels have been observed to be similar to that of solution phase hybridization [204, 231–233].
The method for discrimination of complementary and non-complementary targets used with the
affinity capture gel in this thesis was that used in the MAGIChip system. In the MAGIChip
system, the gel pads are first incubated with solutions containing DNA targets. During this step,
DNA targets move through the gel by diffusion, with some delay due to interactions with the
immobilized probe. This increases the time required for the targets to move into and saturate the
gel pads, given by τH,:
110
sol
DHKh
Km
+=
1ττ (5)
where τD is the characteristic time for the targets to move into the gel by diffusion alone, m is the
immobilized probe concentration, K is the thermodynamic association constant and hsol is the
target solution concentration. This process responsible for slowing the time required to saturate
the gel pads is termed "retarded diffusion".
Following this step, the gel pads are washed and incubated with buffer, and the targets diffuse
out of the gel pads. The rate at which the DNA targets diffuses out of the gel is governed by the
degree of interaction between the DNA targets and immobilized probe. The loss of signal J(t) as
targets are removed from the capture gel by diffusion at time t is described by:
W
t
oeJtJτ
−
≅)( (6)
where Jo is the initial fluorescence signal following reaching hybridization at thermodynamic
equilibrium of hybridization, τw is the characteristic washing time for the target to wash off the
probe immobilized gel:
KmDW ττ = (7)
where τD is the characteristic time of the target by diffusion alone, m is the probe concentration
and K is the association constant.
From Equation (6), discrimination between complementary and mismatch/non-complementary
targets is achieved based on the difference in the association constant between perfect and
mismatch/non-complementary targets [204, 229–233]. Targets that form perfect duplexes are
delayed to a larger degree versus those which form non-perfect duplexes. The ratio between
fluorescence intensity of perfect duplexes and mismatches increases as a function of time.
Increasing stringency conditions during the wash step (increase wash temperature, denaturant)
can reduce the total time needed to discriminate between complementary and mismatched targets
[229, 234].
111
In our system, DNA targets are moved through the affinity capture gel by electrophoresis, and
the time required for targets to move through the gel is based on its electrophoretic mobility,
which can be modified by the presence of the immobilized probe given by Equation (4) (Section
3.0.9).
Figure 3.40 demonstrates such a difference in rates by tracking the fluorescence signal of
labelled complementary target in a gel containing complementary and non-complementary
probes for the gel system used in this work. Here, an average fluorescence intensity was
calculated from quantitative values generated from profile plots from confocal microscope
images taken of the capillary at different times during the washing process. This tracks the rate
at which the complementary target elutes off the capillary and how the immobilized probe affects
such rate. These results demonstrate that the probe immobilized gel system presented in our
thesis follows Equation (6), and behaves similarly to the MAGIChip 3D gel system.
y = 0.947e-0.0201x
R2 = 0.9803
y = 1.0062e-0.0409x
R2 = 0.9966
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30 40 50 60 70 80
Time (min)
No
rmali
zed
Flu
ore
scen
ce (
AU
)
complementary probe non-complementary probe
Figure 3.40: Decrease in average fluorescence intensity as labelled DNA targets wash out of the
capillary following injection of the DNA target into affinity capture gels containing complementary (�) and non-complementary (x) probe. Fluorescence intensity is normalized against the initial fluorescence
intensity. Experimental conditions: Affinity capture gel: 10%T, 5%C, 2 µM β-actin probe, 2 µM non-β-actin probe.
Injection conditions: 10 µL, 250 nM Cy5- 50 nt target, electrokinetic injection at 133 Vcm-1
for 20 minutes. Incubation Time: 5 mins. Wash conditions: 133 Vcm
-1 at 25 °C with 1xTBE/PVP. The data was obtained
from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars represent 1 standard deviation of three trials.
The Mathies group has also used similar affinity capture gel chemistry inside an integrated
microfluidic device for the purification of PCR amplicons for downstream sequencing [96]. The
112
capture gel chamber used in their microfluidic device was 1 mm x 1 mm, and samples were
delivered into the chamber via a 100 µm wide channel.
In their system, Mathies demonstrated the dynamic capture of DNA target as it was introduced
electrokinetically into the capture gel by optimizing the electric field and hybridization
temperature. The change in free DNA (S) is governed by the on/off rate of the duplex formed
between the target and immobilized probe as well as the electrophoretic velocity of the free DNA
target by electrophoresis [96]:
),()(]:[]][[),( txSx
xECSkCSktxSt
sbf∂
∂−+−=
∂
∂µ (8)
where S is the concentration of free ssDNA, C is the concentration of the immobilized probe, µ is
the mobility of the DNA, E(x) is the field strength at position x and kf and kb are the association
and dissociation rate constants, respectively.
Binding of DNA targets was maximized by changing temperature such that the association
constant was maximized relative to the dissociation constant while not exceeding the melt
temperature. Electrokinetic stacking was also used to increase the concentration of DNA targets
during injection into the affinity capture gel. The geometry of the affinity capture chamber was
such that the DNA targets experienced a field drop from 600 Vcm-1 to 60 Vcm-1 as the DNA
targets moved from the 100 µm channel into the 1 mm wide capture chamber. This resulted in
electrokinetic stacking of the DNA targets due to a drop in electrophoretic mobility [96].
However, this work only reported the use of the purification method on PCR reaction products,
where the major component was expected to be the target of interest and discrimination with
non-complementary target may not have been of substantial concern.
The next sections of this thesis expand on such earlier studies by Mathies and others. A
systematic examination of different gel formulations on the amount of target captured was
carried out. The influence of gel formulation on the capture of DNA targets has not been
reported in the literature. Differences in gel formulation may 1) change the effect of
electrokinetic stacking during the injection step, 2) change how the oligonucleotide probes are
incorporated, and therefore the accessibility of probes to target for hybridization, and 3) change
how the DNA targets migrate though the gel.
113
3.5.7 Effects of Gel Formulation on the Concentration of Target Injected
Electrokinetic stacking may increase the concentration of the DNA target as it is introduced into
the affinity capture gel. Changes in the gel formulation resulted in changes in the electrophoretic
mobility of the DNA targets through the gel, increasing the concentration of the target injected
into the gel during electrokinetic injection.
The concentration of the dye-labelled 150 nt target injected into the capillary was observed to be
dependent on the gel formulation. Based on the factorial analysis (Appendix F1), the factors
which had a significant effect (95% confidence) were the concentrations of monomer,
crosslinker and oligonucleotide probe.
Figure 3.41 shows the concentration levels for DNA target injected into the affinity capture gel
as a function of different gel formulations. The data of Figure 3.41 show that the concentration
of material injected into the capillary increased as the total amount of monomer and crosslinker
increased.
0
50
100
150
200
250
300
7.5%/1% 7.5%/5% 12.5%/1% 12.5%/5%
Gel Formulation (%T/%C)
Concentr
ation o
f T
arg
et
Inje
cte
d (
nM
)
Probe (0.5 uM) Probe (3 uM)
Figure 3.41: Concentration of Target Injected. Values corrected for differences in fluorescence intensity
as a function of different gel formulations. Affinity capture gel: Conditions as prescribed in factorial design. Injection: 10 µL of 170 nM (low probe) 500 nM (high probe) target. Electrokinetic injection at 133 Vcm
-1 for 20 minutes. The data was obtained
from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence
intensity to concentration using a calibration curve.
114
The positive effect observed for increased oligonucleotide probe loading was due to the sample
containing a larger concentration of target; the target-to-probe ratio was adjusted to be the same
at the different probe concentrations that were examined.
Figure 3.42 shows the difference in electrophoretic mobility of the DNA targets through the
different gel formulations, demonstrating a correlation between electrophoretic mobility and the
quantity of target that was injected. A larger difference in electrophoretic mobility between
DNA target in solution and gel would stack the targets, resulting in an increase in the
concentration.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
7.5%/1% 7.5%/5% 12.5%/1% 12.5%/5%
Gel Formulation (%T/%C)
DN
A M
obili
ty (
x 1
04 c
m2/V
s)
Figure 3.42: Electrophoretic mobility of 150 bp target using different gel filled capillaries with different gel formulations. Gel formulations used were as previously prescribed, with radical initiator concentrations of
TEMED/APS of 10%/10%. Mobility calculated based on the time required to travel 2.6 cm along the capillary.
Injection conditions, 10 µL, 0.5 µM Cy5-150 bp target, 86 Vcm-1
, 15 s. Run conditions, 143 Vcm-1
, 1x TBE/PVP buffer. PMT gain 400 mV. Error bars represent 1 standard deviation of three trials.
The quantity of material introduced into a gel filled capillary by electrokinetic injection, Q, is
given based on the following equation [235]:
)1(2 απµ
−= rCL
tVQ
iiep (9)
where µep is the electrophoretic mobility of the DNA target, Vi is the injection field strength, ti is
the injection time, L is the capillary length, C is the concentration of the target solution, r is the
115
radius of the capillary and α is a correction term to account for the interstitial space, geometric
obstruction factor and volume fraction of electrolyte component of having a gel filled capillary.
Changes in electrophoretic mobility of the analyte will affect the quantity of material introduced
into the capillary. Since the mobility of the targets through the different gel formulations were
not the same, the injection time was adjusted so that enough time was allowed for the DNA
target to move through the entire length of the capillary. This reduced any differences in the
quantity of material injected between the different gel formulations.
3.5.8 Effects of Gel Formulation on the Quantity of Target Captured
Following injection of DNA targets into the gel, unbound material was removed from the
capillary column by 'washing' the capillary with buffer. The affinity capture gel was 'washed' to
remove any unbound or non-complementary material. From the factorial analysis (Appendix
F3), a positive effect was considered significant (95% confidence) for increasing monomer
content, while a negative effect was observed on increasing the amount of crosslinker, TEMED
concentration and an interaction between monomer content and crosslinker and monomer
content and APS concentration.
The response levels for the different gel formulations are presented graphically in Figure 3.43. It
is important to note that the wash time was adjusted based on the difference in electrophoretic
mobility of the targets. This avoided variations that would have occurred as a result of
insufficient time for material to wash off the gel.
The observed results can be due to two possibilities: 1) the accessibility of the oligonucleotide
probes that are incorporated into the gel is different at the different gel formulations, and 2)
changes in the gel structure might affect the conformation of the DNA targets as they migrate
through the porous gel.
116
0
50
100
150
200
250
300
350
400
450
7.5% / 1% 12.5% / 1%
Gel Formulation (%T/%C)
Am
ount
of
Targ
et
Captu
red (
fmol)
Probe (0.5 uM) Probe (3 uM)a)
0
50
100
150
200
250
300
350
400
7.5% / 5% 12.5% / 5%
Gel Formulation (%T/%C)
Am
ount
of
Targ
et
Captu
red (
fmol)
Probe (0.5 uM) Probe (3 uM)b)
Figure 3.43: Amount of material captured by affinity capture gel. Values were corrected for differences
in fluorescence intensity as a function of different gel formulations. Affinity capture gel: Conditions as prescribed in factorial experiment. Injection: 10 µL of 170 nM (low probe) or 500 nM (high probe) Cy5-150 nt target. Electrokinetic injection at 133 Vcm
-1 for 20 minutes.
Incubation time: 5 mins at 10 °C. Wash Step: electrophoresis at 133 Vcm-1
for 25 mins at 10 °C with 1xTBE/PVP. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries
and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.
117
3.5.9 Effects of Polymerization of Polyacrylamide
Polymerization of acrylamide in the presence of crosslinker occurs initially at many nucleation
sites where the monomer material is incorporated into growing centers. These gel centers have a
higher density of crosslinker than the remainder of the polyacrylamide gel. This is due to the
higher reactivity rate of the crosslinker as compared with acrylamide monomer. The increased
crosslinker content creates gel centers which are densely crosslinked [206, 217, 236–238].
Crosslinks are formed between growing polymer centers that are in close proximity as monomer
material is depleted. This second step forms the porous network within the polyacrylamide gel
[205, 217, 237, 239–241].
The dimension of the pore network (average pore size, volume occupied by the pores, pore
surface area) can be influenced by the initial monomer content. For example, gels with a larger
average pore size can be obtained where the initial monomer concentration or radicals is low. A
smaller number of growth centers is generated, and the centers are allowed to grow to a larger
size prior to being crosslinked to one another. This results in a pore network with a larger
average pore size. This is shown schematically in Figure 3.44(a). Conversely, gels with smaller
average pore size can be obtained when the number of growth centers is increased (i.e.
increasing monomer or radical concentration). The growth centers are smaller when crosslinked
together, resulting in a pore network with smaller average pore size. This is shown
schematically in Figure 3.44(b).
It is important to note that polyacrylamide gel consists of a distribution of pores of different
dimensions. Kremer et al. examined the mode, mean and variance of pore sizes formed for
different concentrations of cationic polyacrylamide gels [242]. The initial monomer
concentrations they examined were higher than used in our experiments (20%-30%T, 0.4-
1.2%C). General trends observed were that the mode, mean and variance in pore sizes decreased
with increasing acrylamide and crosslinker concentration. Increasing total monomer
concentration from 20 to 30%T (at 0.3%C) decreased the mode from 7.3 nm to 2.8 nm, mean
pore size 16.8 nm to 10 nm and variance from 400 nm to 169 nm. Increasing crosslinker content
from 0.4 to 1.2%C (at 15%T) showed a decrease from mode 9.9 nm to 5.4 nm, mean 12.4 nm to
6.7 nm and variance from 204 to 64 nm [242].
118
Figure 3.44: Schematic representation of polyacrylamide gel and how oligonucleotide probes are
incorporated into the gel. a) indicates how gels with large pores are formed and b) indicates how gels with small pores are formed. ssDNA with hairpin structures are presented in the center and illustrates the difference between how DNA might move through the different gel structures; a) unhindered via Ogston,
and b) stretched by reptation.
For the affinity capture gels used in this thesis, the oligonucleotide probe may not be accessible
for hybridization with target if these are incorporated inside gel centers. Conversely, if they are
incorporated onto the surface area inside the pore network, then they are available for interaction
with target. Changes in the initial monomer concentration can influence the size of the gel
centers, changing the proportion of oligonucleotide probes present on the surface of the pore
network versus inside the gel centers. This can change the total quantity of probe available for
hybridization.
Additionally, changes in the average pore size can affect how the DNA targets migrate through
the polyacrylamide gel. The mechanism by which DNA targets move through a porous gel is
dependent on the radius of gyration and the pore size. The volume occupied by a strand of DNA
is taken as a sphere with a radius given by the radius of gyration, Rg, of the DNA. The radius of
gyration is the root-mean-square of the distance of the center of mass of the polyelectrolyte and
the individual monomer segments. If Rg is smaller than the pore size of the polyacrylamide gel
(Figure 3.44a), then the DNA moves unhindered through the gel by the Ogston process, where
the gel acts as a sieve and movement is based on the probability that the DNA molecule will find
119
a pore large enough to accommodate its passage through the gel [243–246]. If Rg is larger than
the pore size (Figure 3.44b), then DNA moves by a reptation process, and must weave through
the gel by stretching and relaxing as it moves through the pores [243–246]. Migration of the
ssDNA through the gel by reptation may eliminate any secondary structures, opening up the
target region so that it available for hybridization with the immobilized probe.
In solution, ssDNA can adopt a discrete number of conformations, dependent on temperature, pH
and salt concentration [244, 247]. Most commonly, ssDNA will form intramolecular base pairs,
either with bases in close proximity (short range folding) or with bases further apart on the strand
(long range folding). Short range folding is kinetically favoured and expected to dominate under
all conditions over long range interactions. Short range base pairing also prevents these bases
from participating in long range associations. Additionally, forming long range base pair
interactions will contract the negatively charged DNA molecule, increasing charge repulsion.
Long range interactions also decrease the conformational entropy. At alkaline or low ionic
strength conditions, ssDNA can become unfolded and behave as randomly coiled, flexible
polyelectrolytes [248–250]. The ssDNA may take on more compact forms under high ionic
strength conditions due to charge screening that is provided by the salt [248].
A number of calculated hairpin structures for the 150 nt target was previously shown in Figure
3.19 (Section 3.3). The structures predict that the complementary region of the target participate
in the formation of a hairpin structure. Ogston migration of ssDNA through the polyacrylamide
gel would retain these hairpin structures, leaving the target region unavailable for hybridization.
Conversely, if the pore size of the polyacrylamide gel was smaller than the Rg of the ssDNA, the
ssDNA would extend and weave through the gel. This would linearize the target region, making
it available for hybridization with a consequently higher amount of target captured.
From the results of the factorial experiment, affinity capture gels made with higher monomer
concentrations resulted in a larger quantity of DNA target captured versus gels made with lower
monomer concentrations. The results might be due to a combination of a difference in the
availability of the oligonucleotide probe inside the affinity capture gel and differences in how the
ssDNA targets move; with Ogston migration in gels prepared from lower monomer
concentrations, and by reptation when moving through gels prepared using the higher monomer
concentrations.
120
3.5.10 Effect of Probe Availability As a Function of Gel Formulation
Figure 3.45 presents data about the amount of oligonucleotide captured by hybridization as a
function of different gel formulations.
0
200
400
600
800
1000
1200
12.5%/5% 12.5%/1% 7.5%/5% 7.5%/1%
Gel Formulation (%T/%C)
Am
ount
of
Targ
et
Captu
re (
fmol)
Figure 3.45: Amount of target captured for a 19 nt probe/target pair at different monomer and crosslinker
levels as examined in the factorial analysis. Affinity capture gel: 0.5 µM SMN probe. TEMED and APS used were at 10% w/v and v/v, respectively.
Injection: 10 µL, 0.5 µM Cy5 complementary target. Electrokinetic injection at 133 Vcm-1
for 10 min. Incubation time: 5 mins at 10 °C. Wash step: electrophoresis at 133 Vcm
-1 for 15 min at 10 °C with
1xTBE/PVP. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following
correlation of average fluorescence intensity to concentration using a calibration curve.
The shorter 19 nt target was used to assess whether the availability of oligonucleotide probe
altered with gel formulation. The use of 19 nt target avoided issues of sterics and possible
differences in migration mechanism through the gel in contrast to the 150 nt ssDNA. The
amount of material captured is assumed to reflect the availability of probes that are accessible for
hybridization. No statistically significant differences were observed in the concentration of
target captured for the different gel formulations, suggesting that the amount of probe available
was not altered. Assuming that the quantity of target was sufficient to saturate all available
probes, and that only hybrids remained after washing, then the percentage of probe incorporated
was 40% ± 5% of the original amount of probe in the original pre-polymerization mixture.
121
3.5.11 Effect of Gel Formulation on Migration of DNA Targets
An estimate of the average pore size was attempted using the different gels that were made. The
intention was to establish some evidence about the processes of migration that occurred in the
different gel formulations. The calculation of pore size was based on the method reported by Lo
and Ugaz [251]. The pore size can be estimated by identifying the critical DNA fragment size
where the mechanism of migration through a particular gel transitions from Ogston to reptation
[189, 251, 245]. Under the Ogston regime, the porous gel acts like a sieve, and the plot of log of
mobility (µ) normalized by free solution mobility (µo) varies linearly with gel concentration c
with KR retardation coefficient [217, 244]:
cK R
o
−=
µ
µlog (10)
Figure 3.46 shows the relationship of log(µ/µo) for different gel formulations using known
sequences of various lengths from a dsDNA ladder. Here, the initial linear portion corresponds
to DNA fragments that move through the gel under a Ogston regime. The point where linearity
deviates is considered the point where DNA fragment begins to move through the gel by
reptation. The Rg of the DNA fragment calculated at this point can be used to provide an
estimate for pore size of the gel. Reported values of free solution mobility reported in the
literature ranged from 3.46x10-4 cm2sV-1 to 3.8 x10-4 cm2sV-1 [251–253]. The average of the
values, 3.63 x 10-4 cm2sV-1, was used as the free solution mobility used in the data shown in
Figure 3.46.
122
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0 50 100 150 200 250
Base Number
log(u
/uo)
12.5%/5% 12.5%/1% 7.5%/5% 7.5%/1%
Figure 3.46: log(µ/µo) versus number of bases for DNA fragments from a Low Range DNA Gel ladder
(Fast Ladder) using TOPRO3 in different gel formulations. Capillary Gel: Different gel formulations as described in factorial analysis with 1 µM TOPRO-3. Injection:
10 µL of ladder incubated with 10 µM TOPRO3 (1:1), 5 seconds, 570 Vcm-1
. Run: 93 Vcm-1
in 1xTBE/PVP/1µM TOPRO3. Acquisition settings: PMT Gain 400 mV, sampling rate 1Hz.
The radius of gyration of a polymer can be approximated using equation 11 [252]:
−
−
+
−×=
−p
L
DDD
D
g
D
eL
p
L
p
L
ppLMR 16631
3)(
32
2 (11)
where p is the persistence length (3 nm (ssDNA), 50 nm (dsDNA)) [254][253], LD is the contour
length (0.34 nm/base (dsDNA), 0.43 nm/base (ssDNA)) [251]. Table 3.9 lists the estimated pore
radius for the different gel formulations.
123
Table 3.9: Determination of pore size from the Ogston plot presented in Figure 3.46. The range of DNA fragments where log(µ/µo) deviates from linearity is assumed to be the size range where DNA transitions
from Ogston to reptation. The radius of gyration of the dsDNA fragments was calculated by Eq (11), where persistence length for dsDNA was 50 nm, and contour length of DNA was 0.34 nm/base.
Gel Formulation
(%T/%C)
Fragment range where DNA deviates from Ogston
(bp)
Rg (nm) = Pore radius
12.5%/5% 20-35 1.9-3.4
12.5%/1% 35-50 3.4-4.7
7.5%/5% 75-100 7-9.2
7.5%/1% 100-150 9.2-13.4
The gel concentrations for some of the gel formulations used in this work in a similar range as
reported by other groups. Lo and Ugaz reported a pore radius of 5.57 nm at 12%T/1.3%C and a
radius of 9.6 nm for a gel at 6%T/0.7%C gel [251]. Rousseau also reported pore sizes for gels
higher than 8%T/5%C to be under 10 nm [255]. Tombs examined pore size of gels by
electrophoresis of protein markers through different gels. Based on the diameter calculated by
molecular weight, they reported a pore diameter of 4 nm and 7 nm at 12.5%/5% and 7.5%/5%,
respectively [256].
Pore size has been directly examined by others by means of electron microscopy. Rüchel and
Blank obtained SEM images of the freeze-dried PAAm gels, and reported pore sizes based on
SEM images to be on the order of 2-15 µm for gels in 2.5%T-4%T/5% C gels [257–259]. These
values are several orders of magnitude larger than those determined by electrophoretic methods.
Results from SEM images imply that large particles such as a 130 nm diameter virus particle and
E. coli cells (0.5x2 µm) could migrate through the gels. However, it was demonstrated that these
particles could not migrate through the gel. E. coli could only move through very dilute, liquid
agarose like gels (0.03%). It is theorized that although larger pores are observed under SEM,
they are interconnected by much smaller channels as determined by the electrophoresis results
[259].
The radius of gyration of the 150 nt ssDNA was calculated to be 7.7 nm. At the lower monomer
concentration, the pore size of the gel is larger than the Rg of the ssDNA. This results in the
DNA targets moving through the gel relatively unhindered. This limits the portion of the DNA
where the target region is accessible to the probe. However, at the higher monomer
concentration gels, the pore size is less than the DNA, meaning that a larger proportion of the
124
ssDNA targets must reptate through the gel, eliminating secondary structure and increasing the
availability of the target region for hybridization.
A compromise takes place between availability of the target region for hybridization and the
quantity of DNA that can migrate through the gel. As previously mentioned, increasing
crosslinker content will decrease mode and average pore size of the gel. It is possible that this
can create pores so small that the DNA target no longer can access oligonucleotide probes in that
area of the gel. Additionally, pores might be of such dimensions that only a limited number of
DNA strands can fit inside. Decreasing the size of the pore would further reduce the number of
targets that might enter, reducing the contact of probe with target. This can lead to a decrease in
the amount of target captured for gel concentrations where the crosslinker content was increased.
Additionally, the radical initiator can also influence the final polyacrylamide gel structure [216,
217]. It was noted from the factorial analysis that a negative effect was observed for increasing
TEMED concentration as well as an interaction effect between monomer concentration and APS
was present. TEMED catalyzes the generation of radicals with APS and enhances the
polymerization rate by a factor of 3 [216–218]. Increasing the number of radicals present in the
pre-polymer solution can initiate a larger number of such growing radical centers during the
initial polymerization phase, resulting in gels with smaller average pore size, and may further
limits accessibility of the DNA target to regions of the gel.
Relative changes in probe density for the different gels can be calculated based on the
accessibility of 19 nt target and the average pore size. These relative changes are listed in Table
3.10. The calculation was done based on the number of pores that can fit in a cube of volume
with sides 1000 x 1000 x 1000 nm cube for a gel of the lowest monomer concentration
(7.5%T/1%C). The number of pores was determined assuming that they were packed side by
side in the cube. Probe density was calculated based on the amount of probe available from data
obtained for 19 nt oligonucleotide target binding from Figure 3.45.
125
Table 3.10: Probe density based on 0.5 µM probe in the original monomer solution. Values were calculated based on the number of pores that could fit in a 1000x1000x1000 nm cube at the lowest gel
formulation (7.5 %T/1 %C). The calculation assumes that the pore volume for the remaining gel formulation was the same as the lowest monomer concentration and number of pores for the remaining
gel formulations were calculated as such. The amount of probe was determined based on previous experiments examining the percentage of probe incorporated, and the accessibility of the probe from
experiments performed with 19 nt targets in Figure 3.45.
Gel Formulation
(%T/%C)
Number of Pores (x10
5)
Volume total (x10
8
nm3)
Surface Area Total (x10
8
nm2)
Probe density( x
10-21
nmoles/nm
2)
Ratio probe
density (versus
7.5%/0.1%)
7.5%/1% 0.52-1.6 5.2 1.2 - 1.7 1.81-3.24 1
7.5%/5% 1.6-3.6 5.2 1.7 - 2.2 3.24-4.48 0.69-0.76
12.5%/1% 12-32 5.2 3.3 - 4.6 6.67-8.77 0.35-0.37
12.5%/5% 32-180 5.2 4.6 - 8.3 8.77-12.8 0.21-0.25
For the other gel formulations, the number of pores present in the gel was calculated assuming
they occupied the same volume as the lowest gel concentration. For example, the volume
occupied by the pores was 5.2x108 nm3 at 7.5%T/1%C. It was assumed that the pores in the
7.5%T/5%C gel would occupy the same volume, and the number of pores was the pore volume
divided by the volume of each pore. Surface area and probe density for the other gel
formulations were determined assuming that the total pore volume was the same for all gel
formulations. Plieva reported a 7% decrease in pore volume for the same magnitude increase in
monomer concentration due to water vapour uptake [260]. The values of probe density therefore
represent the maximum probe density. Since the pore volume decreases with increasing
monomer concentration, it is expected that the actual probe density is lower than that calculated.
Based on the relative changes in probe density, gels with smaller pores result in larger total
surface areas, but the probe density decreases for increasing gel concentration, suggesting that
higher gel concentrations should lead to a decrease in amount of target captured. However, as
shown in Figure 3.43 an increase in the concentration of target captured was observed for the
higher monomer concentrations. This results suggests the importance of forcing ssDNA strands
to migrate by reptation, opening the target region for interaction with the immobilized probes
and thereby increasing the amount of material retained by the affinity capture gel.
3.5.12 Effect of Stringency Conditions on Percent Recovery and Purity during Washing Step
Following injection of target into the affinity capture gel, discrimination between complementary
and non-complementary targets occurred during the washing step. Here, targets inside the
126
affinity capture gel are moved out of the capillary by electrophoresis. Targets which interact
with the immobilized probe experience a modification of electrophoretic mobility as indicated by
Equation (4) (Section 3.0.9), and will take longer to move through the capillary compared to
targets which do not interact with the probe. Over time, the ratio of the amount of
complementary to non-complementary target remaining inside the capillary should increase. The
rate at which this increase occurs can be modified by changing the stringency conditions of this
washing step. Increasing stringency can decrease the time required to achieve a defined
signal/noise ratio [229].
The effect of increasing stringency by increasing temperature and addition of formamide into the
buffer on percent recovery and purity was examined using a three level factorial experiment.
These experiments were done using target solutions containing complementary and non-
complementary targets. The data from the factorial analysis (Appendix G3) confirmed that
increasing formamide content improved purity, and that there was an interaction effect between
formamide and temperature.
Figure 3.47 shows the percent recovery and percent purity for mixtures of targets as a function of
different temperature and formamide content. An improvement in percent purity as formamide
and temperature was increased was due to improvement of removal of non-complementary
material while complementary targets were retained.
Temperature can effect the dissociation/association rate of the DNA duplex and the stability of
the DNA duplex. The dissociation/association rates both increase with temperature [96, 229].
Glazer et al. examined association rates (ka) and dissociation rates (kd) at 22 and 45 °C for
perfect duplexes and a mismatch (20 nt). In this work the values of ka were similar for both the
complementary and the mismatched target, and increased by a factor of 3 at the higher
temperature. The kd of the mismatched target was larger than the complementary target by a
factor of 2, and both increased by a factor of 24 with increasing temperature [261]. Bishop et al.
developed a mathematical model based on the finite element method, and demonstrated that the
association constant increased by an order of magnitude from 320-340 K, while the dissociation
constant changed from 10-10 to 10-4 for the same temperature change for a 20 bp duplex [262].
The goal during the washing step was to maximize recovery by ensuring that the bound
complementary targets did not dissociate from the immobilized probe. It was desirable to keep
127
the dissociation constant low while still being able to discriminate between complementary and
non-complementary target. Therefore, the washing step was also examined at a low temperature
(10 °C). This was thought to reduce dissociation of complementary target, which might provide
a positive effect on recovery. However, from the factorial analysis, temperature alone did not
have a significant effect on recovery.
Temperature and formamide in combination can be used to disrupt the stability of the duplex
formed between target and probe, with formamide forming competing hydrogen bonds with the
DNA targets [263]. Increasing stringency in the capillary would weaken the stability of duplexes,
reducing the interaction between target and probe, allowing for materials to move along the
capillary column.
It has been reported that formamide lowers the melt temperature by 0.63 °C per percent
formamide added [264]. The amounts used in this work therefore results in a drop of 6.3 °C at
10 % v/v and 15.8 °C at 25%, resulting in an expected melt temperature for the complementary
target from 65 °C to 59 °C and 49 °C, respectively. The highest temperature condition during
the wash step was 40 °C. This was still well below the melt temperature for the highest
formamide content that was used so as to minimize the amount of complementary material that
could be lost. Formamide concentrations less than 30% v/v have been shown to not affect
association constants [265].
128
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0%v/v formamide 10%v/v formamide 25%v/v formamide
Recovery
(%
)
10 C 25 C 40 C
0
20
40
60
80
100
120
0% v/v formamide 10% v/v formamide 25%v/v formamide
Purity
(%
)
10 C 25 C 40 C
Figure 3.47: Percent recovery and percent purity of sample following washing of the affinity capture gel.
Data represents the average from the duplicates defined in the factorial design. Affinity capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and
12 nM of non-complementary target. Electrokinetic injection at 181 Vcm-1
for 20 minutes. Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm
-1 for 25 minutes at 10 °C, 25 °C, 40 °C
using 0%, 10% and 25% v/v formamide of 1xTBE/PVP. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to
concentration using a calibration curve.
129
Figures 3.48 and 3.49 show the profile plots from confocal microscope images of the affinity
capture gels showing the position of the non-complementary target and complementary target in
the capillary following the wash step at the different stringency conditions of 0%v/v formamide;
10 °C and 25%v/v formamide; 40 °C, respectively. It was observed that some complementary
was lost at the higher stringency, but a larger proportion of non-complementary target was lost in
the same amount of time. As stated previously, discrimination between complementary and non-
complementary targets is based on the degree of interaction between the DNA strand and
immobilized probe. Figure 3.48 shows the efficacy of increasing the stringency condition on the
non-complementary target component. Here, increasing formamide content decreases the degree
of duplex formation with the probe, which allowed the non-complementary target to be removed
from the capillary following the same wash time. Additionally, in figure 3.49, it can be observed
that the complementary target is still retained in the capillary even after increasing formamide
content. However, some complementary material was removed and the plug representing the
complementary material has moved forward through the capillary. This suggests an effect of
formamide on the complementary target, where it has disrupted some base pair formation and the
decrease in mobility is not as large.
In this experiment, the non-complementary target was longer (500 nt) than the complementary
target (150 nt). Therefore, the expected mobility was slower than the 150 nt complementary
target. The measured mobility of the non-complementary target was 50 ± 3 µm/s, and that for
the 150 nt target was 69 ± 1 µm/s through an unmodified polyacrylamide gel of the same
monomer concentration.
The change in mobility of the DNA targets through the affinity capture gel versus mobility
through unmodified gel is related to the degree of interaction between complementary and non-
complementary targets. The mobility of targets was calculated by observing the centre of the
fluorescence peak along the capillary and the time applied during the wash step. The mobility
was calculated as 9.4 µm/s for the complementary target and 12 µm/s for the non-complementary
target. From Equation (4), the Ka was calculated as 6.3x106 M-1 and 3x106 M-1 for the
complementary and non-complementary target, respectively. This was calculated for the lowest
stringency condition.
130
The conclusions from the data presented in Section 3.5 are that a larger amount of target was
captured by gels that were made with higher acrylamide monomer concentrations due to the
ssDNA target moving through the gel by reputation. The gel formulation that showed the
maximum amount of target captured was at 12.5 %T total monomer concentration and 1%
crosslinker content, which is a balance between availability of the target sequence and the
quantity of probe that is accessible in pores. The maximum purity was achieved when the
stringency condition was set at 25%v/v formamide and 40 °C temperature during the wash step.
It is these conditions that were applied in experiments examining the purification of targets of
150, 250 and 400 nt length in the presence of non-complementary target as presented previously
in Section 3.4.
131
0
1000
2000
3000
4000
5000
6000
7000
8000
0 0.5 1 1.5 2 2.5 3 3.5 4
Distance (cm)
Flu
ore
scen
ce In
ten
sit
y (
AU
)
a)
0
500
1000
1500
2000
2500
3000
3500
0 0.5 1 1.5 2 2.5 3 3.5 4
Distance (cm)
Flu
ore
scen
ce In
ten
sit
y (
AU
)
b)
Figure 3.48: Fluorescence profile plots of capillaries taken from the outlet to inlet end generated from
confocal microscope (Chipreader) images tracking the non-complementary target following affinity capture and a subsequent wash step using two different stringency conditions.
Affinity capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and 12 nM of non-complementary target. Electrokinetic injection at 181 Vcm
-1 for 20 minutes. Incubation time: 5
minutes at 10 °C. Washing step: electrophoresis at 181 Vcm-1
for 25 minutes, at (a) 10 °C with 1xTBE/PVP and (b) 40 °C with 25%v/v formamide/1x TBE/PVP.
132
0
2000
4000
6000
8000
10000
12000
14000
0 0.5 1 1.5 2 2.5 3 3.5 4
Distance (cm)
Flu
oro
scen
ce In
ten
sit
y (
AU
)
a)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
0 0.5 1 1.5 2 2.5 3 3.5 4
Distance (cm)
Flu
ore
scen
ce In
ten
sit
y (
AU
)
b)
Figure 3.49: Profile plots of capillaries from the outlet to inlet end from confocal microscope (Chipreader) images of the complementary target following affinity capture the wash step in the affinity capture and a
subsequent wash step using two different stringency conditions. Affinity capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and 12 nM of non-complementary target.
Electrokinetic injection at 181 Vcm-1
for 20 minutes. Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm
-1 for 25 minutes, at (a) 10 °C with 1xTBE/PVP and (b) 40 °C with 25%v/v
formamide/1x TBE/PVP.
133
3.6 Selective Concentrating of Oligonucleotide Targets by Step Elution From Affinity Gels
An examination was completed of the potential for collecting and concentrating the captured
targets from affinity gels by step elution using heating. In previous work, captured targets were
eluted by heating the entire capillary column above the melt temperature of hybrids during
electrophoresis. The target was released from the affinity gel column in a volume at least equal
to the geometric volume of the capillary, i.e. a relatively large volume. The interest in the use of
step-elution was to stack targets into a smaller volume than that of the capillary, thereby
increasing the concentration of the targets on elution from the capillary.
In step elution, only one portion of the capillary is heated at a time during electrophoresis.
Hybridized targets in the heated region denature and the single-stranded material that is released
from the gel collectively moves in the affinity capture gel under the electric field. The heater is
then advanced along the capillary, sequentially denaturing any hybridized targets in a new region
of the capillary while previously denatured targets are carried along by electrophoresis. By
appropriately matching the rate at which the resistive heater was moved across the capillary to
the mobility of the oligonucleotide targets, the eluting targets could be stacked into a smaller
volume than that of the affinity capture capillary, increasing the concentration of the targets.
Figure 3.50(a) presents data that represents the elution of captured oligonucleotide targets from
an affinity capture gel by step elution. The electrophoretogram tracks the fluorescence intensity
of the Cy5-labelled targets through a window in the capillary column near the elution end as a
function of time. The peak that was observed is indicative of the stacked oligonucleotide targets
eluting from the capillary column. Figure 3.50(b) represents a control experiment using identical
elution conditions, for a gel that made use of non-complementary probes. The complementary
probe used in these experiments was for the SMN sequence and the non-complementary probe
was the β-actin probe.
Table 3.11 summarizes the results of the concentrating effect of step elution as a function of the
length of the eluted gel, i.e., the length of the capillary that was subjected to the step elution
process. The integrated peak area, peak height and peak width of the eluting peak was calculated
using Origin Pro 8.0. The integrated peak area represents the quantity of material eluted during
the stacking experiment. The volume of the eluted material was calculated from the peak width
134
and mobility of the oligonucleotide target. The enhancement factor was calculated as the
quantity of material eluted (peak area) contained in the volume of the eluting peak divided by the
concentration of the same material eluted in the original volume for the length of capillary
eluted. Typically, the enhancement factor is reported as the ratio of peak heights after
amplification and before amplification. However, these experiments were conducted using a low
concentration (50 nM) of probe and target. This was due to saturation of the PMT detector by
the peak of enriched material eluting off the capillary without the use of neutral density filters
and lowering sensitivity. At the same instrument setting, no signal above background noise was
observed from dye-labelled targets captured on the capillary prior to step elution. Rather than
correcting for the signal by developing an instrument correction factor, enhancement ratio was
calculated using peak area, and would minimize the introduction of additional sources of error.
135
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
0 50 100 150 200 250
Time (s)
Flu
ore
scence I
nte
nsity (
AU
)
a)
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
0 50 100 150 200 250 300 350 400 450 500
Time (s)
Flu
ore
scence I
nte
nsity (
AU
)
b)
Figure 3.50: Electrophoretograms comparing the relative fluorescence intensities (concentrations) of short oligonucleotide target by step elution from complementary and non-complementary probes. a)
Affinity capture gel: 50 nM SMN probe, 10% LAAm. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150 Vcm
-1. b) Affinity capture gel: 50 nM β-actin probe, 10% LAAm. Target injection: 10 µL 5 µM
Cy5-SMN target for 1 min at 150 Vcm-1
. Capture step: 10 min, 1xTBE/PVP running buffer, 150 Vcm-1
. Concentrating step: coverage length: 25 mm; step size: 250 µm; step rate: 86 µms
-1; Voltage: 96 Vcm
-1;
Acquisition settings: ND 4, 8 and 16 filters, PMT gain 400 mV. Sampling rate: 10 Hz.
136
Table 3.11: Summary of results of the concentrating effect of step elution of complementary target as a function of elution length. Volume of the eluting targets was calculated based on the peak width and
mobility of the oligonucleotide, which was 86 µms-1
at 96 Vcm-1
. Error represent 1 standard deviation of three trials.
Affinity capture gel: 50 nM SMN probe, 10% LAAm affinity capture gel. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150 Vcm
-1. Capture: electrophoresis for 10 min at 150 Vcm
-1 in 1x TBE/PVP
running buffer. Step elution for concentrating effect: coverage length: 12.5, 25 and 37.5 mm; step size: 250 µm; step rate: 86 µms
-1; Voltage: 96 Vcm
-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain
400 mV. Sampling rate: 10 Hz
Step Elution Length
Volume of
Capillary Eluted
(nL)
Peak Area (AUs)
Peak Width at half max
height (s)
Peak Height (AU)
Volume (nL)
(based on peak width
and mobility)
Concentration of eluted targets in original volume (AU/nL)
x10-3
Conc. of stacked targets (AU/nL)
Enhance-ment Factor
12.5 mm
98.5 0.32 ± 0.07
6.27 ± 0.98
0.047 ± 0.01
4.2 ± 0.9 3.2 ± 0.7 0.08 ± 0.01
24 ± 4
25 mm 197 0.58 ± 0.11
4.15 ± 0.63
0.13 ± 0.03
2.8 ± 0.6 2.9 ± 0.6 0.21 ± 0.06
71 ± 12
37.5 mm
296 0.45 ± 0.26
1.24 ± 0.2
0.5 ± 0.3
0.84 ± 0.43 2.0 ± 0.9 0.81 ± 0.50
393 ± 73
Examining the data in Table 3.11, it can be noted that the peak widths increased for decreasing
elution lengths. Peak widths should increase as the eluting targets move through the capillary
due to longitudinal band broadening. However, as shown schematically in Figure 3.51a, the
experiment was set up so that the eluting targets travelled the same distance for the different
elution lengths examined; the final peak widths were expected to be similar if longitudinal
diffusion was the major contributor to band broadening. Therefore, the data indicates that
application of the heating element also affects band broadening of the oligonucleotide targets as
they travel through the capillary column.
The application of the heating element increases the temperature of the gel at one region of the
capillary (effective heating zone of approximately 0.8 mm). Increasing the temperature of the
gel increases the viscosity of the gel in that area and creates a temporary local discontinuity
where the mobility of the oligonucleotide targets was higher in that region of the capillary [266–
268]. Once past the heated region, the eluted target band would slow as viscosity of the solution
increased. The consequence was that the targets stacked along one side of the heated zone
boundary, and this effect was anticipated to reduce some of the dilution caused by longitudinal
broadening [171–173]. Further band broadening occurred as the targets moved through the
capillary to the detector after step elution, diluting the concentrated targets.
137
Figure 3.51: Schematic diagram of different permutations of the step elution sweeps where a) the resistive heating element was started at the same point at the injection end of the capillary, and the
terminal position was varied. In b), the step elution again swept through different distances, but stopped at the same position along the capillary.
In the case of the shortest elution length (12.5 mm), the eluted targets travelled the longest from
the end of step elution to the detector when compared to the longer elution lengths. This
subjected the eluting targets to the largest degree of band broadening, resulting in the largest
peak width for the shortest elution length.
It may also be possible that stacking the oligonucleotide targets into a small contained volume on
the capillary can create its own localized field similar to what occurs in isotachophoresis. In ITP,
targets with similar mobilities stack in a concentrated zone and become the charge carrier in that
region of the capillary, and a localized field is develops in the zone [172, 173]. However, in ITP,
the analyte is prepared such that no background electrolyte is present and the analytes serve as
the charge carrier in the electrophoresis experiment and plays a role in determining the local field
strength of the channel. In our experiment, the concentration of the 1x TBE buffer used is 90
mM Tris, the maximum concentration of the stacked peak is 20 µM, which means that the charge
carrier during electrophoresis is still the 1x TBE buffer.
138
Table 3.12 summarizes the results for the step elution experiments where the resistive heating
element was stopped at the same position along the capillary for all lengths that were thermally
scanned (depicted in Figure 3.51(b)). The eluted targets travelled the same distance to the
detector after step elution in all three cases that are shown in Figure 3.51(b), eliminating
differences in band broadening that occurred outside of the heated zone.
Table 3.12: Summary of results from step elution of complementary target as determined from data obtained from the experiment depicted in Figure 3.51b. Results for integrated Peak Area, Width, Height
were calculated using Origin Pro 8.0 Volume of the eluting targets was calculated based on the peak width and mobility of the oligonucleotide, which was 86 µms
-1 at 96 Vcm
-1. Error represent 1 standard
deviation of three trials. Affinity capture gel: 50 nM SMN probe, 10% LAAm affinity capture gel. Target injection: 10 µL 50 nM
Cy5-SMN target for 1 min at 150 Vcm-1
. Capture: electrophoresis for 10 min at 150 Vcm-1
in 1x TBE/PVP running buffer. Step elution for concentrating effect: coverage length: 12.5, 25 and 37.5 mm; step size: 250 µm; step rate: 86 µms
-1; Voltage: 96 Vcm
-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain
400 mV. Sampling rate: 10 Hz
Step Elution Length
Peak Area (AUs)
Peak Width at half max
height (s)
Peak Height (AU)
Volume of Peak (nL)
Concentration of eluted targets in original volume (AU/nL)
X10-3
Conc. of stacked targets (AU/nL)
Enhancement Factor
12.5 mm
0.17 ± 0.05
0.37 ± 0.09
0.40 ± 0.09
0.25 ± 0.03
1.7 ± 0.5 0.52 ± 0.12
300 ± 29
25 mm 0.34 ± 0.07
1.2 ± 0.5
0.29 ± 0.10
0.81 ± 0.45
1.7 ± 0.3 0.36 ± 0.13
224 ± 128
37.5 mm
0.52 ± 0.39
0.46 ± 0.34
0.31 ± 0.36
0.93 ± 0.41
1.7 ± 1.3 0.59 ± 0.42
419 ± 205
The trend of peak widths observed in Table 3.12 is reversed from that observed in Table 3.11.
The shortest step elution length resulted in the least broadening. By changing the starting
position of the resistive heater along the capillary, the total capillary column processing time was
shortest for the shortest elution length. The reduction of time on the column minimized
opportunity for band broadening.
The enhancement factors reported in Table 3.12 showed no obvious trend. The enhancement
factor was not statistically different at 95% confidence for the different elution lengths that were
examined. This might be due to variations in matching of the electrophoretic mobility of the
oligonucleotide and the scanning rate of the external heater; an effect that would become more
significant when using longer capillary lengths. The shortest elution length (12.5 mm) provided
the best precision and reproducibility in enhancement factor and was used in the remainder of the
experiments. The compromise would be that use of a shorter region of elution would lower the
quantity of target that would be accessible. Experiments performed examining a range of step
139
sizes (125 to 500 µm) showed no difference on the results (peak width, area, height) of the step
elution process (Appendix H).
3.6.1 Concentrating the 150 nt, 250 nt and 400 nt Targets by Step Elution
Tables 3.13 to 3.15 present the percentage Recovery and Purity that was achieved for samples
containing 150 nt, 250 nt, 400 nt targets and 500 nt non-complementary target processed by
affinity capillary gel electrophoresis and concentrating by step elution. These experiments were
done using the same conditions as outlined in Section 3.4. The percentage Recovery and Purity
were calculated based on the amount of complementary and non-complementary target in the
eluting peak on the capillary. The results were obtained offline versus in real time since two
targets (complementary and non-complementary) were measured and it was not possible to
measure Cy3 and Cy5 channels concurrently in real-time.
Table 3.13: Summary results for Recovery and Purity from affinity capture gel for mixtures containing varying amounts of 150 nt complementary and 1.5 pmol non-complementary targets. The Recovery and
Purity were calculated from quantitative concentration data for the eluting peak by use of calibration curves. Errors represent propagated error following correlation of average fluorescence intensity to
concentration using a calibration curve. Affinity capture gel: 3 µM β-actin probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic injection for 20 min at 133 Vcm
-1. Incubation time 5 min. Wash step: electrophoresis for 25 min at 133 Vcm
-1, 45 °C,
with 25% v/v formamide/1X TBE/PVP. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms
-1; Voltage: 96 Vcm
-1; Acquisition settings (Cy5): PMT gain 500 mV, translation speed:
50 µms-1
, scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS Original Target
Solution By Selective Concentration
Amount of 150 nt
Complementary Target (fmol)
Recovery (%)
Purity (%)
Amount of Complementary target captured
(amol)
Amount of Non-
complementary material
captured (fmol)
Recovery (%)
Purity (%)
Enhance-ment
10 100 0.8 106 ± 16 1.4 ± 0.3 1.0 ± 0.2 6.9 ± 1.8
10 ± 2
5 100 0.4 68 ± 5 1.4 ± 0.3 1.4 ± 0.1 4.7 ± 1.1
14 ± 1
1 100 0.08 1.39 ± 7.4 1.4 ± 0.3 0.1 ± 0.7 0.1 ± 0.5
1.5 ± 8
140
Table 3.14: Summary results for Recovery and Purity for mixture containing varying amounts of 250 nt of complementary and 1.5 pmol non-complementary targets by affinity capture gel. Errors represent propagated error following correlation of average fluorescence intensity to concentration using a
calibration curve. Affinity capture gel: 3 µM β-actin probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic injection for 30 min at 133 Vcm
-1. Incubation time 5 min. Wash step: electrophoresis for 40 min at 133 Vcm
-1, 45 °C,
with 25% v/v formamide/1X TBE/PVP. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 66 µms
-1; Voltage: 96 Vcm
-1; Acquisition settings (Cy5): PMT gain 500 mV, translation speed:
50 µms-1
, scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS Original Target
Solution By Selective Concentration
Amount of 250 nt
Complementary Target (fmol)
Recovery (%)
Purity (%)
Amount of Complementary target captured
(amol)
Amount of Non-
complementary material
captured (fmol)
Recovery (%)
Purity (%)
Enhance-ment
100 100 7 690 ± 96 0.88 ±0.2 0.7±0.1 44 ± 9 7 ± 1
50 100 3.6 290 ± 38 0.88 ±0.2 0.43 ±0.8 20 ± 5 6 ± 1
10 100 0.8 100 ± 42 0.88 ±0.2 1.0 ±0.4 10.2 ±
4.8 15 ± 6
Table 3.15: Summary results for Recovery and Purity for mixture containing 400 nt and 1.5 pmol non-
complementary targets by affinity capture gel. Errors represent propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.
Affinity capture gel: 3 µM uidA probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic injection for 40 min at 133 Vcm
-1. Incubation time 5 min. Wash step: electrophoresis for 50 min at 133 Vcm
-1, 45 °C, with
25% v/v formamide/1X TBE/PVP. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 52 µms
-1; Voltage: 96 Vcm
-1; Acquisition settings (Cy5): PMT gain 500 mV, translation speed: 50
µms-1
, scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS Original Target
Solution By Selective Concentration
Amount of 400 nt
Complementary Target (fmol)
Recovery (%)
Purity (%)
Amount of Complementary target captured
(amol)
Amount of Non-
complementary material
captured (fmol)
Recovery (%)
Purity (%)
Enhance-ment
50 100 3.6 373 ± 44 1.0 ± 0.2 0.75 ± 0.09
25.7 ± 4.4
8 ± 1
10 100 0.8 48 ± 16 1.0 ± 0.2 0.48 ± 0.16
4.3 ± 1.6
7 ± 2
1 100 0.08 0.26 ± 0.30 1.0 ± 0.2 0.03 ± 0.03
0.02 ± 0.03
0.4 ± 0.4
From the results presented in this section, it was demonstrated that concentrating the 19 nt
oligonucleotide targets by step elution provides an enhancement factor of 300 ± 29 at a step
elution length of 12.5 mm as the targets elute off the capillary. Additionally, band broadening is
minimized when the targets are eluting under the heating element, due to changes in mobility as
a function of gel viscosity and the development of a local field strength as the targets are stacked.
Based on the results presented in Tables 3.13-3.15, the lowest quantity of material that could be
processed and purified with the affinity capture gel was 1 fmol, 10 fmol and 1 fmol for the 150
nt, 250 nt and 400 nt targets, respectively.
141
In comparison to the results shown previously in Tables 3.3-3.5 in Section 3.4, where the
recovery and purity of the method was calculated where the targets were not concentrated, the
recovery by selective concentrating was observed to be lower, but the purity was improved by a
factor from 6 to 15 by selective concentrating versus selective purification without any
concentrating.
3.7 Delivery of Concentrated Targets into Microfluidic DNA Biosensing Platform
3.7.1 Design Aspects for Sample Transfer from the Capillary to the Microfluidic Biosensing Platform
The affinity capture gel was intended to serve to selectively purify and concentrate
oligonucleotide targets for delivery into a microfluidic-based DNA biosensor that was previously
described by our research group [269][185]. The following experiments made use of a 19 nt
oligonucleotide target, which was the target length for which the microfluidic biosensor had been
designed. Preliminary experiments using 150, 250 and 400 nt DNA sequences that were injected
directly into the microfluidic based biosensing platform did not result in any appreciable
hybridization signal even though the short target sequence was within the longer sequence. This
was thought to be due to folding of the longer DNA targets, which would likely block
hybridization of the short target sequence with probes that were immobilized on the glass
substrate surface.
The microfluidic biosensing platform was modified to include an interconnect for delivery of the
effluent from the affinity capture gel capillary. The interconnect was designed such that the
capillary was positioned orthogonally on top of a microfluidic channel containing pads of
immobilized oligonucleotide probes. This design was chosen over a design where the capillary
would be in-plane to the microfluidic device. Figure 3.52 illustrates the physical challenge for
the interfacing of a capillary in different orientations to the microfluidic channel. Orienting the
capillary in-plane to the microfluidic channel would result in a large difference between the
diameter of the capillary and the height of the microfluidic channel. The height of the
microfluidic channel was 8 µm, while the diameter of the capillary used here was 160 µm (the
smallest diameter of fused silica capillary that was commercially available with an I.D. of 100
µm). An in-plane configuration would physically impede the delivery of material from the
142
capillary column to the microfluidic channel, with challenges including the matching of
volumetric flow and dilution of a stacked elution band at leading and trailing edges. Orienting
the capillary column to be orthogonal to the microfluidic channel eliminated these issues in the
case where the diameter of the delivery channel of the capillary was situated within the width of
the microfluidic channel (185 µm).
In order to create the orthogonal capillary-microfluidic interconnect, an empty piece of fused
silica capillary was positioned using an xyz micromanipulator stage so that the inner diameter of
the capillary was touching the microfluidic channel on the microfluidic template. PDMS was
poured over this setup and cured on a hotplate.
This method of creating an interconnect is similar to other methods reported in the literature.
Other methods typically have been reported for applications using glass-based microfluidics
chips, and involve creation of a hole that will house the capillary in PDMS or in glass [270–273].
The ports are then plugged with a fitting or flange tubing followed by insertion of the capillary
[271, 274, 275]. Some methods also glued the capillary to the interconnect with epoxy-based
resins [272, 273, 276]. The interconnects described in these publications were for connections to
microfluidic devices where fluid was moved through the system by pressure flow. The use of
pressure flow necessitated additional features for the interconnect to be leak-proof, to minimize
dead volume and to withstand pressures from 140 to 2000 kPa [270–274, 277].
143
Figure 3.52: Schematic diagram illustrating two possible orientations for creating an interconnect
between the capillary column and the microfluidic channel. The interconnect can be created by orienting the capillary (a) orthogonal to the microfluidic channel and (b) in-plane with the microfluidic channel. The
area that is shaded in blue represents the filled area of the capillary and microfluidic channel.
For the microfluidic device used in the work of this thesis, analytes were moved by
electrophoresis. Issues associated with leakage caused by pressure flow were not of significant
concern. Dead volume in the system would result in distortion of the eluting plug of
oligonucleotide targets at the interface port. This was minimized by ensuring that the template
capillary and the affinity gel capture capillaries were as flat as possible [270, 271]. Figure 3.53
shows line scans along the channel of the microfluidic device following delivery of
oligonucleotide targets. The fluorescence signal observed represents the hybridization of the
Cy5-labelled 19 nt complementary targets with the immobilized probe on the glass surface
following injection followed by washing of the concentrated target from the capillary into the
microfluidic channel. The absence of a fluorescence signal from labelled oligonucleotides at the
location of the interconnect port indicates that oligonucleotide was not lost in the interconnect
144
region. The experiment was conducted using the setup described in Figure 2.5. Briefly,
complementary 19 nt targets were captured using the affinity capture gel and concentrated by
step elution. Immediately after step elution, the capillary was trimmed to 2 cm such that the
concentrated oligonucleotide was right near the outlet end of the capillary. The capillary was
then inserted into the previously prepared biosensing platform through the interconnect made
during the casting of the PDMS chip. A potential applied across the inlet end of the capillary
and the end of the microfluidic channel delivered the concentrated oligonucleotide targets from
the affinity capture capillary into the microfluidic channel. As the concentrated oligonucleotide
targets move through the microfluidic channel, they hybridize with oligonucleotide probes
imobilized on the surface of the epoxy-modified glass slide. After the experiment was
completed, the fluorescence intensity across the microfluidic channel was scanned using an
epifluorescent microscope (Alpha). The fluorescence intensity observed corresponds to
complementary targets hybridized to the immobilized probe spots.
Figure 3.53: Line scans of the microfluidic channel of the DNA biosensing platform following delivery of
fluorescently-labelled complementary target by selective concentration. Affinity capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL 5 nM A647 SMN target, electrokinetic injection for 1 min at 150 Vcm
-1. Capture: electrophoresis for 10 min at 150 Vcm
-1 with 1x
TBE/PVP running buffer. Concentration step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms
-1; Voltage: 96 Vcm
-1; Delivery of concentrated targets into microfluidic biosensing platform: 500 V,
10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms
-1 scan rate: 50 Hz.
The interconnect was not always functional at the time of assembly of some of the microfluidic
devices, and would fail after repeated use. Failure was typically observed for devices that had
145
been subjected to approximately 6 cycles of delivery of oligonucleotides, hybridization and
regeneration of the microfluidic chip. Confirmation that the interface was at fault was confirmed
by testing for the presence of the immobilized oligonucleotide probes. This was done by
injecting complementary oligonucleotide targets through the reservoir wells, bypassing the
interconnect. Hybridization confirmed that the immobilized oligonucleotide probes were still
present and selective. Imperfect alignment of the capillary with the microfluidic channel during
the casting process and the repetitive insertion and removal of the capillary during replicates may
have resulted in physical damage of the interconnect, most probably introducing debris that
blocked flow and prevented the delivery of stacked oligonucleotides into the microfluidic
channel.
Selective concentrating of the oligonucleotide targets was done within the capillary column
before coupling to the microfluidic platform. This was done since it was found that the mobility
of the oligonucleotide targets through the capillary was more reproducible when the electric field
was applied to the capillary alone, rather than the capillary-to-microfluidic device. Concentrating
the oligonucleotide targets relied on an accurate measure of the mobility of material moving
through the affinity capture gel. Given the manual handling that was required to define the
length of the capillary column, the positioning of the electrodes, and the positioning of the
capillary in the interconnect region, it was not a surprise that there was significant variability in
the distribution of the electric field between the capillary and microfluidic chip when these were
connected.
Since the concentrating of oligonucleotide targets was done on the capillary, it was necessary to
stop the elution while the plug of target remained inside the capillary. A portion of the capillary
was trimmed prior to interfacing with the microfluidic platform to minimize the distance the
eluted targets needed to travel from the capillary to the microfluidic device, thereby minimizing
longitudinal diffusion.
The field strength used in the capillary-microfluidic system was similar to that reported
previously in successful hybridization experiments where hybrids were formed with immobilized
19 nt probe strands in microfluidic channels. The field strength suitable for hybrid formation as
reported by Erickson et al. was in the range of 110 Vcm-1 to 350 Vcm-1. Higher voltages
146
increased stringency conditions due to increased Joule heating and shearing effects, but resulted
in substantial reduction in the extent of hybrid formation [269].
The interconnect coupled a gel filled capillary and a buffer filled microfluidic channel.
Therefore a discontinuous buffer system existed resulting in different local field drops across the
capillary and microfluidic channel. The applied voltage was influenced when the field strength
across the capillary or the microfluidic channel was measured individually. Therefore, the local
field drops across the capillary and microfluidic device were estimated based on resistance.
The voltage that was required to achieve a set current was separately measured across the
capillary and the microfluidic chip. The relative resistance of the capillary in comparison to the
microfluidic channel was determined and the field drop across the two different devices was then
estimated. The resistance of the gel filled capillary was measured to be approximately half of the
resistance of the microfluidic channel. Therefore, the field strength across the capillary and
microfluidic device at a total voltage applied of 500 V was 62 Vcm-1 and 222 Vcm-1,
respectively. The voltage across the microfluidic channel was in a range that was suitable for
hybridization of fully complementary and one base pair mismatch targets [269].
3.7.2 Delivery of Oligonucleotide Targets to the Microfluidic Biosensing Platform by Direct Injection and by Selective Concentration
A comparison was completed of the response of the DNA biosensor to quantities of
complementary targets delivered by direct injection and by selective concentrating using the
capillary column. These experiments were completed to explore whether an improvement in the
response of the biosensor could be achieved by use of the capillary column.
Figure 3.54(a) shows the profile (background subtracted) of the fluorescence intensity along the
microfluidic channel following the introduction of labelled complementary target. The
fluorescence profile is consistent with hybridization with the immobilized oligonucleotide probes
along the microfluidic channel. Figure 3.54(b) demonstrates the selectivity of hybridization by
replacing one of the oligonucleotide probe spots with a non-complementary sequence. The
complementary target was labelled with AlexaFluor 647 (A647) fluorescent dye. The A647 dye
has similar excitation and emission wavelength as Cy5 and was used without introducing any
changes to the experiment.
147
Figure 3.54: Line scans of the fluorescence intensity along a microfluidic channel of the DNA biosensing platform following delivery of complementary target by selective concentration. a) both pad probe spots
are complementary to the SMN target sequence, and b) one probe pad is complementary (SMN) and the second is non-complementary (β-actin probe).
Affinity capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: a) 10 µL 1 nM A647 SMN target, b) 10 µL 1nM A647 SMN target, 1 µM Cy3 β-actin target, electrokinetic injection for 1 min at 150 Vcm
-1. Capture: electrophoresis for 10 min at 150 Vcm
-1 in 1x TBE/PVP running buffer. Concentration
step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms-1
; Voltage: 96 Vcm-1
; Delivery of purified and concentrated targets into microfluidic biosensing platform: 500 V, 10 minutes, 1x TB/PVP/20
mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms-1
scan rate: 50 Hz.
148
Figure 3.55 presents the response of the biosensor to the direct injection of different quantities of
complementary target through the interconnect port. The data was generated from scans
obtained similar to Figure 3.54. Here, the fluorescence intensity signal from one "pad" was
taken into account, and the average intensity of the fluorescence signal as well as the integrated
fluorescence intensity were calculated.
y = 0.053x + 0.0142
R2 = 0.9997
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12 14 16 18 20
Quantity of Complementary Oligonucleotide Injected (femtomoles)
Avera
ge F
luore
scence I
nte
nsity (
AU
)
a)
y = 2.1087x + 1.041
R2 = 0.9994
0
5
10
15
20
25
30
35
40
45
50
0 2 4 6 8 10 12 14 16 18 20
Quantity of Complementary Oligonucleotide Injected (femtomoles)
Inte
gra
ted F
luore
scence S
ignal (A
U)
b)
Figure 3.55: Response of the microfluidic based DNA biosensing platform to quantities of
complementary target a) average fluorescence intensity signal level and b) integrated fluorescence intensity. Different concentrations of DNA were mixed in 10% LAAm gel and injected into an empty fused silica capillary using a syringe to CE adapter. Delivery of complementary oligonucleotide into microfluidic
biosensor: 500 V, 10 minutes, 1xTB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms
-1, scan rate: 50 Hz. Error bars represent 1 standard deviation of three trials.
Known concentrations of the oligonucleotide targets were mixed with a 10% linear
polyacrylamide solution that was then injected into the same lengths of capillary used in the pre-
concentration experiments (2 cm) using a syringe outfitted with a capillary-to-syringe luer
149
adapter. The complementary targets were then delivered into the biosensor device via the
interconnect port. This approach allowed for the delivery of a known quantity of oligonucleotide
targets to the microfluidic biosensor, with subsequent hybridization under the same experimental
conditions as experienced when delivery was performed by step elution. No measurable response
was observed when quantities lower than 0.98 fmole were injected into the detection system.
Table 3.16: Summary of data for the response of the microfluidic DNA biosensing platform to delivery of complementary targets by selective concentrating using the affinity capture gel. The amount of target
injected into the affinity capture gel by electrokinetic injection from the original target solution is shown in parenthesis. The equivalent quantity was determined based on correlation to the concentration-response
curve of Figure 3.55. The enhancement factor was calculated based on the ratio of the equivalent quantity and quantity of material injected. Errors represent 1 standard of three trials expect for Equivalent Quantity Determined from Calibration Curve, which is propagated error from correlation with calibration
curve. Affinity capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL A647 SMN target,
electrokinetic injection for 1 min at 150 Vcm-1
. Capture: electrophoresis for 10 min at 150 Vcm-1
in 1x TBE/PVP running buffer. Concentrating Step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms
-1; Voltage: 96 Vcm
-1; Delivery of purified and concentrated targets into microfluidic biosensing
platform: 500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms
-1 scan rate: 50 Hz.
Original Target Concentration, and (Quantity
of Target Injected)
Average Fluorescence
Intensity (AU)
Integrated Fluorescence Signal (AU)
Equivalent Quantity
Determined from Calibration
Curve (fmol)
Enhancement Factor
0.1 nM (0.30 ± 0.04 fmol)
0.070 ± 0.01 2.4±0.4 (1.0±0.2, 0.7±0.1)
(3.3±0.8,2.3±0.4)
0.5 nM (1.5 ± 0.2 fmol)
0.13 ± 0.03 5.9±1.3 (2.2±0.5, 2.2±0.5)
(1.5±0.4, 1.5±0.4)
1 nM (3.0 ± 0.3 fmol)
0.81 ± 0.17 35±7 (15±3, 16±3) (5±1,5±1)
Table 3.16 presents the response of the microfluidic biosensing platform following the delivery
of the concentrated oligonucleotide. The values for average fluorescence intensity and integrated
fluorescence were then correlated with the calibration curve in Figure 3.55 and an equivalent
quantity was determined. This value represents the quantity of material needed to achieve the
same response if it was delivered without concentrating.
The enhancement factor is the ratio between the equivalent quantity (determined from the
calibration curve) versus the amount of material that was injected electrokinetically into the
affinity capture gel from the original target solution (in parenthesis). The amount of material
injected was calculated from steady-state fluorescence measurement of solutions of target before
and following electrokinetic injection into the capillary. Based on the results, 31% ± 4% of the
complementary target was injected into the capillary by electrokinetic injection. This value was
obtained using solutions of 10 nM A647-labeled oligonucleotide targets. Note that the
150
percentage of material injected electrokinetically into the affinity capture gel did not appear to be
affected by the concentration of the target solution.
The results indicate that the limit of detection (LOD) of the microfluidic biosensor could be
lowered to 0.1 nM or 0.3 fmol of material by delivery by concentrating the target. Based on the
results in Figure 3.55, the LOD of the biosensor by direct injection of the oligonucleotide targets
was 1 fmol of material. The enhancement affect calculated was 3. This value is lower than the
enhancement factor determined in the previous section.
A discontinuous system exists between the gel filled capillary and buffer filled microfluidic
device since the resistance in the two channels is different. There is a difference in mobility of
the oligonucleotide targets moving through gel filled capillary and the microfluidic channel. The
mobility of the oligonucleotide target through the affinity capture gel was measured at 115 ± 6
µms-1 at a field strength of 93 Vcm-1. The mobility of the oligonucleotide target through the
microfluidic channel was measured at 354 ± 27 µms-1 at a field strength of 100 Vcm-1. During
the concentrating and hybridization experiments, the oligonucleotide targets experienced an
increase in velocity as they eluted from the capillary into the microfluidic channel. This likely
caused de-stacking of the eluted material, reducing the enhancement effect.
3.7.3 Response of Microfluidic Biosensing Platform to Mixtures of Targets Delivered by Direct Injection and Following Selective Concentration
Figure 3.56 presents the response of the microfluidic biosensor for complementary and non-
complementary oligonucleotide targets by direct injection and after the targets were purified and
concentrated on the affinity capture gel. Selective concentrating on the original sample
improved the limit of detection of the biosensor to 0.5 nM of complementary material (A647-
SMN) in the presence of a 2000 fold excess of non-complementary material (Cy3-β-actin).
151
0
0.05
0.1
0.15
0.2
0.25
0.5 nM/1 uM 1 nM/1 uM 2 nM/1 uM 1 nM/0.5 uM 1 nM/2 uM
[complementary] / [non-complementary] in original target solution
Avera
ge F
luore
scence I
nte
nsity (
AU
)
direct injection selective concentratinga)
0
1
2
3
4
5
6
7
8
9
0.5 nM/1 uM 1 nM/1 uM 2 nM/1 uM 1 nM/0.5 uM 1 nM/2 uM
[complementary] / [non-complementary] in original target solution
Inte
gra
ted F
luore
scence I
nte
nsity (
AU
)
direct injection selective concentratingb)
Figure 3.56: The response of the microfluidic biosensing platform for samples containing complementary
and non-complementary target comparing delivery with and without selective concentrating. Direct Injection: mixture of A647 SMN and Cy3 β-actin target in 10% LAAm gel. Selective concentrating:
Affinity capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL of A647 SMN and Cy3 β-actin target, electrokinetic injection for 1 min at 150 Vcm
-1. Capture: electrophoresis for 10 min at
150 Vcm-1
in 1x TBE/PVP running buffer. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms
-1; Voltage: 96 Vcm
-1; Delivery of oligonucleotide targets into microfluidic biosensing
platform: 500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms
-1 scan rate: 50 Hz. Error bars represent 1 standard deviation of three trials.
152
For these experiments, the target solutions were made in 1x TBE buffer (rather than deionized
water in the previous experiment that examined the response to only complementary target). The
targets were made in buffer to reduce the amount of non-complementary target being injected
into the capillary.
The experiments were originally performed under the same conditions as those in used for the
experiments with complementary target only. The amount of non-complementary target
remaining inside the capillary was 1.0 ± 0.2 fmole (originally 10 pmole). To further reduce the
amount of non-complementary target, the experiment was then modified to include a second
washing step for 10 minutes at 150 Vcm-1. This reduced the amount of non-complementary
target inside the capillary to 0.67 ± 0.1 fmole. The largest reduction of the non-complementary
target was observed when the sample was made in 1x TBE buffer; the amount of non-
complementary target in the capillary was reduced to below 0.1 fmol (the fluorescence intensity
was below the lowest data point for the calibration curve used). This decrease in the amount of
non-complementary target retained in the capillary was thought to be due to a decrease in the
amount of material injected into the capillary.
The quantity of material injected into the capillary by electrokinetic injection can be influenced
by the conductivity of the sample solution:
2
1
2
1
2
1
sample
sample
sample
sample
sample
sample
c
c
vel
vel
Q
Q= (12)
where Q is the quantity of material injected, vel is the velocity of the analyte ions in the system
and c is the concentration of the sample. The velocity of the analyte ions in the sample is
dependent on its concentration of the analyte and the conductivity of the buffer used. This
implies that a different quantity of material can be injected into the capillary for a sample
containing the same concentration of analyte, but made in buffers of different conductivities
[278].
More material is injected into the capillary if the conductivity of the targets in the sample is less
than that of the background electrolyte in the capillary [278, 279]. A lower conductivity buffer
can also result in stacking of the targets as they enter the capillary through field amplified sample
stacking (FASS) [163, 280, 281].
153
Deionized water is often used to provide the largest concentrating effect by field amplified
stacking [172]. This was done to help improve the amount of material injected into the capillary
for the low target concentration examined. However, when the sample contained both
complementary and non-complementary targets, a large amount of the non-complementary target
was also injected into the affinity capture gel. During the concentrating process, this led to the
concentrating of both complementary and non-complementary target in the eluent. Experiments
were done to examine the influence of increasing stringency conditions by increasing formamide
content and temperature, and by extending wash times to twice of what was initially used. Even
with these measures the data still indicated significant retention of the non-complementary target
by the gel. Increasing the conductivity of the buffer reduced the total quantity of
oligonucleotides injected, which aided in reducing the amount of non-selective adsorption of the
non-complementary targets on the affinity capture gel, and this limited the amount of non-
complementary targets being concentrated during the elution step.
The amount of material injected as measured by spectrofluorimetry was 6.0% ± 0.2% for the
complementary target and 15.8 ± 0.9 % for the non-complementary target. This was calculated
as the difference in fluorescence intensity of the sample solution in the original solution before
and after injection into the affinity capture gel.
Due to the presence of immobilized probe, the electrophoretic mobility of the DNA target inside
the get was modified as suggested by Equation (4) (Section 3.0.9), and was decreased. The
difference between Electrophoretic mobility of the complementary and non-complementary
target might have influenced the total quantity of material introduced into the affinity gel. Figure
3.57 shows an experiment using Cy3 labeled dT-20 targets. The amount of target injected was
measured as a difference of before and after injection was decreased with increasing probe
concentration inside the gel.
154
400
600
800
1000
1200
0 0.5 1 1.5 2
Concentration of Probe (uM)
Am
ou
nt
of
Targ
et
Lo
ad
ed
(fm
ol)
Figure 3.57: Effect of the probe concentration on the amount of target introduced into the affinity gel by electrokinetic injection. Affinity gel: Varying concentrations of dA20 probe (1.8 µM, 0.45 µM, no probe) in
a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL sample containing 0.5 µM Cy3-dT20 at 267 Vcm
-1 for 60 seconds. The amount of target in the original sample was 2.5 pmol. Error bars
represent 1 standard deviation of three trials.
The non-complementary target remaining in the capillary was also concentrated with the
complementary target during step-elution. The amount of the concentrated non-complementary
targets in the eluent was calculated to be 0.57 ± 0.06 fmol and did not appear to vary as a
function of the amount of non-complementary target tested in the original target solution. Table
3.17 summarizes the performance of the affinity capture gel in the processing of samples
containing complementary and non-complementary.
Table 3.17: Summary of the performance of the two delivery methods. Percent recovery is based on the proportion of the amount of target delivered to the microfluidic biosensing platform from of the original
starting sample. The values for delivery by direct injection were calculated based on the concentration of the targets in the original sample. The values used for the delivery selective concentrating were
calculated based on the response of the biosensing platform. The enrichment factor is the ratio of the percent complementary target with and without selective concentrating. Errors represent propagated
error resulting from calculating derived values. Direct
Injection Selective
Concentrating [complementary] /
[non-complementary] in original target
solution
Amount complementary
/ non-complementary
target
Recovery (%)
Purity (%)
Recovery (%)
Purity (%)
Enrichment Factor
0.5 nM/1 µM 5 fmol/10 pmol 100 0.05 15 ± 5 56 ± 16 1117 ± 333
1 nM/1 µM 10 fmol/10 pmol 100 0.1 18 ± 3 73 ± 11 731 ± 114
2 nM/1 µM 20 fmol/10 pmol 100 0.2 17.3 ± 0.9 86 ± 4 429 ± 22
1 nM/0.5 µM 10 fmol/5 pmol 100 0.1 13.3 ± 0.4 70 ± 2 699 ± 18
1 nM/2 µM 10 fmol/20 pmol 100 0.1 3.5 ± 0.9 46 ± 11 457 ± 114
While step elution reduced the total quantity of complementary material being delivered into the
microfluidic biosensor in comparison to direct injection, the material delivered through the
155
interconnect port in step elution contained a higher proportion of complementary material. The
performance of the affinity capture gel in terms of percent recovery and percent complementary
target delivered was not observed to be influenced by the amount of non-complementary target
present except for the case where the sample solution contained 1 nM complementary and 2 µM
non-complementary targets. Here, the percentage of complementary material delivered from the
original sample was observed to be less than the other cases. This decrease in performance was
not due to the delivery of a large amount of non-complementary to the microfluidic chip.
However, the large amount of non-complementary material could affect the affinity capture gel
in its capture of complementary targets. Further mixtures using higher concentrations of non-
complementary material were not examined.
3.7.4 Response of Microfluidic Biosensing Platform to Delivery of Concentrated Oligonucleotide Targets With and Without Purification
An examination was completed of the response of the microfluidic biosensing platform to
concentrated samples that were processed with or without purification. Other methods of
purification of nucleic acids typically involve the capture, concentration and elution of all nucleic
acid material, which may include non-complementary targets. Here, the work examined the
response of the biosensing platform for such a scenario, where all nucleic acid targets present in
a sample were concentrated and delivered into the microfluidic biosensing platform [88, 150,
282].
In these experiments, the affinity capture gel was modified such that it contained two different
selective oligonucleotide probes, one for the A647-SMN target and the other for the Cy3-β-actin
target. The oligonucleotide length of both targets was selected to be the same so that mobility
would be similar. The microfluidic biosensing platform contained pads of immobilized probes
that were for the SMN target only. Since two targets were captured by the affinity capture gel,
both targets were concentrated during elution and delivered into the biosensing platform.
Table 3.18 reports the position of the material eluting from the capillary column corresponding
to the A647 and Cy3 labelled targets. The similarity of location of the two targets suggests that
both targets were of the same mobility and that targets were not already separated upon elution.
156
Table 3.18: Position and peak width of the eluted targets from the concentrating of the two captured targets along the capillary (from the injection end). Error represent 1 standard deviation of three trials. Affinity capture gel: 10% LAAm, 100 nM SMN and 5 µM β-actin probes. Target injection: 10 µL of 1 nM
A647-SMN and 1 µM Cy3-β-actin targets, electrokinetic injection for 1 min at 150 Vcm-1
. Capture: electrophoresis for 10 min at 150 Vcm
-1 in 1x TBE/PVP running buffer. Concentration step: coverage
length: 12.5 mm; Step rate: 86 µms-1
; Voltage: 96 Vcm-1
; Acquisition settings (Alexa647): PMT gain 700 mV, translation speed: 50 µms
-1, scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV,
Pinhole: 60 µm, 1 FPS
Position after concentrating
Peak Width (µm)
A647-SMN Target 17.1 ± 0.7 mm 437 ± 100
Cy3-β-actin Target 17.3 ± 0.8 mm 542 ±109
Table 3.19 presents information about the complementary material eluting from the capillary
column during the concentrating step under two systems: the capture gel contained SMN probe,
and the capture gel contained both SMN and β-actin probes. The data suggests that capture of
the complementary target was not influenced by the presence of the second probe in the affinity
capture gel. Any differences observed between selective concentrating and non-selective
concentrating in the response of the microfluidic biosensing platform would be due to the
delivery of a larger amount of non-complementary material by non-selective concentrating of all
targets.
Table 3.19: Quantitative information of the eluted A647-SMN target during stacking from gels which containing only SMN probe (selective concentrating) and containing both β-actin and SMN probe
(concentrating only). Errors represent 1 standard deviation of three trials. Affinity capture gel: Selective concentrating: 10% LAAm with 100 nM SMN probe. Concentrating only:
10% LAAm with 100 nM SMN and 5 µM β-actin probes. Target injection: 10 µL of 1 nM A647-SMN and 1 µM Cy3-β-actin targets, electrokinetic injection for 1 min at 150 Vcm
-1. Capture: electrophoresis for 10
min at 150 Vcm-1
in 1x TBE/PVP running buffer. Concentrating Step: coverage length: 12.5 mm; Step rate: 86 µms
-1; Voltage: 96 Vcm
-1; Acquisition settings (Alexa647): PMT gain 700 mV, translation speed:
50 µms-1
, scan rate, 50 Hz.
Peak width (nL) Peak height
(AU) Peak area (AU)
Selective Concentrating
3.67 ± 0.96 0.47 ± 0.03 3.5 ± 0.95
Concentrating Only
3.0 ± 0.5 0.55 ± 0.07 3.98 ± 1.3
Figure 3.58 presents a comparison of the response of the microfluidic biosensing platform to
three methods of target preparation: without any processing (direct injection), concentrating of
all oligonucleotide targets (concentrating only) and by selective purification and concentrating of
the target of interest (selective concentrating).
The results indicated that the response of the biosensing platform for a mixture of targets
delivered by concentrating all oligonucleotide targets in the sample was similar to that observed
157
for the material on direct injection from the capillary without any concentrating. This
demonstrates the advantage of using selective concentrating.
Based on previous results shown in Table 3.2 in Section 3.0.6, it was noted that the probe sites
were saturated at target concentrations of 10% of the probe concentration. The use of higher
concentrations of non-complementary target would not be expected to recovery a difference in
the results since the probes were already saturated.
Although the response of the biosensing platform to the non-selective delivery of material was
observed to be similar to that of direct injection, it is important to note that the actual amounts
delivered were different. For example, the concentration of the material being delivered to the
microfluidic device by concentrating of all oligonucleotide targets for the initial mixture of 1 nM
complementary in 1 µM non-complementary target was determined to be 17 ± 2 nM
complementary target to 59 ± 4 µM non-complementary target.
158
0
0.05
0.1
0.15
0.2
0.25
0.5 nM/1 uM 1 nM/1 uM 2 nM/1 uM
[A647-SMN] / [Cy3-Bactin] in original target solution
Avera
ge F
luore
scence I
nte
nsity (
AU
)
direct injection concentrating only selective concentratinga)
0
1
2
3
4
5
6
7
8
9
0.5 nM/1 uM 1 nM/1 uM 2 nM/1 uM
[A647-SMN] / [Cy3-Bactin] in original target solution
Inte
gra
ted F
luore
scence I
nte
nsity (
AU
)
direct injection concentrating only selective concentratingb)
Figure 3.58: Comparison of the response of the microfluidic biosensing platform (containing probe for
SMN) for a sample containing A647-SMN and Cy3-β-actin targets as prepared by selective concentrating and concentrating of the all oligonucleotide targets. Delivery of oligonucleotide targets by selective
concentrating was done as previously described. Delivery of oligonucleotide targets by non-selective pre-concentration was done using 10% LAAm affinity capture gels which contained 100 nM SMN probe and 5
µM β-actin probe. Error bars represent 1 standard deviation of three trials.
159
Chapter 4 Future Directions
4.1 Determination of Oligonucleotide Probe Incorporated into Affinity Capture Gel
One response parameter that was examined as part of fractional factorial analysis was the amount
of oligonucleotide probe incorporated into the polyacrylamide gel. The fluorescence intensity of
a fluorophore attached to the oligonucleotide probe was measured to determine the amount of
probe remaining on-column following polymerization and pre-conditioning of the affinity gels.
However, reaction between the fluorophore and radical initiator as well as scattering of
fluorescence as a function of the polymer at different gel formulations affected the fluorescence
intensity, and an absolute measure of the quantity of oligonucleotide probe could not determined
by fluorescence.
One possible alternative method to reliably quantify probe that becomes incorporated into the gel
may be the use of radiolabelled DNA [283]. Oligonucleotides can be labelled with 32P isotopes
and a quantitative radioactive signal can be measured using scintillation counter.
Additionally, examination of samples following reaction between the oligonucleotide probe and
radical initiator by capillary gel electrophoresis for possible cleavage reaction products showed
the presence of a second, slower eluting peak, indicating some reaction had taken place. It would
be interesting to analyze the compounds in these peaks using CE-MS to confirm the identity of
the eluent in the electrophoretograms.
4.2 Further Factorial Experiments on Gel Formulations
The factorial design used in this thesis was a quarter fractional factorial design. Such a design is
typically used as an initial survey experiment in order to quickly screen for significant factors out
of a large number of factors without the need to perform a large number of experiments.
However, main factor effects can be confounded by possible interaction effects due to the nature
of the fractional factorial design. The trends identified from factorial analysis were that amount
of target captured increased as monomer content increased, while it decreased with increasing
crosslinker content. This preliminary result may suggest non-linearity in the response surface,
160
and a better model may be obtained by performing a full factorial experiment that examines a
larger range of monomer and crosslinker content.
It was proposed that changes in average pore size of the gel stretches out the ssDNA, resulting in
more targets being captured. A set of experiments could be considered to confirm this
hypothesis, and one approach may be to modify a hairpin structure into a Molecular Beacon
(MB). MBs are strands of ssDNA designed that have a stem-loop structure, and these has are
commonly used as molecular switches to detect binding of complementary nucleic acid
sequences [284, 285]. The loop portion reports the presence of a strand of complementary
oligonucleotide, while the stem consists of 5-7 nt base paired intramolecularly [284, 285].
Attached at the ends of the ssDNA strand are a fluorophore and a quencher [284, 285]. When the
MB is in its stem-loop conformation, the fluorophore and quencher are brought close together
spatially [284, 285]. Fluorescence emission by the fluorophore is reduced due to non-radiative
resonant energy transfer to the quencher. Since the resonant energy transfer is distance
dependent, separation of the fluorophore from the quencher allows for an increase in
fluorescence emission to be measured [284, 286]. It may be possible to adopt a MB motif into
the ssDNA target. Addition of a fluorophore and quencher such that it flanks the hairpin
structure present in the ssDNA can be used to measure any differences in fluorescence emission
intensity caused by folding or extension as the oligonucleotide reptates through the gel.
4.3 Improvements to Capillary-Microfluidic Platform
One issue encountered with the current design of the capillary to microfluidic platform is that of
destacking of the concentrated targets. The experimental work indicated that an enhancement
factor of about 300 of the eluted targets can be achieved. However, due to differences in
mobility and longitudinal band broadening, the stacked targets are diluted as they exit the
capillary into the microfluidic device due to a larger mobility of the oligonucleotide targets in the
microfluidic channel.
It may be possible to limit the de-stacking of the eluting oligonucleotide targets by adjusting the
velocity of the oligonucleotide targets inside the capillary and microfluidic channel. This could
be done by modifying the resistance of the channels, either by changing the geometry or the
buffer system. Changing the resistance would alter the local field drops across the discontinuous
system such that current is constant throughout the system [172, 280]. For example, increasing
161
the diameter of the capillary increases current flow, lowering resistance of the capillary [267].
The use of higher conductivity buffers can also affect mobility, as field strength is inversely
proportional to conductivity [163, 287].
Here, the system would need to be modified such that microfluidic channel has a higher
conductivity than the affinity gel capillary. The local field strength would be lower inside the
microfluidic channel such that the mobility is decreased versus the capillary, reducing the
dilution effect. An additional stacking system can also be included into the microfluidic chip to
stack the diluted targets prior to delivery to the immobilized probe pads. For example,
isotachophoresis can be employed. Targets eluted off the capillary can be stacked on a side
microfluidic channel prior to being delivered into the main microfluidic channel.
For this thesis, the length of target was quite limited due to the constraints inherent in the
biosensing platform to offer efficient hybridization. It is desirable to test the response of the
biosensing platform for delivery of the longer DNA targets. Issues of sterics that limit
hybridization efficiency may be alleviated if the oligonucleotide probes used with the
microfluidic biosensing platform were complementary to the ends of the target of interest, rather
than being complementary to the middle of the strand.
4.4 Moving Towards DNA Targets in Complex Matrices
Since the intent for the selective concentrating technique was towards the processing of real
samples, there was also an interest in the utility of this technique with samples contained in a
complex matrix. Preliminary experiments were performed where a sample of ground beef was
processed using pre-treatment steps commonly applied to such sample matrices
(homogenization, filtration, centrifugation, dialysis) to generate a complex matrix buffer.
Fluorescently labelled ssDNA oligonucleotide (20 nt) targets were used to spike these samples
and were used as samples to be processed by the affinity capture gel by electrokinetic injection.
Based on the preliminary results, it was observed that only a small portion of the DNA was could
be injected into the capillary; this small portion was retained by the affinity capture gel. This
observation could be due to injection bias. As noted in Equation 12, charged ions with faster
electrophoretic mobilities will preferentially load into the capillary during electrokinetic
injection. It can be assumed that a large number of high mobility ions are present in the complex
162
matrix. At the initial moments of electrokinetic injection, any DNA targets immediately near the
inlet end of the capillary will be injected into the capillary. However, once depleted, the higher
mobility ions will be injected preferentially, limiting the injection of DNA moving into the
capillary from bulk solution. This preliminary result suggests limitations of processing samples
of high salt content is of limited utility and that a step to remove such other high mobility species
is required.
163
Chapter 5 Conclusions
This thesis presented a method for the selective purification and concentrating of DNA targets
for delivery into a channel-based microfluidic DNA biosensing platform. The selective
purification was done by capillary affinity gel electrophoresis. Oligonucleotide probes were
immobilized by covalent attachment in polyacrylamide gels located inside fused silica
capillaries. Single-stranded DNA targets were injected into the polyacrylamide gel by
electrokinetic injection. Complementary targets were retained in the affinity capture column due
to interaction with the immobilized probe while non-complementary targets were removed from
the column. Release of the captured DNA targets was performed by step elution to concentrate
the DNA targets. The use of localized heating created an elution zone, and was applied in small
steps along the length of the capillary to stack the targets during elution. The volume of the
stacked targets was much smaller than the geometric volume of the capillary and the original
sample volume. Stacking experiments performed with 19 nt oligonucleotide demonstrated that
the targets were stacked into a volume of 0.25 nL inside the capillary from an original volume of
elution of 98.5 nL. This provided for sample in a volume that could be introduced into a
microfluidic device. The enhancement factor of the stacked targets in the capillary was 300 ±
29.
A thorough evaluation of the selective capture of a 150 nt DNA target in a complicated mixture
was completed. The investigation considered the effects of gel formulation, and different
loading and elution conditions on recovery and purity. This was addressed systematically and
guided by a factorial analysis.
From factorial design experiments, differences in gel formulation were examined for the
incorporation of probe and amount of target captured. The amount of probe incorporated into the
affinity capture gel was dependent on the original concentration used in the pre-polymer solution
and was not influenced by differences in gel formulation. It was identified that a higher amount
of complementary target was retained in the affinity capture gels made with higher
concentrations of monomer. This was thought to be a result of such gels having a smaller
average pore size than the radius of gyration of the ssDNA target examined, causing the ssDNA
target to move through the gel by reptation. It was proposed that movement through the gel by
164
reptation eliminates the hairpin structures present in the ssDNA, this made the probe region on
the target available for hybridization with the gel immobilized probe, resulting in a higher
amount of capture of the complementary target by the affinity capture gel.
This selective concentrating method was applied to a series of DNA targets of different lengths,
19 nt, 150 nt, 250 nt and 400 nt. Improvement in purity was achieved, but recovery was reduced
was reduced for longer sequences. The recovery of the method ranged from 0.5 to 4% for the
PCR targets, while it was 13 to 18% for the 19 nt oligonucleotide target. The purity was
calculated to be up to 44% for the PCR target and up to 86% for the 19 nt target. This was an
improvement in purity of 15 fold and 1100 fold in comparison to the original samples for the
PCR targets and 19 nt oligonucleotide, respectively. The lowest concentration of the 150, 250
and 400 nt targets that saw an advantage by selective concentration was 1 nM of complementary
in 150 nM non-complementary target. The lowest concentration of the 19 nt oligonucleotide
target that could be processed to see advantage in selective purification and concentration was
0.5 nM complementary target in 1 µM non-complementary target.
A capillary-to-microfluidic biosensing platform was developed and an interconnect was used to
deliver target analyte from the capillary column into the microfluidic device. This platform was
used for the delivery of purified and concentrated 19 nt targets from sample solutions containing
a mixture of non-complementary targets. The capillary column selectively concentrated the
targets, which were then delivered into a microfluidic based biosensor device for hybridization.
Performance was determined as the difference in response of the biosensor following a number
of different sample introduction methods. It was shown that selective concentrating provided the
best signal by the biosensor. The capillary column provided an improvement of purity, moving
from a value for the unseparated mixture of 0.01% to a final stacked band from the column of
50%. This correlated with a recovery that decreased from 100% to 20%. Selective concentrating
was shown to allow for the detection of a 0.5 nM in 1 µM non-complementary solution, which
was a result that could not be achieved by direct injection of sample into the biosensing system.
165
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Appendices
A. Factorial Design Experiment
A factorial design experiment is a method to systematically examine the effects of a number of
explanatory variables (factors) on a response. Experiments are run where the level of all the
factors is varied according to a design matrix and a response is measured. The levels for each of
the factors are changed concurrently in each treatment in a factorial experiment. This approach
is different from a one-factor-at-a-time (OFAT) approach where only one factor is changed while
all others are held constant [288, 289]. A factorial analysis can usually be completed in less
experimental runs than for OFAT and allows for interaction effects between factors to be
identified. Additionally, the level of precision obtained from a factorial experiment is greater
than that of an OFAT experiment. Table A1 shows an example of a design matrix for a three
factor, 2 level factorial experiment.
Table A1: A sample design matrix for a 3 factor, 2 level factorial experiment, using factors A,B and C.
A B C AB AC BC ABC
1 + + + + + + +
2 + + - + - - -
3 + - + - + - -
4 + - - - - + +
5 - + + - + + -
6 - + - - + - +
7 - - + + - - +
8 - - - + - + -
The two level factorial design examines the change in response for each factor between two
levels, a high and a low level. Here, the high level is denoted by ‘+’, where it can refer to a
higher concentration of reagent, and ‘-‘, referring to a low level of reagent used for each factor.
The two level factorial design is the most common since this approach minimizes the number of
runs to complete a full data set collection. The two level factorial experiment assumes that the
response surface is linear. However, the two level design can be modified to account for non-
linearity [288].
In the example given, the response due to each factor is replicated four times at each level with a
total experiment run of 8 experiments. In contrast, for an OFAT experiment, the same level of
precision would require 24 experimental runs [289].
183
Additionally, a factorial design experiment can identify interaction affects between factors.
Interaction effects are those that combine to change the response, in addition to the main factor
effects. As a hypothetical example, consider that both A and B increase the response by 20 and
10 points, respectively, when examined at the high level. The expected response for the
experiment when both factors are run at high levels would be 30. A larger or smaller response
than what is expected would indicate an interaction between the two factors at the high level, and
indicate that A and B are not fully independent variables. Interaction effects are denoted in the
design matrix in Table A1 as AB, AC, BC and ABC. For example, the column AB examines the
interaction between the factors A and B. The level for each of the interaction terms is a product
of the signs of the two main factors whose interaction is being examined. For example, in
experiment number 3, the interaction factor for A and B is ‘-‘. A ‘+’ for an interaction factor
indicates that both factors, in this case A and B, are both being examined at the same level, either
both high or both low, while a ‘-‘ means that the two factors are at different levels. A three factor
interaction is the response of the 2 factor interaction term with a main factor term. For example,
for the three-factor interaction ABC, it can be either between AB x C or A x BC or AC x B
[289].
A1. Fractional Factorial Designs
The number of experiments required for a 2 level factorial design experiment is 2k, where k is the
number of factors. As the number of factors being examined increases, the total number of
required experiments is increased. Clearly a large number of experiments requires time and can
become costly [288, 289]. The large number of experiments is required to determine the
contribution of higher order interaction terms. For a 26 design, 64 experimental runs are
required. However, only six degrees of freedom out of total 63 are used to estimate main affects,
while 42 are associated with interactions of three or more factors [288]. Oftentimes higher order
interactions are found to be negligible in contributing to the response [288, 289].
For initial screening experiments, it may be more practical to complete a sub-set of the total
number of experiments to obtain information about main and two factor interactions even if
information about higher order interactions is lost. Such fractional factorial designs are obtained
by confounding main factor terms with higher order interaction terms. The estimated effect of
the confounded term becomes the sum of the main effect and the effect of the interaction term.
184
For example, the factor D can be accommodated in the interaction term ABC. Therefore, the
effects due to the main factor and the interaction terms cannot be separated. The ABC term is
called an alias of D.
By confounding one factor with a higher order interaction term, the total number of experimental
runs needed is 2(n-1) instead of 2n. Since the total number of experimental runs is reduced by half,
this is known as a half fractional design. A quarter fractional design is obtained by confounding
two main factors, reducing the total number of experimental runs by a quarter.
A2. Design Matrix for Quarter Fractional Factorial Design
A quarter fraction design allowed for the identification of major main effects without extensive
experimentation. Here, the terms for the radical initiator systems, TEMED and APS, were
confounded with two interaction terms, AxC and BxC. This design is a 25-2, resolution III
design, meaning that some of the main effects were confounded with two-factor interactions
[288].
Aside from confounding the terms for TEMED and APS, the quarter fractional design also
introduced other confounding terms in the design matrix. In the factorial design, the term for
TEMED was confounded with the interaction term for AC, meaning D = AC. The generator
relationship is more commonly expressed as I = ACD, where I is the identity term, which
represents a column of ‘+’. A column squared gives its identity squared (AxA=A2=I). Any
column multiplied by the identity remains the same. Aliased relationships between other factors
can be determined by multiplying that factor with the generator term. For example, factor A is
aliased with the interaction term CD (IxA = ACD x A, A = A2CD, A=CD) [289]. Table A2
shows the generator term used to generate the quarter fraction as well as the aliased or
confounded terms in this design.
Table A2: Alias structure for the fractional factorial experiment.
Term Aliased Term
Generator I=ACD, I=BCE, I=ABDE
A CD + BDE + ABCE
B CE + ADE + ABCD
C AD + BE + ABCDE
D AC + ABE + BCDE
E BC + ABD + ACDE
AB DE + ACE + BCD
AE BD + ABC + CDE
185
A3. Choice of Factors and Levels
The ‘+’ levels for the factors were selected to accommodate practical constraints. The onset time
for polymerization following addition of the radical initiator was observed to decrease when the
concentration of the monomer or the initiator increased. The upper level of monomer and
initiator was chosen to allow enough time to inject the polymerization solution into the capillary
before gelation occurred. Additionally, high concentrations of monomer would mean that the
mobility of the longer DNA targets would be decreased, increasing the total processing time.
The lowest monomer concentration chosen was based on results examining EOF suppression
methods of EOF presented in Appendix C. This experiment was done to assess the effect of two
different EOF suppression methods to reduce loss of the modified polyacrylamide gel during
electrophoresis. Loss of the probe in such a manner would alter the results negatively. The
lowest gel concentration tested, 7.5%T, did not show any significant loss of the gel immobilized
oligonucleotide probes during electrophoresis and was selected as the lowest monomer
concentration for the factorial analysis.
186
B. Synthesis of DNA Targets
B1. Construction of 250 bp Target
The 250 bp target was generated by combining the 150 bp target with the 100 bp target in a
ligation reaction using DNA ligase. Prior to the ligation reaction it was necessary to replace the
hydroxyl group on the 5’ end of the PCR products with a phosphate group using T4
polynucleotide kinase and ATP. The ligation reaction involved the formation of a
phosphodiester bond between the 5’-phosphate and the 3-hydroxyl termini of two adjacent
double-stranded DNA fragments, with water being a reaction by-product [290].
The commercial oligonucleotide primers were produced by solid phase synthesis, which placed a
hydroxyl group on the 5’ end of the oligonucleotide target instead of a phosphate group [291].
During PCR, these synthetic primers bound to the DNA target, marking the region for the DNA
polymerase to provide replication. Since the synthetic primers became part of the amplicons, the
5’ end must also have a hydroxyl group. Phosphorylation of the amplicons was therefore
required to replace the hydroxyl group with a phosphate group for the ligation reaction to occur.
This was done using ATP catalyzed by T4 polynucleotide kinase.
Figure B1 shows the agarose gel electrophoresis of the products following the ligation reaction.
A profile plot of the lane containing the ligation product is also included to better highlight the
bands.
187
0
5
10
15
20
25
30
35
40
45
50
100 120 140 160 180 200 220 240
pixel number
Av
era
ge
Flu
ore
sc
en
ce
In
ten
sit
y (
AU
)
Figure B1: Top: Agarose (2% w/v) gel electrophoresis of the ligation reaction products: 1) Fermentas 100 bp DNA ladder, 2) 150 bp DNA target, 3) 100 bp DNA target, 4) to 5) reaction product of the ligation
reaction. Background subtraction using a rolling ball of diameter 100 pixel was used. Run conditions: 100 V for 1 hour in 1x TBE (pH 8.0) buffer. Bottom: profile plot of Lane 5.
188
The gel showed bands from the original starting material, plus a series of product bands that are
identified in Table B1:
Table B1: A summary of the assignment of the bands observed from the gel in Figure B1.
DNA length (bp)
Possible Assignment
87 100 bp
158 150 bp
235 100 bp +150 bp
297 150 bp +150 bp
In this reaction the two pieces of DNA used in the ligation reaction were blunt ended, which
would result in a total of four permutations in terms of how two pieces of DNA could couple:
100 bp +100 bp, 100 bp + 150 bp, 150 bp + 100 bp, 150 bp + 150 bp. From Figure B1, the
bands correspond to the original starting DNA sequences; one for the 100 bp +150 bp and one
for the 150 +150 bp. A band corresponding to the ligation of the 100 bp +100 bp was expected.
It is possible that the reaction did occur, but the yield was very low and was not observed on the
gel. The bands due to the other products were also very weak. The significant signals that were
observed for the original DNA sequences suggest that the yield of the reaction was low.
The part of the gel corresponding to the band at 250 bp was excised. The DNA was extracted
using a commercially available kit (Qiagen QIAquick PCR Purification Kit) and PCR was
performed. Since both DNA targets were subjected to the T4 polynucleotide kinase reaction, the
lane that corresponds to the desired target (250 bp) would contain two different products, 150 bp
+100 bp and 100 bp +150 bp. Each product could be amplified preferentially using a different
set of primers.
Figure B2 shows the agarose gel separation of the PCR products. The possibility of having two
different targets of the same length was noted since the target of interest could be located on
different strands depending on the arrangement of the two targets. However, since only one
target could be amplified using one set of primers, the amount of the desired target would be in a
large excess over the undesired combination.
189
Figure B2: Agarose (1% w/v) gel electrophoresis of the PCR from the bands around 250 base excised from the ligation reaction product . 1) Fermentas 100 base DNA ladder, 2) PCR Control of 100+150 bp reaction, 3)+4) PCR reaction for 100+150 bp product, 5) PCR Control for 150+100 bp reaction, 6) PCR
Reaction for 150+100 bp. Run conditions: 100V for 50 minutes in 1x TBE (pH 8.0) buffer.
B2. Construction of 400 bp Target
The choice of the 400 bp target sequence based on the uidA gene was made due to previous
experience in our group with this target. Additionally, the genomic sequence of E. coli is readily
available.
Genomic DNA from E. coli was extracted from the cells using a commercially available kit
(Qiagen DNeasy Blood and Tissue Kit). The DNA was then subjected to a double restriction
enzyme digest to cut a 1000 bp fragment from the region of the genomic E. coli DNA
corresponding to the uidA gene. The 1000 bp fragment was used as the DNA template in PCR
to generate the 400 bp target. The sequence information about the uidA gene was obtained from
the NCBI Entrez Genome Project (Escherichia coli str K12 Mg1655 K12) [292].
The initial double restriction enzyme digest was done because it was not known if PCR on the
genomic DNA target would generate the 400 bp target from the 4 Mbp length DNA. The
restriction enzymes were chosen so that the DNA would be cut at equidistant length from both
ends of the target region. Figure C3 shows the gel electrophoresis of the reaction product
190
following the double restriction enzyme digest. A broad range of targets from 424 to 4236 bp
length were detected. The results suggest that the restriction enzymes recognized and cut various
regions of the genomic DNA, resulting in generation of fragments of different lengths along with
the one of interest.
Figure B3: Agarose (0.5% w/v) gel electrophoresis of the PCR from the bands that were about 250 base
that had been excised from the ligation reaction product. 1) Fermentas 1 kpb DNA ladder, 2) E. coli genomic DNA 3) E. coli genomic DNA following double restriction enzyme digest. Run conditions: 100 V
for 1 hour in 1x TBE (pH 8.0) buffer.
A band of about 1000 bp was excised and purified using the gel purification kit. The purified
DNA was then subjected to amplification by PCR using the primers designed to amplify a 400
bp length target. The primers were selected using PrimerQuest, which is web-based software
available online from Integrated DNA Technologies. The design criteria were that the target
region was in the middle of the strand of DNA, the GC content of both primers were 50%, with a
Tm of 60 °C and a length of 24 bases. The latter three criteria were the default settings in the
PrimerQuest software and were the recommended for primer design for PCR [293, 294].
B3. Confirmation of DNA Targets
Figure C4 shows the agarose gel electrophoresis of all the DNA targets used for the experiments.
191
Figure B4: Agarose gel of the various targets used for the experiments. Left: 1% w/v agarose gel.
Lane 1: Fermentas 100 bp DNA ladder, 2: 100 bp target, 3: 150 bp target, 4: 250 bp target. Run conditions: 50 V for 2 hours in 1xTBE (pH 8.0). Right: 1% w/v agarose gel. Lane 1: 400 bp DNA target,
2: 100 bp ladder. Run conditions: 100 V for 1 hour in 1xTBE (pH 8.0)
The lengths of the DNA targets as calibrated using the DNA ladder were 96 bp, 148 bp, 273 bp
and 404 bp. Additionally, the DNA targets were sequenced to confirm that the target region was
present. Table B2 provides the sequences of the targets, and the highlighted segment indicates
the location of the conserved region representing the desired hybridization sequence.
192
Table B2: Sequence of the DNA targets. Legend R = A,G; Y = C,T; M = A,C; K = G,T; S = C,G; W = A,T; H = A,C,T; B = C,G,T; V = A,C,G; D = A,G,T; N = A,C,G,T. The sequence for the 100 nt used in the
ligation product was provided by Dr. Paul Piunno. Desired Target Length
Length Obtained (nt)
Sequence (5’ to 3’)
100 nt 79 CAC ATA ACT CGC TTG CAG TTG ACT TTG ACC GGG AGG CTG AAG AAA TGG CAC CCT TTG CTG CTG TGA ACT GTA GCC CAG A
150 nt 100 ARA YKG KYR TTT YMK WRG GTA TWW RGC AGT MMC CGC CCA GCM GGT YMG GCG CAR GGT GGC ATG GGG GRG GCA MMC CCT CGT AAT GGG CAC AGT GTG GGM Y
250 nt
187 GSM TGT AGM TYM TGA RGG TAG TCA RGC AGT TCC CGC CCA GCC AGG TCT AGG CGC AGG GTG GCA TGG GGG AGG GCA TTC CCC TCG TAG ATG GGC ACA GTG TGG GTG ATC CAC ATA ACT CGC TTG CAG TTG ACT TTG ACC GGG AGG CTG AAG AAA TGG CAC CCT TTG CTG CTG TGA ACT GTA GCC CAG A
400 nt
393 CTG CGT ATG AGT GMM WST YTS ACA TCA CCA TTG GCC ACC ACC TGC CAG TCA ACA GAC GCG TGG TTA CAG GCT TGC GCG ACA TGC GTC ACC ACG GTG ATA TCG TCC ACC CAG GTG TTC GGC GTG GTG TAG AGC ATT ACG CTG CGA TGG ATC CCG GCG TAG TTA AAG AAA TCA TGG AAG TAA GAC TGC TTT TTC TTG CCG TTT TCG TCG GTA ATC ACC ATT CCC GGC GGG ATA GTC TGC CAG TTC AGT TCG TTG TTC ACA CAA ACG GTG ATA CGT ACA CTT TTC CCG GCA ATA ACA TAC GGC GTG ACA TCG GCT TCA AAT GGC GTA TAG CCG CCC TGA TGC TCC ATC ACT TCC TGA TTA TTG ACC CAC ACT TTG CCG TAA TGA TGG ACC GCA ATA
Figure B5 shows capillary gel electrophoresis sequencing traces and the related quality graphs
for the 150 nt, 250 nt and 400 nt targets. Details for the sequence of the 100 nt target were
provided by Dr. Paul Piunno. The quality graph is a plot of the quality of each base call as a
function of the base number. The error probability of each assignment can be calculated by the
following [295]:
pQ 10log10−= (B1)
where Q is the quality of the base call and p is the estimated error probability for that base call.
For example, a base call having a probability of 1/1000 of being incorrect is assigned a quality
value of 30 [296].
The length of the targets as provided from sequencing did not match with the lengths as obtained
by gel electrophoresis.
193
a)
b)
194
c)
Figure B5: CGE traces and quality graphs of the base calls for a) 150 nt target, b) 250 nt target and c)
400 nt target.
Sanger sequencing is based on a modified version of PCR. In the reaction mixture, fluorescently
labelled dideoxy nucleotide triphosphates are mixed with normal deoxy-nucleotide triphosphates
used for building the complement strand in PCR. The dideoxy nucleotide lacks a hydroxyl group
on the 3’ position of the deoxyribose sugar that is necessary for the formation of a
phosphodiester bond with the incoming nucleotide base. The polymerase reaction for a fragment
of DNA terminates when the dideoxy-nucleotide is incorporated into the complementary strand
instead of a deoxy-nucleotide. The reactions lead to a mixture of DNA strands having different
lengths, each terminated with a fluorescent label corresponding to the base at the end of the DNA
fragment when extension of that fragment completed. The mixture of the DNA fragments are
then size separated by capillary gel electrophoresis. Detection of the fragments is done by laser
induced fluorescence where detection makes use of the four wavelengths that correspond to the
four labelling dyes.
Ideally, the electrophoretogram of the material from the sequencing reaction would have all the
individual fragments fully resolved without overlapping peaks. However, it is very common to
see the first 50 bases of the sequencing reaction appear as noisy or unevenly spaced eluents.
This can be due to the anomalous migration of the very short fragments, and may be
compounded by the changes in mobility of the DNA due to the attachment of fluorescent dyes.
195
Detection of unreacted dye-terminator molecules may also convolute the electrophoretogram
[295, 297].
Near the end of the sequencing run, band broadening due to longitudinal diffusion may result in
poorly resolved peaks. In addition, the relative change in mass when adding additional base
pairs for the longer DNA fragments in the sequencing reaction is relatively small, making it
difficult to separate out fragments of different lengths for the long reaction fragments [295, 297].
The formation of hairpin structures of the DNA fragments may alter electrophoretic mobility,
allowing folded sequences to appear earlier in the separation run. These are observed as
compression artifacts, where two overlapping peaks are observed in the trace and may lead to
problems in base calling [295, 297].
Such compression artifacts as well as poorly resolved peaks in the early portion of the
electrophoretograms were observed, and this led to ambiguities in base calling and incorrect
length information. Additionally, the primers used in the sequencing reaction were not
accounted for in the sequencing data, which further decreased the apparent length obtained from
Sanger sequencing. Therefore, the agarose gel data presented in Figure B4 is a better indicator
of the length of the DNA targets. The sequencing information confirmed the sequence integrity
for the hybridization site within the longer DNA strands.
B4. Examination of Sequence for the 150 nt Target
A difference was observed between the expected hybridization sequence and the sequencing
results that were obtained for the 150 nt target.
Expected b-actin sequence (previously determined by Dr. Paul Piunno)
AGG ATG GCA TGG GGG AGG G
b-actin sequence from sequencing (from TCAG sequencing facility)
ARG GTG GCA TGG GGG RGG C
A single base pair inconsistency was noted between the expected sequence and the sequence
determination. There was concern that a base pair mismatch had the potential to influence the
performance of the affinity gels.
196
A displacement chromatography experiment was done to confirm the selectivity of the affinity
gels. In displacement chromatography, the analyte of interest is first attracted onto the
chromatography column using an immobilized probe that has affinity to the analyte of interest.
Following this, a displacer compound is introduced into the column. This displacer compound
has a higher binding affinity towards the probe versus the analyte of interest. Therefore, as the
displacer moves through the column, it begins to displace the analyte, allowing it to elute [298,
299]. I had previously examined displacement chromatography for a 40 nt length
oligonucleotide target where the central 20 base was complementary to the strand with a 20 nt
fully complementary target [177]. Displacement chromatography using the same
oligonucleotide attachment chemistry was also used by Zangmeister et al. for the displacement
of a 20 nt target using a 10 nt displacer [300].
Figures B6 and B7 demonstrate the effect of introducing a complementary and non-
complementary displacer (20 nt) into an affinity capture gel where the 150 nt length DNA target
had already been captured. In Figure B6, a small increase in fluorescence intensity can be
observed following the introduction of a displacer that is complementary to the probe sequence
used. This suggests that some of the previously captured material had been displaced, and
moved under the influence of the applied electric field.
197
4000
5000
6000
7000
8000
9000
10000
0 0.5 1 1.5 2
distance (cm)
flu
ore
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y (
AU
)
0 min
3 min
9 min
Figure B6: Profile plots from the inlet to outlet end of the capillary taken from confocal fluorescence
microscope images (Chipreader) tracking elution progress following the application of a complementary displacer to the affinity column where a Cy5 labelled 150 nt length DNA target was already captured. The displacer sequence was injected electrokinetically under the following conditions: 5 µL 20 µM, 267 Vcm
-1,
15 s; following which a potential of 167 Vcm-1
was applied. Confocal images were taken following electrophoresis for 3 and 9 minutes. Probe sequence: 5’ CCC TCC CCC ATG CCA TCC T 3’ Target
sequence: 5’ AGG ATG GCA TGG GGG AGG G 3’ Displacer sequence: 5’ AGG GTG GCA TGG GGG AGG G 3’
3000
3500
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4500
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5500
6000
6500
7000
0 1 2 3 4
distance (cm)
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ore
scen
ce i
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y (
AU
)
0 min
3 min
6 min
Figure B7: Profile plots from the inlet to outlet end of the capillary taken from confocal fluorescence
microscope images (Chipreader) tracking elution progress following the application of a non-complementary displacer to the affinity column where a Cy5 labeled 150 nt length DNA target was
already captured. The displacer sequence was injected electrokinetically Confocal images were taken following electrophoresis for 3 and 9 minutes. Probe sequence: 5’ CCC TCC CCC ATG CCA TCC T 3’ Target sequence: 5’ AGG ATG GCA TGG GGG AGG G 3’ Displacer sequence: 5’ ACA GGG TTT CAG
ACA AAA T 3’
198
The 150 nt sequence would have a lower binding affinity towards the probe than the 20 nt fully
complementary sequence. The additional length of DNA target would increase steric hindrance
and electrostatic repulsion between the target and probe versus that of the shorter oligonucleotide
displacer. Additionally, since the experiment was operated under an electric field, the longer
DNA target would experience a shearing force due to the electrophoretic force under the applied
field [269]. This would put a strain on the interaction between the hybridized target and probe.
The lack of a displacement affect when a non-complementary target was used as a displacer as
shown in Figure B7 suggests that a competitive displacement effect was present in the results of
Figure B6.
If a base-pair mismatch was introduced between the captured target and probe sequence, a much
greater displacement effect would result following the introduction of a fully complementary
displacer. This is demonstrated in Figure B8, where a significantly higher increase in
fluorescence intensity was observed. Here, a base pair mismatch was deliberately introduced
between the target and probe, while the displacer was fully complementary to the probe
sequence. Based on these displacement experiments, there is high confidence that the original
sequence that was supplied by Dr. Piunno was correct.
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
0 1 2 3 4
distance (cm)
flu
ore
scen
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nsit
y (
AU
)
0 min
3 min
9 min
bkg
Figure B8: Profile plots from the inlet to outlet end of the capillary taken from confocal fluorescence microscope images (Chipreader) tracking the progress following the application of a complementary
displacer to the affinity column where a Cy5 labeled 150 nt DNA target that contained a single base pair mismatch to the probe was already captured. Confocal images were taken following electrophoresis for 3
and 9 minutes. Probe sequence: 5’ CCC TCC CCC ATG CCA CCC T 3’ Target sequence: 5’ AGG ATG GCA TGG GGG AGG G 3’ Displacer sequence: 5’ AGG GTG GCA TGG GGG AGG G 3’
199
C. Generation of Single Stranded DNA Targets
The targets were generated by symmetric PCR, and the DNA existed in double stranded form.
Therefore, prior to the injection of the DNA sample into the capillary, the targets were first
denatured. Denaturation was accomplished by heating the DNA sample at 95 °C for 5 minutes,
followed by rapid cooling of the solution by putting it on ice. Heating disrupts the hydrogen
bonding between the complementary base pairs, separating the two strands and the rapid cooling
in ice is thought to slow the separated strands from re-annealing with one another.
Complete denaturation of PCR fragments of 0.6 kbp to 3.2 kbp have been reported in 30 seconds
at 98 °C, and 5 seconds for fragments 2 kbp and longer. Extended heat denaturation of up to
10 minutes did not appear to affect the quality of the DNA. Renaturation of the DNA in ice or at
RT was not observed in buffers without MgCl2 after 6 hours [301].
The denaturation of DNA was confirmed by measuring the hyperchromicity of DNA as it went
from double to single stranded state. The molar absorptivity at 260 nm of double stranded DNA
as calculated based on the individual molar absorptivity of the nucleotides is about 70% higher
than the molar absorptivity of the measured DNA strand [302]. The main contributor to this
hypochromicity is thought to be due to the stacking of the base pairs in the DNA [303]. The
electronic transitional dipoles of the stacked bases are ordered parallel to one another when the
DNA is in its double stranded form and the absorbance shifts to higher energy bands [303, 304].
There is an increase in absorption of bands at 200 nm and lower, while there is a decrease in the
absorption at 260 nm [305]. When DNA is heated, the interactions between the bases are
disrupted, leading to a disordering of the bases and an increase in absorption at 260 nm [304,
306]. The maximum increase in hyperchromicity due to denaturation of DNA has been reported
to be between 34-40% [302, 306, 307].
Figure C1 shows the absorbance at 260 nm in a solution of 150 bp DNA targets before and after
heating at 95 °C. A 400 µL solution of unlabeled 150 bp dsDNA target was heated to 95 °C for 5
minutes and then the absorbance was measured. The solution was then transferred to a
microcentrifuge tube and immediately put on ice for a total of 20 minutes, with the absorbance
measured at 10 minutes intervals.
200
0.9
0.95
1
1.05
1.1
1.15
1.2
1.25
1.3
initial 5 min 95 C 10 min ice 20 min ice
No
rmali
zed
Ab
so
rban
ce (
AU
)
Figure C1: Normalized absorbance of a solution of 13 µg/mL 150 bp DNA in Tris-HCl (pH 7.0) before and after denaturation by heat and following rapid cooling on ice. The absorbance values are normalized to the initial absorbance readings prior to heat denaturation. Error bars represent 1 standard deviation of
three trials.
An experiment was done using food colouring dye to confirm that the change in absorbance was
not due to a change in volume from heating the solution at 95 °C.
A decrease in absorbance following cooling in ice was observed, suggesting some of the DNA
had re-associated. This may be due to the solution not being cooled rapidly enough. The
volumes of solution used in these experiments were 400 µL. The experiment was done such that
solutions were transferred between a microcentrifuge tube and a cuvette at room temperature for
measurement. The relatively large volume of solution may have negatively impacted how
quickly the solution was cooled, allowing some of the DNA targets to re-anneal. Smaller
volumes of the target DNA solutions of up to 20 µL were used during the actual capture
experiments to allow for faster heating and cooling of the solutions.
201
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80000
0 20 40 60 80 100 120 140 160
Time (s)
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y (
AU
)
TOPRO3 + DNA TOPRO3
Figure C2: Denaturation of the 13 µg/mL 150 bp target in Tris-HCl (pH 7.5) at 95 °C as detected using
an intercalating dye, (5 µM) TO-PRO3. Excitation: 642 nm, Emission: 660 nm. The blue trace is the response of the intercalating dye alone to temperature. Error bars represent 1 standard deviation of three
trials.
Figure C2 shows the fluorescence intensity of the DNA intercalating dye TO-PRO3 when mixed
with a solution containing the 150 bp DNA target after denaturation at 95 °C. TO-PRO3 is an
intercalation dye derived from thiazole orange [308]. Fluorescence enhancement due to
increased quantum yield is observed from interactions with double stranded and single stranded
DNA, but the enhancement is about 2 times larger for dsDNA [308, 309]. The dye interacts with
DNA by base-stacking, groove binding and electrostatic interaction with the phosphate
backbone. However, it has been suggested that intercalation is preferred at low dye-to-base pair
ratios (0.20) [310, 311]. The dye-to-base pair ratio in this work was approximately 0.1.
The fluorescence intensity decrease observed in Figure C2 was due to the drop in quantum yield
of the fluorescent dye as the double stranded DNA target denatured. Figure C2 also includes data
representing the emission of the intercalating dye in bulk solution as a function of temperature.
The termination of the fluorescence intensity change after 140 seconds suggests complete
denaturation of the target at this point. The fluorescence intensity remained higher than that for
the unassociated state of the dye due to some interaction of the dye with single stranded DNA.
202
Short, single stranded DNA targets can also be produced by the method of asymmetric PCR.
Asymmetric PCR was also investigated for target production, but it was found that symmetric
PCR followed by heating/cooling produced significantly more target.
A number of reports in the literature that examined real samples have made use of symmetric
PCR and generated single stranded DNA targets in the same fashion as used herein (by heat) [24,
35, 85, 199, 312]. Therefore, symmetric PCR was used to generate the targets in the work of this
thesis.
203
D. Suppression of Electroosmotic Flow (EOF) in Capillary Affinity Capture Gels
EOF was suppressed in these experiments because this process has the ability to physically eject
the affinity gel from the capillary. There are two common methods for the suppression of EOF
inside the capillary: covalent modification of the silanol wall on the inside of the capillary, or by
dynamically coating a polymer on the wall that prevents the generation of an electrical double
layer.
The use of a covalently immobilized coating for the suppression of EOF in a capillary was first
reported by Hjerten [313]. This work reported a bifunctional linker which was covalently
bonded onto the fused silica surface while providing a vinyl group that allowed for a layer of
polyacrylamide to be covalently bound to the wall. The bifunctional linker used was 3-
methacryloxypropyltrimethoxysilane (MPS). The methoxy groups in 3-
methacryloxypropyltrimethoxysilane reacted with the silanol groups in the glass wall, while the
free acryl group was available for polymerization with polyacrylamide to form a thin layer of
polymer covalently bound to the wall [313].
Figure D1: Structure of 3-methacryloxypropyltrimethoxysilane.
An example of the dynamic coating process is based on use of polyvinylpyrrolidone (PVP)
which is continuously present in flow solutions to suppress EOF. The structure of PVP is shown
in Figure D2.
ON
CH3
CH3 nn Figure D2: Structure of polyvinylpyrrolidone.
204
The use of PVP for the suppression of EOF in capillary electrophoresis was demonstrated by
Gao and Yeung [314]. It has been proposed that the PVP associates with the wall of the
capillary through non-covalent interactions with the silanol groups on the silica surface. PVP is
considered a hydrophobic polymer, but the hydrophilic carbonyl groups can hydrogen bond with
the silanol groups [314, 315]. The hydrophobic nature of PVP suggests that water is a poor
solvent, and therefore PVP is not easily desorbed by aqueous buffer systems [315].
Both methods for EOF suppression were examined to determine the optimal method for the
operation with the affinity gel in capillaries. Figure D3 shows the time-dependence of
fluorescence signal from fluorescently labeled probes that were incorporated in affinity gels.
Electrophoresis was run for a set amount of time, and data was collected to compare MPS and
PVP modified capillaries. No significant difference between the MPS and PVP modified
capillaries in terms of change in fluorescence signal was observed over the course of the
electrophoresis experiment. If EOF was not adequately suppressed, then a decrease in
fluorescence intensity would have been observed as the fluorescent polyacrylamide affinity gel
eluted from the column. It was concluded that both methods suppressed EOF and provided
physical stability to the polyacrylamide affinity gel in the capillary. PVP was used for the
remainder of the experiments to suppress EOF since it was the simpler method for
implementation.
205
0
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4000
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6000
before 15 min 500V 10 min 1000V 20 min 1000V
Avera
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)MPS
PVP
Figure D3: Time-dependent experiments tracking the fluorescence intensity of the Cy3 labeled
oligonucleotide probe incorporated into the affinity gel to examine the effectiveness of two different methods for EOF suppression. The fluorescence intensity following polymerization of the gel was
measured, and then was subsequently measured following electrophoresis for a set amount of time at various applied voltages. The column contained 7.5%T 6%C polyacrylamide gel with 2 µM Cy3-β-actin oligonucleotide probe. The data was obtained from confocal fluorescence images (Chipreader) of the
capillaries. Error bars represent 1 standard deviation of three trials.
The data shown in Figure D3 was collected by fluorescence imaging of the same capillary a
number of times. It was necessary to determine any undesired affect of photobleaching of the
fluorescent dyes as a function of the number of times the capillary was scanned. Data
representing an investigation of photobleaching effects is displayed in Figure D4. A marginal
decrease in the fluorescence intensity from the capillary was observed when scanning the same
capillary a number of times. The decrease was small, representing only about a 3% loss in
fluorescence intensity after scanning the capillary four times. Therefore, the affect of
photobleaching was considered inconsequential in the interpretation of experimental results,
particularly since capillaries were scanned less than four times in all subsequent studies.
206
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0.98
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1 2 3 4
Number of Scans
Norm
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(A
U)
a)
0.9
0.91
0.92
0.93
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0.95
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0.98
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1 2 3 4
Number of Scans
Norm
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)
b)
Figure D4: Change in fluorescence intensity of a) Cy3 and b) Cy5 fluorescent dyes as a function of the number of times it was scanned under the confocal fluorescence microscope. The Cy3 channel was scanned at 5% laser power, and the Cy5 channel was scanned at 40% laser power. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries. Error bars represent 1
standard deviation of three trials.
207
E. Factorial Analysis for Probe Incorporated into Affinity Capture Gel
E1. Table of Results
Table E1 shows the relative change in the amount of probe before and after the pre-conditioning
step based on the factorial design. Following the polymerization of the affinity capture gel, the
fluorescence intensity of the capillary was measured. The capillary was then subjected to a pre-
conditioning step where electrophoresis was run at a low voltage (74 Vcm-1) for 15 min.
Table E1: The relative difference in concentration of Cy3-labeled oligonucleotide probe measured following polymerization and after pre-conditioning of the affinity capture gels.
Std Order
Original Concentration
(µM)
Concentration After
Polymerization (µM)
Concentration After Pre-
conditioning (µM)
Percent Decrease in
Fluorescence Intensity (%)
1 9 0.5 0.36 0.36 0.32 0.28 87.32 78.59
2 10 3.0 3.08 2.74 2.89 2.25 93.86 82.00
3 11 0.5 0.42 0.43 0.40 0.44 93.76 101.51
4 12 3.0 1.98 1.58 1.63 1.39 82.22 87.93
5 13 0.5 0.26 0.31 0.23 0.26 86.52 85.67
6 14 3.0 3.15 2.94 3.07 2.47 97.26 84.03
7 15 0.5 0.51 0.44 0.37 0.38 72.75 86.06
8 16 3.0 3.48 2.32 2.81 2.54 80.60 109.25
Rather than using the absolute fluorescence intensity to identify the amount of oligonucleotide
probes incorporated into the affinity capture gel, the percentage loss in fluorescence intensity
before and after pre-conditioning was used. This loss in fluorescence intensity represents the
amount of the dye-labelled oligonucleotide probe washed off the affinity capture gel after pre-
conditioning.
E2. Factorial Analysis for Percentage of Probe Incorporated
E2.1 Analysis of Results
Results of the fractional factorial experiment were analyzed by three methods to determine
which factors had a large affect on the quantity of probe that was incorporated into the gel. The
methods were: calculation of the magnitude of effect each factor by comparison with standard
error; analysis of variance (ANOVA); and by comparison to a normal probability distribution
plot. The results were calculated using Minitab.
208
E2.2 Pareto Effects Plot
Figure E1 shows the magnitude effect of each factor in a Pareto effects plot. Here, the
standardized effect that each factor has on the response is ranked from largest to smaller. Any
effect beyond the line that is shown in Figure E1 is significantly larger than experimental error
with 95% confidence (α=0.05). The line is the t-value at the 97.5th quantile (1-α/2) for a t-
distribution with the number of degrees of freedom of the error term in the ANOVA table.
Figure E1: Pareto Chart plotting standardized effects of each factor for the percentage of probe
incorporated into the affinity capture. Factors with magnitude greater than shown by the line represent effects larger than experimental variation with 95% confidence.
E2.3 Magnitude of Effects
The following shows the raw output for the magnitude of effects as calculated by Minitab from
the fractional factorial experiment for the percentage of probe incorporated into the affinity
capture gel.
209
Estimated Effects and Coefficients for %probe (coded units)
Term Effect Coef SE Coef T P
Constant 88.084 2.407 36.59 0.000
%T -0.629 -0.315 2.407 -0.13 0.899
%C 2.355 1.178 2.407 0.49 0.638
Probe 3.120 1.560 2.407 0.65 0.535
TEMED 6.912 3.456 2.407 1.44 0.189
APS -1.644 -0.822 2.407 -0.34 0.742
%T*%C -3.559 -1.780 2.407 -0.74 0.481
%T*APS 7.128 3.564 2.407 1.48 0.177
S = 9.62889 PRESS = 4592.72
R-Sq = 41.15% R-Sq(pred) = 0.00% R-Sq(adj) = 0.00%
The magnitude of change for each factor is the difference between the averages of the responses
between the plus level of the factor and the minus level of the factor:
−+ −= yyeffectMain (E1)
The influence of interaction effects are calculated similarly [289]. To determine which factors
have a significant affect, the magnitude can be compared with the estimated standard error of the
experiment, which is calculated by:
2
2
1)( S
nEffectse
k= (E2)
Where the overall variance estimate is calculated by:
∑∑= =
−−
=
k
i
n
j
iijkyy
nS
2
1 1
22 )()1(2
1 (E3)
where n is the number of replicates for each of the 2k runs in the design, k is the number of
factors, y is the response at that run/replicate and y is the mean of the response for that
experimental run. The standard error is multiplied by the appropriate t-statistic for a particular
confidence interval and degree of freedom. Any effect larger than this value cannot be attributed
to experimental variation at the selected confidence level [288, 289].
210
E2.4 ANOVA Table
The data from the fractional factorial experiment was also analyzed by ANOVA and the raw
output from Minitab is shown below.
Analysis of Variance for %probe (coded units)
Source DF Seq SS Adj SS Adj MS F P
Main Effects 5 264.65 264.655 52.93 0.57 0.722
2-Way Interactions 2 253.89 253.893 126.95 1.37 0.308
Residual Error 8 741.72 741.724 92.72
Pure Error 8 741.72 741.724 92.72
Total 15 1260.27
Unusual Observations for %probe
Obs StdOrder %probe Fit SE Fit Residual St Resid
15 15 80.598 94.926 6.809 -14.328 -2.10R
16 16 109.254 94.926 6.809 14.328 2.10R
R denotes an observation with a large standardized residual.
ANOVA is a method for comparing whether mean responses between two or more populations
are different. For a factorial design, the comparison is between the mean response that each
factor has on the response versus that of the experimental noise that is present. This is done by
first calculating the total variance of the experiment. For clarity, the equation shown is for a two
factor, two level experiment involving A and B.
n
yySS
ki j
n
k
ijkT 2
22
1
2
1 1
2 K−
= ∑∑∑
= = =
(E4)
The variance is then distributed amongst all the different factors being examined, including
interaction terms. For example, for a two factor, two level experiment with factors A and B, the
sum of squares attributed to factor A and B is calculated by the square of the difference of the
response between the high and low levels of the factor, divided by the total number of
experimental runs:
n
BABABABASS
kA 2
][ 2−+−−+++ −−+= (E5)
n
BABABABASS
kB 2
][ 2−−−++−++ −−+= (E6)
The variance attributed to the interaction term, AB, is calculated similarly by:
211
n
BABABABASS
kAB 2
][ 2+−−+−−++ −−+= (E7)
Where k is the number of factors, n is the number of replicates performed while.
Any variance not assigned to a term in the model is assigned to the error term and is assumed to
be random error associated with the experiment. An F-statistic is used to determine whether
there is a statistical difference between the variance explained by a particular factor relative to
the variance associated with experimental error:
ABBATE SSSSSSSSSS −−−= (E8)
The sum of squares is divided by the degrees of freedom to obtain the mean square. Each term
has one degree of freedom. The total number of degrees of freedom in the experiment is 2kn-1.
The number of degrees of freedom in the error term is the remainder not assigned to a term in the
model.
An F-statistic is used to compare the variation among groups with variation within groups. The
F test is then performed considering the variation between means versus the mean square of the
error (variation within a group):
E
R
MS
MSF =0 (E9)
P-values less than 0.05 would indicate that the population means are different. This would imply
for the work of this thesis that a particular parameter for gel preparation is statistically significant
to the response [288].
E2.5 Normal Probability Plot
A further method for determining which factors are significant is based on comparison to a
normal probability plot or a normal quantile plot. Effects that are negligible are normally
distributed, with the mean being a value of zero and variance σ2 [316]. Figure 3.43 shows the
normal probability plot for the amount of probe incorporated. The seven data points seen on the
graph are the orthogonal terms in the design matrix (five main factors and the two interaction
factors) as shown in Table B2.
212
Figure E2: Normal probability plot of the effect of the factors as compared to a to a line representing
normally distributed data (in blue). Those highlighted in red are considered statistically significant. The data points are the orthogonal terms in the design matrix (five main factors and the two interaction
factors) as shown in Table A2.
E2.6 Examination of Model Adequacy
It is necessary to examine the fitted model to ensure that it provides an adequate approximation
to the true system, and to verify that none of the least squares regression assumptions are
violated.
A check of the assumption of normality is done by constructing a normal probability plot of the
residuals. If the residuals plot provides data that lies approximately along a straight line, then the
normality assumption is satisfied. When this plot indicates a problem, the response variable is
often transformed as a remedial measure.
A plot of the residuals ei versus the predicted response yi should appear randomly scattered on
the display, suggesting that the variance of the original observations is constant for all values of
y. If the variance of the response depends on the mean level of yi, then this plot will often
exhibit a funnel-shaped pattern and can suggest a need for transformation of the response
variable y.
213
A histogram plot of the residuals checks the normality of the variance. It should appear evenly
distributed around mean zero and indicates if random error is normally distributed.
A plot of residuals in time, or run order, versus each of the individual regressors is also done.
Non-random patterns on these plots would indicate model inadequacy.
Figure E3 shows all four plots. The diagnostic plots did not indicate any deviations from the
least squares model.
Figure E3: Statistical diagnostic plots of data distributions from factorial experiment that investigated
percentage of probe that was incorporated in gels.
E3. Amount of Probe Incorporated
The following is a summary of data generated by Minitab for analysis of the fractional factorial
experiment for the response labelled as ‘Amount of Probe Incorporated’. The data were taken
from the ‘Concentration after Pre-conditioning’ column in Table E1, transformed using a log10
function. The diagnostic plot of the original, unmodified data showed a funnel shape in the
residual versus predicted plot, which prompted the transformation of the data.
214
Figure E4: Pareto Chart plotting standardized effects of each factor on the amount of probe incorporated.
Factors with values greater than the line represent effects larger than anticipated by experimental variation at 95% confidence.
Magnitude of Effects
Estimated Effects and Coefficients for log probe amt (coded units)
Term Effect Coef SE Coef T P
Constant -0.06160 0.01212 -5.08 0.001
%T 0.03772 0.01886 0.01212 1.56 0.158
%C 0.02246 0.01123 0.01212 0.93 0.381
Probe 0.84750 0.42375 0.01212 34.96 0.000
TEMED 0.10395 0.05197 0.01212 4.29 0.003
APS -0.14382 -0.07191 0.01212 -5.93 0.000
%T*%C 0.06442 0.03221 0.01212 2.66 0.029
%T*APS 0.04369 0.02184 0.01212 1.80 0.109
S = 0.0484902 PRESS = 0.136319
R-Sq = 99.38% R-Sq(pred) = 95.53% R-Sq(adj) = 98.84%
ANOVA Table
Analysis of Variance for log probe amt (coded units)
Source DF Seq SS Adj SS Adj MS F P
Main Effects 5 3.00667 3.00667 0.601334 255.75 0.000
2-Way Interactions 2 0.02423 0.02423 0.012116 5.15 0.036
Residual Error 8 0.01881 0.01881 0.002351
Pure Error 8 0.01881 0.01881 0.002351
Total 15 3.04971
215
Figure E5: Normal probability plot of the effect of the factors as compared to a line representing normally distributed data (in blue). Those highlighted in red are considered statistically significant. The data points are the orthogonal terms in the design matrix (five main factors and the two interaction factors) as shown
in Table A2.
Figure E6: Statistical diagnostic plots of data distributions from factorial experiment that investigated the
amount of probe that was incorporated into gels.
216
F. Amount of Target Captured
F1. Data Reflecting the Quantity of Target Captured
Table F1 summarizes data about the capture of complementary target by the affinity capture gel
at various points during experiments. The responses reported were: the concentration of target
injected, concentration captured by the gel and concentration remaining inside the capillary
following elution. The fluorescence intensity measured following elution was below the lowest
value of the calibration curve and was set as zero.
Table F1: Summary of results from fractional factorial experiment on the following responses: Concentration of target in affinity capture gel following electrokinetic injection (Inject); Concentration of
targets captured following the wash step (Load); Concentration of targets remaining in the capillary following elution step (After Elute).
Standard Order Inject (nM) Load (nM) After Elute (nM)
1 9 66 74 18 19 0 0
2 10 189 208 38 42 0 0
3 11 104 95 8.5 9.5 0 0
4 12 227 216 31 32 0 0
5 13 131 119 53 65 0 0
6 14 212 235 73 92 0 0
7 15 116 148 35 45 0 0
8 16 273 254 55 48 0 0
F2. Concentration of Target Injected
The following is a summary of data generated by Minitab for analysis of the fractional factorial
experiment for the response labelled as ‘Concentration of Target Injected’. The data were taken
from the Inject column in Table F1. The diagnostic plots shown in Figure F3 did not indicate
any deviations from the least squares model.
217
Figure F1: Pareto Chart plotting standardized effects of each factor for the concentration of
complementary target injected. Factors with values greater than the line represent effects larger than anticipated by experimental variation at 95% confidence.
Magnitude of Effects
Estimated Effects and Coefficients for Injected (coded units)
Term Effect Coef SE Coef T P
Constant 6000.00 85.35 70.30 0.000
%T 1012.50 506.25 85.35 5.93 0.000
%C 650.00 325.00 85.35 3.81 0.005
Probe 3175.00 1587.50 85.35 18.60 0.000
TEMED -137.50 -68.75 85.35 -0.81 0.444
APS 175.00 87.50 85.35 1.03 0.335
%T*%C -37.50 -18.75 85.35 -0.22 0.832
%T*APS 262.50 131.25 85.35 1.54 0.163
S = 341.413 PRESS = 5935000
R-Sq = 98.04% R-Sq(pred) = 87.51% R-Sq(adj) = 96.32%
ANOVA Table
Analysis of Variance for Injected (coded units)
Source DF Seq SS Adj SS Adj MS F P
Main Effects 5 46311250 46311250 9262250 79.46 0.000
2-Way Interactions 2 281250 281250 140625 1.21 0.348
Residual Error 8 932500 932500 116563
Pure Error 8 932500 932500 116563
Total 15 47525000
218
Figure F2: Normal probability plot of the effect of the factors as compared to a line representing normally distributed data (in blue). Those highlighted in red are considered statistically significant. The data points are the orthogonal terms in the design matrix (five main factors and the two interaction factors) as shown
in Table A2.
Figure F3: Statistical diagnostic plots of data distributions from factorial experiment that investigated the
concentration of target injected into gels.
219
F3. Amount of Target Captured
The following is a summary of data generated by Minitab for analysis of the fractional factorial
experiment for the response labelled as ‘Concentration of Target Captured’. The data were taken
from the Load column in Table F1, transformed using a log10 function. The diagnostic plot of
the original, unmodified data showed a funnel shape in the residual versus predicted plot, which
prompted the transformation of the data.
Figure F4: Pareto Chart plotting standardized effects of each factor on the log10 of amount of
complementary target captured. Factors with values greater than the line represent effects larger than anticipated by experimental variation at 95% confidence.
220
Magnitude of Effects
Estimated Effects and Coefficients for log load (coded units)
Term Effect Coef SE Coef T P
Constant 1.53899 0.01223 125.79 0.000
%T 0.41781 0.20890 0.01223 17.07 0.000
%C -0.19735 -0.09868 0.01223 -8.07 0.000
Probe 0.28408 0.14204 0.01223 11.61 0.000
TEMED -0.15552 -0.07776 0.01223 -6.36 0.000
APS 0.04435 0.02217 0.01223 1.81 0.108
%T*%C 0.01100 0.00550 0.01223 0.45 0.665
%T*APS -0.06075 -0.03037 0.01223 -2.48 0.038
S = 0.0489399 PRESS = 0.194722
R-Sq = 98.54% R-Sq(pred) = 85.20% R-Sq(adj) = 97.27%
ANOVA Summary
Analysis of Variance for log load (coded units)
Source DF Seq SS Adj SS Adj MS F P
Main Effects 5 1.28146 1.28146 0.256291 107.01 0.000
2-Way Interactions 2 0.01524 0.01524 0.007622 3.18 0.096
Residual Error 8 0.01916 0.01916 0.002395
Pure Error 8 0.01916 0.01916 0.002395
Total 15 1.31586
Figure F5: Normal probability plot of the effect of the factors as compared to a line representing normally distributed data (in blue). Those highlighted in red are considered statistically significant. The data points are the orthogonal terms in the design matrix (five main factors and the two interaction factors) as shown
in Table A2.
221
Figure F6: Statistical diagnostic plots of data distributions from factorial experiment that investigated the
amount of target captured by gels.
222
G. Evaluation of Hybridization and Stringency Conditions
G1. Effectiveness of Gels for Purification of Target
Table G1 summarizes the results for the recovery and purity of the sample following
examination of a number of hybridization and stringency conditions using a three level factorial
analysis experiment.
Table G1: Summary of results from a 3-level factorial analysis for factors that affect hybridization and stringency. The response %Recovery (complementary target alone) was performed using solutions
containing Cy5-150 nt target only. The factors examined were A (hybridization time) and B (wash voltage). %Recovery and % Purity (complementary and non-complementary targets) were done using
solutions containing Cy5-150 nt and Cy3-non-complementary targets. The factors examined were A (wash temperature) and B (formamide content).
Standard Order
A B % Recovery (complementary
target alone)
% Recovery (complementary
and non-complementary
targets)
% Purity (complementar
y and non-complementary
targets)
1 10 1 1 1.46 1.83 1.44 1.35 66 64
2 11 1 2 2.48 1.28 1.59 0.94 75 97
3 12 1 3 1.77 1.45 1.14 1.57 85 82
4 13 2 1 1.92 2.99 1.51 1.68 62 58
5 14 2 2 1.77 1.72 1.53 1.14 89 91
6 15 2 3 1.66 1.55 1.03 1.53 86 81
7 16 3 1 1.28 1.25 1.58 1.81 61 60
8 17 3 2 1.67 1.22 1.72 1.56 68 68
9 18 3 3 1.74 2.08 1.35 0.63 88 94
G2. Hybridization Time and Wash Voltage for Samples Containing Only Complementary Targets
The following is a summary of data generated by Minitab for analysis of the three level factorial
experiments for the response identified as percent recovery on varying hybridization time and
wash voltage for samples containing only complementary target. The data were taken from the
%Recovery (complementary target only) column in Table G1. Since the experiment was based
on a three level factorial design, only the ANOVA table and model diagnostic plot was generated
by Minitab. The diagnostic plots shown in Figure G1 did not indicate any deviations from the
least squares model.
223
ANOVA Table:
Analysis of Variance for recovery, using Adjusted SS for Tests
Source DF Seq SS Adj SS Adj MS F P
Blocks 1 0.0027 0.0027 0.0027 0.01 0.910
Time 2 0.4707 0.4707 0.2354 1.19 0.352
Wash V 2 0.0328 0.0328 0.0164 0.08 0.921
A*B 4 1.3273 1.3273 0.3318 1.68 0.246
Error 8 1.5762 1.5762 0.1970
Total 17 3.4098
S = 0.443877 R-Sq = 53.77% R-Sq(adj) = 1.77%
Figure G1: Statistical diagnostic plots of data distributions from three level factorial experiments that investigated the %Recovery of target captured by gels.
G3. Stringency Conditions for Samples Containing Complementary and Non-complementary Targets
The following is a summary of data generated by Minitab for analysis of the three level factorial
experiments for the responses identified as percent recovery and purity on varying wash
temperature and formamide content for samples containing complementary and non-
complementary targets. The data were taken from the %Recovery and %Purity (complementary
and non-complementary targets) columns in Table G1. The diagnostic plots shown in Figure G2
and G3 did not indicate any deviations from the least squares model.
224
ANOVA Table for Percent Recovery:
Analysis of Variance for recovery mix, using Adjusted SS for Tests
Source DF Seq SS Adj SS Adj MS F P
Blocks 1 0.0018 0.0018 0.0018 0.02 0.898
A 2 0.0327 0.0327 0.0164 0.16 0.855
B 2 0.3777 0.3777 0.1889 1.84 0.220
A*B 4 0.3683 0.3683 0.0921 0.90 0.508
Error 8 0.8199 0.8199 0.1025
Total 17 1.6004
S = 0.320137 R-Sq = 48.77% R-Sq(adj) = 0.00%
Figure G2: Statistical diagnostic plots of data distributions from three level factorial experiments that investigated the percent recovery of target captured by gels.
225
ANOVA Table for Percent Purity:
Analysis of Variance for purity, using Adjusted SS for Tests
Source DF Seq SS Adj SS Adj MS F P
Blocks 1 20.06 20.06 20.06 0.60 0.462
A 2 93.78 93.78 46.89 1.39 0.303
B 2 1972.11 1972.11 986.06 29.28 0.000
A*B 4 560.89 560.89 140.22 4.16 0.041
Error 8 269.44 269.44 33.68
Total 17 2916.28
S = 5.80350 R-Sq = 90.76% R-Sq(adj) = 80.37%
Unusual Observations for purity
Obs purity Fit SE Fit Residual St Resid
3 75.0000 84.9444 4.3257 -9.9444 -2.57 R
12 97.0000 87.0556 4.3257 9.9444 2.57 R
R denotes an observation with a large standardized residual.
Figure G3: Statistical diagnostic plots of data distributions from three level factorial experiments that investigated the percent purity of target captured by gels.
226
H. Effect of Step Size in the Step Elution Process
Step elution was accomplished by moving the resistive heating element across the capillary in
discrete steps. The physical size of the heating element was approximately 0.8 mm. One
consideration was whether a length scale of steps at the size scale of the heating element would
avoid discontinuities in the heating zone. Therefore, the effect of changing the step size was
examined.
Since the step rate must be matched to the electrophoretic mobility of the DNA targets, the
heating time was also adjusted when the step size was altered. For example, the larger steps
were held for a longer time than the smaller steps before the heating element was advanced. Step
sizes were selected based on the minimum time needed for elution to take place, and the
maximum amount of time before gel breakdown. For the 19 nt targets it was observed that about
1 second was required for elution to be completed. Additionally, continuous heating of the gel at
one spot of the capillary caused the loss of current across the gel capillary in some cases. This
was observed to coincide with formation of small voids or bubbles inside the capillary where the
heater element was positioned, and occurred about 50% of the time when the gel was heated for
more than about 6 seconds. The range of step size tested was 125 to 500 µm, with heating times
of 1.1 to 4 seconds.
Figure H1 shows the peak area, height and width of eluted oligonucleotide targets obtained by
step elution from the same length of capillary for a variety of different step sizes. These results
were obtained using a step elution length of 25 mm versus the 12.5 mm stated as optimal
previously. These experiments were performed concurrently with the previous set of
experiments prior an optimal elution length was determined. The results indicate no statistically
significant differences in peak height or area for steps of the size scale of the heating element,
indicating that elution would be continuous and unfluctuating.
227
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
peak area peak height
Flu
ore
sce
nce
In
ten
sity (
AU
)
125 um 250 um 500 uma)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
peak width
tim
e (
s)
125 um 250 um 500 umb)
Figure H1: Comparison of peak area, height and width as a function of step size used during step elution
across a column length of 25 mm. Data for a) integrated Peak Area, Height and b) Peak Width were calculated using Origin Pro 8.0.
Affinity capture gel: 50 nM SMN probe, 10% LAAm affinity capture gel. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150 Vcm
-1. Capture: electrophoresis for 10 min at 150 Vcm
-1 in 1x TBE/PVP
running buffer. Step elution: step size: 125 µm, 250 µm and 500 µm; Step rate: 86 µms-1
; Voltage: 96 Vcm
-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain 400 mV. Error bars represent 1 standard
deviation of three trials.