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Towards a Surface Microarray based Multiplexed Immunoassay on a Digital Microfluidics Platform
by
Uvaraj Uddayasankar
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Chemistry University of Toronto
© Copyright by Uvaraj Uddayasankar 2010
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Towards a Surface Microarray based Multiplexed Immunoassay on a Digital Microfluidics Platform
Uvaraj Uddayasankar
Master of Science
Department of Chemistry University of Toronto
2010
Abstract
The use of digital microfluidics (DMF) for sample handling in a microarray immunoassay was
investigated. A two plate DMF device was used, with the top plate being used for the
immobilization of antibodies for a sandwich immunoassay. A patterning procedure was
developed for the top plate to expose patches of glass that were chemically modified, using
silane chemistry, to allow for the covalent immobilization of antibodies. For creating
microarrays, a set of parallel microchannels were used for the high density patterning of proteins
onto the functionalized surface of the top plate. This patterning procedure was optimized to
ensure the reproducibility of the immobilization and the physical integrity of the top plate.
Preliminary work for a multiplexed immunoassay such as verification of cross-reactivity and
detection schemes was also conducted. This work represents the initial efforts towards a
microarray immunoassay on DMF, which has the potential to improve high throughput analysis.
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Acknowledgments
First and foremost, I would like to extend my sincerest gratitude towards my supervisor, Dr.
Aaron Wheeler, whose support and guidance throughout my project has been invaluable. I would
also like to thank Dr. David McMillen for serving as a second reader to review my MSc. thesis.
I would also like to thank the entire Wheeler Microfluidics Group for making it a fun and very
interesting place to work. Their support and willingness to help will always be appreciated. I
would especially like to thank Alphonsus Ng for all the training and help with digital
microfluidics and also for all the creative discussions, both in the lab and outside.
I would like to thank Matthew Kofke (University of Pittsburgh) and Shell Ip (Prof. Gilbert
Walker’s lab) for their help with fabrication and use of gold nanohole array substrates. I would
like to thank Wen Li (Kelly) Chen (Prof. Craig Simmons’ lab) for help with the protein
microarrayer. In addition, I would like to thank Dr. Henry Lee, Dr. Yimin Zhou (ECTI clean
room) and Dr. Aju Juggesur (Electron Beam Nanolithography Facility) for all their help in
microfabrication training.
Finally, I would like to thank my parents for their support and guidance in all my endeavors.
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Table of Contents
Abstract………………………………………………………………………….......................... ii
Acknowledgements……………………………………………………………………………... iii
Table of Content……………………………………………………………………………….... iv
Table of Tables……..……………………………………………………………………........... vii
Table of Figures………………………..…………………………………………………..........viii
1 Introduction .........................................................................................................................1
1.1 Background information ...............................................................................................2
1.1.1 Immunoassays .......................................................................................................3
1.1.2 Digital microfluidics ............................................................................................ 10
1.2 Literature review ......................................................................................................... 15
1.2.1 Multiplex immunoassays in microfluidic systems ................................................ 15
1.2.2 Immunoassays on DMF devices ........................................................................... 17
1.2.3 Surface based assays on DMF .............................................................................. 17
1.3 Research outline .......................................................................................................... 18
Chapter 2 .................................................................................................................................. 21
2 Surface immobilization strategies for capture antibodies .................................................... 21
2.1 Introduction ................................................................................................................ 21
2.2 Materials and methods ................................................................................................ 22
2.2.1 Materials .............................................................................................................. 22
2.2.2 Patterning of DMF top plates ............................................................................... 23
2.2.3 Surface functionalization ...................................................................................... 24
2.2.4 Verification of surface modification. .................................................................... 25
2.2.5 Capture antibody immobilization ......................................................................... 25
2.2.6 Immunoassay ....................................................................................................... 26
2.3 Results and discussion ................................................................................................. 27
2.3.1 Surface immobilization on DMF devices .............................................................. 27
2.3.2 Immobilization via physisorption ......................................................................... 27
2.3.3 Top plate patterning ............................................................................................. 30
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2.3.4 Surface functionalization ...................................................................................... 32
2.4 Conclusion .................................................................................................................. 34
3 Immunoassay on digital microfluidic device ...................................................................... 35
3.1 Introduction ................................................................................................................ 35
3.2 Materials and Methods ................................................................................................ 36
3.2.1 Materials .............................................................................................................. 36
3.2.2 Device fabrication ................................................................................................ 37
3.2.3 Device operation .................................................................................................. 37
3.2.4 Optimization of parameters .................................................................................. 38
3.2.5 Single analyte immunoassay ................................................................................ 39
3.3 Results and discussion ................................................................................................. 40
3.3.1 Effect of surface patterning on DMF .................................................................... 40
3.3.2 Optimization ........................................................................................................ 43
3.3.3 Single analyte immunoassay ................................................................................ 44
3.4 Conclusion .................................................................................................................. 45
Chapter 4 .................................................................................................................................. 46
4 Preliminary work for the implementation of multiplexed immunoassays ............................ 46
4.1 Introduction ................................................................................................................ 46
4.2 Materials and methods ................................................................................................ 47
4.2.1 Materials .............................................................................................................. 47
4.2.2 Verification of cross reactivity ............................................................................. 48
4.2.3 Optimizing array parameters ................................................................................ 49
4.2.4 Surface patterning of antibodies ........................................................................... 50
4.2.5 Verification of detection scheme .......................................................................... 52
4.2.6 Sandwich immunoassay ....................................................................................... 53
4.3 Results and discussion ................................................................................................. 53
4.3.1 Verification of cross reactivity ............................................................................. 53
4.3.2 Patterning of top plate for microarrays ................................................................. 55
4.3.3 Surface patterning of antibodies ........................................................................... 56
4.3.4 Verification of Tyramide signal amplification scheme. ......................................... 58
4.4 Conclusion .................................................................................................................. 61
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Chapter 5 .................................................................................................................................. 62
5 Conclusions and Future work ............................................................................................. 62
5.1 Conclusions ................................................................................................................ 62
5.2 Future work ................................................................................................................ 63
References ............................................................................................................................. 65
vii
List of Tables TABLE 1 PROS AND CONS OF COVALENT AND NON-COVALENT IMMOBILIZATION OF ANTIBODIES
ONTO SOLID SURFACES. .........................................................................................................5TABLE 2 EFFECT OF THE SIZE OF THE GLASS SPOT ON DROPLET MOVEMENT ON ACTUATION
ELECTRODES WITH SIDE LENGTH 2.25 MM. VOLTAGE USED FOR DROPLET MOVEMENT: 100 V PP (AC, 18 KHZ). MOVABLE INDICATES THE DROPLET WAS ABLE TO COMPLETELY CROSS THE HYDROPHILIC PATCH. ..........................................................................................................41
TABLE 3 EFFECT OF THE SPACING BETWEEN SPOTS IN A 4 X 4 ARRAY ON DROPLET MOVEMENT. MOVABILITY INDICATES IF A DROPLET MOVEMENT WAS ABLE TO COMPLETELY MOVE ACROSS THE MICROARRAY. ..............................................................................................................56
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List of Figures FIGURE 1 GENERAL SCHEMATIC OF A SANDWICH IMMUNOASSAY PERFORMED ON A FLAT SURFACE.
IT DERIVES ITS NAME FROM THE FACT THAT THE ANALYTE IS “SANDWICHED” BETWEEN TWO ANTIBODIES. .........................................................................................................................4
FIGURE 2 THE STREPTAVIDIN-BIOTIN BASED IMMOBILIZATION SCHEME. THE BIOTIN MOLECULES ARE COVALENTLY IMMOBILIZED ONTO A SURFACE WITH THE HELP OF A LINKER. .....................6
FIGURE 3 SCHEME DEPICTING THE ACTUAL REACTION VOLUME (INTERFACIAL REGION) AVAILABLE FOR SURFACE IMMUNOASSAY REACTIONS. SURFACE BASED REACTION RATES ARE LIMITED BY THE DIFFUSION OF MOLECULES FROM THE BULK SOLUTION TO THE INTERFACIAL REGION. ........7
FIGURE 4 REACTION SCHEME FOR LUMINOL, WHICH REACTS WITH HRP IN THE PRESENCE OF HYDROGEN PEROXIDE TO PRODUCE A PRODUCT (3- AMINOPHTHALATE), ACCOMPANIED BY THE EMISSION OF A PHOTON. ........................................................................................................8
FIGURE 5 REACTION SCHEME OF TYRAMIDE-FITC CONJUGATE, WHICH REACTS WITH HRP IN THE PRESENCE OF HYDROGEN PEROXIDE TO PRODUCE AN ACTIVATED TYRAMIDE RADICAL, WHICH CAN THEN BIND TO NEARBY PROTEINS VIA THEIR TYROSINE RESIDUES. ....................................8
FIGURE 6 SIDE VIEW SCHEMATICS OF DIGITAL MICROFLUIDIC DEVICES (A) TWO PLATE DMF DEVICE (B) ONE PLATE DMF DEVICE. ...................................................................................10
FIGURE 7 (A) GENERAL SETUP OF A DMF DEVICE (B) VARIOUS FLUID MANIPULATIONS ON DMF .13FIGURE 8 DIGITAL MICROFLUIDIC DEVICE FOR PERFORMING SURFACE BASED IMMUNOASSAYS. ...19FIGURE 9 TOP PLATE FABRICATED USING LIFT-OFF BASED MICROFABRICATION IN ORDER TO
INCORPORATE A COVALENT IMMOBILIZATION SCHEME ONTO DIGITAL MICROFLUIDIC DEVICES. ..........................................................................................................................................22
FIGURE 10 DOSE-RESPONSE CURVES FOR IMMUNOASSAYS PERFORMED AT DIFFERENT CAPTURE ANTIBODY CONCENTRATIONS. THE IMMUNOASSAYS WERE PERFORMED BY MANUALLY SPOTTING ALL REAGENTS. THE DATA CORRESPONDS TO A SINGLE TRIAL, AND A DIFFERENT SLIDE WAS USED FOR EACH EXPERIMENT. .............................................................................29
FIGURE 11 FLUORESCENT IMAGE OF AN IMMUNOASSAY SPOT, DEMONSTRATING THE NON-UNIFORM SPOT MORPHOLOGY OF IMMUNOASSAYS PERFORMED USING PHYSICALLY ADSORBED CAPTURE ANTIBODIES ON A CYTOP SURFACE. .....................................................................................30
FIGURE 12 MICROFABRICATION PROCEDURE FOR THE PATTERNING OF DMF TOP PLATES IN ORDER TO INCORPORATE A COVALENT IMMOBILIZATION SCHEME FOR SURFACE BASED IMMUNOASSAYS. .................................................................................................................31
FIGURE 13 SURFACE FUNCTIONALIZATION SCHEME FOR IMMOBILIZING BIOTIN ONTO A GLASS SURFACE. ............................................................................................................................32
FIGURE 14 FLUORESCENCE INTENSITY OF FLUORESCAMINE ON THE DIFFERENT MODIFIED SURFACES (N=4). ERROR BARS ARE ± 1 S.D. ........................................................................33
FIGURE 15 DOSE RESPONSE CURVES FOR IMMUNOASSAYS PERFORMED USING SPECIFICALLY (WITH STREPTAVIDIN) AND NON-SPECIFICALLY (WITHOUT STREPTAVIDIN) IMMOBILIZED CAPTURE ANTIBODIES. THE DATA CORRESPONDS TO A SINGLE TRIAL. ..................................................34
FIGURE 16 PASSIVE DISPENSING IN DIGITAL MICROFLUIDIC DEVICES. THE DIFFERENT ROWS IN THE FIGURE DEPICT THE SEQUENTIAL STEPS IN ORDER TO GET PASSIVE DISPENSING. .....................42
FIGURE 17 OPTIMIZING THE NUMBER OF WASH STEPS IN ORDER TO GET COMPLETE REMOVAL OF PASSIVELY DISPENSED SOLUTION. THE FLUORESCENCE INTENSITY HAS BEEN NORMALIZED TO THE INITIAL FLUORESCENCE INTENSITY. THE DATA CORRESPONDS TO A SINGLE TRIAL. THE
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FLUORESCENT IMAGES ABOVE THE PLOT DEPICT THE ACTUAL IMAGE OBSERVED THROUGH THE MICROSCOPE. ......................................................................................................................43
FIGURE 18 OPTIMIZATION OF THE NUMBER OF DROPLET CYCLES REQUIRED FOR MAXIMUM SURFACE REACTION. EACH CYCLE INVOLVES THE DROPLET MOVING ACROSS THE FUNCTIONALIZED SPOT AND BACK TO THE STARTING ELECTRODE. THE DATA POINTS ARE AVERAGED FORM THREE TRIALS AND THE ERROR BARS ARE ± 1 S.D. ....................................44
FIGURE 19 DOSE-RESPONSE CURVES OF IMMUNOASSAYS PERFORMED ON A DMF DEVICE. ALL SAMPLE HANDLING STEPS WERE PERFORMED ON DMF. THE DATA POINTS CORRESPOND TO A SINGLE TRIAL. .....................................................................................................................45
FIGURE 20 REACTION SCHEME USED TO DETECT THE PRESENCE OF BIOTIN LABELED CAPTURE ANTIBODIES ON THE SURFACE. .............................................................................................49
FIGURE 21 SCHEMATIC OF A TOP PLATE THAT HAS BEEN PATTERNED WITH MICROARRAYS (4 X 4). EACH MICROARRAY IS RESPONSIBLE FOR ONE SAMPLE ASSAY. ..............................................49
FIGURE 22 THE USE OF MICROCHANNELS TO PATTERN PROTEINS ONTO THE PATTERNED TOP PLATES. THE FINAL MICROARRAY IMAGE DEPICTS THE FINAL GOAL OF THIS ASSAY, WHICH IS TO DETECT 3 PROTEINS SIMULTANEOUSLY. ..........................................................................51
FIGURE 23 CROSS REACTIVITY ASSESSMENT OF THE CAPTURE ANTIBODIES FOR LACTOFERRIN AND PLASMINOGEN. THE DATA POINTS CORRESPOND TO A SINGLE TRIAL. .....................................54
FIGURE 24 CROSS REACTIVITY ASSESSMENT OF THE DETECTION ANTIBODIES FOR LACTOFERRIN AND PLASMINOGEN. THE DATA POINTS CORRESPOND TO A SINGLE TRIAL. ..............................55
FIGURE 25 THE USE OF PLURONICS TO CONFINE PROTEIN IMMOBILIZATION ONTO THE FUNCTIONALIZED GLASS SPOTS. (A) PROTEIN PATTERNING WITHOUT PLURONICS ADDED TO SOLUTION (B) PROTEIN PATTERNING WITH PLURONICS ADDED TO SOLUTION. IT SHOULD BE NOTED THAT THE PATTERNED GLASS SPOTS WERE LARGER THAN THE CHANNEL, AND THIS FACT IS SHOWN IN THE FIGURE (ESPECIALLY IN (B)) AS NOT THE ENTIRE GLASS SPOT IS FLUORESCENT. ....................................................................................................................57
FIGURE 26 FLUORESCENCE INTENSITY OF THE COLUMNS OF A MICROARRAY, WITH EACH COLUMN CONSISTING OF A SINGLE TYPE OF PROTEIN. THE DATA IS AVERAGED FROM 3 TRIALS AND THE ERROR BARDS CORRESPOND TO ± 1 S.D. ..............................................................................59
FIGURE 27 SURFACE MICROARRAY BASED SANDWICH IMMUNOASSAY FOR PLASMINOGEN. DETECTION WAS PERFORMED USING TYRAMIDE SIGNAL AMPLIFICATION SCHEME. EACH DATA POINT IS THE AVERAGE OF 3 TRIALS AND THE ERROR BARS ARE ± 1 S.D. ................................60
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Chapter 1
1 Introduction The immunoassay is a technique that uses the sensitivity and specificity of antibody-antigen
interactions for the detection of relevant analytes. It is used for the quantification of proteins and
small molecules in a number of different fields such as medical diagnostics [1], proteomics [2],
food safety [3], environmental monitoring [4] and drug development [5].
Immunoassays in medical diagnostics can be used for the early diagnosis of cancers by detecting
proteins, known as biomarkers, that are associated with the diseased state [6]. Early diagnosis is
important; for example, lung cancer is often not diagnosed until late in the disease, and only 15
% of patients that are diagnosed with lung cancer survive 5 years after diagnosis [7]. By
detecting biomarkers indicative of lung cancer or other diseases long before the symptoms arise,
it would be possible to improve the prognosis of the patient [8]. Some of the difficulties in early
detection arise from the lack of specificity of a single biomarker for a disease. For an accurate
diagnosis, a panel of multiple biomarkers needs to be analyzed [9], which would increase the
operational cost as each biomarker requires its own immunoassay. To overcome these
challenges, most diagnostic techniques are moving towards multiplexed modes of analysis.
Multiplexed immunoassays allow for the simultaneous detection of multiple proteins from a
single sample. This is accomplished by immobilizing the various assays onto miniaturized
platforms such as microbeads, or spots in a microarray. Each element (i.e. microbead or spot) in
the miniaturized platform is encoded, allowing the immunoassay signal obtained from each
element to be matched with a specific analyte. Microarrays involve the spatial encoding of the
different antibodies on an array, which can easily be analyzed by imaging methods. The
development of various techniques for microarray fabrication and analysis has allowed this
technique to be widely adopted for multiplexed assays [2]. High throughput sample handling for
these microarrays are usually performed by robotic systems, but this solution is only available to
wealthy laboratories, and requires significant maintenance efforts and a large laboratory
footprint.
One way of circumventing problems associated with conventional immunoassays is
miniaturization in microfluidic systems. The most common microfluidic paradigm relies on
2
networks of enclosed micron-dimension channels. At these small scales, fluids exhibit laminar
flow—i.e., fluidic streams flow parallel to each other and mixing occurs only by diffusion [10].
Microfluidic immunoassays offer at least three advantages over conventional methods [11]: (1)
increased surface-area-to-volume ratios speeds up antibody-antigen reactions; (2) smaller
dimensions reduces the consumption of expensive reagents and precious samples; and (3)
automated fluid handling can improve reproducibility and throughput. These advantages can
potentially improve the performance and reduce the operating cost of conventional
immunoassays.
While microchannel based microfluidics is the most widely used format, a relatively new form of
microfluidics, known as digital microfluidics (DMF) [12], has the potential to improve
microfluidic sample handling for high throughput applications. Digital microfluidics facilitates
the manipulation of small volume droplets (nL - µL) over an array of electrodes covered with a
dielectric. Using only electric fields (no moving or mechanical parts involved), a number of fluid
handling procedures such as dispensing, merging, mixing and splitting are possible. In addition,
the technique allows for the discrete control of droplets rather than the continuous flow of fluid
(as in microchannels) which facilitates the simultaneous control of multiple droplets,
significantly improving the throughput of the system. Despite the advantages this system offers,
there has been only a limited number of studies for immunoassays in DMF. This thesis describes
the initial work performed in order to integrate a microarray based immunoassay, selective for
lung cancer biomarkers, onto a digital microfluidic platform to improve the efficiency of
microfluidic immunoassays.
1.1 Background information
This section will discuss some of the important aspects of immunoassays and digital
microfluidics that need to be understood for purposes of this thesis. In addition, the literature
associated with multiplexed immunoassays in microfluidics systems will be reviewed.
3
1.1.1 Immunoassays
Immunoassays are a set of biochemical techniques that are used for the identification and
quantification of proteins. It is a ligand-binding assay that relies on the selective binding of a
class of proteins, known as antibodies (Abs), to the analyte of interest, sometimes referred to as
the antigen (An). This binding event is then transduced into a signal by a number of different
detection schemes. The signal can then be processed to yield information regarding the analyte.
Immunoassays can be classified into main two types: heterogeneous and homogeneous. In
heterogeneous immunoassays, antibodies are immobilized on a solid support and interact with
the antigen at the boundary layer. In this format, unbound antibodies and other reagents can be
easily removed. In homogenous immunoassays, antibodies interact with antigens in solution. In
this case, the bound and unbound antibodies are discriminated based on physical [13-14] or
chemical [15] changes arising from the binding event. Heterogeneous and homogeneous
immunoassays can be further divided into competitive and non-competitive modes. In
competitive mode, target antigens (from the sample) compete with exogenous labeled antigens
for a limited number of antibody binding sites. Thus, the generated signal is inversely
proportional to the antigen concentration. This mode is particularly important for small antigens
with limited number of binding sites. In non-competitive mode, antigens are captured by an
excess of antibodies and are detected after subsequent binding of a second set of labeled
antibodies that bind to the antigen at a different epitope (binding site). This format is referred to
as a “sandwich” immunoassay (Figure 1), in which the signal is proportional to the antigen
concentration. This mode is only compatible with large analytes (>1000 Da) that have more than
one epitope [11].
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Figure 1 General schematic of a sandwich immunoassay performed on a flat surface. It derives
its name from the fact that the analyte is “sandwiched” between two antibodies.
1.1.1.1. Sandwich immunoassays
The sandwich immunoassay offers one of the highest selectivity for an immunoassay. This high
selectivity is because it involves being bound by two different antibodies (at two different
epitopes) in order to give a positive signal.
A general set-up for a sandwich immunoassay involves an antibody being immobilized onto a
solid support, and this antibody is referred to as the capture antibody. The solid support could be
a number of materials such as microbeads, planar surfaces (e.g. plastic well plates, glass slides,
etc.) and membranes. The antibodies could be immobilized onto these surfaces either covalently
or non-covalently (also known as physisorption). The pros and cons of these methods are
outlined in Table 1.
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Table 1 Pros and cons of covalent and non-covalent immobilization of antibodies onto solid
surfaces.
Mode of attachment Pros Cons
Covalent: Involves the reaction between an active group on the surface and a functional group on the protein (amines, hydroxyls and thiols). Primarily a single point attachment.
- Increased stability - Ability to orient
antibodies - Can tailor surfaces to
improve antibody conformations on the surface, such as the introduction of spacers to improve flexibility of antibodies
- Covalent
modification requires extra steps, which adds time and cost to the assay
Non-covalent: Primarily involves the interaction between a hydrophobic surface and hydrophobic portions of the proteins. Results in a multipoint attachment to the surface. Some surfaces are hydrophilic in nature and rely on hydrogen bonding and ionic interactions for immobilization
- Simple and fast; does
not require special surface treatments.
- Membranes allow for high density of immobilization due to increased surface area
- Requires greater
concentration of antibody for immobilization
- Less stable; possibility of leaching
- Greater chance of surface induced denaturation.
One variation on the covalent immobilization scheme is the use of the biotin-avidin system to
immobilize antibodies onto a surface (Figure 2). In this scheme, biotin is covalently immobilized
onto the surface with the help of a linker, followed by immobilizing a monolayer of avidin, or
one of its variants (streptavidin, neutravidin) onto the biotin layer. Because the proteins have 4
binding sites, the surface immobilized avidin can bind other biotinylated molecules which, for
immunoassays, could be biotin labeled antibodies. The avidin-biotin interaction is one of the
strongest non-covalent interactions (Kd = 10-15), making the immobilization essentially
6
irreversible. There a number of advantages to this approach of immobilization: (1) it provides a
generalized surface over which all antibodies can be immobilized without harsh conditions (2)
the high affinity of the avidin-biotin system allows the use of low concentrations of antibody for
immobilization and (3) it prevents the direct interaction of the antibody with the surface,
preventing surface induced denaturation of the antibody.
Figure 2 The streptavidin-biotin based immobilization scheme. The biotin molecules are
covalently immobilized onto a surface with the help of a linker.
Sample handling for surface based immunoassays require the following steps: (1) delivery of
sample solution to the surface immobilized antibody and incubation (2) washing away of
unbound analytes and other proteins (3) delivery of detection antibody solution to the surface and
incubation (4) washing away of unbound detection antibody (5) detection.
These are the sample handling techniques that benefit from microfluidics. In the macroscale, the
solution delivery procedures are typically performed by automated robotic systems, which
require µL to mL volume of solutions. Microfluidic systems tend to reduce the samples required
to nL to µL volume of solutions. In addition, automation of microfluidic systems allow for
controlling multiple samples simultaneously for high throughput sample handling.
Mixing is an important function for surface based immunoassays. The kinetics of the reaction
between the surface immobilized antibodies and the analyte are slow, because they are diffusion
limited. The initial immunoreaction occurs between the immobilized antibodies and the analyte
that is present in the interfacial region (Figure 3). Once depleted, the reaction is then limited by
7
the diffusion of analyte from bulk solution to the interfacial region. In macroscale systems, this
reaction rate is accelerated by mixing. Microfluidic systems overcome this limitation because
they confine the fluid in micron dimension channels, reducing the diffusion distance from bulk
solution to the interfacial region and thus increasing the reaction rate.
Figure 3 Scheme depicting the actual reaction volume (interfacial region) available for surface
immunoassay reactions. Surface based reaction rates are limited by the diffusion of molecules
from the bulk solution to the interfacial region.
The detection of sandwich immunoassays is primarily done using fluorescent labels or enzymes.
The amount of detection antibody present on the surface is proportional to the amount of analyte
bound by the capture antibody. Thus, by detecting the presence of the detection antibodies, the
amount of analyte can be determined. Enzyme based detection schemes provide an amplification
strategy, which helps to improve the sensitivity of the assay. In this thesis, horseradish
peroxidase (HRP) was used to label all detection antibodies. This enzyme was used for
luminescence and fluorescence based detection.
Luminescence is based on the reaction of luminol with HRP, in the presence of hydrogen
peroxide (Figure 4). The 3-Aminophthalate formed relaxes from an excited electronic state,
accompanied by the emission of a photon of light. The signal is not localized to one area as the
excited 3-Aminophthalate molecules are free to diffuse away from the enzyme before relaxing to
the ground state.
8
NH
NH
O
O
NH2
+ HRP + H2O2 + 2 OH-
NH2 O
O
OH
OH+ 2 H2O + N2 + hυ
Luminol 3-Aminophthalate
Figure 4 Reaction scheme for luminol, which reacts with HRP in the presence of hydrogen
peroxide to produce a product (3- Aminophthalate), accompanied by the emission of a photon.
The fluorescence detection scheme is based on the reaction of tyramide labeled dyes with HRP
in the presence of hydrogen peroxide. This scheme is unique as the amplification is localized,
allowing it to be used in imaging based detection schemes. The amplification scheme is referred
to as Tyramide Signal Amplification (TSA) and is based on oxidation of tyramide to a radical
that is allowed to react with tyrosine residues of proteins (Figure 5). Due to the high reactivity of
the tyramide radical, it has a short lifetime in solution preventing it from diffusing over large
distances. This allows the signal to be localized and be used in applications that require high
spatial resolution.
O
CH3
NH
O OH
S
NH
OH+ H2O2
Protein CH3 OH
ProteinCH3
OH
O
CH3
NH
O OH
S
NH
OH + O2
HRP
Figure 5 Reaction scheme of tyramide-FITC conjugate, which reacts with HRP in the presence
of hydrogen peroxide to produce an activated tyramide radical, which can then bind to nearby
proteins via their tyrosine residues.
9
1.1.1.2. Multiplexed immunoassays
As mentioned previously, multiplexed immunoassays enable the detection of multiple proteins
simultaneously from a single sample. Heterogeneous immunoassays are typically used because
multiplexing requires the antibodies be attached to encoded solid phases (such as microbeads or
spots on a microarray).
Surface based immunoassays facilitate the performance of multiplex immunoassays in
the form of microarrays. In microarrays, the different antibodies for the various targets are
spatially encoded by immobilizing them at different locations on the array. Antibody microarrays
were some of the first platforms developed for multiplexed immunoassays, as most of the
principles and instrumentation used for DNA microarrays were transferable. But there are certain
unique challenges for antibody microarrays due to the heterogeneity of antibodies and also the
variance in terms of specificity and binding properties. Microarrays are fabricated using
automated fluid handlers (protein plotters) capable of spotting small volumes of solutions (pL -
µL) at high spatial densities (pitch of array is usually > 200 µm). They tend to be at high
densities such that a smaller sample volume would be able to cover the entire array. While
automated fluid handling is the most widely used method, there are other techniques that could
be used for protein patterning such as microfluidic networks [16], photolithography based
methods [17] and microcontact printing [18].
The sample handling is similar to those of solid phase immunoassays, with the final
analysis performed primarily by imaging methods. Fluorescence is the most common detection
scheme for multiplexed immunoassays due to the high resolution and sensitivity possible with
fluorescent labels. While fluorescence is sensitive for most purposes, there are a number of
amplification schemes available to improve the sensitivity. Enzyme based detection schemes are
highly sensitive, but the signal generated is usually not localized because the products of the
enzymatic reaction are free to diffuse away from the enzyme. Certain enzyme based
amplification schemes such as the tyramide signal amplification, which allow the signal to be
amplified and also localized at the vicinity of the enzyme, allowing its use in microarrays.
10
1.1.2 Digital microfluidics
Digital microfluidics is a relatively new form of microfluidics in which droplets of fluid (nL –
mL) are manipulated over an array of electrodes that has been covered with a dielectric[12].
There are two common configurations for digital microfluidic devices; a two plate configuration
and a one plate configuration (Figure 6). The electrodes responsible for droplet movement are
usually referred to as the driving electrodes, and the setup also includes a ground electrode to
complete the circuit. Devices usually have a 2 dimensional array of electrodes patterned on a
solid surface across which droplets can be manipulated.
Two Plate DMF Device
(a)
(b)
One Plate DMF Device
Figure 6 Side view schematics of digital microfluidic devices (a) Two plate DMF device (b) one
plate DMF device.
11
DMF devices have some advantages over channel based techniques for immunoassays. First,
DMF allows the control of discrete droplets of solution rather than a continuous flow of solution.
This allows for easier control of multiple solutions without the risk of cross contamination.
Second, microchannels can be clogged either due to impurities in solution or defects during
fabrication. DMF relies on an array of electrodes to move the droplets across a 2D surface. In the
case of a defect on an electrode, the array based nature allows the droplets to be re-routed, thus
reducing the failure rate of assays.
1.1.1.3. Theory of droplet movement
The forces that affect droplet movement in DMF can be divided into two categories: (1) driving
forces that tend to move the droplet and (2) resistive forces that impede droplet movement.
The driving forces have sometimes been referred to as electrowetting-on-dielectric (EWOD) and
dielectrophoresis (DEP) but are most generally referred to as being electrostatic forces. EWOD
applies for conductive liquids on hydrophobic surfaces, and is based on the fact that applying an
electric field on a hydrophobic surface would make the surface hydrophilic. When the electrode
adjacent to the one with the droplet is charged, a surface tension gradient is formed. The
capillary force due to this surface tension gradient is thought to be responsible for droplet
movement. DEP is responsible for the movement of non-conductive liquids, and in this scheme
the non uniform electric field that exists between a charged and uncharged electrode generates a
force on the droplet, moving it onto the charged electrode.
Electrostatic forces, F, explain droplet movement for all liquids (i.e. conductive and non
conductive).
F = qE,
where q is the charge on a particle and E is the electric field around the charge.
In this scheme, droplet movement is attributed to the electric forces generated on the charges in a
droplet meniscus (in case of conductive liquids) or on dipoles inside of a droplet (in case of
dielectric liquids) when an electric field is applied adjacent to the droplet. While the exact cause
12
of droplet movement is still being investigated by a number of groups, the electrostatic forces
serves as a general scheme that explains the movement of all liquids on DMF.
The resistive forces include capillary forces arising from differential surface tension and friction
due to the viscosity of solutions. The differential surface tension can arise either from defects on
the surface or from purposeful modification of the surface. Most techniques for optimizing
droplet movement serve to create a situation where the driving force is larger than the resistive
forces, thus allowing the droplet to be moved.
1.1.1.4. DMF device fabrication and operation
The devices are fabricated onto solid supports, with the most common support being glass slides.
Metals such as chromium and gold are deposited onto the glass slides and patterned using
photolithography in order to form the array of electrodes used for droplet actuation.
The next component of a digital microfluidic device is the dielectric layer. The main purpose of
the dielectric layer is to prevent direct contact between the droplet and electrode, as the high
voltages used for droplet movement would lead to electrolysis of the solutions. Parylene-C is
used as a dielectric layer for our devices due to the ease of deposition of this material and other
favorable characteristics.
Once the dielectric layer is deposited, the surface is made hydrophobic by depositing a layer of
hydrophobic polymer. The main purpose of this layer is to have minimal resistance to droplet
movement. A number of different fluorinated polymers can be used and the two most common
ones are Teflon AF and CYTOP, both of which can be spin coated onto the devices.
The top plate consists of a single, continuous electrode that serves as the ground electrode to
complete the circuit. This is usually made of ITO because it is transparent and allows the
visualization of the droplet movement and also helps in the integration of optical detection
methods. The top plate is covered with a thin layer of fluorinated polymer to minimize the
resistance to droplet movement.
The spacing between the top plate and bottom plate is controlled with the help of spacers that can
be made of a number of different materials. Double sided tapes are most commonly used as they
13
are easy to apply and the thickness can be controlled by varying the number of tapes used. For
smaller gaps (<70 um) between the two plates, a number of other alternatives are available such
as photoresist, which is patterned to be present at the edges of the bottom plate, and the top plate
is placed on top.
Figure 7a represents the general setup of a digital microfluidic device and Figure 7b represents
some of the common fluid handling techniques on DMF. The actuation voltages are applied to
the electrodes via the contact pads. Function generators are used to generate the voltage, which is
amplified before being applied to the device.
(a)
(b)
Figure 7 (a) General setup of a DMF device (b) Various fluid manipulations on DMF
14
1.1.1.5. Biological solutions on DMF
As previously mentioned, the surfaces of DMF devices are covered with a hydrophobic polymer
to facilitate droplet movement, but these surface pose a challenge for the movement of biological
solutions. In this case, a biological solution is a solution that consists of one or more
biomolecules such as proteins, cells, DNA, etc. It is a well known phenomenon that proteins can
stick to hydrophobic surfaces through hydrophobic interactions, and since the device surface is
hydrophobic material, it is prone to being fouled by proteins and other molecules present in the
biological solution. This not only complicates analytical methods, but it would also inhibit
droplet movement. That is because the patch of adsorbed protein would present a low surface
energy spot for the droplet to adhere to, contributing to a resistive force for droplet movement.
A number of strategies have been employed to overcome this difficulty. One involves immersing
the aqueous droplets in oil. In this method, the droplet can be moved around without it having
any contact with the surface, as a thin layer of oil lies in between the droplet and the surface of
the device. Although this method works well, there are some concerns such as the extraction of
non polar components from the biological solution into the oil phase, the increased complexity of
fabrication required to confine the oil in the DMF device and also the inability to move oil
miscible liquids required for certain applications. The other strategy uses solution additives to
reduce non-specific adsorption on DMF surfaces. The most commonly used additive is an
amphiphilic tri-block copolymer commercially known as Pluronics. The mechanism of operation
is usually attributed to the temporary coating of the hydrophobic DMF device surface with
pluronics; with the hydrophobic end towards the device and the hydrophilic part of the molecule
facing the solution. This prevents any proteins present in solution from adhering to the surface.
This approach has proven effective in a number of different applications in our laboratory, and is
currently the method of choice in our work for moving biological solutions.
15
1.2 Literature review
1.2.1 Multiplex immunoassays in microfluidic systems
Microfluidics has been used as a sample handling system for multiplexed immunoassays based
on both surface microarrays and microbeads. For surface microarrays, various microchannel
designs have been used for the delivery of reagents to the microarrays such as channels that are
able to individually address each row of a microarray [19], or microfluidic chambers, or a series
of them, that can cover an entire array [20-21]. The microfluidic chamber approach is attractive
as it allows a single sample inlet to deliver reagents to the whole array, but there are some
limitations for their design. Large aspect ratios (i.e. wide and shallow) of these chambers need to
be avoided because they tend to buckle under their own weight, causing it to block the flow. In
addition, the size of the chamber needs to be small in order to retain the benefits of microfluidics.
Fluid flow in these systems is accomplished by the use of syringe pumps, requiring microlitre
volume of fluids.
One unique application of microfluidics for microarrays was introduced by Delamarche et al
[16], in which a set of parallel microfluidic channels, referred to as microfluidic networks
(µFNs) were used to pattern the capture antibodies onto surfaces. This allowed the patterning of
strips of antibodies onto the surface at high densities. One set of µFNs was used to deliver the
capture antibodies to the surface. After removing the first set of µFNs, a second µFN was aligned
perpendicular to the initial set, and this was used to deliver the samples and other reagents of the
immunoassay to each of the strips of antibodies patterned. This method was effective at
generating microarrays, allowing it to be quickly adapted for various other multiplexed
immunoassay applications in microfluidics [22-28]. Most of the applications with the use of
microfluidic networks involved only pressure or capillary based approaches for fluid flow, but
the use of electrokinetic flow for sample handling has also been used on surfaces that have been
patterned using the microfluidic networks [27]. The use of electrokinetic control of fluid flow
allows for a more automated approach to fluid handling and does not require valves in order to
control fluid flow. A more complex design was developed by Kartalov et al. [29], in which the
patterning and subsequent immunoassay steps were performed using the same microfluidic
device (i.e. two different µFNs were not required). The design involved interconnected channels
16
that were controlled by set of pneumatic valves. These valves allowed the user to control the
injection sequence of the solutions required for immunoassays. While offering a method for high
throughput analysis, the integration of valves in microfluidic devices complicates device
fabrication and also requires the integration of mechanical components such as pumps.
As an alternative to surface microarrays, some microfluidic immunoassays use beads that are
physically confined to a region on the microfluidic chip. Multiplexed assays can be performed by
having different beads trapped at different positions on a microfluidic chip [30-31], or by
differentially labeling the beads such that the various assays can be distinguished [32-33] . The
reagents and other solutions are introduced to these confined regions using microfluidic
channels. The beads are usually confined with pillar-like structures that prevent the beads from
passing through, but still allow the flow through of solutions. Bead based approaches have a
number of advantages in that they can be produced in bulk, making their production relatively
cheap and also batch to batch variations are reduced. In addition, microbeads have a larger
surface area available for reaction which increases the sensitivity and reaction rate of the
immunoassay. The challenges in the bead based approach includes the increased complexity of
fabricating devices with pillar like structures and also the clogging of the microchannels with the
beads is a concern.
While physically confined beads are one approach for bead based multiplexing in microfluidics,
the separations capability of microfluidics can also be used for this purpose. Due to the reduced
size of microchannels, it is possible to create systems in which the beads are confined to a single
line, allowing them to be analyzed in a manner similar to flow cytometry, as was demonstrated
by Klostranec et al [34]. Some limitations with this approach are that the channels need to be
created narrow such that the beads flow in a single line, increasing the probability of the beads
clogging the channel. In addition, high speed detectors are required in order to analyze each
bead. The bead-by-bead analysis also leads to long analysis times for a large number of beads.
17
1.2.2 Immunoassays on DMF devices
Digital microfluidics is a relatively recent technology when compared with microchannels. Most
of the bioanalytical work performed on DMF until now has focused on solution phase processes
such as enzyme assays [35-38] and DNA based applications [39-42]. To date, three
heterogeneous immunoassay methods based on digital microfluidics have been reported, all of
which rely upon particles suspended in droplets [43-45]. In the first approach, an open plate
device was used, with the antibodies immobilized onto latex beads suspended in an aqueous
droplet. The droplet was suspended in a fluorinated oil and electric fields was used to move the
droplet around and mix the various reagents required for the assay. Immunoassays were first
carried out on two plate DMF devices by Sista et al [44]. In this work, the immunoassays were
performed on suspended magnetic beads, onto which the capture antibody was immobilized.
DMF was used for all sample handling methods such as mixing the magnetic beads with the
sample and the various washing steps associated with the assay. There was another recent
attempt for integrating magnetic bead based immunoassays on DMF [45], along with a unique
detection scheme based on a Superconducting Quantum Interference Device (SQUID)
gradiometer. But in this case, a one plate DMF device was used rather than the two plate
configuration.
1.2.3 Surface based assays on DMF
The first surface based assay on DMF was a DNA hybridization assay develop by Malic et al
[46]. In this system, the top plate of a two plate DMF device was fabricated out of gold and the
hydrophobic layer on it was patterned such that the gold surface was exposed at certain areas.
The bare gold surfaces were then used for the surface immobilization of DNA strands and DMF
was used to deliver the various assay solutions to these spots. They were able to detect three
different DNA sequences, with each sequence requiring one drop of sample solution.
18
1.3 Research outline
As was seen in the literature review (Section 1.2), almost all research conducted towards
integrating multiplexed immunoassays into microfluidic systems has involved the use of
microchannels. This approach has brought about a number of improvements such as reduced
sample consumption, automation, faster and more sensitive immunoassays. But the use of
microchannels for high throughput sample handling has been limited. These could be because
microfluidic devices for high throughput applications require the incorporation of valves, along
with mechanical pumps, into the device architecture. These valves serve to prevent cross-
contamination between samples, but their integration into the device would complicate the
device fabrication and operation. The use of electrokinetic control for fluid handling has also
been demonstrated in high throughput applications, and these systems do not require externally
controlled valves. Electrokinetic control of flow is a promising technique, but they do have some
disadvantages as well. Electrokinetic pumping requires high voltages that are directly applied to
the solutions. This prevents long term operation of the devices due to the electrolysis of the fluid.
In addition, by applying potentials directly to the liquid, there is a risk of joule heating, which
causes the temperature to vary during the assay [47].
As an alternative to channel microfluidics, DMF can be used for high throughput sample
handling for multiplexed immunoassays in microfluidics. DMF controls discrete droplets of
liquid using only electric fields, avoiding the need for valves in order to keep different samples
from mixing. In addition, the array based nature of DMF makes the devices more versatile. If a
single path is not functioning, the droplets can be re-routed through a number of other routes
[48]. This reduces the failure rate of the assays.
Immunoassays on DMF platforms have only been demonstrated on suspended beads. These are
very practical for single analyte detection, but are not well suited for multiplexed detection. In
addition, the use of DMF for surface assays has also been limited, with the only application
being for DNA hybridization assays.
The main goal of the project described in this thesis is the integration of a microarray based
immunoassay into a digital microfluidic device for the detection of at least three proteins. The
19
multiplexed immunoassay would target three proteins: Lactoferrin, plasminogen and
ceruloplasmin, which are known biomarkers for lung cancer.
The proposed final device design is shown in Figure 8. The bottom plate of the device comprises
the digital microfluidics component that will be used for all sample handling steps for a
sandwich immunoassay, including: (1) delivery of analyte-containing solutions to the
microarrays and incubation; (2) washing of unbound analyte; (3) delivery of enzyme-labeled
detection antibodies to the microarray; (4) washing away of unbound detection antibodies; and
(5) delivery and washing of the enzyme’s fluorescent substrate. The top plate of a DMF device is
used as the surface onto which the immunoassay components are immobilized. This part was
patterned using microfabrication techniques in order to accommodate the covalent
immobilization of the capture antibodies.
Figure 8 Digital microfluidic device for performing surface based immunoassays.
This thesis describes the preliminary work conducted towards achieving the above described
system.
20
All surface based assays require a robust and reproducible method to immobilize the proteins
onto the surface. Chapter 2 will discuss the strategies employed to immobilize antibodies onto
surfaces in digital microfluidic devices.
The modified surfaces were then tested for their compatibility with droplet movement in digital
microfluidic devices. Chapter 3 describes these experiments and also demonstrates a proof of
principle immunoassay for a single analyte on the modified surfaces.
The final goal of the device is for multiplexed immunoassays, and Chapter 4 describes some of
the initial work done towards setting up the surface immobilization schemes and immunoassays
for multiplexed detection.
21
Chapter 2
2 Surface immobilization strategies for capture antibodies
2.1 Introduction
Surface based assays, such as immunoassays, have been widely incorporated into microfluidic
systems [49]. The higher surface area to volume ratio of microfluidic devices increases the
sensitivity of surface based assays and also the reduced diffusion distances in microfluidics
increases the rate of surface based reactions. While surface chemistry for the immobilization of
biomolecules in microfluidic channels has received a lot of attention [50], surface modifications
of DMF devices are not as prevalent.
Surface modifications on DMF devices need to take into consideration the importance of the
surface on droplet movement. A change in the surface hydrophobicity tends to add a resistive
force towards droplet movement, making it harder to move the droplets. Another challenge with
the surface of digital microfluidic devices is that they are covered with fluorinated polymers,
which require harsh conditions to directly modify them.
To this date, two main biomolecule immobilization methods have been demonstrated on DMF
devices. The first involves the physisorption of proteins onto the hydrophobic surfaces of the
digital microfluidic device [51-52]. The second method allows for a covalent immobilization of
molecules and is based on a lift-off based patterning scheme that was demonstrated by Malic et
al [46]. In the latter scheme, a gold coated glass slide was used as the top plate of a DMF device.
The hydrophobic layer (Teflon) on top of the gold was patterned such that there were patches of
exposed gold. The patches were made small enough to not disturb droplet movement and these
gold patches could be used for biomolecule immobilization. It should be noted that the lift-off
based patterning schemes have previously been demonstrated as a method to pattern proteins on
silicon based surfaces [53-54], but these surface were not compatible with DMF as none of them
were conductive.
22
In this chapter, the above mentioned immobilization schemes were investigated for their use in
surface based immunoassays. The first scheme relies on the physisorption of antibodies onto a
hydrophobic Cytop layer. The second scheme relies on the covalent immobilization of antibodies
onto a surface that has been patterned using a lift-off technique. The above mentioned patterning
scheme was demonstrated for gold surfaces, but these surfaces are not compatible with a
fluorescence mode of detection, as fluorescence is quenched by gold surfaces [55]. A modified
patterning scheme was thus developed in order to obtain a substrate as shown in Figure 9. This
patterned slide was both compatible with DMF and also provided a surface onto which proteins
can be covalently immobilized for fluorescence based immunoassays.
Figure 9 Top plate fabricated using lift-off based microfabrication in order to incorporate a
covalent immobilization scheme onto digital microfluidic devices.
2.2 Materials and methods
2.2.1 Materials
Bovine serum albumin (BSA), absolute ethanol, glacial acetic acid, aminopropyl triethoxysilane
(APTES), dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), triethylamine,
anhydrous dimethylformamide, fluorescamine and human lactoferrin were obtained from Sigma-
Aldrich (Oakville, ON, CA). Sulfuric acid, 30 % hydrogen peroxide, biotin, streptavidin and
23
SuperSignal pico chemiluminescent substrate were obtained from Fischer Scientific (Ottawa,
ON, CA). Biotin labeled anti-lactoferrin was obtained from Genway Biotech (San Diego, CA,
USA). HRP conjugated anti-lactoferrin was purchased from Immunology Consultants
Laboratory (Newberg, OR, USA). Tyramide signal amplification (TSA) fluorescein system was
purchased from Perkin-Elmer (Melville, NY, USA)
Clean room reagents and supplies included Shipley S1818 photoresist and MF-321 developer
from Rohm and Hass (Marlborough, MA, USA), Chromium pellets from Kurt J Lesker (Toronto,
ON, Canada), Teflon AF from DuPont (Wilmington, DE, USA), Fluorinert FC-40 from Sigma-
Aldrich (Oakville, ON, Canada) and CYTOP from Bellex International (Wilmington, DE, USA)
Indium tin oxide (ITO) coated glass slides were obtained from Delta technologies (Stillwater,
MN, USA).
All protein solutions were made from the same aqueous buffer: 10 mM sodium phosphate (pH
7.4), 2 mg/mL BSA, 25 v/v% glycerol and 0.05 wt% Pluronics L64, and will be referred to as
“Buffer A”. Buffers without the BSA and glycerol would be referred to as “Buffer B”
2.2.2 Patterning of DMF top plates
The top plate of the DMF device was chosen as the surface onto which the antibodies will be
immobilized, the reasons for which will be discussed later. Two different kinds of top plates
were fabricated: an un-patterned top-plate (as is used conventionally with DMF), and a patterned
top-plate, developed specifically for the purpose of immunoassays. The un-patterned top plate
comprises an ITO slide coated with a hydrophobic polymer, and the patterned top plate was
fabricated such that it included regular patterns of exposed glass surrounded by a hydrophobic
and conductive layer (Figure 9).
To fabricate the un-patterned top plates, ITO-glass slides were spin coated with a 100 nm thick
layer of CYTOP, followed by a hard bake at 160o
Patterned top-plates were fabricated by photoresist lift-off patterning, as follows. Clean glass
slides were spin coated with a 2.5 µm thick layer of Shipley S1818 photoresist. The photoresist
was exposed to UV light (365 nm, 10 s, 16 mW/cm
C for 15 minutes.
2) through a photomask using a Karl Suss
24
mask aligner. After developing in MF-321, 10 nm of chromium was deposited onto the
substrates using an electron beam evaporator. A 100 nm layer of CYTOP was then spin-coated
on the chrome surface and the substrate was baked on a hot plate at 115o C for 10 minutes. The
slides were then sonicated in acetone for 5 minutes to perform the lift-off, followed by a 5
minute sonication in methanol. The slides were then thoroughly rinsed with deionized water and
dried under a stream of nitrogen. The slides were hard baked on a hot plate at 160o
2.2.3 Surface functionalization
C for 15
minutes.
The patterned top plates had glass surfaces that can be functionalized for the covalent
immobilization of proteins. A biotin-streptavidin based immobilization method (section 1.1.1.1)
was used to immobilize the biotinylated capture antibodies onto the surface, and this surface was
prepared as described below.
The glass surfaces of the patterned top plates were cleaned by immersing the slides in a piranha
solution (1:1 v/v, 30 % hydrogen peroxide and concentrated sulfuric acid) for 30 minutes,
followed by a rinse in deionized water, after which the slides were dried under a stream of
nitrogen. A silanization solution was prepared, which comprised 2 % (v/v) APTES in 95 %
ethanol acidified to a pH of 4.5 using acetic acid after the addition of the silane. The solution was
allowed to stand at room temperature for 5 minutes, and then the patterned top plates were
immersed in the silanization solution for 1 hour at RT, after which they were washed twice with
95 % ethanol (each wash comprised of 5 minute incubations in ethanol) and then twice with
deionized water. The substrates were dried under a stream of nitrogen, after which they were
baked in an oven at 120o
For biotin modification of the exposed glass patches, the biotin NHS ester was synthesized in
situ and allowed to react with the amine modified glass surface. A 1 mM solution of biotin in
anhydrous dimethylformamide was mixed with 2 molar equivalents each of DCC and NHS and
was allowed to stir for 2 hours. The APTES modified slides were then immersed in this solution,
along with 0.1 % (v/v) of triethylamine, and the reaction was allowed to take place overnight at
room temperature. After the reaction, the slides were washed twice by sonicating them for 5
C for 1 hour.
25
minutes in dimethylformamide. The slides were then rinsed with water, dried under nitrogen and
stored in a dessicator until used.
The streptavidin layer was created immediately prior to use. To create a streptavidin layer on the
surface, a solution of 10 µg/mL streptavidin was formed in Buffer A. The streptavidin solution
was spotted on the biotin-modified surfaces and was allowed to incubate in a humidified
chamber for 2 hours, after which the slides were washed 3 times in 0.05 % Tween-20 solution,
and finally rinsed with water.
2.2.4 Verification of surface modification.
To test the surface modification, a fluorescence assay using fluorescamine was performed. A 1
mg/mL fluorescamine solution was prepared in anhydrous dimethylformamide, which consisted
of 0.1 % (v/v) triethylamine. The solutions were spotted on three different surfaces: (1) un-
functionalized glass surface, (2) APTES modified glass surface and (3) biotin modified surface.
The reaction was allowed to proceed for 1 hour, after which the slides were washed twice with
dimethylformamide and finally rinsed with water and dried under a stream of nitrogen. The
fluorescence intensity was recorded using a Pherastar well plate reader (BMG Labtech, Durham,
NC).
2.2.5 Capture antibody immobilization
Two different immobilization strategies were used for capture antibody immobilization,
depending on the type of top plate. Immobilization onto the un-patterned top plates were
accomplished by physisorption, while the immobilization on the streptavidin functionalized top
plates were through the biotin-streptavidin interaction.
Capture antibodies were immobilized on un-patterned top plates through physisorption. A 1
mg/mL solution of the capture antibody was prepared in a 10 mM phosphate buffer. The solution
was then spotted on the required areas on the un-patterned, Cytop coated, top plate. Typically the
spots were placed along a line towards the center of the top plate, with an inter-spot spacing of
4.5 mm. The substrate bearing the droplets was allowed to incubate in a humid chamber for 2
26
hours, after which the spots were washed with a 0.05 % Tween-20 solution, and a final rinse with
deionized water.
For immobilization of biotin labeled antibodies onto the streptavidin layer (patterned top plates),
the capture antibody was dissolved at 100 µg/mL in Buffer A. The solutions were spotted (~ 1
uL) onto the functionalized patches of the patterned top plate and were allowed to incubate for 1
hour in a humid chamber at RT. The slides were then rinsed with 0.05 % Tween 20 solution,
followed by a rinse in deionized water.
2.2.6 Immunoassay
Immunoassays that were carried out to test the surfaces were performed by spotting the reagents
onto the surface using a pipette. The following procedures were followed for these
immunoassays.
3 µL of the analyte solution (varying concentrations) were spotted onto the capture antibody spot
on the slide. The solution was allowed to incubate at RT for 30 min in a humidified chamber.
The substrates were washed twice with a 0.05 % Tween-20 solution followed by a final rinse
with water. 3 µL aliquots of the detection antibody were spotted on each spot of the slide. A
similar protocol to the analyte incubation was followed for the detection antibody incubation and
washing steps. The detection antibodies were labeled with horseradish peroxidase (HRP), and
thus the detection step involved an extra step in which the enzyme substrates were introduced.
Depending on the detection mode (luminescence or fluorescence), different procedures were
followed as is described below.
Luminescence
The substrate solution was prepared by mixing equal volumes of the luminol solution with the
peroxide solution, as provided in the SuperSignal pico kit. A 3 µL drop of the luminol/peroxide
mixture was dispensed on each of the immunoassay spots on the substrate (top plate of DMF
device). The top plate was then aligned with a 384 well plate such that each immunoassay spot
corresponded to the location of well, and the entire assembly was placed inside the well plate
reader. The luminescence intensity was recorded every 30 seconds for 5 minutes.
27
Fluorescence
A tyramide signal amplification scheme was used for the fluorescence detection. The substrate
solution was prepared by diluting the fluorescein tyramide conjugate (1:50) in the diluents
included in the TSA fluorescein kit. A 3 µL drop of the substrate solution was pipetted onto the
immunoassay spot and incubated in the dark for 20 minutes. The solutions were then washed
thoroughly with deionized water, and the surface was dried before imaging the slide in an
epifluorescence microscope (Leica, Germany) coupled to a CCD camera. The fluorescent image
was then processed and analyzed using ImageJ (NIH, Bethesda, MD, USA).
2.3 Results and discussion
2.3.1 Surface immobilization on DMF devices
There are two main surfaces onto which the capture antibodies could be immobilized in digital
microfluidic devices; the top plate and the bottom plate. While both of them present the same
surface chemistry (i.e., Teflon-AF), there are several advantages for choosing the top plate. First,
the top-plate is simpler to fabricate and thus easier to replace than the grid of actuation
electrodes; second, if the top-plate surface becomes flawed in the process of printing, drying, and
rinsing the capture antibody spots, electrolysis is avoided (in initial trials using the bottom plate,
which is covered with an insulator, electrolysis was sometimes observed); and third, the surface
area of the capture antibody spot is approximately the same size as the actuation electrodes, but
is small relative to the size of the ground electrode, and therefore is less likely to interfere with
its role in actuation. It should be noted that the top plate was also chosen by Malic et al [46] for a
surface based DNA hybridization assay, possibly for the same reasons.
2.3.2 Immobilization via physisorption
The first immobilization method that was investigated was the physisorption of antibodies onto a
hydrophobic surface. This method is common in immunoassays performed in well plates, and is
one of the simplest methods of immobilization as it requires no pre-treatment of the surface [56].
It is also attractive for digital microfluidics as the device surfaces are hydrophobic.
28
Two hydrophobic polymers, Teflon AF and Cytop, were tested as surfaces onto which antibodies
can be immobilized by physisorption. It was observed that the Teflon AF was not as robust as the
Cytop surface for the immobilization and washing steps. Upon dipping Teflon AF coated slides
into wash solutions, tearing of the Teflon layer was observed, making it unusable for DMF.
Cytop, on the other hand, was more stable towards the various immobilization and washing
steps. The increased robustness of Cytop has also been observed by other research groups [53,
57], and it is also known that Cytop has much better adhesion to most substrates when compared
to Teflon [57]. Thus, Cytop was used as the hydrophobic layer in all top plates used for protein
immobilization by physisorption.
Two different concentrations of capture antibody solutions (1 mg/mL and 0.1 mg/mL) were
investigated to determine which one gave a proper surface for immunoassays. The dose-response
curves for immunoassays on these surfaces are presented in Figure 10. As shown, the intensities
and sensitivity of the assay are much higher when a high concentration of capture antibody is
used. It can also be seen that the signal arising from non specific binding (0 mg/mL of capture
antibody) is not much lower than the specific signals obtained for the 0.1 mg/mL concentration
of immobilized antibody, indicating that higher concentrations of capture antibodies are required
for immobilization by physisorption, This observation was also obtained by other studies that
used physisorption based immobilization [56].
29
Figure 10 Dose-response curves for immunoassays performed at different capture antibody
concentrations. The immunoassays were performed by manually spotting all reagents. The data
corresponds to a single trial, and a different slide was used for each experiment.
Another factor that is especially important for multiplexed immunoassays is the immobilized
spot’s morphology. This is because most detection schemes associated with surface microarrays
involve imaging the microarray and then using image analysis to derive quantitative data [58].
An irregular spot complicates the analysis, leading to larger uncertainties in the measurement.
The morphology of the spots obtained by physisorption of capture antibodies was visualized
using a fluorescently labeled detection antibody, and the image is shown in Figure 11. As shown,
there are “islands” of high fluorescence intensity, especially at the edge of the spot. These
“islands” of fluorescence are common artifacts of physisorbed proteins and they are caused by
protein aggregation [59].
0
5
10
15
20
25
30
35
40
0 2 4 6 8 10 12
Lum
ines
cenc
e In
tens
ity (a
.u.)
x 10
000
Concentration of Lactoferrin (ug/mL)
1 mg/mL Capture Antibody
0.1 mg/mL Capture Antibody
0 mg/mL Capture Antibody
30
Figure 11 Fluorescent image of an immunoassay spot, demonstrating the non-uniform spot
morphology of immunoassays performed using physically adsorbed capture antibodies on a
Cytop surface.
Because of the drawbacks associated with physisorption based immobilization described above,
a covalent immobilization scheme was developed.
2.3.3 Top plate patterning
The patterning scheme developed is summarized in (Figure 12). This patterning scheme enabled
the covalent immobilization of proteins onto a glass surface. Glass surfaces have already been
demonstrated to be compatible with surface based immunoassays [60], and a number of different
schemes for immobilization have already been developed. In addition, the patterning scheme is
relatively simple in that only a single photolithographic step is required, avoiding the
complications of patterning multiple layers of photoresist.
31
Figure 12 Microfabrication procedure for the patterning of DMF top plates in order to
incorporate a covalent immobilization scheme for surface based immunoassays.
The final design comprises a glass slide on which a thin layer of chromium and Cytop are
patterned. The thickness of the metal was chosen such that it was conductive enough to serve as
a ground electrode, but also was transparent to visualize droplet movement. Chromium was
chosen as the metal since it has good adhesion for glass surfaces, and also does not require harsh
conditions (especially temperature) for deposition. Cytop was chosen as the hydrophobic
material because of its robustness [53], which can withstand the various fabrication procedures
involved.
32
2.3.4 Surface functionalization
After patterning the top plates, the exposed glass regions were functionalized to immobilize the
capture antibodies. A streptavidin-biotin based immobilization scheme was used for the
immobilization of biotinylated capture antibodies (as described in section 1.1.1.1.). The surface
was first functionalized with biotin (Figure 13) before a layer of streptavidin was deposited on
top of it.
Figure 13 Surface functionalization scheme for immobilizing biotin onto a glass surface.
The surface modification was verified by the use of a fluorescamine labeling assay.
Fluorescamine is an amine reactive dye that is fluorescent only when conjugated to primary
amines. This assay is advantageous because any non-specifically bound dyes are not fluorescent,
reducing the occurrence of false positives. The fluorescence intensity of fluorescamine on the
various modified surfaces is shown in Figure 14. As expected, the fluorescence intensity
originating from bare glass surfaces is low compared to that originating from the APTES
modified surface (which has primary amines). The fluorescence intensity originating from
APTES – biotin coated surfaces is lower, because most of the primary amines are now
conjugated to biotin. These results indicate that the surface was successfully modified.
33
Figure 14 Fluorescence intensity of fluorescamine on the different modified surfaces (N=4).
Error bars are ± 1 S.D.
The next step of the surface functionalization was the formation of a layer of streptavidin on the
immobilized biotin, followed by the introduction of biotinylated capture antibodies. To verify
that the antibody layer was formed by specific biotin-streptavidin interactions rather than
adsorbing non-specifically, an immunoassay was performed on two biotinylated surfaces, one of
which was incubated with streptavidin and the other was not. The result is shown in Figure 15.
The intensity of the dose-response curve is higher for the surface that was incubated with
streptavidin, which indicates that the streptavidin layer either helps to increase the amount of
immobilized antibodies, or improve the conformation of the immobilized antibody. Both effects
lead to a more robust layer of immobilized antibodies.
0
5
10
15
20
25
30
35
40
Glass APTES Biotin
Fluo
resc
ence
inte
nsity
(a.
u.)
Thou
sand
s
34
Figure 15 Dose response curves for immunoassays performed using specifically (with
streptavidin) and non-specifically (without streptavidin) immobilized capture antibodies. The
data corresponds to a single trial.
2.4 Conclusion
This section described two different immobilization schemes for the immobilization of
antibodies onto a DMF surface. The top plate was chosen as the surface onto which the
antibodies would be immobilized and it was demonstrated that the covalent method of
immobilization was more favorable, when compared with the physisorption based
immobilization scheme. Based on the results of these experiments, the patterned top plate will be
used for surface based immunoassays on DMF.
0
10
20
30
40
50
60
0.0 5.0 10.0 15.0
Lum
ines
cenc
e in
tens
ity
x 10
000
Concentration of lactoferrin (µg/mL)
With Streptavidin
Without Streptavidin
35
Chapter 3
3 Immunoassay on digital microfluidic device
3.1 Introduction
Digital microfluidics has been demonstrated as a sample handling technique for a number of
different bioassays [61], but surface based immunoassays on DMF are not well studied. There
are a number of advantages that DMF can bring towards surface immunoassays. First, the ability
to control discrete droplets allow for handling multiple samples on a single device without any
cross reactivity. Second, only electric fields are required for droplet movement, allowing for
easier automation. This would enable the control of multiple samples simultaneously which is
important for high throughput applications. Third, mixing in digital microfluidics has been
demonstrated, and can be achieved simply by moving the droplet in a front and back motion
[62]. The mixing is an important aspect of surface assays as most of them are diffusion limited
(which results in slow reaction kinetics).
The most recent work in our group has demonstrated a proof-of-principle immunoassay in which
human IgG was used as the analyte [52]. This served to demonstrate the feasibility of DMF for
immunoassays. Some of the parameters investigated were the composition of solution to move
protein solutions on DMF and the various incubation and washing parameters for DMF based
immunoassay. But the immunoassay was performed using the physisorption method of
immobilization for capture antibodies. As mentioned in the previous chapter, physisorption
based immobilization has certain disadvantages when used for multiplexed immunoassays. Thus,
the incorporation of the patterned top plates into a DMF device was investigated in this chapter.
This chapter reports the studies that were undertaken to determine the ability to move droplets
over the patterned top plates. In addition, a single analyte immunoassay for lactoferrin was
demonstrated, with the capture antibodies being immobilized onto the streptavidin layer as
described in the previous section (Chapter 2).
36
3.2 Materials and Methods
3.2.1 Materials
Bovine serum albumin (BSA), absolute ethanol, glacial acetic acid, aminopropyl triethoxysilane
(APTES), dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), triethylamine,
anhydrous dimethylformamide, fluorescamine and human lactoferrin were obtained from Sigma-
Aldrich (Oakville, ON, CA). Sulfuric acid, 30 % hydrogen peroxide, biotin, streptavidin and
SuperSignal pico chemiluminescent substrate were obtained from Fischer Scientific (Ottawa,
ON, CA). Biotin labeled anti-lactoferrin was obtained from Genway Biotech (San Diego, CA,
USA). HRP conjugated anti-lactoferrin was purchased from Immunology Consultants
Laboratory (Newberg, OR, USA). Tyramide signal amplification (TSA) fluorescein system was
purchased from Perkin-Elmer (Melville, NY, USA)
Clean room reagents and supplies included Shipley S1818 and S1811 photoresists and MF-321
developer from Rohm and Hass (Marlborough, MA, USA), Chromium pellets from Kurt J
Lesker (Toronto, ON, Canada), CR-4 Chromium etchant from Cyantek (Fremont, CA, USA),
AZ-300T photoresist stripper from AZ electronic materials (Somerville, NJ, USA), Parylene-C
dimer and A-174 silane from Specialty coating systems (Indianapolis, IN, USA), Teflon AF from
DuPont (Wilmington, DE, USA), Fluorinert FC-40 from Sigma-Aldrich (Oakville, ON, Canada)
and CYTOP from Bellex International (Wilmington, DE, USA). ). Indium tin oxide (ITO) coated
glass slides were obtained from Delta technologies (Stillwater, MN, USA).
All solutions that were used in the digital microfluidic devices were made from the same
aqueous buffer: 10 mM sodium phosphate (pH 7.4), 2 mg/mL BSA, 25 v/v% glycerol and 0.05
wt% Pluronics L64, and will be referred to as “Buffer A”. Buffers without the BSA and glycerol
would be referred to as “Buffer B”
37
3.2.2 Device fabrication Bottom plate fabrication
Bottom plates of digital microfluidic devices were fabricated at the Emerging Communications
and Technology Institute (ECTI) cleanroom facilities and the masks used for photolithography
were printed at Pacific Arts and Design Inc. (Markham, ON, CA).
Glass slides (3” x 2”) were thoroughly rinsed with acetone and methanol followed by a 10
minute immersion in piranha solution (30 % hydrogen peroxide and concentrated sulfuric acid
1:1 v/v). The slides were then washed with water and dried under a stream of nitrogen. A 250 nm
thick layer of chromium was deposited onto the cleaned slides using an electron beam
evaporator. Photolithography and wet etching were used to pattern the chrome layer to create the
array of electrodes required for DMF as described elsewhere [63]. Briefly, the chrome slides
were spin-coated with a 1.5 µm thick layer of Shipley S1811 photoresist, followed by UV
exposure (365 nm, 10 s, 16 mW/cm2) through a photomask using a Karl Suss (Germany) mask
aligner. After developing the slides with MF-321, the chromium was etched using CR-4 etchant
to transfer the pattern from the photoresist to the metal layer. Finally, the substrates were
immersed in AZ-300T to strip the remaining photoresist from the surface. The electrode array
was then coated with a 7 µm layer of Parylene C using a chemical vapor deposition system. Prior
to parylene coating, the substrates were treated with A-174 silane as per the supplier’s
instructions, to improve parylene’s adhesion to the glass surface. The devices were then spin-
coated with a 100 nm thick layer of Teflon using a 1 % (w/v) solution of Teflon AF in Fluorinert
FC-40 followed by a final bake step at 160o
Top plate
C for 15 minutes.
The top plate fabrication protocol is described in Section 2.2.2.
3.2.3 Device operation
Devices were assembled with the top plate (either patterned or un-patterned) and a patterned
bottom plate separated by a spacer formed from two pieces of double-sided tape (~140 µm
thick). Droplets were sandwiched between the two plates and actuated by applying electric
potentials between the top electrode and sequential electrodes on the bottom plate. The driving
38
potential of 100 Vpp
3.2.4 Optimization of parameters
were generated by amplifying the output of a function generator operating at
18 kHz, and were applied manually to exposed contact pads (each pad connected to one
actuation electrode) on the bottom plate surface. The devices used here had ten 4.5 x 4.5 mm
reservoir electrodes mated to a 17 x 4 array of 2.25 x 2.25 mm actuation electrodes, with
interelectrode gaps of 50 µm. Each “unit” droplet covered a slightly larger area than a single
actuation electrode such that the volume in each droplet was ~ 650 nL.
Size of exposed glass patches
The effect of the size of the exposed glass spots on droplet movement was optimized by
qualitatively observing the movement of a droplet over patches of different sizes. Patterned top
plates were fabricated with the exposed glass patches having different dimensions. The following
dimensions were used: 0.25 x 0.25 mm , 0.5 x 0.5 mm, 0.75 x 0.75 mm, 1 x 1 mm, 1.5 x 1.5 mm,
2 x 2 mm.
The device was then assembled as described in section 3.2.3, and Buffer A was used as the test
solution. The droplets were actuated over the patterned spots, and then removed from the spots.
The ability of the droplet to go across the patterned spot was qualitatively observed.
Washing protocols
As will be discussed later, passive dispensing usually occurs on the patterned patches of the top
plate. The following experiment was conducted to determine the number of droplets required to
completely displace any passively dispensed solution.
A device was assembled with a top plate that had patches of dimension 0.75 x 0.75 mm. A
solution of 1 mM fluorescein was introduced into the reservoir, and a 650 nL drop dispensed
from it. The droplet was moved over and across the patch, and the fluorescence intensity of the
passively dispensed drop was measured using an epifluorescence microscope. The passively
dispensed droplet was then displaced with droplets of water, with the fluorescence intensity
being recorded after each wash.
39
Incubation time for immunoassays
The top plates were fabricated and functionalized with streptavidin as per the protocols described
in section 2.2.3. Biotinylated anti-lactoferrin antibody was then immobilized as described in
section 2.2.5.
A solution of lactoferrin (1 µg/mL in Buffer A) was incubated over the spots for 30 minutes. The
slide was then rinsed with a 0.05 % Tween 20 solution, followed by a rinse with water and dried
under a stream of nitrogen. The top plate was then used to assemble a device as described in
section 3.2.3. A solution of 5 µg/mL of HRP labeled anti-lactoferrin (in Buffer B) was
introduced into the reservoir of the device. Droplets were dispensed from the reservoir and then
incubated over the spots for a specific number of cycles (5, 10, 15). One cycle involves moving
the droplet across the patch and back to the starting electrode. The top plate was then removed
and washed with a 0.05 % Tween 20 solution, followed by a rinse with water and dried under a
stream of nitrogen.
The spots were then incubated with the luminescent substrate solution as described in section
2.2.6.
3.2.5 Single analyte immunoassay
The device was assembled with a top plate that consisted of functionalized patches with
dimensions: 0.75 x 0.75 mm. The surface was functionalized with anti-lactoferrin antibodies as
mentioned above.
For the immunoassay, a 650 nL droplet of analyte (lactoferrin) solution was dispensed from the
reservoir electrode, and moved over the modified spot on the surface. The droplet was moved
around this spot for 20 cycles, after which it was moved away and the surface was then washed
with Buffer A (using DMF). The washing step involved the dispensing three droplets of the wash
buffer, and sequentially moving it to the immunoassay spot. The third wash buffer droplet was
cycled around this spot 20 times, and then removed. A similar procedure was followed for the
droplet used to introduce the detection antibody for the immunoassay. The detection antibodies
were labeled with horseradish peroxidase (HRP), and thus the detection step involved an extra
40
step in which the enzyme substrates were introduced. A luminescence detection scheme was
used for the immunoassay, and this was performed as described below.
The DMF device was first aligned with a 384 well plate, such that each immunoassay spot
corresponded to the location of a well. The substrate solution was prepared as described in
section 2.2.6. A 3 µL aliquot of the substrate solution was placed on one of the reservoir
electrodes. 650 nL daughter droplets were then dispensed and moved to immunoassay spots. The
signal was read similar to that described in section 2.2.6.
3.3 Results and discussion
3.3.1 Effect of surface patterning on DMF
One aspect of the patterning process that was investigated is the size of the exposed glass
patches. The effect of the size of the glass openings on droplet movement was investigated by
trying to move a droplet of buffer over the region and noting if the droplet could be moved out of
the hydrophilic patch. The results are listed in Table 2. The general trend observed is that as the
size of the hydrophilic patch increases (while keeping the actuation electrode at constant size) the
droplet movement over the glass spot was slower. This is an expected result, as the hydrophilic
patch is considered to provide a resistive force (because of surface tension) to droplet movement,
and increasing its size would increase the resistive force. It should be noted that the data obtained
in Table 2 applies only for the conditions under which it was tested ==actuation voltage 100 Vpp
(AC, 18 kHz); and constant electrode size. Increasing the actuation voltage may allow droplets to
move across larger openings, but the use of higher voltages increases the likelihood of
electrolysis, which would make the device unusable. Thus, for an AC voltage of 100 Vpp
(18
kHz) and an actuation electrode size of 2.25 x 2.25 mm, robust droplet movement is possible
over glass patches of up to 0.75 mm x 0.75 mm in size. While droplet movement was observed
over patches of 1 mm x 1 mm in size (19.75 % hydrophilic area), the droplet movement was
observed to be qualitatively slower.
41
Table 2 Effect of the size of the glass spot on droplet movement on actuation electrodes with
side length 2.25 mm. Voltage used for droplet movement: 100 V pp (AC, 18 KHz). Movable
indicates the droplet was able to completely cross the hydrophilic patch.
Side length of hydrophilic
patch (mm)
Percentage of Area covered by
hydrophilic patch (relative to
actuation electrode)
Movable?
0.25 1.23 Yes
0.5 4.94 Yes
0.75 11.11 Yes
1 19.75 Yes*
1.5 44.44 No
2 79.01 No
* Qualitatively observed to be slower to move
One effect that is observed when droplets are driven over glass openings on a hydrophobic
surface is passive dispensing of a droplet (Figure 16) [51]. When a droplet is moved away from
the exposed glass, a small volume of the solution is left behind. This occurs because of the
differences in surface energy between the hydrophilic spot and a hydrophobic surface, and is
observed for exposed glass and also for spots of physisorbed antibodies. The passive dispensing
on the physisorbed protein patches was not reproducible (likely an effect of the heterogeneity of
the patches, see Figure 11), but passive dispensing observed on the patterned patches were
uniform.
42
Figure 16 Passive dispensing in digital microfluidic devices. The different rows in the figure
depict the sequential steps in order to get passive dispensing.
While passive dispensing is not an insurmountable problem, it is inconvenient, as the passively
dispensed droplet must be removed before the next solution for the immunoassay is introduced.
This requires additional rinsing steps to remove the residual fluid from the spot. In a manner
similar to what was described by Barbulovic-Nad et al. [51], the following experiment was
performed to determine the number of droplets required to completely displace a passively
dispensed droplet. An aqueous droplet consisting of fluorescein was passively dispensed and
then iteratively displaced with droplets of deionized water, recording the fluorescence intensity
after each wash (Figure 17). It was determined that two droplets were required to reduce the
fluorescence intensity to less than 5 percent of the initial intensity. Thus, all washing steps for
assays performed on the modified slides made use of more than 2 droplets of wash buffer.
43
Figure 17 Optimizing the number of wash steps in order to get complete removal of passively
dispensed solution. The fluorescence intensity has been normalized to the initial fluorescence
intensity. The data corresponds to a single trial. The fluorescent images above the plot depict the
actual image observed through the microscope.
3.3.2 Optimization All solutions used on DMF were formed in10 mM sodium phosphate containing 2 mg/mL BSA,
0.05 % (w/w) Pluronics L64 and 25 % glycerol (pH 7.4). Sodium phosphate buffers the pH, BSA
and Pluronics reduce non-specific adsorption, and glycerol reduces the evaporation rate.
Immunoassays in the macroscale are usually static, making the surface based immunoreactions
diffusion limited. It has been shown that this diffusion limited process can be accelerated by
mixing or vortexing the solutions [56]. In DMF, a mixing effect can be generated by
continuously moving a droplet, rather than leaving it static, over the immunoassay spot [62]. To
optimize the number of droplet movements for maximum signal, the reaction between a protein
immobilized on the surface (via a capture antibody), and an HRP labeled antibody in solution
was monitored as a function of the number of droplet movements (each movement involves a
44
back and forth motion). The results of the experiment are shown in Figure 18; the signal
saturates above 15 droplet movements. Thus, all immunoassay steps were performed by moving
the solution at least 20 times over the immunoassay spot.
Figure 18 Optimization of the number of droplet cycles required for maximum surface reaction.
Each cycle involves the droplet moving across the functionalized spot and back to the starting
electrode. The data points are averaged form three trials and the error bars are ± 1 S.D.
3.3.3 Single analyte immunoassay
Using the optimized conditions described above for droplet handling and solution composition,
an immunoassay for lactoferrin was performed using DMF for fluid handling. The covalent
method of immobilization was also compared to an immunoassay performed on a top plate onto
which the antibodies were immobilized by physisorption. The dose-response curve for these
assays is shown in Figure 19. The signal intensities and sensitivities obtained for the
immunoassay performed on the covalently immobilized antibodies are higher. The higher
intensities observed could be due to an increased concentration of antibodies immobilized on the
surface or a more favorable orientation of antibodies immobilization using the streptavidin layer.
In addition to demonstrating the improved performance of the covalent method of
immobilization, this experiment also demonstrated the feasibility of using DMF for fluid
handling in immunoassays.
78899
101011111212
0 5 10 15 20
Lum
ines
cenc
e in
tens
ity
(a.u
.)Th
ousa
nds
Number of droplet cycles
45
Figure 19 Dose-response curves of immunoassays performed on a DMF device. All sample
handling steps were performed on DMF. The data points correspond to a single trial.
3.4 Conclusion
In this section, the effect of the size of the glass patches on droplet movement was investigated,
and it was found that reproducible droplet movement is achieved when the hydrophilic patch is
less than 20 % the area of the actuation electrode. In addition, various steps associated with a
DMF immunoassay were investigated such as the washing protocols and incubation times. A
proof of principle immunoassay of lactoferrin was conducted, and it was determined that the
covalent mode of immobilization provided for a more robust platform when compared to the
physisorption mode of immobilization. These experiments served as a starting point in order to
develop surface based immunoassays on DMF.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0
Lum
ines
cenc
e in
tens
ity (a
.u.)
x 10
000
Concentration of Lactoferrin (ug/mL)
Covalently immobilizedPhysisorbed
46
Chapter 4
4 Preliminary work for the implementation of multiplexed immunoassays
4.1 Introduction
Microarray immunoassays allow for the detection of multiple proteins simultaneously by having
different antibodies immobilized in an array. Usually a single droplet of sample is used to cover
the entire array, with most of the sample handling usually performed in robotic systems in order
to perform all sequential steps required for immunoassays. Integrating all sample handling into
microfluidic systems would bring about a number of advantages such as reduced sample
consumption, faster reaction rates and increased throughput of analysis.
In addition to the task of integrating the immunoassay with a microfluidic device, certain
preliminary studies need to be conducted to verify/optimize the multiplexed immunoassays.
First, in multiplexed immunoassays, cross reactivity is a major concern. Cross reactivity refers to
an antibody reacting with an antigen towards which it is not supposed to be selective. This is
usually a concern when large arrays are used as the probability of two proteins sharing a
common binding site (or epitope) increases as the number of proteins increases. Second, array
fabrication requires methods that allow for the patterning of proteins at high resolutions. A
higher resolution allows for more proteins to be fit into a certain area, allowing for higher
multiplexing capabilities. Third, microarray spots tend to be small in size such that multiple spots
can be placed in a single array. The small size decreases the signal obtained from microarrays,
requiring high sensitivity detection schemes.
This chapter describes the work conducted to address the above concerns. The cross-reactivity of
plasminogen and lactoferrin antibodies towards all three proteins (ceruloplasmin, lactoferrin and
plasminogen) was investigated. For protein patterning, a microfluidic based patterning scheme,
first introduced by Delamarche et al [16], was used and its compatibility with the surface of a
DMF top plate was established. In order to obtain higher sensitivities, a enzyme based
47
amplification scheme known as Tyramide Signal Amplification (TSA) was investigated for its
compatibility with the arrays patterned onto the DMF top plates.
4.2 Materials and methods
4.2.1 Materials
Bovine serum albumin (BSA), absolute ethanol, glacial acetic acid, aminopropyl triethoxysilane
(APTES), glycidoxypropyl trimethoxysilane (GPTMS), dicyclohexylcarbodiimide (DCC), N-
hydroxysuccinimide (NHS), triethylamine, anhydrous dimethylformamide and human lactoferrin
were purchased from Sigma-Aldrich (Oakville, ON, Canada). Sulfuric acid, 30 % hydrogen
peroxide, biotin, streptavidin and SuperSignal pico chemiluminescent substrate were purchased
from Fisher Scientific (Ottawa, ON, Canada). Biotinylated anti-ceruloplasmin, biotinylated anti-
plasminogen, human plasminogen and horseradish peroxidase (HRP) conjugated anti-
plasminogen were purchased from Abcam Inc. (Cambridge, MA, USA). Biotinylated anti-
lactoferrin and human ceruloplasmin were purchased from Genway Biotech (San Diego, CA,
USA). HRP conjugated anti-lactoferrin was purchased from Immunology Consultants
Laboratory (Newberg, OR, USA). Tyramide signal amplification (TSA) fluorescein system was
purchased from Perkin-Elmer (Melville, NY, USA)
Clean room reagents and supplies included Shipley S1818 and S1811 photoresists and MF-321
developer from Rohm and Hass (Marlborough, MA, USA), Chromium pellets from Kurt J
Lesker (Toronto, ON, Canada), CR-4 Chromium etchant from Cyantek (Fremont, CA, USA),
AZ-300T photoresist stripper from AZ electronic materials (Somerville, NJ, USA), Parylene-C
dimer and A-174 silane from Specialty coating systems (Indianapolis, IN, USA), Teflon AF from
DuPont (Wilmington, DE, USA), Fluorinert FC-40 from Sigma-Aldrich (Oakville, ON, Canada),
CYTOP from Bellex International (Wilmington, DE, USA), SU-8 2100 and SU-8 developer
from MicroChem (Newton, CA, Canada) and Sylguard-184 (polydimethylsiloxane (PDMS) kits)
from Dow Corning (Midland, MI, USA). Indium tin oxide (ITO) coated glass slides were
obtained from Delta technologies (Stillwater, MN, USA).
All solutions that were used in the digital microfluidic devices were made from the same
aqueous buffer: 10 mM sodium phosphate (pH 7.4), 2 mg/mL BSA, 25 v/v% glycerol and 0.05
48
wt% Pluronics L64, and will be referred to as “Buffer A”. Buffers without the BSA and glycerol
would be referred to as “Buffer B”
4.2.2 Verification of cross reactivity
A patterned top plate was fabricated with glass spots of dimension 0.75 x 0.75 mm. The surfaces
were functionalized with epoxy silane using the following procedure.
The silanization method for these substrates was similar to the one followed for APTES
(described in section 2.2.3), but the silanization solution comprised 2 % GOPS in 95 % ethanol
containing 16 mM acetic acid. After silanization and washing steps (similar to those with
APTES), the slides were baked in an oven at 150o C for 1 hour, after which the slides were
stored in a dessicator. For protein immobilization, a 100 µg/mL solution of the protein was
formed in Buffer B, and was incubated with the epoxy surface overnight at 4 o
Using the above method, three proteins, ceruloplasmin, plasminogen and lactoferrin, were
immobilized onto 4 spots each. These immobilized proteins were incubated with all antibodies
(both capture and detection antibody), such that each protein was individually interrogated with
the antibodies for plasminogen and lactoferrin. This was performed by incubating the
immobilized proteins with a 10 µg/mL solution of the antibody (in Buffer A) for 30 minutes. The
surfaces were then rinsed with 0.05 % Tween 20, followed by a rinse with water and dried under
a stream of nitrogen.
C. The slides were
then rinsed with water and remaining active sites were blocked with a 1 mg/mL solution of BSA
(in Buffer B), for 1 hour at RT.
The detection antibodies were already labeled with HRP, allowing them to be easily detected. In
order to detect the presence of the biotinylated capture antibodies, the surfaces were incubated
with streptavidin that was labeled with HRP (Figure 20). The detection was then performed with
a luminescent substrate, similar to that described in section 2.2.6.
49
Figure 20 Reaction scheme used to detect the presence of biotin labeled capture antibodies on
the surface.
4.2.3 Optimizing array parameters
In order to accommodate the top plates for microarrays, a microarray design (4 x 4 array) was
patterned on the top plates (Figure 21)
Figure 21 Schematic of a top plate that has been patterned with microarrays (4 x 4). Each
microarray is responsible for one sample assay.
50
Top plates were fabricated with the exposed glass patches having different array dimensions. The
following pitches (center to center distance) were tried: 0 mm, 0.35 mm, 0.45 mm and 0.55 mm.
The device was then assembled as described in section 3.2.3, and Buffer A was used as the test
solution. The droplets were actuated over the patterned spots, and removed from the spots. The
ability of the droplet to go across the patterned spot was qualitatively observed and recorded.
4.2.4 Surface patterning of antibodies
A microchannel based patterning scheme is used to pattern the different antibodies onto the
functionalized top plate of a DMF device. The general scheme for this patterning scheme is
demonstrated in Figure 22. The array demonstrated in the figure depicts the final patterning
scheme required to detect three proteins, but the experiments described below deals with just two
proteins, lactoferrin and plasminogen, which was used as a starting point.
In the patterning scheme, a PDMS based microchannel is aligned with the microarray spots that
were patterned onto the top plate. Different solutions can be flowed through the different
channels without any cross contamination. In this manner, columns of different proteins can be
immobilized onto a surface with high resolution.
51
Figure 22 The use of microchannels to pattern proteins onto the patterned top plates. The final
microarray image depicts the final goal of this assay, which is to detect 3 proteins
simultaneously.
The microchannels used to deliver antibodies to the surface were formed from PDMS using the
replica molding technique[64]. Briefly, a positive-relief master was formed from glass slide as
follows. Microscope glass slides were washed with acetone and methanol, followed by a 10
minute immersion in piranha solution. The slides were then rinsed in water, and dried under a
stream of nitrogen. A 100 µm layer of SU-8 2100 was spin coated onto the glass slides, and pre
baked at 65o C for 5 min and at 95 o C for 30 min. The photoresist was exposed to UV radiation
through a photomask using a Karl Suss (Germany) mask aligner (365 nm, 15 s, 16 mW/cm2).
The slides were then post baked at 65o C for 5 min and at 95 o C for 12 min. The photoresist was
developed in an SU-8 developer for 10 minutes, after which the slides were rinsed with isopropyl
alcohol, dried under a stream of nitrogen and baked at 150o
The surface pattern from the master was transferred to PDMS by replica molding. The PDMS
pre-polymer mixture was prepared in a 10:1 ratio by weight (10 parts pre-polymer to 1 part
curing agent), and degassed in a vacuum dessicator. The prepolymer mixture was then poured
over the master, which was held in a plastic petri dish, and baked in an oven at 70
C for 15 minutes.
o C for 2 hours.
52
The PDMS was allowed to cool before it was cut out and peeled away carefully form the master.
The reservoirs on the PDMS were created using a blunt 18 gauge syringe needle. Right before
use, the PDMS surfaces were washed with deionized water and the surface was cleaned further
by applying scotch tape and then removing it. The PDMS microchannels were aligned with the
patterns on the chromium based top plate with the help of a microscope (Figure 22). The
channels were then sealed by the application of pressure, to allow for conformal contact based
seal.
4.2.5 Verification of detection scheme
A fluorescence based enzymatic detection scheme is to be used in the microarrays. The
following experiments were conducted in order to determine their feasibility for this purpose.
In order to determine the localization of the signal, microchannels were used to pattern the
various columns of the microarray with different proteins (analytes). The surfaces of the
microarray were functionalized with epoxy silane (as described in section 4.2.2.) such that
proteins can be directly immobilized onto the surface. The array was patterned using the system
described in section 4.2.4, with the first column consisting of immobilized plasminogen, the
second column consisted of immobilized lactoferrin, and the last two columns consisted of BSA
(which served as the negative control). The solutions were filled into the microchannels and
allowed to incubate for 2 hours in a humidified chamber. After the incubation, the PDMS
channels were removed from the chromium based top plate under a continuous stream of
deionized water. The slides were then incubated with a 1 mg/mL solution of BSA for 1 hour,
after which the slides were washed with a 0.05 % Tween 20 solution, followed by a rinse in
water.
The microarray was then interrogated with an HRP labeled anti-lactoferrin antibody. This was
done by spotting a 3 µL solution of the antibody solution (in Buffer A) onto the microarray and
incubating them in a humidified chamber at RT for 1 hour. The slides were washed with a 0.05
% Tween 20 solution, followed by a rinse in water.
53
The microarrays on the slide were incubated with 3 µL of the Tyramide-FITC solution as
described in section 2.2.6. The imaging was also performed similar to that described in section
2.2.6.
4.2.6 Sandwich immunoassay
The patterned top plate (microarray design) was functionalized with biotin and streptavidin as
was described in section 2.2.3. Microchannels were then used to pattern various biotinylated
capture antibodies onto the surface using the procedure described in section 4.2.4. The patterning
scheme was as follows: the first column consisted of plasminogen antibody, the second column
consisted of BSA, the third column consisted of lactoferrin antibody and the final column
consisted of BSA.
The following procedure was followed for the immunoassay. Plasminogen solutions of varying
concentrations (0 µg/mL, 0.1 µg/mL, 1 µg/mL, 10 µg/mL) were spotted onto the microarrays,
and incubated in a humidified chamber for 30 minutes. The slides were washed with a 0.05 %
Tween 20 solution, followed by a rinse in water. A solution consisting of a mixture of detection
antibodies (for plasminogen and lactoferrin) at a concentration of 5 µg/mL was spotted over the
arrays and incubated for 30 minutes. The slides were washed with a 0.05 % Tween 20 solution,
followed by a rinse in water. The spots were then incubated with a FITC-tyramide solution and
processed as described in section 2.2.6.
4.3 Results and discussion
4.3.1 Verification of cross reactivity
One concern with multiplexed immunoassays is the possibility of cross reactivity, i.e. non-
specific interaction of an antibody with analytes other than its designated antigen. For
multiplexed immunoassays, it is important to verify the extent of cross-reactivity of each
antibody for the various antigens in the assay.
To probe cross-reactivity in the system described here, analytes (lactoferrin, plasminogen and
ceruloplasmin) were immobilized onto epoxy functionalized glass slides, and each antigen was
54
allowed to react with each of the antibodies (both detection and capture antibody). For a perfect
system with no cross-reactivity, positive signal would only be observed for the correct antibody-
antigen pair. The results of the experiment are shown in Figure 23 (capture antibodies) and in
Figure 24 (detection antibodies). It can be seen from the plots, that only the correct antigen-
antibody pair gave positive signals, indicating that there is no (or very little) cross-reactivity
between the various antibodies and antigens in this assay.
Figure 23 Cross reactivity assessment of the capture antibodies for lactoferrin and plasminogen.
The data points correspond to a single trial.
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
Ceruloplasmin Lactoferrin Plasminogen
Lum
ines
cenc
e In
tens
ity (a
.u.)
x 10
000
Protein Immobilized on Surface
Lactoferrin Capture Antibody
Plasminogen Capture Antibody
55
Figure 24 Cross reactivity assessment of the detection antibodies for lactoferrin and
plasminogen. The data points correspond to a single trial.
4.3.2 Patterning of top plate for microarrays
As described in section 3.3.1, the size of the hydrophilic patch is an important parameter for
droplet movement, so the effect of a patterned array on droplet movement was also investigated.
It would be beneficial to have the largest possible area for the array, and it was previously
determined that the largest size that allowed droplet movement (for the given voltage and
electrode size) was 1 mm x 1 mm. Therefore, the 1 x 1 mm spots were divided into a 4 x 4 array,
and the effect of the pitch of the array on droplet movement was investigated. Droplets were
moved over the arrays of varying pitch, using a driving voltage of 100 Vpp
Table 3
(AC, 18 KHz), and
the movability of the droplets were evaluated and the results listed in
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
Ceruloplasmin Lactoferrin Plasminogen
Lum
ines
cenc
e In
tens
ity (a
.u.)
Thou
sand
s
Protein Immobilized on Surface
Lactoferrin Detection Antibody
Plasminogen Detection Antibody
56
Table 3 Effect of the spacing between spots in a 4 x 4 array on droplet movement. Movability
indicates if a droplet movement was able to completely move across the microarray.
Pitch (Center to
center spacing)
(mm)
Percentage of area covered
by hydrophilic patch (with
respect to total array size)
Movable?
0.55 27.70 Yes
0.45 39.06 Yes
0.35 59.17 Yes*
0 100.00 Yes*
* Qualitatively to be slower to move
It was observed that as the pitch of the array decreased, droplet movement became more
sluggish, with a pitch of 0.45 mm being the smallest possible for reproducible droplet movement.
Thus, all top plates fabricated for multiplexed assays had a 4 x 4 array with a pitch of 0.45 mm
and each spot measuring 0.25 x 0.25 mm.
4.3.3 Surface patterning of antibodies
Like the single-antigen assays, for multiplexed assays, streptavidin was used to functionalize the
surfaces for antibody immobilization. But a significant new challenge existed for multiplexed
assays: different antibodies had to be patterned proteins onto different spots in the array. A
microchannel patterning method, introduced by Delamarche et al [16], was used for this purpose.
In this method, 4 parallel microchannels were aligned over the 4 columns of the microarray
patterned on the top plate. Each channel was long enough to cover all eight arrays on a single top
plate, allowing for simultaneous patterning of all arrays.
One concern for the microchannel-based patterning scheme is non-specific binding of capture
antibodies onto the Cytop layers between the functionalized glass spots. One strategy used to
57
overcome this problem was including Pluronics as an additive in the capture antibody solution.
To test the effectiveness of this strategy, solutions of HRP labeled streptavidin (with and without
Pluronics) were flowed through the channels and allowed to immobilize onto the biotin
functionalized glass spots. The spots were then visualized using FITC labeled tyramide and the
resulting fluorescent images are shown in Figure 25 . As shown, the surface exposed to solution
containing Pluronics had minimal non-specific adsorption. In addition, the entire spot is not
fluorescent because the microchannel used for patterning did not cover the entire spot. This
indicates that the microchannels are properly sealed, and that there is no leakage. Thus, the
channel method is effective in delivering protein solutions to be patterned on the top plate and
that inclusion of Pluronics additives is useful for immobilizing proteins only onto the
functionalized spots.
Figure 25 The use of pluronics to confine protein immobilization onto the functionalized glass
spots. (a) Protein patterning without pluronics added to solution (b) Protein patterning with
pluronics added to solution. It should be noted that the patterned glass spots were larger than the
channel, and this fact is shown in the figure (especially in (b)) as not the entire glass spot is
fluorescent.
58
4.3.4 Verification of Tyramide signal amplification scheme.
Microarrays usually use fluorescence based detection scheme in order to have a spatially
resolved signal from the microarray. Our microarray used an enzyme based amplification
scheme known as the Tyramide signal amplification (TSA) scheme. This detection scheme relies
on the oxidation of tyramide to a reactive radical by HRP. The radical generated is very reactive
with tyrosine residues in proteins, and it usually reacts with proteins in the vicinity of HRP. The
high reactivity of the radical prevents it from diffusing and reacting with other proteins that are
far away from the desired spot.
To test the TSA detection scheme, microchannels were used to pattern the various analytes onto
epoxide-functionalized arrays, and each array was interrogated with a single antibody, with the
expected result being that only one column of a microarray would show a fluorescent signal (i.e.
the correct antigen-antibody pair). The result of this experiment for an array containing
lactoferrin and plasminogen as analytes and a HRP labeled lactoferrin antibody for detection is
shown in Figure 26 . The result obtained was unexpected, as fluorescence was observed on the
positions corresponding to all of the analytes (not just lactoferrin). Interestingly, the fluorescence
intensities originating from the spots adjacent to the lactoferrin spots were higher than that from
the spot that was two columns away. Moreover, of the two columns containing BSA (negative
controls which should presumably have identical and minimal cross-reactivities to the analyte),
the fluorescence intensity from the column closest to the analyte was higher than that of the
column furthest from the analyte. These data suggest that the system has not been properly
spatially optimized, such that activated tyramide-dye molecules diffuse far away from the
desired spot prior to reacting, thus reducing the spatial resolution of the signal [65].
59
Figure 26 Fluorescence intensity of the columns of a microarray, with each column consisting of
a single type of protein. The data is averaged from 3 trials and the error bards correspond to ± 1
S.D.
To further probe the hypothesis outlined above, a sandwich immunoassay for plasminogen was
performed. In this experiment, the capture antibodies were immobilized onto a streptavidin-
functionalized array using the microchannel method. The capture antibody for plasminogen was
on the first column of the array, followed by a negative control (BSA), the lactoferrin antibody
was immobilized on the third column, and the last column was also a negative control.
Plasminogen and its detection antibody were then sequentially introduced and the assay
visualized using the TSA system. The results of this experiment are shown in Figure 27. As
shown, although the signal associated with the plasminogen antibody is higher than the rest and
the plasminogen signal also increases as the concentration of plasminogen is increased,
increasing fluorescence as a function of plasminogen concentration is observed in the other spots
as well.
The intensity profiles in Figure 27 indicate that the activated tyramide diffuses away from the
site of production. The gradient in fluorescence intensity suggests that most of the reaction takes
place on the plasminogen spots, as the signal decreases as a function of distance from the
0
200
400
600
800
1000
1200
1400
Plasminogen Lactoferrin BSA BSA
Fluo
resc
ence
Int
ensit
y (a
.u.)
Immobilized protein
60
plasminogen spot. Since it was previously determined that cross reactivity is not a concern with
the antibodies and it was also shown that there is no leakage between the channels used for
patterning, the only reasonable explanation is the diffusion of activated tyramide away from the
spot where it is generated. Most microarrays that use the TSA system for detection have spots
that are much farther apart (1 mm between spots) [66], indicating that the spacing used in our
array (0.45 mm) should be increased or the tyramide concentration and reaction time optimized
for our array parameters.
Figure 27 Surface microarray based sandwich immunoassay for plasminogen. Detection was
performed using tyramide signal amplification scheme. Each data point is the average of 3 trials
and the error bars are ± 1 S.D.
0
5
10
15
20
25
30
0 0.1 1 10
Fluo
resc
ence
inte
nsity
(a.u
.)
Concentration of plasminogen (ug/mL)
Plasminogen
BSA
Lactoferrin
BSA
Capture antibody Identity:
61
4.4 Conclusion
It was determined that the antibodies used in this experiment have minimal cross reactivity with
each other, and are thus compatible for multiplexed detection. In addition, the microchannel
based patterning scheme proved to be compatible with the top plate of a DMF device, allowing
for the facile creation of high density microarrays. The tyramide based amplification scheme was
found to have certain problems with regards to spatial resolution. The next step in this work
would be directed towards improving the spatial resolution of the tyramide signal amplification
scheme, and eventually using this system to perform multiplexed immunoassays on a DMF
platform.
62
Chapter 5
5 Conclusions and Future work
5.1 Conclusions
In this thesis, work was done towards implementing a multiplexed immunoassay on a digital
microfluidics platform. This approach has the potential to overcome some of the challenges
presented for high throughput analysis, and also would improve immunoassays because of the
advantages of miniaturization, i.e., lower reagent use, faster detection times and improved
sensitivities.
The first step towards this goal was to develop a surface immobilization scheme for the capture
antibodies. Two different approaches were considered, (1) physisorption of antibodies onto
Cytop and (2) patterning of the top plate and subsequent covalent immobilization of the
antibodies using the biotin-streptavidin system. The covalent immobilization proved to be a
better strategy as lower concentrations of antibodies are required for immobilization and it also
provided a more reproducible method of immobilization.
The parameters of top-plate patterning, which included choice of metal and hydrophobic layer
and also the designs used for patterning, were investigated. It was determined that thin chromium
layers provided sufficient conductivity and adhesion to the glass substrates to serve as the ground
electrode. Cytop was chosen as the hydrophobic material because of its stability towards the
patterning and functionalization steps. The effect of the size of the patterned spots on droplet
movement in DMF was also investigated and a patch that was 20 % the size (or lower) of the
actuation electrode was the largest over which the droplets could be moved. For multiplexed
immunoassays, a 4 x 4 array was fabricated, with a total area that was 20 % of the actuation
electrode. The pitch (center to center spacing) was optimized for droplet movement, and it was
determined that a pitch size of 0.45 mm (which corresponds to 60 % hydrophilic area with
respect to the entire microarray dimensions) was ideal for robust droplet movement.
The next step was to pattern the antibodies onto the surface for a multiplexed immunoassay. The
microchannel patterning method developed by Delamarche et al [16] was used for this purpose.
63
It was demonstrated that the microchannel structures can be effectively used to deliver proteins
onto the patterned surfaces of the top plate, with no cross-contamination or non-specific binding
observed.
The cross-reactivities of the antibodies and antigens involved in the assay were evaluated, and it
was determined that there is no significant cross-reactivity for the antigens and antibodies used
here, which should allow for them to be used for the multiplexed assay. A sandwich
immunoassay for plasminogen was performed using the antibodies immobilized on a microarray.
But the signal obtained did not follow the expected results, as spots that were not related to the
antigen-antibody interaction possessed significant fluorescence intensity. This effect was
attributed to the lack of localization of the TSA system, which suggests that future work should
focus on the optimization of the TSA system to improve signal localization.
5.2 Future work
The next step of this work should focus on the optimization of the detection scheme (TSA
system) for the microarrays. It is known that the spatial resolution, and thus the signal
localization, can be improved by reducing the concentration of the tyramide dye conjugate and
also by reducing the incubation time. In addition, the spacing of the array (pitch) can also be
increased to reduce the negative impact of diffusion of the activated tyramide radicals.
Once a reliable detection scheme is established, the various DMF sample handling parameters
such as active incubation time (droplet movement over the microarray) and washing steps should
be optimized for the microarrays. These optimized conditions can be used to perform
immunoassays on the microarray using DMF for fluid handling. Analytical parameters such as
limit of detection, sensitivity, reproducibility and linear range of detection can then be
determined.
The long-term goals for the project lie in two main areas:
(1) Automation for high throughput analysis:
Automation is one of the key advantages of digital microfluidics, and the Wheeler group is
working towards developing a robust system for automated control of many droplets in parallel.
64
An ideal device would be able to control multiple samples simultaneously, resembling a robotic
screening platform in which many assays are implemented in parallel via automated dispensers,
mixers, and aspirators. The work directed towards this goal would involve designing devices
that can be interfaced with the automation hardware, and also designing devices capable of
holding multiple samples such that multiple assays can be performed simultaneously.
(2) Clinical sample evaluations:
The use of DMF for evaluating clinical samples, such as blood, has been demonstrated [67]. But
most applications rely on the use of systems in which aqueous droplets are immersed in an oil
matrix. We speculate that this is not a tenable strategy for surface-based analyses as there is not
sufficient contact between the droplets and the surface (and moreover, exposure to oil will likely
degrade the activity of immobilized species). On the other hand, systems such as those used in
the Wheeler group (i.e., with air as matrix rather than oil) are known to suffer from problems
related to surface fouling and droplet movement irreproducibility [68]. Thus, effort should be
directed to improving the movement of complex solutions on devices with minimal fouling, and
to develop strategies allowing for reduced actuation times to reduce contact with device surface,
passive dispensing of solutions into the patterned spots such that there is minimal contact with
device surface, and optimization of solution additives such as Pluronics to reduce non-specific
adsorption.
65
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