microfluidic systems for bioreporting, separations ... · pdf fileepa star edc-’06...

EPA STAR EDCEPA STAR EDC--’’0606

Microfluidic Systems for Bioreporting, Separations, Vibrational Spectroscopy, & Microcantilever

Sensing of EDCs(EPA – 83274001)

Mike Sepaniak, University of Tennessee,Department of Chemistry &

Center for Environmental Biotechnology;Oak Ridge National Laboratory

Backg

round

Electrophoretic Separations in Environmental AnalysisSERS in Environmental Analysis

Nanomechanical Bio-MEMS Sensing

Future WorkConcept of an Integrated Fluidic Platform for EDC Analysis


Nanomechanical Bio-MEMS SensingMeasurement principles

t

l

MC-based measurements

∆z =3l2 (1-ν) ∆σ / Yt2

∆z – tip displacement∆σ – differential stress changel & t – length & thicknessY- Young’s modulusν - Poisson’s ratio

∆z

Nanostructures

Cavitand receptors

Thin organic films

Bioaffinity phases

Nanostructuring the MCsurface prior to MRP coating

greatly enhances responses

Analytes – Extremely versatile (metals, VOCs,proteins, drugs, pollutants, DNA, etc.)

Detection


IgG IgG IgG IgG IgG

IgGIgGIgGIgGIgG IgGIgG

IgGIgGIgG

IgG

Following initial exposure (above), protein-protein &

protein-surface interactions and charge effects can

influence magnitude and direction of bending

Non-specific and Specific Binding of Proteins(Toward Bioaffinity Approach to Selectivity in

nanomechanical sensing of EDCs)

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(nm

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Protein 1

2

3

4

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2 – IgG on smooth surface3 – IgG on nanostructured surface4 – IgG on functionalized, nanostructured surface

“tensile

” (vs

“compressive

”)

bending

Stable receptors require anchoring; e.g. Glutaraldehyde or Protein A


Chiral Discrimination Using Ab-Mediated Cantilever Nanomechanics

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D /L-AA

1

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34

5

6

1 – 50 mg/L L-tryptophan (anti-L-AA)

2 – 50 mg/L L-phenylalanine (anti-L-AA)

3 – 50 mg/L D-tryptophan (anti-L-AA)

4 – 50 mg/L D-phenylalanine (anti-L-AA)

5 – 50 mg/L L-phenylalanine (IgG)

6 – 50 mg/L D-phenylalanine (IgG)

3 & 4 no response with“wrong” Ab (exceptionalselectivity)

Linear calibration and > 99% ee capability


Calibration Using Antibody Functionalized MCs

Toxin A Cytokine

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0.1ppm0.05ppmHIL

PBS(pH 7)

R2 = 0.9904

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Concentration of HIL-1beta (ppm)

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R2 = 0.9904

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Concentration of HIL-1beta (ppm)

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600ppb

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y = 0.0012x + 0.0525R2 = 0.9998

00.10.20.30.4

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Concentration (ppb)R

espo

nse(

V)

Low ppb concentrations can be detected


Distributed Selectivity Approach via Differentially Coated, VCSEL-Interrogated MCAs

Laser array pitch matches that of MC array; lasers –850 nm, 5 mW typicallyscanned over MC array in less than one second


Distributed Selectivity Response Signatures(Signatures of EDCs should be possible)

Time (Sec)0 10 20 30 40 50 60

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Data @ one/sec

Both Equilibrium & kinetic phenomenainfluence shape following 30 sec injection

Each MC in the arrayproduces a characteristic response that depends on the MRP & analyte

GC phase 1Polymer phase 1

CD macrocycleCalix macrocycle

Etc.


Distributed Selectivity & ANN Training & Performance

Input Middle

Output

Wim Wmo Ws are weighting factors

ANN 3-Layer Feed-Forward, Back-Propagation Diagram

ANN Training For Dioxane (4th analyte)

Five snapshots of training showing zeroing-in on 1.0 probably that

analyte is DIOXANE

44,000 training iterations

10 min. training time (computer!)

Most components in 10 analytetest suite identified with > 95%Does not identify CHCl3 forwhich not trained


SERS in Environmental AnalysisThe Optical Process

Anti-Stokes Scatter

Stokes Scatter

VibrationalSignatures

Raman scattering is an information rich process but, unfortunately, it is also very inefficient


SERS - Why

Small inherent cross sections of the normal Raman process lead to poor sensitivity, however Raman signals can be greatly enhanced by SERS and resonance effects (enhancements > 1012 have been reported in rare cases)

Commonly used optical methods fluorescence & absorbance are “information poor” while Raman methods provide detailed vibration information (unambiguous identification when needed)

SERS - Why NotLimited qualitative & quantitative reproducibilityLimited dynamic rangeHigh sensitivity is not very uniform

surface/nanostructure/phenomenon


SERS – What is it

I Raman is proportional to product of EM field and polarizability of molecule

Electromagnetic model ( “E” ) -> Radiation-induced polarization in nanometer-dimensioned precious metals enhances field (E) experienced by proximal analytes; metal type (Ag & Au), dimensions and shape, and spatial arrangement are very important


SERS Project GoalsBy technique and substrate advances, address these

analytical limitations and apply to EDCs

Our Advanced substrates are metal – polymer nano-composites substrates formed by vapor depositing Ag or Au onto:

15 nm 0.2 Å/s

Moldable PDMS Films

Spontaneous generatedsubstrates with randommorphologies

E-Beam Resist Pillars

Lithographically preparedsubstrates with controllablemorphologies


Comparison of SERS Substrates

Ag-PDMS Ag-E-Beam Pillars

A

B

C

D

A

B

C

D

Rh6G on different EBL

patterns

Different EBL-created pillarpatterns yield variations in SERS activity

0

10000

20000

30000

450 1200 1950Wavenumber (cm-1)

Ag-PDMS

Ag- Glass

4-Aminobenzoic acid (PABA)

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10000

20000

30000

450 1200 1950Wavenumber (cm-1)

Ag-PDMS

Ag- Glass

4-Aminobenzoic acid (PABA)

Our substrates significantly out-perform traditional Ag islands on glass


SERS “On-The-Move” Improves PerformanceTranslating Stationary

SERS -Mapping

Reproducibility:Intra-Well RSD: <7%Inter-Well RSD: <10%

NH 2

HS

Spinning(translating)

Rastoring(static)p-ATPSTT-SERS


Electrophoretic Separations (CE) in Environmental AnalysisElectrophoretic Separations (CE) in Environmental Analysis

Separation technique based on the mobility differences in an electric field for ions injected into narrow-bore capillaries

Neutrals can be separated based on differential association with chargedrunning buffer additives (e.g., CDs)

Running buffer is electrokinetically pumped through the capillary

Mobility scales roughly with charge to mass ratios of the ions

Capillary format can be changed to a lab-on-a-chip platform usinglithographic techniques

* Cartoon of CE separation

CE Characteristics: efficient,fast, versatile, diminutive


+

- - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - -

-

+++++++++++++++ +++++++++++++++

++++++++++++++++++++++++++++++

NeutralsEOF

-OOC

-OOC

-OOC

-OOC

Capillary Electrophoretic Separations of Neutrals by “CDCE”

Observed Mobilitiesµeof =µCD =µCMCD =µobs(N) = depends on inclusion

Wide windows very desirableWide windows very desirablemigration time

Creates an elution window

µeof = µneutrals= µCD

Inclusion in the cavity depends on the chemical and physicalproperties of both the CD and Analyte. There are several dozen different commercial types of CDs and we have also

chemically modified native CDs; hence a plethora of CD “cocktails”can be tailored to meet EDC separation challenges


Examples of Prior Pseudo-phase CE Separations of Environmentally Significant Mixtures

mixture of ten mycotoxins

mixture of PCBs

alkyl substituted PAHs


Conceptual Design & Early Progress


Conceptual Design & Early ProgressV buffer reservoir

V bioreportertiter well Vwaste-1

Vwaste-2

bioluminescenceplate reader

objectiveChannels (typically 50x100 µm)

created in PDMS usingphotolithography; footprint

size of microscope slide

PDMS has been used to house living cells

Center for Environmental Biotechnology Bioreporter System

Engineered by fusing genetic regulatoryelements to a reporter gene (yeast & mammalian cells systems)Inducer is typical analyte of interest (e.g., EDC)Regulatory protein activates transcription oflight emitting system (currently measurementsmade in titer well format)

Suitable manipulation of applied voltages (Vs) allows

flow control for sampling, injection,and separation/detection

Vwaste-3



V buffer reservoir


Vwaste-2


objectiveTimed

electrokinetic fillingof offset with < 1 µL

of well contents

Vwaste-3



V buffer reservoir


Vwaste-2


objective

Injection performed,cross channels cleared,the separation proceeds

Vwaste-3


Conceptual Design & Early ProgressExample CE separation of EDCs

V buffer reservoir


Vwaste-2 Vwaste-3

V buffer reservoir


Vwaste-2 Vwaste-3

Separation of kaempferol (a), genistein & daidzein (b),

apigenin (c) and DES (d) usinga running buffer with

β-CD & SDS pseudo-phases.

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15 20 25 30tim e

A.U

. @ 2

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(a)

(c)(d)

(d)

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(d)(d)

Separation of bisphenol A (a), 17b-esradiol (b), 17 a-

ethynylestradiol (c) and DES (d)using a running buffer with β-

CD & SDS pseudo-phases.

Chemical Changes


Conceptual Design & Early ProgressV buffer reservoir


Vwaste-2


objective

Diversion into detection channels (NMC & SSC)

Vwaste-3


Conceptual Design & Early ProgressDistinctive SERS Spectra of EDCs using Ag-PDMS substrates

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Bisphenol A

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(A.U

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V buffer reservoir


Vwaste-2 Vwaste-3

V buffer reservoir


Vwaste-2 Vwaste-3

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Apigenin

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Genistein

Bands unique to each EDC are evident


Conceptual Design & Early ProgressSERS Spectra of EDCs using EBL nanofabricated substrates

25 nm of Ag was deposited onto nano-dimensional hexagon, square, and elliptical (2 aspect ratio) pillars; Apigenin SERSactivity greatest on square pattern

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HCC EC2-1:3 EC2-1:4 CC4-1:1

Pattern

Ban

d A

rea

698-

763

cm-1

(A.U

.)Apigenin

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HCC EC2-1:3 EC2-1:4 CC4-1:1

Pattern

Ban

d A

rea

698-

763

cm-1

(A.U

.)Apigenin


Conceptual Design & Early ProgressSERS using EBL nanofabricated substrates (in channel)

EBL nanopillarmorphologies resorufin

band intensities

A

Information-rich in-channel

Raman spectrum

B

CEBL patterns

in channel

V buffer reservoir


Vwaste-2 Vwaste-3

V buffer reservoir


Vwaste-2 Vwaste-3


Conceptual Design & Early ProgressSERS Studies of EDC Chemical Fate Using EBL Nanofabricated Substrates

DES (30 min UV) DES (30 min UV)

DES (fresh DES (fresh solnsoln.).)

DES (photo prod.?)DES (photo prod.?)

DES (solid)DES (solid)

?


Conceptual Design & Early ProgressNanomechanical sensing of EDCs

MCA functionalized with macrocyclic receptors

V buffer reservoir


Vwaste-2 Vwaste-3


objective

nano-mech

anical

channel (NMC)

V buffer reservoir


Vwaste-2 Vwaste-3


objective

nano-mech

anical

channel (NMC)

Different equilibrium & kinetic responses based on analyte & MRP;distributed selectivity with pattern recognition possibilities


Conceptual Design & Early ProgressNanomechanical sensing of EDCs

Responses to EDCs for MC with anImmobilized h-ER-β on surface V buffer reservoir


Vwaste-2 Vwaste-3


objective

nano-mech

anical

channel (N

MC)

V buffer reservoir


Vwaste-2 Vwaste-3


objective

nano-mech

anical

channel (N

MC)

Further bioaffinity approach studies are being performed(literally as I speak)

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17 β-Estradiol

Bisphenol A

PBS blank


Future Work (next 12 month period):

Continue surveying targeted EDCs for nanomechanical and SERS responsesMCA modifications (phases and nano structuring) to enhance responses & selectivityAdvanced SERS substrates to enhance sensitivityFate studies of DES and other EDCs

Develop conditions for on-chip separations for various target EDC combinationsPerform correlation studies involving mating our advanced methods with bioluminescence reporting in titer well formatWork toward integrating the individual analytical components and field portability

Key Members of Research Team:UT-Chemistry – Maggie Connatser, Kasey Hill, Jenny Oran

Pampa Dutta, Marco DeJesusUT-CEB – Gary Sayler, John Sanseverino, Melanie Eldridge


Questions?

Key Members of Research Team:UT-Chemistry – Maggie Connatser, Kasey Hill, Jenny Oran

Pampa Dutta, Marco DeJesusUT-CEB – Gary Sayler, John Sanseverino, Melanie Eldridge


Field-Ready Potential for These Technologies


Key Issue - SelectivityAchieving Selectivity Analysis

MCA

extra


Achieving Selectivity In AnalysisWith analyte sensitive sensors (e.g. MCs) these spectroscopic &

chromatographic analytical attributes are not present. However, selectivity is attainable through the clever

uses of molecular recognition phases (MRPs).

MCA

extra

Two viable approaches

Use of highly selective MRPs with large binding constants:

• Bioaffinity • Selective chelates for metal ions• Issues of reversibility & robustness

Use of moderately selective Phases in arrays for “distributed selectivity”:

• Based on response signatures• Pattern recognition• Issues of molecular recognitioncontrast and differential coating


Nanostructured Interfaces Enhance Responses

Analyte SAM (smooth)

SAM (nano)

17 nm (smooth)

17 nm (nano)

50 nm (nano)

2,3-DHN 290 4.8 31 0.99 0.025

2,7-DHN 250 7.5 39 1.0 0.039

Tolazoline 300 17 214 13 4.9

Benz. Acid 1500 140 250 42 18

36 X 39 X

MCA

extra

Example of the dramatic improvement in LODs (in ppm) with: - nanostructuring

- thicker films (phases are modified cyclodextrins)


MCA

extraBioaffinity Interaction on Different Surfaces

Antihuman IgG - human IgG

Our dealloyed surfaces not only provide larger signals, but also better reversibility. Studies are currently underwayto investigate mechanism


Cantilever Deflections Can Also be Usedfor DNA Hybridization Studies

The direction of cantilever motion is opposite during

immobilization and hybridization of ss-DNA on

two different surfaces (dealloyed gold, smooth

gold).

Inconsistencies maybe related to the complexity of surface charge, adsorbate-

surface, adsorbate-adsorbate, and adsorbate

rearrangement effects.

MCA

extra


MCA

extra Metal Ion Detection

• Reasons for heavy metal detection- Highly toxic- Bioaccumulation

• Traditional detection methods- Chromatography- Flow-injection systems- Atomic absorption - Electrochemistry- Sensors

(e.g., microcantilever MEMS)

CH 2 CH 2H 2 N SH

CO2HCH2CH2HS

(CH 2 )10HO 2C SH

CH 2CH 2 CH 2HO SH

(CH 2) 11 OHHS

CH 2 CH 2H3 C SH

(CH 2) 17 MeHS

CH 2CH 2 CH 2HS SH

(CH 2) 8HS SH

CO 2 HCHCH 2

NH 2

HS

AET

MPA

Cyst

MUA

MP

MUD

OTD

PDT

ODT

PTCH 2 CH 2H 2 N SH

CO2HCH2CH2HS

(CH 2 )10HO 2C SH

CH 2CH 2 CH 2HO SH

(CH 2) 11 OHHS

CH 2 CH 2H3 C SH

(CH 2) 17 MeHS

CH 2CH 2 CH 2HS SH

(CH 2) 8HS SH

CO 2 HCHCH 2

NH 2

HS

AET

MPA

Cyst

MUA

MP

MUD

OTD

PDT

ODT

PT

• Take advantage of thiol-gold binding to form SAMs of monodentated ligands

EPA STAR EDCEPA STAR EDC--’’0606LiCl CsCl CoCl2 CuCl2 AlCl3 CrCl30

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LiCl CsCl CoCl2 CuCl2 AlCl3 CrCl30

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LiCl CsCl CoCl2 CuCl2 AlCl3 CrCl3LiCl CsCl CoCl2 CuCl2 AlCl3 CrCl30

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Metal-Ligand Interaction & MC Response

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MCA

extra

Selectivity patterns For MCs with

SAMs of different monodentated

ligands


SERS

extra Sample Translation Technique; Effect on Various Analytes

300

800

1300

1800

2300

2800

150 350 550 750 950 1150 1350 1550 1750 1950

Wavenumbers (cm-1)

Inte

nsity

(Cou

nts)

Stationary STT

Riboflavin

0

5000

10000

15000

20000

25000

30000

35000

150 350 550 750 950 1150 1350 1550 1750 1950 2150 2350 2550 2750 2950

Wavenumbers (cm-1)

Inte

nsity

(Cou

nts)

Stationary STT

Folic Acid

500

1500

2500

3500

4500

5500

6500

7500

150 350 550 750 950 1150 1350 1550 1750 1950

Wavenumbers (cm-1)

Inte

nsity

(Cou

nts)

Stationary STT

8-Hydroxyquinoline

100

600

1100

1600

2100

2600

3100

150 350 550 750 950 1150 1350 1550 1750 1950

Wavenumbers (cm-1)

Inte

nsity

(Cou

nts)

Stationary STT

Tetracaine

0

5000

10000

15000

20000

25000

30000

35000

150 350 550 750 950 1150 1350 1550 1750 1950 2150 2350 2550 2750 2950

Wavenumbers (cm-1)

Inte

nsity

(Cou

nts)

Stationary STT

Rhodamine 6G

300

800

1300

1800

2300

2800

150 350 550 750 950 1150 1350 1550 1750 1950

Wavenumbers (cm-1)

Inte

nsity

(Cou

nts)

Stationary STT

Benzoic Acid(BA)

Samples centeredon spinner then moved

off-center a few hundred µm &

spun at leastseveral hundred RPMto yield STT spectra


Quantitative Performance of STT-SERS With Ag-PDMS Substrates

SERS

extra

1.0E+04

1.1E+04

1.2E+04

1.3E+04

1.4E+04

1.5E+04

1.6E+04

1.7E+04

1.8E+04

1.9E+04

2.0E+04

0.0E+00 2.0E-05 4.0E-05 6.0E-05 8.0E-05 1.0E-04

Concentration(M)

Ban

d Ar

ea 7

83 ±

(35)

cm

-1 (A

. U.)

R6G Linear Fit Poly. (R6G)

Y=4.3± (0.6)E+08 X +9.5± (0.5)E+03R2=0.98

Rh6G

y = 5.290±(0.009)E+08x + 5.06±(0.4)E+02R2 = 1.0

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0.0E+00 2.0E-06 4.0E-06 6.0E-06 8.0E-06Concentration (M)

Ban

d Ar

ea 5

73.4

1-64

2.98

cm

-1(A

.U.)

R6G Linear (R6G)

Rh6GExperimental values for:LOL: 1.0x10-5 MLOD: 1.0x10-9 M

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0.0E+00 2.0E-06 4.0E-06 6.0E-06 8.0E-06 1.0E-05Concentration (M)

Ban

d A

rea

1342

.15-

1443

.67

cm-1 (A

.U.)

p-ABA Reg

Y=1.57±(0.03)E+09X+4±(1)E+02R2=1.0

0

5000

10000

15000

20000

150 350 550 750 950 1150 1350 1550 1750 1950

Wavenumbers (cm-1)

Stationary STT

p-ABA

Experimental values for:LOL: N.R.LOD: 1x10-8 M


Saccharomyces cerevisiae Yeast Estrogen Screen

Bioextra

Routledge and Sumpter. 1996. Environ. Toxicol Chem. 15:241-248


Microtiter Plate Layout1:2

1:10

1:10

1:10

1:10

Bioextra


Our EPA STAR work is focused on development of advanced, materials-based, integrated analytical methodologies for EDC analysis

Objectives:

Develop conditions for capillary then on-chip electrokinetic separations

Optimize nanocomposite-based SERS substrate morphologies for greatest activity and survey responses

Evaluate nanomechanical response signatures using functionalized MCAs- traditional molecular recognition phases - bioaffinity phases for EDCs

Integrate bioreporter yeast conditions for total systems with bioluminescence-based screening of EDCs and the aforementioned advanced analytical technologies (initially in titer-plate to microfluidic configuration; ultimately a totally integrated platform )

microfluidic systems for bioreporting, separations ... · pdf fileepa star edc-’06...

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