integration of chip-based sample preconcentration into the ... of chip-based sample.pdfdan yee....

Integration of Chip-based Sample Preconcentration into the µChemLabTM/CB

Analysis Platform

Victoria A. VanderNoot, Boyd Wiedenman, YolandaFinstchenko and Julia Fruetel

Sandia National Laboratories

P.O. Box 969, Livermore, CA 94551-0969

HPCE 2003San Diego, CA

Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company,for the United States Department of Energy under contract DE-AC04-94AL85000.

dmlollSAND2003-8005P

µChemLab™ Separation PlatformAerosol

Collector

WaterAnalysis

SamplePrep

µChemLab™/CBLiquid Analysis

Module

Dual Channel Microchip Separations

Laser Induced Fluorescence

Detection

Microfluidic Delivery

•Application of electric fields moves fluids

µChemLab™ Separation Platform

Two Channel DeviceCapillary Gel Electrophoresis (CGE) Channel

Beckman 14-200 SDS GelReversed polarity

Capillary Zone Electrophoresis (CZE) Channel10 mM phytic acid, pH 9.5 containing 2 mM DAPS (zwitterionic detergent)Normal polarity

Fluorescamine labelingFluorogenic dyeFast-reacting, amine specificEx/Em 390 nm/480nm

Detection limitsnM for CGE

Approaches to Preconcentration

• Solid Phase Extraction (on-going efforts for the miniaturization of SPE)

• Electrokinetic Trapping (P411-T: Novel Miniaturized ProteinPreconcentrator Based On Electrokinetic Trapping, Anup K. Singh; Daniel J. Throckmorton; Brian J. Kirby)

• Salt-Bridge[1] J. Khandurina, T.E. McKnight, S.C. Jacobson, L.C. Waters, R.S. Foote, J.M. Ramsey, Analytical Chemistry 72 (2000) 2995-3000[2] R. S. Foote, J. Khandurina, S. C. Jacobson, J. M. Ramsey, Preconcentrationof Proteins on Microchips for Enhanced Detection, Poster presentation at HPCE 2001

Integration into the µChemLabTM/CB

Silicate layer Approach:Cover glass

Conduction via very small bore channels

Sol-gel silicate layer allows current to pass

Etched channels

Large molecules (i.e. proteins) retained

Cover glassAlternative Approach:

•Eliminate silicate layer

•Makes use of narrow gap and natural surface roughness

Large molecules (i.e. proteins) retained

Etched channels

Jean Pierre Alaire, Stephen C. Jacobson, B. Scott Broyles, Timothy E. McKnight, Christopher T. Culbertson and J. Michael Ramsey, ElectroosmoticallyInduced Hydraulic Pumping on Microchips. µTAS 2001. J. Michael Ramsey and A. van den Berg. ed.s, (Kluwer Academic, Amsterdam), (2001), pp. 127-128.

Chip Layout

W

P1

P2

S SW

B

Fill

Close-up of the Preconcentrator

Region

Imaging Normal and PreconcentratedInjections in CGE

P2

SW S

P1

Normal Mode P1 PreconcentrationMode

S P1

P2 PreconcentrationMode

S P2S SW

CGE Separations Using Normal and Preconcentrated Injections

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 50 100 150 200 250 300

Time (s)

Fluo

resc

ence

Sig

nal (

AU

)

No preconcentration

60 s preconcentration

20 nM Lactalbumin,20 nM Ovalbumin

Peak Areas in CGE Separations Increase as a Function of Preconcentration Duration

800

1300

1800

2300

2800

3300

3800

4300

4800

5300

0 50 100 150 200 250 300 350

Time (s)

Fluo

resc

ence

No preconcentration


50 nM Lactalbumin,50 nM Ovalbumin


***

***

***

Sub-Nanomolar Detection Limits Using CGE Separations and Preconcentration

2100

2300

2500

2700

2900

3100

3300

3500

3700

3900

4100

0 100 200 300Time (s)

Fluo

resc

ence

500 pM LAC500 pM OVA

LAC

OVA

Characterization of Performance as a Function of Gap Width

Gap width• Isotropic etch depth used to generate

gaps of different widths • For comparison purposes, all

preconcentrations were held to 60s

Cover glass

variable etch depth

00.5

11.5

22.5

33.5

44.5

7 9 11 13 15 17 19Gap Width (µm)

Pre

conc

entra

tion

Cur

rent

(µ

A)

02468

1012141618

7 9 11 13 15 17 19Gap Width (µm)

Appr

oxim

ate

Prec

once

ntra

tion

Fact

or

*Single Batch Data *Single Batch Data

Preconcentration Using CZE

Elimination of EOF is required• no bulk flow of fluid through the gap

∴residual EOF could lead to pressure generation

Coating with linear polyacrylamide

O NH2

( )n

O

Si

OO

O

O

Si

OO O

Si

OO O

( )m n>>m

O

CONH2CONH2CONH2

O O

CONH2CONH2CONH2

O O

CONH2CONH2

O

CONH2 CONH2CONH2CONH2 CONH2CONH2CONH2 CONH2

SAM applicationUV light polymerization

•Julie Fruetel, Victoria VanderNoot, Jay West, Brian Kirby, Ernest Hasselbrink and Timothy Shepodd, Laser-polymerized thin-film coating for protein analysis by CGE in a microchip, HPCE 2002

Imaging Normal and PreconcentratedInjections in CZE

P2

SW S

P1

Normal Mode P1 Preconcentration ModeS SW S P1

CZE Separations Using Normal and Preconcentrated Injections

1000

1200

1400

1600

1800

2000

2200

2400

2600

0 100 200 300 400Time (s)

Rel

ativ

e Fl

uore

scen

ce

No Preconcentration

60 s Preconcentration

Running buffer: 5 mM phosphate, pH 8 containing 5 mM CAS-U zwitterionic detergentReverse polarity

Summary

•Preconcentration has been incorporated in the chip design with no additional processing steps during chip fabrication

•Successfully demonstrated with both CGE and CZEPreconcentration factors of 10-20x are routinely achievable in only 60s

Coating and/or elimination of EOF is essential for CZE separations

Buffer conditions in CZE will need to be optimized to achieve both good separation efficiency and the elimination of EOF

AcknowledgementsRobert S. Foote and J. Michael Ramsey at Oak Ridge National Labs for their valuable insights

&Nicole Baryla and Charles A. Lucy for supplying us with a sample of CAS-U zwitterionic detergent

Brian HollidayBrent Horn

Susan JamisonBill Kleist

Ron RenziGeorge Sartor

George Schubert

Isaac ShokairJamie Stamps

Mary Clare StoddardVictoria VanderNoot

Jay WestBoyd Wiedenman

Dan Yee

FundingDept. of Energy Chemical and Biological National Security Program

Gabriela ChiricaWen-Yee ChoiMark ClaudnicEvelyn CruzScott Ferko

Yolanda FintschenkoJulie Fruetel

The µChemLab Technical Team

Integration of Chip-based Sample Preconcentration into the µChemLabTM/CB Analysis PlatformµChemLab™ Separation PlatformApproaches to PreconcentrationChip LayoutImaging Normal and Preconcentrated Injections in CGESub-Nanomolar Detection Limits Using CGE Separations and PreconcentrationCharacterization of Performance as a Function of Gap WidthPreconcentration Using CZEImaging Normal and Preconcentrated Injections in CZESummaryAcknowledgements

integration of chip-based sample preconcentration into the ... of chip-based sample.pdfdan yee....

Documents