Microchip electrophoresis

Microchip electrophoresis

Analytical Strategy, 06.10.2015

Petra S. [email protected]

www.dittrich.ethz.ch

Overview

1. Why miniaturization of analytical instruments?

2. Fabrication of microchips

3. Capillary zone electrophoresis

4. An alternative solution

Microdevices

RW heads

Pacemaker

Intelligent clothing

Displays, electronic gadgets

Sensors: ChemPro©100i(1: humidity sensor, 2:ion mobility spectrometer, 3: pressureSensor, 4,5: Micro gas sensor, 6: air flow, 7: pump unit)

Diagnostic chips

iSTAT

Micro fuel cell

Why miniaturization ?

microchip

Blood sugar, diabetes Blood monitoring Pregnancy test

Point-of-care devices

Cytometer on a cell phone

What is microfluidics?

It is the science and technology of systems that process ormanipulate small (10–9 to 10–18 litres) amounts of fluids, using channels with dimensions of tens to hundreds of micrometres.*

*G.M. Whitesides: The origin and the future of microfluidics, Nature 2006, 442, 368.

Platform („chip“): cm

50 μm

Advantages of miniaturized instruments

• Reduction of space and volumesà small instruments, portableà economic sample consumption,e.g. safer work with toxic compounds

• Reduction of synthesis and analysis timeà faster transport of compoundsàmore efficient reaction, e.g. controlled heatingà parallelization of processes

length



• Integrationà Automation of individual steps, e.g. sample pretreatment

Injection Separation DetectionPretreatment

Heating/Reaction/Incubation/…






• Sensitive detectionà e.g. optical methods like fluorescence spectroscopy



• Sensitive detectionà e.g. optical methods like fluorescence spectroscopy

• “Size Matters”à Handling of small objects and liquid volumes D. DiCarlo, L.P. Lee





Fabrication of microchips

12

Idea

13

1 cm

5 cm

5 cm


Lithography Mask(Cr-Glass or film)

Master:silicon wafer

Soft lithography(molding)

Bonding, Interfaces

Choice of materialsDesignDrawing

PMMAPDMS (Silicone) /Glass

GlassSilicon

PDMS (Silicone)/Glass

14

1 cm

5 cm

5 cm


Lithography Mask(Cr-Glass oder film)



Bonding, Interfaces


15

1 cm

5 cm

5 cm

SiPhotoresistPhotomask

UV-light





Bonding, Interfaces


16

1 cm

5 cm

5 cm

SiPhotoresist





Bonding, Interfaces


17

1 cm

5 cm

5 cm

25 µm





Bonding, Interfaces


18




Bonding, Interfaces


1 cm

5 cm

5 cm


19

Multilayer soft lithography

Fluid layer

Control layer

Integration of dynamic features: valves, pumps

pneumatic orhydraulic actuation

ü fluid controlü metering of pL volumesü reaction chambers of pL volumesü high parallelization- but: flexible materials required

Incubation chamber

Microscope

Syringe pumps

20

Experimental Setup

Bio-Analyzer, Agilent Techn.commercial product since1999

Example of a commercial instrument for electrophoretic separationofRNA, DNA, proteins and cytometry (cell analysis)

Commercial instrument

Capillary zoneelectrophoresis (CZE)

Microchipelectrophoresis

Electrophoresis

Separation of charged molecules/particles in an electric field due to their different electrophoretic mobility

InjectionIntroduction of smallsample volumes into the separation channel

SeparationDetectionspecific/unspecificdetermination of theseparation bands

Microchip:very small, definedinjection volumereproducible

Microchip:faster separation due tohigher electric fieldstrengths;;prevention of Joule heatingdue to large surface-to-volume ratio

Microchip:detection like conv. CZE

Integration with othersynthetic/analytic steps

Example movie and photographs

Courtesy of A. Manz

1989

1993

Calliper

Electrophoresis vs. Electroosmosis

from: Bioanalytical Chemistry, A. Manz, N. Pamme, P. S. Dittrich, D. Iossifidis

V

uep

ueo Electroosmosis:Bulk movementof the liquid

(ueo: electroosmoticmobility)

Electrophoresis:Movement of thecharged particles(ui: electrophoreticmobility)

Velocity of migration in capillary electrophoresis, withE: electric field strength):

vi,tot = (ui + ueo )E

vi,tot = (−ui + ueo )E

(for cations)(for anions)

uapp

Theory: Electrophoresis

vi = ui E

Velocity of ions (vi in cm/s):

cations

vi = -ui E anions

E: Electric field strength (V/cm) ui: Electrophoretic mobility (cm2/(sV))z: Valence number of ionse: Elementary electric chargeF: Faraday constant (96  485 C/mol)D: Diffusion coefficientη: Viscosity (Pa s)r: RadiusElectrophoretic mobility:

(for small ions)

For large molecules (Stoke‘s law):

ui =zi e6πηri

àhighly charged, small analyte:high electrophoretic mobility

• Cause: electric double layer, interface of stationary/mobile phase• e.g. deprotonated silanol groups at stationary phase, ions in mobile phase

• Stern layer:specifically bound molecules“adsorbed” molecules

• Diffusive layer:solvated cations undergo Brownian motion(can be considered as point charges)electrostatic forces: exponential decayof potential

• Bulk:solvated ions, charges equalized

• electric double layer thickness : < 500 nm

Electroosmotic flow

Theory: Electroosmosis

veo = ueo E

Electroosmotic velocity:E: Electric field strength(V/cm) ueo: Mobility coefficient EOF

(cm2/(sV))ζ: Zeta-potential (Potential between capillarywall and solution, about 0.1 V)ε: Dielectric coefficient

ueo = εζ4πη

e.g. water/25°C: 7.8 x 10 -3 cm2 V-2 s-1 ζ

stamp-likeprofile(compare to the parabolic flow profile of pressure-driven flow):

Electroosmotic flow

• Negatively charged analytes, e.g. DNA fragmentsà Move towards cathode slower than EOF

• Positively charged analytes, e.g. proteins àMove faster towards cathode than EOF

• To improve resolution, EOF is sometimes suppressed by coatings

• Additionally, coatings provide better reproducibility

from: Bioanalytical Chemistry, A. Manz, N. Pamme, D. Iossifidis

V

uep

ueo

• Reduction of EOF by(i) Separation at low pH (protonated Si-OH groups)(ii) Chemical coatings e.g. polyacrylamide etc.(iii) Dynamic coatings e.g. PEG)(iv) Choice of buffers, e.g. high ion concentration

Injection

• Hydrodynamic or electrokinetic injection• Advantage ofmicrochip electrophoresis: Injection of small volumes in nL andpL range !

EOF / EP

Injection

Summarized in M. Vlckova, Dissertation UBasel, 2008

Bharadwaj et al. Electrophoresis 2002, 2729

Separation: Channel length

t = Lvi,tot

= L2

(u i + u eo)UAnalysis time:

1060 V/cm, FITC-Aminosäuren, pH9.0 Tris Puffer

L: Length of separation channelU: Applied voltage

Racetrack effect

S. GhosalAnnu. Rev. Fluid Mech.2006. 38:309–38

electrical field enhanced racetrackeffect:shorter distance– faster velocityat the inner side of the curve

about 1/3 of original width

Santiago and coworkers

But: High voltages results in large temperature gradients(Joule heating), which limits the resolutionAdvantage of small microchannels: Heat reduced due tolarge surface to volume ratio

t = Lvi,tot

= L2

(u i + u eo)U

σ dif2 = 2Dt = 2DL2

(u i + u eo)U

...22222 ++++= adsinjTdiftotal σσσσσ

Analysis time:

Peak broadening due to diffusion:

Peak broadening in total:

Number of theoretical plates:

Ideally, high voltages are applied

σ: standard deviation

Separation: Limitations

N = L2

σ total2 =

uappU2D

(in electropherogramm:N=5.54 (tR/w1/2)2=16(tR/wB)2

Detection

– Absorption (UV, Diodenarraydetector), difficult in capillaries– Fluorescence, often unspecific;; labeling andcalibration required;;but: highly sensitive and quantitative

– Electric conductivity and related techniques– MS, e.g. ESI-MS

J. S. Mellors, V. Gourbounov, R.S. Ramsey, J. M. Ramsey,Anal. Chem. 80 (2008), 6881-6887

www.agilent.com

Example: Electrophoresis of N-glycans

MicrochipSubstrate: Glass, coated with poly(acrylamide)Separation channel: 22 cm long, tapered channels in turns

InjectionPinched injection

SeparationElectric field strength: 1250 V/cm Separation efficiency: up to 700 000 plates, analyses time < 100 s

Detection8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeled glycansExcitation: 488 nm, FITC filter cube, Photomultiplier

Mitra et al., J. Proteome Res. 2013, doi: 10.1021/pr400549e

Work flow

Vanderschaeghe et al., Anal. Chem. 82 (2010), 7408-7415

Example electropherograms

Mitra et al., J. Proteome Res. 2013, doi: 10.1021/pr400549e

Data analysis

Large differences betweenpatient samples before and aftertreatment of the ovarian cancer

Differences to control group unclear

à Microchipelectrophoresis forN-Glycananalysis usefulà Potential for cancer diagnosis, but results are not yet clear

Microchip electrophoresis

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