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
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
• Reduction of space and volumesà small instruments, portableà economic sample consumption,e.g. safer work with toxic compounds
Advantages of miniaturized instruments
• Integrationà Automation of individual steps, e.g. sample pretreatment
Injection Separation DetectionPretreatment
Heating/Reaction/Incubation/…
• 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
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
• Sensitive detectionà e.g. optical methods like fluorescence spectroscopy
• Integrationà Automation of individual steps, e.g. sample pretreatment
Advantages of miniaturized instruments
• Sensitive detectionà e.g. optical methods like fluorescence spectroscopy
• “Size Matters”à Handling of small objects and liquid volumes D. DiCarlo, L.P. Lee
• 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
Advantages of miniaturized instruments
• Integrationà Automation of individual steps, e.g. sample pretreatment
13
1 cm
5 cm
5 cm
Fabrication of microchips
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
Fabrication of microchips
Lithography Mask(Cr-Glass oder film)
Master:silicon wafer
Soft lithography(molding)
Bonding, Interfaces
Choice of materialsDesignDrawing
15
1 cm
5 cm
5 cm
SiPhotoresistPhotomask
UV-light
Fabrication of microchips
Lithography Mask(Cr-Glass oder film)
Master:silicon wafer
Soft lithography(molding)
Bonding, Interfaces
Choice of materialsDesignDrawing
16
1 cm
5 cm
5 cm
SiPhotoresist
Fabrication of microchips
Lithography Mask(Cr-Glass oder film)
Master:silicon wafer
Soft lithography(molding)
Bonding, Interfaces
Choice of materialsDesignDrawing
17
1 cm
5 cm
5 cm
25 µm
Fabrication of microchips
Lithography Mask(Cr-Glass oder film)
Master:silicon wafer
Soft lithography(molding)
Bonding, Interfaces
Choice of materialsDesignDrawing
18
Lithography Mask(Cr-Glass oder film)
Master:silicon wafer
Soft lithography(molding)
Bonding, Interfaces
Choice of materialsDesignDrawing
1 cm
5 cm
5 cm
Fabrication of microchips
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
Bio-Analyzer, Agilent Techn.commercial product since1999
Example of a commercial instrument for electrophoretic separationofRNA, DNA, proteins and cytometry (cell analysis)
Commercial instrument
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
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
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