Stanford Microfluidics Microfluidics Lab Lab Stanford Juan G. - - PowerPoint PPT Presentation

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Stanford Microfluidics Microfluidics Lab Lab Stanford Juan G. Santiago Research Examples: Research Examples: Optimized Geometries: Stanford Microfluidics Lab Stanford University microfluidics.stanford.edu Activities: Diagnostics

slide-1
SLIDE 1

50 µm

1 3 2

Stanford Stanford Microfluidics Microfluidics Lab Lab

Activities: Activities:

Miniature Bioanalytical Systems

  • Capillary zone electrophoresis
  • Capillary isoelectric focusing
  • CE Binding Assays

Research Examples: Research Examples:

Juan G. Santiago Stanford Microfluidics Lab Stanford University microfluidics.stanford.edu Microflow Devices

  • Micromixers
  • Electroosmotic pumps
  • Sample preconcentration
  • On-chip 2D assays

Applications

  • Drug discovery
  • Genetic studies
  • Proteomics
  • BW detection
  • Electronics cooling

Optimized Geometries: Micromixers: Diagnostics

On On-

  • chip 2D Assay:

chip 2D Assay: CIEF and CZE CIEF and CZE simulation experiment simulation experiment

Optimized Injections:

1 µm

slide-2
SLIDE 2

10 µm 10 µm

Electrokinetics Microfluidics at Extreme Scales

Juan G. Santiago Stanford Microfluidics Laboratory Mechanical Engineering Department Stanford University

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SLIDE 3

Outline

(time, concentration, length)

  • Introduction

Microfluidics Electrokinetic (EK) flows

  • Electrokinetic instabilities (time)

Mechanism and model Transition to chaos

  • ITP (concentration)

Sensitivity and practice Extreme concentration scales

  • Nanochannel electrophoresis (length)

Small ion separations DNA sample separation Near-Future Work

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SLIDE 4

Microfluidics

  • Applications

Point-of-care medical diagnostics Bio-weapon detection Pharmaceuticals/drug discovery Environmental monitoring

  • Challenges and Advantages

Reduced reagent use Specificity, robustness Portability vs. sensitivity Integration and automation Potential for parallel analyses

www.nanogen.com Image courtesy www.calipertech.com

0.7 cm

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SLIDE 5

drift

u ∇ > i

,1 ,2 drift drift

u u ≠

Processes in microfluidics

Separation Stacking Hybridization Reaction

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SLIDE 6

Separation in uFluidics

,1 ,2 drift drift

u u ≠

∆

∇

  • diffusivity
  • mobility
  • valence
  • affinity
  • polarizability
  • size/steric force

∆

∆ ∆ ∆

∆

On-chip CE Nanochannel electrophoresis H-filter

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SLIDE 7

Electroosmotic flow

External Electric Field u(y) y x Wall Wall

adsorbed ion y ψ shear plane λD ζ

  • Zeta vs. chemistry?
  • EDL overlap?
  • Condensation?
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SLIDE 8

Electrohydrodynamics and Electrokinetics History

Gilbert, ~1580s Of the attraction exerted by amber Reuss, F.F. 1809. Memoires de ls Societe Imperial des Naturalistes de Moscow. 2:327.

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SLIDE 9

Electrokinetic Microfluidics

Pressure-driven Electrokinetic

Devasenathipathy S. and J. Santiago, Micro- and nano-scale diagnostics, Springer-Verlag, 2003 EOF

+ + + + + + + + + + +

λd λd

+ + + +

λd

Electrophoresis

+ + +

  • ++ +

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

Glass or fused-silica microchannel wall Charge double-layer Deprotonated silanol groups

+

  • 100 um

100 um

  • Electric control (no

moving parts)

  • Switching, valving
  • Low dispersion
  • Integrated w/ separation

techniques

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SLIDE 10

Electrokinetic Instabilities

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SLIDE 11

Complex electrokinetics

  • Sample preconcentration methods

Thermal gradient focusing Field amplified sample stacking Isotachophoresis

  • On-chip two-dimensional assays
  • On-chip CE with unknown or poorly controlled

sample chemistry

  • On-chip mixing and buffer exchange

CE dimension (mm)

1 2 3 4 5 6 ∆x = 4

IEF dimension (mm)

1 3 2

1

Signal (au) Herr, A.E. et al. Analytical Chemistry, Vol. 75, No. 5, pp. 1180-1187, 2003

C A W B D

IEF/EOF

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SLIDE 12

Major Challenge in Heterogenous EK Systems: Instabilities

Unstable, fluctuating concentrations in high- conductivity-gradient case 50 µm

1 mm 100 µm

Flows at Intersections: Particle visualization

100 µm

Axial interface:

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SLIDE 13

σL σH σL σH σL σH ΕH σL σH ΕL

EK Instability Mechanism

ΕL ΕH

  • - -

+++

σL σH σL σH σL σH σL σH σL σH σL σH

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SLIDE 14

Electrokinetic Flow Instabilities

  • Generated by net charge in bulk
  • Velocity scale:

Controlling parameter:

  • scales as
  • EOF coupling produces convective instab.
  • Multiple ion mobilities have severe impact

/ ~

E

E C ρ ε σ σ ≅ ⋅∇ ∆

ΕL ΕH

  • - -

+++ σL σH

µ ε H E E U

a L EV =

Lin, H. et al., Vol. 16, No. 6 Physics of Fluids, p.1922-1935, 2004. Chen, C.-H. et al., J. of Fluid Mechanics, 524, pp. 263 – 303, 2005.

2 2 , max

1

ev e

U d E d Ra D D ε γ σ µ γ

∗ ∗

− ≡ = ∇

  • δ

depth d

0(

1) / E F ε γ γδ −

C ∆

Posner and Santiago, J. of Fluid Mechanics, pp. 1-42, 2006.

5 6

~ 10 10

− −

−

2

1 ( )

j j j j j j j

D c c Dt Pe Pe χ σ φ = ∇ + ∇⋅ ∇

∑ ∑

  • Electromigration

Diffusion

Oddy and Santiago, in press, Physics of Fluids, 2005.

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SLIDE 15

Electrokinetic instabilities

Experiment Model

t = 0.0 s t = 0.5 s t = 1.5 s t = 2.0 s t = 2.5 s t = 3.0 s t = 4.0 s t = 5.0 s t = 1.0 s

Storey, B.D. et al. Physics of Fluids, Vol. 16, No. 6, p.1922-1935, 2004. Lin et al., submitted to J. Fluid Mechanics, 2005.

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SLIDE 16

f [1/sec]

Rae = 675 Rae = 800 Rae = 2,000 Rae = 2,700 Power spectra

EKI in a cross intersection: Experiments

50 um

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SLIDE 17

Temporal Power Spectrum

f [1/sec]

Rae

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SLIDE 18

C2 C2 C1

Correlation plots

C1 Rae=675 Rae=800 Rae=2000 Rae=2600

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SLIDE 19

Isotachophoresis

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SLIDE 20

ITP History

  • Kohlrausch: KRF function in 1897.
  • Tiselius: Moving boudnary electrophoresis, 1930
  • Longsworth: Performed moving boundary electrophoresis in 1939.
  • Martin AJP: Displacement electrophoresis (also called ITP) in 1942

for cation.

  • Everaerts and Martin: First to perform ITP in thin capillaries (200

to 500 micron) in 1963. Used HEC to suppress EOF.

slide-21
SLIDE 21

1998

Highest ITP stacking Run-time: 6-10 min L = 53.5 cm

5500

LE: 10 mM NaOH titrated with H3PO4, TE: 6.13 mM THeACl titrated with H3PO4 NXX-066

Capillary UV

ITP

2000

Run-time: 10-13 min L = 64 cm

200

50 mM phosphoric acid with 20 % acetonitrile, DI water 1-naphthylamine, laudanosine

Capillary UV

FASI

1999

Run-time: 15-17 min L = 25 cm

1000

75 mM phosphate, DI water Bromide, nitrate, bromate

Capillary UV

FASI

1999

Run-time: 5-10 min L = 61 cm

300

1 mM phosphoric acid, 40 mM potassium dihydrogen phosphate Maleic, fumaric acids, bromide, nitrate

Capillary UV

LVSS

1996

Run-time: 4-6 min L = 24.6 cm

1000

45 mM NaH2PO4 and 15 mM Na2HPO4, 60% v/v 1-propanol Dese, Amino

Capillary UV

FASS

1992

First LVSS Run-time: 6-10 min L d) = 65 cm

  • 100 mM MES and 100 mM

histidine, DI water PTH-aspartic acid, PTH-glutamine acid

Capillary UV

LVSS

2005

Run-time: 2 min

500

LE: 250 mM NaCl, TE: 95 mM TAPS, 73 mM TEA Fluorescein

Microchip Fluore- scence

ITP

2002

Run-time: 1-2 min

530

LE: 25 mM imidazole, 20 mM HCl, TE: 160 mM imidazole, 40 mM HEPES eTags

Microchip Fluore- scence

ITP

2003

Narrow sample channel Run-time: 2 min

80

175 mM Phosphate, DI water Fluorescein disodium salt

Microchip Fluore- scence

FASS

2003

Five-channel geometry. Run-time: 1 min.

100

HEPES (0.1 mM and 100 mM), NaCl (0.2 mM and 200 mM) Fluorescein sodium salt

Microchip Fluore- scence

FASS

2001

Six-channel geometry Run-time: 2 min

65

Carbonate (200 μM and 32 mM) FITC-arginine

Microchip Fluore- scence

FASS

1995

First on-chip FASS Run-time: 20 sec

13.8

Sodium tetraborate (0.5 mM and 500 mM) didansyl-lysine

Microchip Fluore- scence

FASS

Ref. Comments SE Electrolyte Sample Microchip/ capillary Detection Mode Method

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SLIDE 22

Sensitivity in Capillary Electrophoresis

(no stacking)

1.E-14 1.E-12 1.E-10 1.E-08 1.E-06 1.E-04 1.E-02 1E-23 1E-21 1E-19 1E-17 1E-15 1E-13 1E-11

molar sensitivity (mol)

concentration (mol/l)

electrochemical detection fluorescence detection UV absorbance detection

UV absorbance mass spectrometric detection UV absorbance with Z-shaped flow cells radiochemical detection conductivity detection with ITP stacking conductivity detection amperometric detection indirect fluorescence detection end-column electrochemical detection thermooptical absorbance

Chen et al. (1996) Belder et al. (2002) Ocvirk et al. (1998)

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SLIDE 23

Leading Ion (LE) Sample Ion Trailing Ion (TE) Counterion not shown Order of mobility

ν

> ν >

ν

Single Interface Isotachophoresis

  • Characteristics
  • Sample zone grows with time.
  • Stable concentration boundaries
  • Final Sample Concentration depends on Leading ion concentration

E E

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SLIDE 24

ITP

(S-) Alexa fluor (1 µM) HEPES (5mM)(TE) HEPES (5mM) NaCl (large excess) (LE)

  • Concentration enhancement greater

than γ

  • Buffer selections allow for both ITP

and FASS

  • ITP-type stacking with CE

separation 50 µm γ = 393

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SLIDE 25

50 µm

Stability under large disturbances Stable over 1000+ diameters Stable across flow geometries

5 mm

50 µm

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SLIDE 26

ITP Optimization

  • Surface Chemistry: Suppressed EOF to minimize

advective dispersions.

  • High gradients: High LE concentration and very low

initial sample concentration (100 fM vs. 1 M or 1015 ratio)

  • Flow control: Injection protocol has

High TE concentration (maximizes local electric Peclet

number)

Requires no manual buffer exchange step (fast ITP to CZE

transition w/ minimal dispersion)

Large effective sample width LE introduced within TE region, not end of capillary (this

reduces time to overtake ITP zone)

  • Achieved 1E6 stacking in < 2 minutes

Jung, B., Bharadwaj, R., Santiago, J.G., in press, Analytical Chemistry, 2005.

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SLIDE 27

ITP and CE

50 µm

1 2 3 4 5 x 10

  • 4

200 400 600 800 1000 x (m) Normalized Intensity

γ = 393

(S-) Alexa fluor (1 µM) HEPES (5mM)(TE) HEPES (5mM) NaCl (large excess) (LE)

Demonstrated >1e6-fold stacking on chip Stacking achieved in less than 120 sec and < 1 cm

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SLIDE 28

CE Sensitivity revisited

10

  • 26

10

  • 24

10

  • 22

10

  • 20

10

  • 18

10

  • 16

10

  • 14

10

  • 12

10

  • 10

10

  • 16

10

  • 14

10

  • 12

10

  • 10

10

  • 8

10

  • 6

10

  • 4

10

  • 2

molar sensitivity [mol] concentration [mol/l]

UV absorbance thermooptical absorbance indirect fluorescence detection end-column electrochemical detection conductivity detection amperometric detection mass spectrometric detection

UV absorbance detection electrochemical detection fluorescence detection

radiochemical detection conductivity detection with ITP stacking On-chip fluorescence detection On-chip electrochemical detection

ITP stacking + High sensitivity detection system ITP stacking + Low sensitivity detection system High sensitivity detection system

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SLIDE 29

72 73 74 75 76 77 78

  • 5

5 10 15 20 25 30 time (sec) CS (pM)

ITP/CE: LIF high sensitivity detection

  • ITP stacking + High sensitivity CE detection system

Optimized LIF w/ 60x objective (NA = 0.9) objective and

PMT

Brief ITP stacking (~40s) followed by CE mode

  • Sample: 100 aM alexa fluor 488 and bodipy

alexa fluor 488 bodipy

1.6×105 2.1×105 CI 15.9 pM 21.4 pM CS,final 100 aM 100 aM CS,initi

al

bodipy

alexa fluor 103

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SLIDE 30

ITP Simulations

E = 500 V/cm

Buffer Counter-ion Trailing Leading

Collaboration with Bijan Mohammadhi, Montpellier University, France

Lin, Storey, Santiago, submitted to Journal of Fluid Mechanics, 2006. Baldessari et al., under preparation, 2006

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SLIDE 31

ITP Simulations, cont.

Modeling by H. Najm, B. Debussere of Sandia Natl. Labs

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SLIDE 32

Nanochannel electrophoresis

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SLIDE 33

Prior Work

Separation of DNA

Petersen/Alarie/Jacobson/ Ramsey

Molecular dynamics simulations

Qiao/Aluru

Ion depletion

Pu/Yun/Datta/Gangopadhyay/ Temkin/Liu

Transport and dispersion of neutral species Griffiths/Nilson,

Dutta/Kotamurthi

Conductance measurements

Stein/Kruithof/Dekker

Analytical studies of flow

Burgeen/Nakache, Rice/Whitehead

Experimental Theoretical

No work on charged species transport No work separations or ion dispersion dynamics

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SLIDE 34
  • Nanofluidics Thick EDL

Enables new functionality Method to determine both valence and mobility

Nanoscale Electrokinetics for small ions

Micro Nano U(y) U(y)

Slower moving +2 ion Faster moving +1 ion h~10 µm λd~20 nm h~100 nm λd~20 nm Ions moving at same speed −

Eapplied EEDL

+ − − + − + − + + + + + −

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SLIDE 35

Concentration Profiles

NEUTRAL NEGATIVE POSITIVE

50 nm 50 nm

Species is in quasi-steady transverse equilibrium

Valence = +2 Valence = +1 Valence = 0

( ( ) ) ( ) exp 1

x S c charged S S x

E z e y y u z FE G kT ε ζ ψ ψ ψ ν µ ζ ⎛ ⎞ − − ⎛ ⎞ = − − ⎜ ⎟ ⎜ ⎟ ⎝ ⎠⎝ ⎠

slide-36
SLIDE 36

Nanochannel Fabrication

3um

  • Techniques:
  • E-beam Lith < 500 nm
  • Photolith > 500 nm
  • Dry Plasma Etching
  • Fusion Bonding

detection area 7mm nanochannel tick marks

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SLIDE 37

Species Transport

slide-38
SLIDE 38

Experimental Results

Pennathur and Santiago. “Electrokinetic transport in nanochannels: 1. Theory,” Vol. 77, No. 21, Analytical Chemistry, pp. 6772-6781, 2005. Pennathur and Santiago “Electrokinetic transport in nanochannels: 2. Experiments,” Vol. 77, No. 21, Analytical Chemistry, pp. 6782-6789, 2005.

slide-39
SLIDE 39

Oligonucleotide Separation

  • Length scales on same order

Nanochannel height EDL thickness DNA length

3 Length Scales:

λM λD L

slide-40
SLIDE 40

Microchannel DNA Separation

Fluorescein DNA (1bp, 10bp, 25bp, 50bp, 100bp)

Conditions:

  • 10 mM Borate

Buffer

  • 1 nm EDL

thickness

  • DNA lengths: 1
  • 34 nm
  • 25 mm separation

length

slide-41
SLIDE 41

Nanochannel DNA Separation

Fluorescein DNA Fluorescein DNA

Conditions:

  • 10 mM Borate

Buffer

  • 1 nm EDL

thickness

  • DNA lengths: 1
  • 34 nm
  • 25 mm separation

length

slide-42
SLIDE 42

Nanochannel DNA Separation

(10 mM λD= 3 nm in 100 nm channel)

Experiments:

  • 5 buffer

concentrations

  • 6 bp lengths
  • 3+ realizations

per exp.

  • ~135 exp.
slide-43
SLIDE 43

Nanochannel DNA Separation

(1 mM λD= 10 nm in 100 nm channel)

Conditions:

  • 1 mM Borate

Buffer

  • 10 nm EDL

thickness

  • DNA lengths: 1
  • 34 nm
  • 25 mm separation

length

slide-44
SLIDE 44

DNA separation in nanochannels

Small ion theory

slide-45
SLIDE 45

Oligonucleotide Separation

  • Length scales on same order

Nanochannel height EDL thickness DNA length

3 Length Scales:

λM λD L

  • Interaction between

Transverse

electromigration

Non-uniform velocity

field/diffusion coupling

Steric interactions with

wall

Polarization @105V/cm

slide-46
SLIDE 46
  • 3D electrokinetic instability modeling
  • ITP modeling

Analytical models for shock width and concentration increase Numerical models including

  • N species
  • Reaction kinetics of buffer
  • pH gradients
  • DNA separation in nanochannels

Single bp resolution DNA separation in nanochannels

  • Roughly 1/3rd of human genome costs was reagents
  • Ultra high sensitivity

Molecular dynamics modeling (w/ Eric Shaqfeh)

  • Combine ITP and nanochannel electrophoresis

Near-Future Work

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SLIDE 47

Acknowledgements

Microfluidics Lab Members:

  • EK instabilities and micromixing
  • Dr. Michael Oddy*
  • Dr. Chuan-Hua Chen *
  • Dr. Hao Lin *
  • Dr. Jonathan Posner *
  • David Hertzog *
  • Sample pre-concentration
  • Rajiv Bharadwaj *
  • Byoungsok Jung
  • David Huber
  • Alexandre Persat
  • Particle tracking & control
  • Klint Rose
  • (Shankar Devasenathipathy) *
  • On-chip CE and nanochannel work
  • Tarun Khurana
  • Dr. Fabio Baldessari
  • Sumita Pennathur *
  • Alexandre Persat
  • Julien Sellier

Funding Sources:

  • NSF PECASE/CAREER Award
  • NIH/NIHLB Proteomics Grant
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SLIDE 48