cell

Single Cell Analysis with an Integrated Electrophoretic/ Electrochemical Chip

Ching-Yu CHANG 1, 2, Tatsuya MURATA2, Yasufumi TAKAHASHI2, Ryota KUNIKATA2, Hitoshi SHIKU2, Hsien-Chang CHANG1, Tomokazu MATSUE2*

redox recycling

secretion

catalyzeS

PoxiPred

Oxidation current

1-Institute of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan2-Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan

conductive substrate

electrode

Electrochemical Single Cell Measurement

Interference: ＋ ＋SNR: －Signal level: －

Interference : －SNR: ＋Signal level: ＋

Interference: － －SNR: ＋ ＋Signal level: ＋ ＋

Interference: ＋ ＋SNR: －Signal level: －

Micro Well Structure

25 m

15 m

5 m

LFVA(low flow velocity area)

100 m

30 m

Active vs. Passive Single Cell Trap

Passive Active

trapping force gravity or hydraulic force electrophoretic or dielectrophoretic force

cell placement random, large scale addressable, limited scale

Stabilization for the trapped cell by

no additional force applied keeping on the voltage

biocompatibility good concerns about the applied electrical field

cell array one kind hybrid

How to take advantages of

active and passive traps?

Pt

25 m

30 m

+2.0 V

SU- 8

30 m

Trapping solution: 0.2 M sucroseTrapping voltage: 2.0 V vs. ITO

Chip Design

electrode

SU-8

tape

ITO

＋－

Cell Manipulation

Electrophoretic trapping

Hydraulic flush

Electrophoretic repelling

－＋

＋－

- 2.0 V

+ 2.0 V

Top View

Steady-state Currents for Microelectrode

nFDCrI 4lim

rL

nFDCrI

4

4lim

Ilim

E

i

disc electrode

recessed disc electrode

parametersn: transferring electron / moleculeF: Faraday constantD: diffusion constantC: substance conc.r: electrode radiusL: recessed depth

Analyst, 2004 (129) 1157-65

Model for Recessed UME on a Conductive Substrate

Anal. Chem., 2007 (79) 5809-16

rl

d

normalized parameterH=l/r, L=d/r

conductive substrate

232

232

, )(ln)(lnln1

)(ln)(lnln

kLjLHiHhHg

fLeLHdHcHba

i

iI

T

TT

a b c d e f

1.79862 0.40135 0.16349 0.1994 1.79815 0.38238

g h i j K

0.67767 0.17304 0.015745 2.01384 0.33559

redox

UME Chip Electrode

Micro Well Electrode Validation

scan rate: 10 mV/sec2E configuration, Ag/AgCl as RE+CE5 mM K3Fe(CN)6 / 0.1 M KClscan direction: 0.6 0 0.6 V

πrL

πnFDCrI

4

4 2lim

L=23 m

< 30 m~ 12.9 m

5.5 nA

7.8 nA

I T =7.8/5.5= 1.42

I T = 1.07 (theoretical)

Measurement of Secreted Alkaline Phosphatase

Pt electrode

NH2ONH2HO + 2H+ +2 e-

p-iminoqulnone (IQ)PAP

0.3 V vs. Ag/AgCl

0.1 V vs. Ag/AgCl

SEAP

p-aminophenylphosphate (PAPP)SEAP

p-aminophenol (PAP)

PAPP

PAPPAP IQ

e-

recombinant HeLa

SEAP: secreted alkaline phosphatase

IQPAP

diffusion

ITO electrode

diffusionPAPP/HEPES

RE+CESolid line

WE

RE+CEDot line

WE

ITO

Redox Recycling on ITO Electrode

PAP 4.7 mM /HEPES (line a &b)PAPP 4.7 mM /HEPES (line c &d)HEPES buffer: HEPES 20 mM, NaCl 153 mM, KCl 5 mM, glucose 5 mM, pH 9.5

scan rate: 20 mV/secscan direction: 0 0.6 0 V

Measuring condition

detection voltage

PAP

PAPP

ALP-Bead Preparation

ALP (0.6U/mL) / HEPESovernight incubation

wash with HEPES,suspend in 2.35 mMPAPP

particle descending

UME RE+CE

latex bead: 10 mHEPES: pH 9.5

+ 0.3 V vs. Ag/AgCl n=3

Single ALP-Bead Measurement

bare bead ALP bead

PAPP PAP

PAPPblkbare iii PAPPAPPALP iii

iPAP depletion

Real-time SEAP Secretion Monitoring

SEAP Cell

Micro well

PAP calibration curve

Conclusion

Cell can be trapped and repelled by electrophoretic force.

Micro well structure can provide a LFVA to stabilize the trapped cell during solution change.

ITO electrode provide a conductive surface for redox recycling and then enhances the response current.

The real-time non-continuous SEAP secretion was observed by this device.

Thanks for your attention …

Entrapment and measurement of a biologically functionalized microbead with a microwell electrode

Ching-Yu Chang, Yasufumi Takahashi, Tatsuya Murata, Hitoshi Shiku, Hsien-Chang Chang* and Tomokazu Matsue* Lab Chip, 2009, 9, 1185–92

1 M H2SO4

1 M NaOH

Pd 微粒電析於 GC 表面之電位窗

不同電位電析 Pd 粒子於 GC 電極上的型態

電極 1

電極 2

電極 3

電極 1

電極 2

電極 3

電極 4

循環伏安法電析 Pd 粒子在 SnO2 電極上的型態

步階電位法電析 Pd 粒子在 SnO2 電極上的型態

電化學法測量不同 Pd 粒子表面型台的面積

電位階昇法：不同電透析條件下 Pd(GOD)/GC 電極於 PBS(pH 7.4) 中的循環伏安圖

酵素電極偵測葡萄糖的檢量線

http://www.cem.msu.edu/~cem252/sp97/ch24/ch24aa.html

Amino Acid Structures

Amino Acid -carboxylic acid -amino Side chain

Alanine 2.35 9.87

Arginine 2.01 9.04 12.48

Asparagine 2.02 8.80

Aspartic Acid 2.10 9.82 3.86Cysteine 2.05 10.25 8.00Glutamic Acid 2.10 9.47 4.07

Glutamine 2.17 9.13

Glycine 2.35 9.78

Histidine 1.77 9.18 6.10

Isoleucine 2.32 9.76

Leucine 2.33 9.74

Lysine 2.18 8.95 10.53

Methionine 2.28 9.21

Phenylalanine 2.58 9.24

Proline 2.00 10.60

Serine 2.21 9.15

Threonine 2.09 9.10

Tryptophan 2.38 9.39

Tyrosine 2.20 9.11 10.07

Valine 2.29 9.72

http://www.cem.msu.edu/~cem252/sp97/ch24/ch24aa.html

pKa Values of Amino Acid