cell
description
Transcript of cell
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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
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Electrochemical Single Cell Measurement
Interference: + +SNR: -Signal level: -
Interference : -SNR: +Signal level: +
Interference: - -SNR: + +Signal level: + +
Interference: + +SNR: -Signal level: -
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Micro Well Structure
25 m
15 m
5 m
LFVA(low flow velocity area)
100 m
30 m
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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?
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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
+-
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Cell Manipulation
Electrophoretic trapping
Hydraulic flush
Electrophoretic repelling
-+
+-
- 2.0 V
+ 2.0 V
Top View
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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
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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
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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)
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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
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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
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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
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Single ALP-Bead Measurement
bare bead ALP bead
PAPP PAP
PAPPblkbare iii PAPPAPPALP iii
iPAP depletion
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Real-time SEAP Secretion Monitoring
SEAP Cell
Micro well
PAP calibration curve
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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 …
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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
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1 M H2SO4
1 M NaOH
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Pd 微粒電析於 GC 表面之電位窗
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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
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循環伏安法電析 Pd 粒子在 SnO2 電極上的型態
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步階電位法電析 Pd 粒子在 SnO2 電極上的型態
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電化學法測量不同 Pd 粒子表面型台的面積
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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
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酵素電極偵測葡萄糖的檢量線
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http://www.cem.msu.edu/~cem252/sp97/ch24/ch24aa.html
Amino Acid Structures
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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