Development of Observation Method of Living Cells and Tissue Stimulated Using Fine/Local/Micro Electrochemical Devices

微細・局所・微量電気化学デバイスを用いた生細胞・生組織の刺激観察法の開発
ー Glutamate Releasing Electrochemical Device グルタミン酸放出電気化学デバイス －Department of Electrical & Electronic Information Engineering 電気・電子情報工学系
Toshiaki Hattori 服部敏明
Introduction
Bio-imagingGlutamate➢ It reveals biological features with analyzing the distribution of chemical species.
⇒ In order to analyze stimulation behavior of cells and tissues,
a combination of stimulation and detection is important.
Detection

Stimulation

・Fluorescence microscope・Ion image sensor

・Physical stimulation
・Electric stimulation
・Chemical stimulation➢a most important excitatory transmitterin central nervous system
The results captured Ca2+ uptake by Glu−-dependent channelon the cell membrane.

Stimulus

TissueDecreasing of hippocampal [Ca2+]o 3

Increasing of [Ca2+]i of astrocytes 2
Stimulus by 500 µM glutamate

Stimulus by 3 mM glutamate(High Ca2+)

Detector

ΔVOut(Relative value)+10 mV(High Ca2+)

CA1

Chemical stimulation is excellent and effective, because it activates only specific receptors.

One of chemical stimulus: Ion-releasing electrochemical devices→ It can release chemicals at any position locally, and at any time by electrical switching.
Previous work: We successfully demonstrated a calcium ion releasing electrochemical
device using polymer coated electrode.1

DG(Low Ca2+)

Pre stim

CA31 mm
35 s

Pre stim

10 s−10 mV(Low Ca2+)[3] Doi, H. et al. in Proc. IEEE SENSORS 2019, Montreal, Canada,Oct. 27–30, 2019; C3LA, 1289.

[2] Jensen, A. M. et al. J. Neurosci. 1991, 11, 1674–1684.

If we will have a local glutamate stimulation device, we could reveal their
biological features in more details.
In this study, we report the glutamate release from Glu−-doped poly(3,4ethylenedioxythiophene).

We report a new glutamate releasing device. This device is unique to be able to release
glutamate by the circuit switch on without external battery.
[1] Hattori, T. et al. in Proc. 4th Asian Symp. Anal. Sci. Sendai, Japan, Sep. 12–13, 2018.

Glutamate ion releasing device
Real-time monitoring of Glu− released from the releasing device

Structure & Principle

A Glu− image sensor is used to evaluate the amount of
Glu− released from the device.

The device

The device

The device potential isshifted to reduction potentialof PEDOT.
⇓PEDOT is reduced,
the Coulomb force between
PEDOT and Glu is lost.
⇓Glu− are released.

Au

Au

PEDOT
Shift toreduction potential

PEDOT:Glu

φ1.6 mm releasing device(PD, 10.5 cycles)
Pt wireGlu− image sensor 4,5
It can monitor 2D distribution concentration of Glu− in real-time.
[4] Mizutani, S. et al. Sens. Mater. 2017, 29, 253–260.
[5] Okumura, Y. et al. in Proc. IMCS 2018, Vienna, Austria, July 15–19, 2018; pp.141–142.

Glu− image sensor

Potential difference / mV

Solution

Solution

0.03

Fabrication methods

mono-sodium L-glutamate(NaGlu)

Potentiodynamic mode

WE

Au disk (φ3 mm)

CE

Platinum wireRE

Ag|AgCl (sat. KCl)

Electrolyte
Appliedvoltage

Change in potential / V0A gold electrode was applied voltage in an aqueous solution
including EDOT and NaGlu.
Potnetiostatic mode

3,4-ethylenedioxythiphene(EDOT)

SwitchEDOT 0.01 M, NaGlu 0.1 Min H2O (3 mL, stirring)−0.9 ~ +1.2 V at 25 mV s−1+1.2 V for 30 min.
for 10.5 or 20.5 cycles+0.050s02 mm

151050Switch ON-5030 s

12060 s240360480600Time / s

Variation just under the releasing device.
15 mV → [Glu−] = 10−6 ~ 10−5 M

Real-time monitoring by the Glu− image sensor.

Effect of different polymerization conditions on the Electrochemical Device
The morphology and conductivity of the device electrode change depending on the polymerization conditions of PEDOT.6 PEDOT:Glu membranes werefabricated by two different V-t modes; ➢Potentiostatic (PS) mode: constant potential, ➢Potentiodynamic (PD) mode: linear triangle scan of potential.
[6] Castagnola, V. et al. Synth. Met. 2014, 189, 7–16.

The trace amount of Glu− released

Optical microscopic images of polymer surface

The amounts of Glu− released from the releasing device are quantitative by
L-Glutamate Assay Kit YAMASA NEO (YAMASA, Japan). Samples wereobtained by closing the following release system in the solution for 5 min.

The surfaces of the device electrode after polymerization were observed.
Bare Au

PD mode(10.5 cycles)

PS modePD mode(20.5 cycles)

Releasing system
50 µm

30Error bar:standard error (n = 3)

Switch500 µm

0.1 M Tris-HCl,
pH 7.5 (100 µL)[Glu−] / µM

φ3 mm releasing
device (WE)20

10Pt wire(CE)Cyclic voltammograms of the releasing devices

0PS mode

PD modePD mode(10.5 cycles) (20.5 cycles)

PS modenon-uniform
irreversiblelower

PD modeuniform
reversible
higherCyclic voltammograms were carried out in aqueous solution including 0.1 M NaGlu.
The potentials were scanned at 0.1 V s−1.
80

PS mode60

Current / µA

Current / µA

6040200-20-40-0.2

1st4th00.2 0.4 0.6 0.8

2nd5th1

3rd
1.2 1.4

Potential / V vs. Ag|AgCl80

PD mode(10.5 cycles)60

Current / µA

80

40
20040
200-20-20-40
-0.2

-40
-0.2

00.2 0.4 0.6 0.8

11.2 1.4

Potential / V vs. Ag|AgCl

Polymer surface
Cyclic voltammogram
The amount of Glu− released

PD mode(20.5 cycles)

00.2 0.4 0.6 0.811.2 1.4

Potential / V vs. Ag|AgCl

In the PS mode, rapid polymerization is carried out since the PEDOT
oxidation potential (1.2 V) is applied immediately. On the other hand, in thePD mode, slow polymerization is carried out since the device potential isgradually increased to the PEDOT oxidation potential. This affects uniformityof the membrane, Cyclic voltammogram, and the amount of Glu− released.
Moreover, in the PD mode, the larger number of cycles result in the larger
amount of Glu− released.

Currently we fabricate the Fine/Local/Micro Electrochemical Stimulation Device for cells and tissues.
Toyohashi University of Technology

Electroanalytical Chemistry Laboratory