APPLICATION OF

ELECTROCHEMICAL METHOD
ON BIOSENSOR

Dyah Iswantini 1 and Tokuji Ikeda 2

1. Biopharmaca Research Center, Department of Chemistry

Faculty of Mathematics and Natural Sciences, Bogor
Agricultural University, Indonesia.
2. Division of Applied Life Sciences, Graduate School of
Agriculture, Kyoto University, Japan
 COOH

COOH HN
1 2
3
9
8
7 4
6 5
HOOC N O

Mg 2+ or Ca 2+
 In vivo Activation Process
GlcA
Glc
PQQ, Mg2+
Q0H2

Q0

PQQ Mg2+

GlcA D-glc

I = x I max
1 : Q0
2 : D-glucose
3 : PQQ
Q0H2 Q0 4 : MgSO4
5 : EDTA
D-Glc GlcA 6 : EDTA
 Sensor of glucose

Out put : current / CM ***

1. Electron acceptor *
2. E. coli cells incubated with PQQ **
3. D-glucose
PMS, Q0, DCIP, DBQ, Fe(CN)63-, or O2
** Activated E. coli cell
*** Concentration of Electron acceptor
 Catalytic activity of mGDH in E.coli
Electron E1/2/ KS/ KM zkcat
Acceptor mV* mM / mM /106 s-1

PMS -81 0.90 0.80 15

Q0 -75 0.45 1.1 6.7
DCIP 73 4.2 0.80 7.1
DCBQ 138 1.2 0.43 16
Fe(CN)63- 200 0.18 3.2 1.6
O2 0.14 <0.05 0.45

Oxidation of D-Glucose using Q0 as an electron acceptor.

* vs. Ag/AgCl
 Determination of number of enzyme

Out put : current / CM ***

Fixed amount of E. coli cells

equilibrate with various CPQQ *
1. Q0 ; CM >> KM
2. D-glucose ; CGlu >> KS

* Partially Activated E. coli cell

Free PQQ

Depends on the CPQQ

 Titration Curve

[ E. Coli ] = 11.3 pM

z : number of mGDH in a E. coli cell

z = [PQQ]eq/[E. coli] = 3.0 x 103
Kcat : in vivo catalytic constan
zKcat = 6.7 Kcat = 2.2 x 103 s-1
 Conclusions
1. An electrochemical method using E. coli cell modified
electrode has proved to be useful for glucose sensor,
monitoring the in vivo holo-mGDH formation, determining in
vivo catalytic activity of the enzyme and the number of m-
GDH in single E-coli K-12 cell.
2. In vivo catalytic activity of mGDH in E. coli cells has been
characterized by the three parameters, catalytic constant of E.
coli cell zkcat, apparent Michaelis constants for D-glucose KS
and for electron acceptors KM. The number of mGDH in a
single E. coli cell z is determined as z = 3.0103, and the in
vivo catalytic constant of mGDH in the cells kcat is calculated to
be kcat = 2.2103 s-1.
3. Deactivation and reactivation process of mGDH in E. coli cells
has been studied and the effect of EDTA on the process has
been discussed.