Anal. Chem. 2003, 75, 910-917
Electrical Communication between Glucose
Oxidase and Electrodes Mediated by
Phenothiazine-Labeled Poly(ethylene oxide)
Bonded to Lysine Residues on the Enzyme Surface
Kazumichi Ban, Takeshi Ueki, Yoshinori Tamada, Takahiro Saito, Shin-ichiro Imabayashi, and
Masayoshi Watanabe*
Department of Chemistry and Biotechnology, Yokohama National University, Yokohama 240-8501, Japan
frequently used to shuttle electrons between FAD and the
electrode under an oxygen-free condition.2-7
A series of glucose oxidase (GOx) hybrids (GOx-phe-
nothiazine-labeled poly(ethylene oxide) (P T-P EO)) ca-
pable of direct electrical communication with electrodes
is synthesized by covalently modifying P T-P EO to lysine
residues on the enzyme surface. The length of the P EO
chain and the number of P T groups are systematically
altered. After the P T-P EO modification, all the hybrids
maintain more than 5 0 % of enzyme activity relative to that
of native GOx, although loss of the activity becomes
greater with increasing P EO chain length. The catalytic
current, icat, is observed at a potential more positive than
0 .5 5 V after the addition of glucose, due to the intramo-
lecular electron transfer (ET) from reduced forms of flavin
adenine dinucletide (FADH2 / FADH) to P T+ that are
electrogenerated at the electrode. The icat value increases
with the number of P T groups, indicating that most of the
modified P T groups act as mediators. The magnitude of
the icat increase depends on the P EO chain length and
reveals a maximum for P T-P EO with the molecular weight
of 3 0 0 0 . In contrast, the icat is almost constant for GOx-
2 -(1 0 -phenothiazyl)propionic acid (P T-P A) hybrids with
more than two P T groups synthesized by covalently
modifying P T-P A to surface lysines, indicating that only a
few key P T groups function as mediators. The maximum
rate constant (1 3 0 s-1 ) for the ET from FADH2 / FADH to
P T+ is obtained for the GOx hybrid modified with five
P T-P EO groups with the molecular weight of 3 0 0 0 .
In terms of the application to enzymatic sensor systems8 and
the electrochemical control of an enzymatic function, enzymes
capable of a direct electron exchange with electrodes have been
arousing great interest. One possible way to provide electrochemi-
cal activity for enzymes is the covalent immobilization of an
electron relay to the FAD unit9,10 or on the surface of GOx.11-16
Heller et al.11 and Schuhmann12 reported the synthesis and
electrochemical properties of GOx with ferrocene (Fc) derivatives
attached to the sugar or acidic amino acid residues on its surface
via spacer chains of different lengths. These studies demonstrated
that redox mediators bound via long, flexible, and hydrophilic
spacer chains to the outer surface of GOx can transfer electrons
to the electrode surface according to the so-called “wipe mecha-
nism” with the enzyme-bound mediators swinging in and out of
the active site of the enzyme. Although the number and the
location of mediators and the length of spacer chains are decisive
factors to achieve efficient mediation properties, they have not
been systematically controlled in the previous studies and their
(2) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins,
I. J.; Plotkin, E. V.; Scott, L. D.; Turner, A. P. F. Anal. Chem. 1 9 8 4 , 56,
667-671.
(3) Degani, Y.; Heller, A. J. Phys. Chem. 1 9 8 7 , 91, 1285-1289.
(4) Bartlett, P. N.; Bradford, V. Q.; Whitaker, R. G. Talanta 1 9 9 1 , 38, 57-63.
(5) Ikeda, T.; Hamada, H.; Senda, M. Agric. Biol. Chem. 1 9 8 6 , 50, 883-890.
(6) Taniguchi, I.; Miyamoto, S.; Tomimura, S.; Hawkridge, F. M. J. Electroanal.
Chem. 1 9 8 8 , 240, 333-339.
(7) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1 9 8 8 , 110, 2615-2620.
(8) Schuhmann, W.; Wohlschl¨ager, H.; Lammert, R.; Schmidt, H.-L.; Lo¨ ffler,
V.; Wiemho¨ fer, H.-D.; Go¨ pel, W. Sens. Actuators, B 1 9 9 0 , 1, 571-574.
(9) Katz, E.; Riklin, A.; Heleg-Shabtai, V.; Willner, I.; Bu¨ ckmann, A. F. Anal.
Chim. Acta 1 9 9 9 , 385, 45-58.
(10) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2 0 0 0 , 39, 1180-1218.
(11) Schuhmann, W.; Ohara, T. J.; Schmidt, H.-L.; Heller, A. J. Am. Chem. Soc.
1 9 9 1 , 113, 1394-1397.
(12) Schuhmann, W. Biosens. Bioelectron. 1 9 9 5 , 10, 181-193.
(13) Badia, A.; Carlini, R.; Fernandez, A.; Battaglini, F.; Mikkelsen, S. R.; English,
A. M. J. Am. Chem. Soc. 1 9 9 3 , 115, 7053-7060.
(14) Ryabov, A. D.; Trushkin, A. M.; Baksheeva, L. I.; Gorbatova, R. K.;
Kubrakova, I. V.; Mozhaev, V. V.; Gnedenko, B. B.; Levashov, A. V. Angew.
Chem., Int. Ed. Engl. 1 9 9 2 , 31, 789-791.
Glucose oxidase (GOx; EC 1.1.3.4) is a dimeric glycoprotein
of 186 kDa containing two tightly bound flavin adenine dinucle-
otide (FAD) cofactors, which catalyze the electron transfer (ET)
from glucose to oxygen accompanying the production of glucono-
lactone and hydrogen peroxide.1 The direct electrical communica-
tion between the redox center (FAD) and electrodes is prevented
because the redox center is located too far from the outermost
surface. Therefore, freely diffusing redox mediators with a more
positive redox potential than that of the redox center have been
(15) Bartlett, P. N.; Booth, S.; Caruana, D. J.; Kilburn, J. D.; Santamaria, C. Anal.
* Corresponding author: (e-mail) mwatanab@ynu.ac.jp.
(1) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J.
Mol. Biol. 1 9 9 3 , 229, 153-172.
Chem. 1 9 9 7 , 69, 734-742.
(16) Battaglini, F.; Bartlett, P. N.; Wang, J. H. Anal. Chem. 2 0 0 0 , 72, 502-
509.
910 Analytical Chemistry, Vol. 75, No. 4, February 15, 2003
10.1021/ac025872t CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/21/2003