Uricase-catalyzed oxidation of uric acid using an artificial electron acceptor and fabrication of amperometric uric acid sensors with use of a redox ladder polymer

Article abstract of DOI:10.1021/ac981168u

Electrochemical oxidation of uric acid catalyzed by uricase (uric acid oxidase, UOx; EC 1.7.3.3) was studied using several redox compounds including 5-methylphenazinium (MP) and 1-methoxy-5-methylphenazinium (MMP) as electron acceptors for UOx, which does not contain any redox cofactor. It was found that MP and MMP were useful to mediate electrons from UOx to an electrode in the enzymatic oxidation of uric acid. A novel redox polymer, poly(N-methyl-o-phenyIenediamine) (poly-MPD), containing the MP units was also found to possess the mediation ability for UOx, and poly-MPD was immobilized together with UOx onto an electrode substrate covered with a self-assembled monolayer of 2-aminoethanethiolate with use of glutaraldehyde as a binding agent The resulting electrode (pory-MPD/UOx/Au) exhibited amperometric responses to uric acid with very fast response of ～30 s, allowing reagentless amperometric determination in a concentration range covering that in the blood of a healthy human being. Kinetic parameters of the apparent Michaelis constant and the maximum current response obtained at the poly-MPD/UOx/Au suggested that electrochemical oxidation of uric acid was controlled by diffusion of uric acid into the enzyme film and that the redox polymer worked well in mediating between active sites of UOx molecules and the electrode substrate.

Full text of DOI:10.1021/ac981168u

Anal. Chem. 1999, 71, 1928-1934
Uric a s e -Ca t a lyze d Ox id a t io n o f Uric Ac id Us in g a n
Art ific ia l Ele c t ro n Ac c e p t o r a n d Fa b ric a t io n o f
Am p e ro m e t ric Uric Ac id S e n s o rs w it h Us e o f a
Re d o x La d d e r P o lym e r
Ta ka hiro Na ka m ina m i, Shin-ic hiro Ito, Sus um u Kuw a ba ta ,* a nd Hiros hi Yone ya m a *
Department of Applied Chemistry, Faculty of Engineering, Osaka University,Yamada-oka 2-1, Suita, Osaka 565-0871, Japan
Electrochemical oxidation of uric acid catalyzed by uricase
uric acid oxidase, UOx; EC 1 .7 .3 .3 ) was studied using
several redox compounds including 5-methylphenazinium
MP ) and 1 -methoxy-5 -methylphenazinium (MMP ) as
due to uric acid at a given reaction time, the original concentration
of uric acid is determined.6,7 Several electrochemical measure-
(
ments have also been reported as the substitute. Potentiometric
8
(
determination of CO
determination of consumption rate of O 9
2
produced by reaction 1 and amperometric
-12
electron acceptors for UOx, which does not contain any
redox cofactor. It was found that MP and MMP were
useful to mediate electrons from UOx to an electrode in
the enzymatic oxidation of uric acid. A novel redox
polymer, poly(N-methyl-o-phenylenediamine) (poly-MPD),
containing the MP units was also found to possess the
mediation ability for UOx, and poly-MP D was immobilized
together with UOx onto an electrode substrate covered
with a self-assembled monolayer of 2-aminoethanethiolate
with use of glutaraldehyde as a binding agent. The result-
ing electrode (poly-MP D/ UOx/ Au) exhibited amperomet-
ric responses to uric acid with very fast response of ∼3 0
s, allowing reagentless amperometric determination in a
concentration range covering that in the blood of a healthy
human being. Kinetic parameters of the apparent Michae-
lis constant and the maximum current response obtained
at the poly-MP D/ UOx/ Au suggested that electrochemical
oxidation of uric acid was controlled by diffusion of uric
acid into the enzyme film and that the redox polymer
worked well in mediating between active sites of UOx
molecules and the electrode substrate.
2
may be useful, but
the results are largely influenced by solution pH and original
concentration of dissolved O . Amperometric determination of
2
13-15
H O is also useful,
2
2
but the potential (>0.4 V vs a saturated
calomel electrode (SCE)) at which anodic oxidation of H O takes
2
2
place is positive enough to cause direct oxidation at the electrode
of uric acid and other components such as ascorbate and
acetoaminophen dissolved in human fluid, resulting in inaccuracy
of the determinations.14,15 To solve this problem, the use of
horseradish peroxidase (HRP) together with UOx has been
proposed for enzymatic reduction of H
2 2
O , and then the ampero-
metric determination was achieved at less positive potentials of
.16,17
2 2
.5-0.15 V vs SCE than that causing oxidation of H O
0
]3- was
In our previous paper, it was reported that [Fe(CN)
6
found to work as an electron acceptor for UOx in place of O
2
in
the reaction given by eq 1 and that the concentration of uric acid
can be determined by a convenient way of oxidizing electrochemi-
]4- at a UOx-immobilized Au electrode
cally the produced [Fe(CN)
polarized at 0.1 V vs SCE.18 In this paper, we will show that several
6
(
(
(
2) Ullman, B.; Wormsted, M. A.; Cohen, M. B.; Martin, D. W., Jr. Proc. Natl.
Acad. Sci. U.S.A. 1 9 8 2 , 79, 5127-31.
3) Yamanaka, H.; Togashi, R.; Hakoda, M.; Terai, C.; Kashiwazaki, S.; Dan, T.;
Kamatani, N. Adv. Exp. Med. Biol. 1 9 9 8 , 431, 13-8.
4) Liang, M. H.; Fries, J. F. Ann. Intern. Med. 1 9 7 8 , 88, 666-70.
It is important to determine the concentration of uric acid
dissolved in human urine and/ or blood to diagnose diseases
caused by disorder of purine biosynthesis and/ or purine catabo-
(5) Simkin, P. A. Ann. Intern. Med. 1 9 7 9 , 90, 812-6.
(6) Dilena, B. A.; Peake, M. J.; Pardue, H. L.; Skoug, J. W. Clin. Chem. 1 9 8 6 ,
lism, such as gout, hyperuricemia, and Lesch-Nyhan syndrome.1-5
3
2, 486-91.
7) Feichtmeir, T. V.; Wrenn, H. T. Am. J. Clin. Pathol. 1 9 5 5 , 25, 833-9.
(8) Kawashima, T.; Rechnitz, G. A. Anal. Chim. Acta 1 9 7 6 , 83, 9-15.
9) Nanjo, M.; Guilbault, G. G. Anal. Chem. 1 9 7 4 , 46, 1769-72.
Several determination methods have been developed using uricase
(
(
uric acid oxidase, UOx; EC 1.7.3.3) which catalyzes in vivo
(
oxidation of uric acid as given by eq 1. Addition of UOx to samples
(
10) Janchen, M.; Walzel, G.; Neef, B.; Wolf, B.; Scheller, F.; Kuhn, M.; Pfeiffer,
D.; Sojka, W.; Jaross, W. Biomed. Biochim. Acta 1 9 8 3 , 42, 1055-9.
11) Uchiyama, S.; Shimizu, H.; Hasebe, Y. Anal. Chem. 1 9 9 1 , 66, 1873-6.
12) Uchiyama, S.; Suzuki, S.; Sato, T. Electroanalysis 1 9 9 0 , 2, 559-61.
13) Markas, A.; Gilmartin, T.; Hart, J. P. Analyst 1 9 9 4 , 119, 833-40.
14) Shaolin, M.; Jinqing, K.; Jianbing, Z. J. Electroanal. Chem. 1 9 9 2 , 334, 121-
UOx
(
(
(
(
uric acid + O2
8 allantoin + CO + H O
(1)
2
2
2
3
2.
in the presence of dissolved O
concentration of uric acid. By determining changes in absorbance
2
causes a decrease in the
(
(
(
15) Motonaka, J.; Miyata, K.; Faulkner, L. R. Anal. Lett. 1 9 9 4 , 27, 1-13.
16) Tatsuma, T.; Watanabe, T. Anal. Chim. Acta 1 9 9 1 , 242, 85-9.
17) Miland, E.; Ordieres, A. J. M.; Blanco, P. T.; Smyth, M. R.; F a´ g a´ in, C. OÄ .
Talanta 1 9 9 6 , 43, 785-96.
*
Corresponding author: (fax) +81-6(6879)7373; (e-mail) yoneyama@
ap.chem.eng.osaka-u.ac.jp.
1) Fox, I. H. Metabolism 1 9 8 1 , 30, 616-34.
(18) Kuwabata, S.; Nakaminami, T.; Ito, S.; Yoneyama, H. Sens. Actuators B 1998,
(
52, 72-7.
1928 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
10.1021/ac981168u CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/13/1999

Ch a rt 1 . Ch e m ic a l S t ru c t u re o f (a ) MP a n d (b )
MMP
Laboratories), and anthraquinone-1-sulfonate (Tokyo Chemical
Industries). All aqueous solutions were prepared using twice-
distilled water.
The ability of redox agents as an electron acceptor for UOx-
catalyzed oxidation of uric acid was tested by measuring the time
course of changes in their absorbance caused by reduction of the
oxidized form of the redox agent. The reaction was initiated by
adding 0.5 mmol dm- uric acid to N
3
-saturated 0.1 mmol dm-3
2
borate buffer (pH 8.5) containing the oxidized form of the redox
agent of desired concentration and 8.3 µmol dm- UOx at 30 °C.
A quartz cell having a light path length of 1 or 0.2 cm was used,
and absorption spectra were measured between 300 and 800 nm
using a diode array spectrophotometer (Hewlett-Packard, 8452A).
A Pt disk plate (0.88 cm diameter) was used as an electrode
substrate for electrochemical oxidation of uric acid, and an Au
disk plate of the same area was used as the electrode substrate
for immobilization of UOx and/ or a redox polymer. The electrode
substrates were polished successively with alumina slurries of 1.0
and 0.3 µm, subjected to ultrasonication in deionized water for 30
3
other redox species including 5-methylphenazinium (MP) and
1
-methoxy-5-methylphenazinium (MMP), whose chemical struc-
tures are shown in Chart 1, also serve as useful electron acceptors
for UOx. The most convenient amperometric uric acid sensors
would be an electrode on which electron mediator molecules are
immobilized together with UOx. Searches for improved electron
mediators for UOx have extended to the use of a novel redox
polymer of poly(N-methyl-o-phenylenediamine) (poly-MPD) con-
taining MP units in its polymer structure, which was synthesized
by methylation of chemically prepared poly(o-phenylenediamine)
26
min, soaked overnight in a piranha solution, and then mounted
(
poly-PD). The poly-PD, which can also be prepared by electro-
in a Teflon electrode holder which restricted the exposed area to
chemical polymerization of o-phenylenediamine, was already
2
0
.27 cm . Note: Piranha solution is a strong oxidant and must be
utilized as a permselective membrane in glucose sensors of high
used with extreme caution! A vapor-deposited Au on a glass plate
having an area of 17.5 cm was also used as the Au substrate for
selectivity.1
9-24
In this study, it will be shown that the poly-MPD
2
works well as a promising electron mediator for UOx and
reagentless uric acid sensors have been successfully prepared by
immobilizing this redox polymer onto cross-linked UOx films on
an Au substrate.
observations of the cross section of the prepared enzyme films
with a scanning electron microscope (SEM; Hitachi, S-800).
Immobilization of UOx and poly-MPD onto the electrode
substrate was accomplished as follows. The Au electrode was
immersed in 1 mmol dm- cystamine aqueous solution for 8 h to
deposit a self-assembled monolayer of 2-aminoethanethiolate,
3
EXPERIMENTAL SECTION
UOx from Candida sp. was commercially available from Wako
3
3
2
followed by casting 30 mm () 120 mm per 1 cm of electrode
Pure Chemicals and used without further purification. Poly-PD
was synthesized according to literature25 with the following partial
modifications. Ammonium peroxosulfate (1 mol dm-3) as an
oxidizing agent dissolved in dimethyl sulfoxide (DMSO) was
added drop by drop to give a final concentration of 170 mmol dm-
to an agitated 50 cm3 of DMSO containing 50 mmol dm-3
o-phenylenediamine monomer. The colorless solution gradually
turned red and a black precipitate of poly-PD appeared. After the
solution was stirred for 12 h, the resulting suspension was filtered,
and the precipitate was washed several times with distilled water
and acetone to remove unreacted monomer and low-molecular-
weight products. Poly-MPD was obtained by N-methylation of poly-
-3
area) of 20 mmol dm phosphate buffer (pH 7.0) containing 8.3
µmol dm-3 UOx, 1.8 g dm-3 poly-MPD, 1 wt % (∼80 mmol dm-3)
glutaraldehyde (GA), and 10 vol % ethanol, and then the electrode
was kept in a glass vessel filled with dry N at 35 ( 1 °C for 6 h
2
3
to allow the binding of amino residues of UOx molecules with
the amino groups of 2-aminoethanethiolate on the Au substrate
via glutaraldehyde. When electrodes having different amounts of
poly-MPD were prepared, the concentration of poly-MPD in the
cast solution was varied without changing the concentrations of
the other components. The resulting electrodes were rinsed well
with phosphate buffer (pH 7.0) to remove weakly adsorbed
species. When only UOx was desired to be immobilized onto the
gold electrode, the same procedure was employed but the
phosphate buffer solution used did not contain poly-MPD and
ethanol. In this paper, the electrodes modified with both UOx and
poly-MPD and those with UOx alone will be denoted as poly-
MPD/ UOx/ Au and UOx/ Au, respectively.
3
PD. Ethanol (50 cm ) containing 2 mmol of monomer units of
poly-PD was mixed with 2 mmol of methyl iodide and refluxed at
6
0 °C for 24 h. Then, the solvent and unreacted methyl iodide
were evaporated under reduced pressure. All other chemicals used
were of analytical grade and obtained from Wako Pure Chemicals
except for thionin (3,7-diaminophenothiazinium) (Aldrich), cys-
tamine (Aldrich), 1-methoxy-5-methylphenazinium sulfate (Dojindo
The enzymatic activity of UOx immobilized on the Au substrate
was determined by measuring the time course of a decrease in
the concentration of uric acid in the presence of dissolved O
2
.6
For this purpose, 2 cm of air-saturated 0.1 mol dm borate buffer
pH 8.5) was put in a quartz cell having a light path length of 1
(
(
(
19) Sasso, S. V.; Pierce, R. J.; Walla, R.; Yacynych, A. M. Anal. Chem. 1 9 9 0 ,
2, 1111-7.
20) Malietesta, C.; Palmisano, F.; Torsi, L.; Zambonin, P. G. Anal. Chem. 1 9 9 0 ,
2, 2735-40.
21) Centonze, D.; Palmisano, F.; Torsi, L.; Zambonin, P. G. Electroanalysis 1994,
, 423-9.
22) Lowry, J. P.; O’Neill, R. D. Electroanalysis 1 9 9 4 , 6, 369-79.
23) Trojanowicz, M.; Geschke, O.; Krawczynski vel Krawczyk, T.; Cammann,
K. Sens. Actuators B 1 9 9 5 , 28, 191-9.
3
-3
6
(
6
cm and then the UOx/ Au or the poly-MPD/ UOx/ Au was
immersed in it. An aliquot of 20 mmol dm- uric acid dissolved
in the borate buffer was added to the quartz cell as to give a
concentration of 0.1 mmol dm-3, and absorbance of the solution
3
6
(
(
(
(
24) Wang, J.; Chen, L.; Liu, J.; Lu, F. Electroanalysis 1 9 9 6 , 8, 1127-30.
25) Premasiri, A. H.; Euler, W. B. Macromol. Chem. Phys. 1 9 9 5 , 196, 3655-66.
(26) Hayes, W. A.; Shannon, C. Langmuir 1 9 9 6 , 12, 3688-94.
Analytical Chemistry, Vol. 71, No. 10, May 15, 1999 1929

time after addition of uric acid, and the spectrum approached that
of reduced MMP, which is shown by a broken line in Figure 1.
Since the injection of uric acid into the solution in the absence of
UOx did not cause any changes in the absorption spectrum, the
results given in Figure 1 should have resulted from such electron-
transfer events that uric acid molecules were oxidized by MMP
with catalysis of UOx.
UOx
MMP + uric acid
8 reduced MMP + products (2)
The inset of the figure shows the time course of changes in
the concentration of MMP, which was obtained from the absor-
bance peak at 510 nm. The initial rate of the enzymatic oxidation
of uric acid was determined to be 46.4 µmol dm- min-1, and the
rate became gradually low with time, approaching the reaction
equilibrium. If the reduced MMP is directly oxidized by electro-
chemical means, the uric acid concentration can easily be
determined from the oxidation currents, as will be shown later.
Unlike flavine adenine dinucleotide (FAD)-containing redox
enzymes such as glucose oxidase and cholesterol oxidase, UOx
does not have any redox cofactor.27,28 Accordingly, the electron
transfer from UOx to oxidized MMP cannot be explained on the
basis of redox reactions of UOx. The following discussion have
3
Fig u re 1 . Changes in absorption spectra of N2-saturated 0.1 mol
-
3
-3
dm borate buffer (pH 8.5) containing 0.25 mmol dm MMP and
3
0
.83 µmol dm- UOx caused by addition of 0.5 mmol dm-3 uric acid.
The time given is that elapsed after addition of uric acid. Broken line
-
3
shows the spectrum of MMP reduced by 0.2 mmol dm sodium
hydrosulfite in the same solution composition. Inset shows time course
of the concentration of MMP obtained from the absorbance peak at
510 nm.
been presented for the case of using O
for UOx. UOx-catalyzed oxidation of uric acid may proceed with
an ordered mechanism where uric acid and O are bound to UOx
2
as an electron acceptor
at 290 nm which is due to uric acid, was monitored. Using a molar
absorptivity of uric acid of 1.22 × 10 dm mol cm-1 the rate of
4
3
-1
2
2
consumption of 1 µmol of uric acid per min () 1 unit) per cm of
to give a trimolecular transitory complex which is dissociated to
give the products.27 An important reaction step in that case is an
activation step of uric acid molecule at UOx prior to transferring
electrode area was determined.
Amperometric responses at the poly-MPD/ UOx/ Au to uric
acid were measured at 0 V vs SCE using a potentiostat (Bioana-
lytical Systems, BS-1) in 0.1 mol dm- borate buffer (pH 8.5). A
one-compartment electrochemical cell was used and it was
electrons to O
2
.28 Considering that MMP can work as an electron
3
acceptor like dissolved O
the case of MMP, too.
2
, the same mechanism may be valid for
2
equipped with a platinum foil of 2 cm and an SCE as a counter
UOx-Catalyzed Electrochemical Oxidation of Uric Acid
with Use of an Electron Mediator. Electrochemical detections
of uric acid using UOx and an electron mediator were carried
out using three kinds of electrolyte solutions: borate buffer
solution (pH 8.5) containing UOx and one of redox agents listed
in Table 1, borate buffer containing only the redox agent, and
unadulterated borate buffer solution. The results obtained for
using p-benzoquinonesulfonate (BQS) as the redox agent are
shown in Figure 2. A Pt working electrode used was polarized at
and a reference electrodes, respectively. In this paper, potentials
referred to SCE will be cited for all cases. The cell was placed in
an incubator controlled at 45 ( 1 °C during the course of
measurements. The electrolyte solution was deoxygenated by
bubbling N
2
for 20 min prior to the measurements. When constant
background currents were obtained, an aliquot of 20 mmol dm-
uric acid dissolved in the borate buffer was added to the electrolyte
solution to give the desired concentration, followed by agitation
of the electrolyte solution for 5 s. The time course of the oxidation
currents in quiescent solution was monitored on an electric
polyrecorder (Toa Denpa, EPR-151A). Steady currents obtained
3
0
.3 V which was positive enough to oxidize the reduced BQS,
i.e., p-hydrobenzoquinonesulfonate (H BQS). As shown by curve
2
a in the figure, if a borate buffer containing BQS and UOx was
used as an electrolyte solution, anodic currents increased with
time after adding uric acid to the solution and reached a steady
value of 20.4 µA cm-2. The results then seem to suggest that BQS
works as an electron mediator between UOx molecules and the
Pt electrode, but anodic currents were also obtained with the use
of the electrolyte solution in the absence of UOx, as shown by
curve b in the figure, though the magnitude of the steady currents
was almost half of that shown by curve a. If the borate buffer
containing neither UOx nor BQS was used, much smaller
oxidation currents, which are related to direct oxidation of uric
acid at the Pt electrode, appeared as shown by curve c. The
oxidation currents obtained in the presence of BQS only were
are termed here as the current response (i
to give the steady currents after the addition of uric acid as the
response time (t ). In the measurements of amperometric re-
r
) and the time required
r
sponses at the UOx/ Au, the same procedure was employed except
that the electrolyte solution containing 1 mmol dm- redox agent
in its oxidized state was used. Cyclic voltammetry was performed
using an electrochemical analyzer (Bioanalytical Systems, BAS-
3
1
00B/ W) connected to a Gateway 2000 computer.
RESULTS AND DISCUSSION
Artificial Electron Acceptor for UOx-Catalyzed Uric Acid
Oxidation. Figure 1 shows changes in absorption spectra caused
2
by addition of uric acid of MMP dissolved in N -saturated borate
buffer containing UOx. As shown in this figure, absorbance peaks
of MMP, which appear at around 380 and 510 nm, decreased with
(
27) Kahn, K.; Tipton, P. A. Biochemistry 1 9 9 7 , 36, 4731-8.
(28) Kahn, K.; Serfozo, P.; Tipton, P. A. J. Am. Chem. Soc. 1 9 9 7 , 119, 5435-42.
1930 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999

indicating that they do not possess any ability for oxidation of
uric acid without UOx. As shown in the table, if redox agents
having redox potentials more negative than BQS were used, direct
oxidation of uric acid was suppressed, as shown for ferricyanide,
MP, and MMP. From the viewpoint of selective detection of uric
acid, MP and MMP are preferable, because only the UOx-
catalyzed uric acid oxidation currents are seen. Redox agents
having even more negative redox potentials than MP and MMP
gave no current response to uric acid, suggesting that the redox
potential of uric acid complexed with UOx is approximately
between -0.21 and -0.34 V. Since the one-electron redox potential
of free uric acid is reported to be 0.02 V,29 uric acid is said to be
activated at UOx by ∼0.2 V for its oxidation.
Ta b le 1 . Dire c t a n d UOx -Ca t a lyze d Ele c t ro c h e m ic a l
Ox id a t io n o f Uric Ac id a
redox
electrode
b
c
d
e
redox
compound
potential / potential/
I
1
/ µA
I
2
/ µA
3
I / µA
-2
-
2
-2
V vs SCE
V vs SCE
cm
cm
cm
r
UOx/ %
FC f
BQS
ferricyanide
MP
MMP
thionin
AQS g
0.28
0.20
0.15
-0.18
-0.21
-0.34
-0.52
0.38
0.30
0.10
-0.08
-0.11
-0.10
-0.10
13.1
20.4
8.93
0.175
0.165
0
5.57
11.7
0.130 0.130
0
0
0
0
5.57
2.55
57.5
42.5
98.5
100
100
0
0
0
0
0
a
Performed under experimental conditions similar to those listed
in Figure 2 except that the redox compound and electrode potential
b
listed were used. Estimated as an intermediate potential between
anodic and cathodic peak potentials in the cyclic voltammogram at Pt
-
3
It took more than 12 min to obtain steady-state currents after
addition of uric acid to an electrolyte solution containing dissolved
UOx and MMP. The time course of changes in oxidation currents
in that case was quite consistent with changes in the concentration
of MMP caused by the enzyme reaction as shown in the inset of
Figure 1. It is then concluded that the current response was deter-
mined by an equilibrium concentration of MMP in its reduced
state in the solution bulk, and the response time by the time
required to attain the equilibrium, as already discussed for the
electrochemical oxidation of cholesterol catalyzed by cholesterol
oxidase.30
electrode taken in N2-saturated 0.1 mol dm borate buffer (pH 8.5)
-
3
c
containing 1 mmol dm redox compound listed. Steady-state oxida-
tion currents of uric acid obtained in the presence of UOx and the
d
redox agent listed. Steady-state oxidation currents of uric acid
e
obtained in the presence of the redox agent only. Steady-state currents
for direct oxidation of uric acid in the absence of UOx and the redox
compound. f Ferrocenecarboxylate. g Anthraquinone-1-sulfonate.
It was shown in our previous paper that the concentration of
uric acid can be determined using UOx-immobilized Au electrodes
in the presence of ferricyanide as a dissolved electron mediator.18
The immobilization of UOx on the electrode was made by binding
amino residues of UOx molecules with amino groups of a self-
assembled monolayer of 2-aminoethanethiolate on the Au elec-
trode substrate with the assistance of glutaraldehyde (UOx/ Au;
see Experimental Section). In the present study, attempts were
made to prepare UOx/ Au electrodes using the same technique.
The resulting electrode was found to have an enzymatic activity
of 9.68 units cm-2. When 0.1 mmol dm uric acid was injected in
a borate buffer containing 1 mmol dm- MMP, oxidation currents
appeared at 0 V, indicating that MMP worked as the electron
-3
Fig u re 2 . Time course of changes in oxidation currents at Pt
3
-
3
electrode after addition of 0.1 mmol dm uric acid to N2-saturated
-
3
-3
0
.1 mol dm borate buffer (pH 8.5) containing (a) 0.83 µmol dm
-3
-3
UOx and 1.0 mmol dm BQS, (b) 1.0 mmol dm BQS, and (c) none
mediator for the immobilized UOx molecules. A steady current
response of 0.072 µA cm-2 was obtained and the response time
of these compounds. Electrode potential, 0.3 V vs SCE.
was a little shorter than 1 min, which was much shorter than that
obtained when dissolved UOx was used (12 min). The following
detection scheme seems valid for the appearance of the fast
response observed. When UOx-immobilized gold electrode is
used, reduction of MMP takes place in the thin UOx film on the
gold electrode, and the resulting reduced MMP is then anodically
oxidized at the electrode substrate before it is diffused away into
the solution bulk. The response time must be determined by the
time needed for uric acid in the whole enzyme film to diffuse to
the immobilized UOx.31
much greater than those obtained by the direct oxidation of uric
acid, indicating that chemical oxidation of uric acid by BQS as an
oxidizing agent took place to give H
the Pt electrode.
2
BQS, which was oxidized at
Since the electron mediator has to pick up electrons from uric
acid bound to UOx selectively without any serious interferences,
the selectivity (rUOx) was evaluated for the redox agents listed in
Table 1 with the simple definition as given by eq 3, where I
1
are
rUOx (%) ) 100(I - I )/ I
(3)
Electrochemical P roperties of P oly-P D and P oly-MP D
and Their Abilities as Electron Acceptors for UOx. If MMP
or MP molecules are immobilized on UOx-covered electrode
substrate, the resulting electrode will work as an amperometric
uric acid sensor which does not require any reagents in the
1
2
1
currents obtained in the presence of both UOx and a redox agent
and I are those obtained in the presence of the redox agent only.
2
According to this definition, BQS gave an rUOx of 42.5%. Similar
tests were carried out for several redox agents other than BQS,
and the results are summarized in Table 1. The redox agents of
ferrocenecarboxylate (FC) and ferricyanide did not give any
(29) Jovanovic, S. V.; Simic, M. G. J. Phys. Chem. 1 9 8 6 , 90, 974-8.
(
30) Nakaminami, T.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1 9 9 7 , 69, 2367-
2.
7
(
31) Nakaminami, T.; Ito, S.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1 9 9 9 ,
71, 1068-76.
2 3
noticeable difference between I and direct oxidation currents I ,
Analytical Chemistry, Vol. 71, No. 10, May 15, 1999 1931

Ch a rt 2 . Ch e m ic a l S t ru c t u re o f (a ) P o ly-P D a n d
b ) P o ly-MP D
(
Fig u re 3 . Cyclic voltammograms of Au electrode coated with (a)
poly-PD (poly-PD/Au) and (b) poly-MPD (poly-MPD/Au) taken in N2-
-
3
-1
measurements. However, it is not easy to immobilize covalently
MMP and MP molecules onto the electrode surface, because they
saturated 0.1 mol dm borate buffer (pH 8.5). dE/dt ) 50 mV s
.
3
The electrodes were prepared by casting 10 mm of ethanol contain-
ing 6 g dm of each polymer onto Au substrates, followed by drying
-
3
do not have any functional groups available for covalent
at 35 ( 1 °C for 5 h.
attachments.3
2-38
Also physical incorporation of MMP and MP
molecules into a cross-linked UOx network on the electrode
substrate is of little significance because of their high solubility
in water.39-43 So we searched for a redox polymer44-49 that can
work as a mediator for UOx when entrapped in the immobilized
UOx layer formed on an electrode substrate.
The redox polymer poly-PD, obtained by oxidative polymeri-
zation of o-phenylenediamine, possesses a ladderlike structure in
which phenazine units are organized as shown by (a) of Chart
∼
-0.65 V at pH 8.5, which seems to be from the phenazine
5
2
moiety, whose redox potential is ∼-0.6 V. Since MP, which
works as the electron acceptor for UOx as shown in Table 1, is
derived by N-methylation of phenazine, attempts were made to
quaternize the nitrogen atom of poly-PD with a methyl group. The
prepared poly-MPD is believed to have the chemical structure as
given by (b) of Chart 2. Cyclic voltammograms of Au electrodes
coated with poly-PD (poly-PD/ Au) and poly-MPD (poly-MPD/
Au) in borate buffer (pH 8.5) are shown in Figure 3. Both
.50,51 It was reported that the poly-PD exhibits an electrical
2
conductivity of ∼10 S cm-1 due to its π-conjugation.52 The
-4
3
electrodes were prepared by casting 10 mm of ethanol containing
polymer also shows redox activities with a redox potential of
g dm- of one of the polymers onto the Au electrode substrate,
3
6
(
(
(
32) Kuwabata, S.; Okamoto, T.; Kajiya, Y.; Yoneyama, H. Anal. Chem. 1 9 9 5 ,
7, 1684-90.
33) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J. Am. Chem. Soc. 1 9 9 2 , 114,
0965-6.
34) Willner, I.; Riklin, A.; Shoham, B.; Rivenzon, D.; Katz, E. Adv. Mater. 1 9 9 3 ,
, 912-5.
followed by drying at 35 ( 1 °C for 5 h. The voltammograms as
shown in this figure were not significantly changed by repeating
cycles for at least 1 h. Both the poly-PD/ Au and the poly-MPD/
Au exhibited redox activities due to the deposited polymers. From
an average of peak potentials of anodic and cathodic waves, the
redox potentials of poly-PD and poly-MPD were evaluated to be
6
1
5
(
(
35) Willner, I.; Katz, E.; Willner, B. Electroanalysis 1 9 9 7 , 9, 965-77.
36) Bardea, A.; Katz, E.; B u¨ ckmann, A. F.; Willner, I. J. Am. Chem. Soc. 1 9 9 7 ,
1
19, 9114-9.
∼
-0.67 and ∼-0.27 V, respectively. The more positive redox
(
(
(
37) Foulds, N. C.; Lowe, C. R. Anal. Chem. 1 9 8 8 , 60, 2473-8.
38) Degani, Y.; Heller, A. J. Phys. Chem. 1 9 8 7 , 91, 1285-9.
39) Iwakura, C.; Kajiya, Y.; Yoneyama, H. J. Chem. Soc., Chem. Commun. 1 9 8 8 ,
potential of poly-MPD results from the presence of an electron-
donating methyl group at the nitrogen atom, although the
N-methylation seems to have occurred in different degrees in the
polymer, as suggested from the appearance of broad redox waves.
If the pH of an electrolyte solution was changed, the redox
potentials of poly-PD and poly-MPD were changed at a rate of
1
019-20.
40) Kajiya, Y.; Sugai, H.; Iwakura, C.; Yoneyama, H. Anal. Chem. 1 9 9 1 , 63,
9-54.
41) Kajiya, Y.; Tsuda, R.; Yoneyama, H. J. Electroanal. Chem. 1 9 9 1 , 301, 155-
4.
42) Kajiya, Y.; Matsumoto, H.; Yoneyama, H. J. Electroanal. Chem. 1 9 9 1 , 319,
85-94.
43) Bartlett, P. N.; Ali, Z.; Eastwick-Field, V. J. Chem. Soc., Faraday Trans. 1 9 9 2,
8, 2677-83.
44) Calvo, E. J.; Danilowicz, C.; Diaz, L. J. Chem. Soc., Faraday Trans. 1 9 9 3 ,
9, 377-84.
45) Calvo, E. J.; Danilowicz, C.; Diaz, L. J. Electroanal. Chem. 1 9 9 4 , 369, 279-
2.
46) Calvo, E. J.; Etchenique, R.; Danilowicz, C.; Diaz, L. Anal. Chem. 1 9 9 6 , 68,
186-93.
47) Hodak, J.; Etchenique, R.; Calvo, E. J.; Shinghal, K.; Bartlett, P. N. Langmuir
9 9 7 , 13, 2708-16.
(
(
(
(
(
(
(
(
4
6
∼
-0.07 V pH-1, indicating that protonation and deprotonation
1
reactions were involved in the redox reactions of both polymers.
Judging from the reported results that similar shifts in the redox
potential were observed for phenazine and MP molecules,52,53 the
protonation and deprotonation seem to a occur at methyl group-
free nitrogen atom in the redox reaction of poly-PD and poly-MPD.
If 0.1 mmol dm- uric acid was added to 0.1 mol dm-3 borate
8
8
8
3
4
buffer (pH 8.5) in the presence of dissolved UOx under polariza-
tion of the poly-MPD/ Au at 0 V, a current response of 80 nA cm-2
1
(
(
(
(
(
48) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1 9 8 9 , 111, 2357-8.
49) Gregg, B.; Heller, A. J. Phys. Chem. 1 9 9 1 , 95, 5976-80.
50) Wu, L.-L.; Luo, J.; Lin, Z.-H. J. Electroanal. Chem. 1 9 9 6 , 417, 53-8.
51) Wu, L.-L.; Luo, J.; Lin, Z.-H. J. Electroanal. Chem. 1 9 9 7 , 440, 173-82.
52) Chiba, K.; Ohsaka, T.; Ohnuki, Y.; Oyama, N. J. Electroanal. Chem. 1 9 8 7 ,
was obtained with a response time of ∼30 min. Since no oxidation
currents were obtained in the absence of UOx, the current
(53) Komura, T.; Funahashi, Y.; Yamaguchi, T.; Takahashi, K. J. Electroanal.
Chem. 1 9 9 8 , 446, 113-23.
2
19, 117-24.
1932 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999

Fig u re 5 . Dependence of the current response (ir) at the poly-MPD/
UOx/Au electrode on the concentration of uric acid in N2-saturated
-
3
0
.1 mol dm borate buffer (pH 8.5) obtained for the poly-MPD/UOx/
Au prepared by using cast solution containing poly-MPD of (0) 1.2,
-3
(
4) 1.5, (b) 1.8, (O) 2.4, (2) 3.3, and (9) 4.2 g dm (see Experimental
Fig u re 4 . Time course of currents at the poly-MPD/UOx/Au obtained
Section). Electrode potential; 0 V vs SCE.
-
3
by stepwise addition of 0.1 mmol dm uric acid in N2-saturated 0.1
-
3
mol dm borate buffer (pH 8.5). Electrode potential, 0 V vs SCE.
ability, the efficiency of which is as high as that of MMP dissolved
in the solution.
response must arise from oxidation of the poly-MPD which picks
up electrons from the UOx-uric acid complex produced in the
solution bulk. The poly-PD/ Au electrode, on the contrary, did not
exhibit any current responses, indicating that poly-PD has a too
negative redox potential of -0.60 V to capture electrons from the
UOx-uric acid complex, the results being consistent with those
listed in Table 1.
Figure 5 shows i
ing different amounts of poly-MPD as a function of the uric acid
concentration. For all cases, the i values increased with increase
r
obtained at the poly-MPD/ UOx/ Au contain-
r
in the uric acid concentration, indicating that the poly-MPD/ UOx/
Au worked as a reagentless amperometric uric acid sensor. The
concentration range shown in Figure 5 covers that of uric acid in
healthy human blood (0.15-0.40 mmol dm-3).3-5,15,54 As recog-
nized in this figure, amperometric response to uric acid was
dependent on the amount of poly-MPD, and the highest sensitivity
of the electrode was obtained at an intermediate amount of poly-
MPD of 1.8 mg dm-3. To discuss the presence of the optimum
Amperometric Response of P oly-MP D/ UOx/ Au. Both
poly-MPD and UOx were immobilized on the Au substrate with
the assistance of a self-assembled monolayer of 2-aminoethanthi-
olate and a cross-linking agent of GA. The prepared electrode is
denoted as poly-MPD/ UOx/ Au. SEM observations of the cross
section of the resulting electrode revealed that an enzyme film
whose thickness was 1.1 ( 0.02 µm was adhered on the Au
substrate. The film was durable against vigorous agitation of the
solution in which the electrode was immersed. However, if the
enzyme film was prepared on a naked Au substrate, it was easily
exfoliated from the electrode substrate, suggesting the importance
of covalent bond formation between the Au substrate and the
enzyme film of UOx via the self-assembled monolayer. The
enzymatic activity of UOx of the poly-MPD/ UOx/ Au electrode
was 8.01 units cm- as determined by consumption rates of uric
acid in the presence of oxygen as an electron acceptor, and the
electrode showed amperometric sensitivities to uric acid, as shown
in Figure 4. The addition of 0.1 mmol dm-3 uric acid under
polarization of the electrode at 0 V gave a significant increase in
amount of poly-MPD, kinetic parameters for the poly-MPD/ UOx/
Au electrode were obtained. Plots of the reciprocals of i
r
as a
-1
function of C
S
(Lineweaver-Burk plots) allowed us to determine
app
the apparent Michaelis constant for uric acid (K
M
) and the
maximum current response (Imax).37,55-58
ir-1 ) (1 + KMappCS-1)I
-1
(4)
max
2
S
where C is the concentration of uric acid. The electrodes prepared
by using various amounts of poly-MPD satisfied the relation given
app
by this equation. Figure 6 shows the obtained K
M
and Imax as
functions of the amount of poly-MPD in the casting solution. The
app
obtained K
M
M
were much larger than the Michaelis constant (K )
-
2
r
anodic currents, and steady currents, i , of 0.066 µA cm were
of dissolved native UOx, which ranges from 10 to 10-1 mmol
dm- and showed a tendency to increase with an increase in the
-2
-
3
obtained within ∼30 s. If another 0.1 mmol dm uric acid was
3
-
3
added (total concentration, 0.2 mmol dm ), currents increased
further and reached again another steady value. The magnitude
(54) Harper, H. A. Review of Physiological Chemistry, 17th Asian ed.; Lange
obtained at 0.1 mmol dm-3 uric acid was comparable to that
Medical: San Francisco, 1979; Chapter 14.
of i
r
(
55) Segel, I. H. Enzyme Kinetics; Wiley-Interscience: New York, 1993; Chapters
and 9-I.
obtained at the UOx/ Au in the presence of dissolved MMP, where
2
0
.072 µA cm- was obtained. The enzymatic activities of poly-
2
(56) Foulds, N. C.; Lowe, C. R. J. Chem. Soc., Faraday Trans. 1 1 9 8 6 , 82, 1259-
MPD/ UOx/ Au (8.01 units cm 2) and UOx/ Au (9.68 units cm-2
-
)
64.
(
(
57) Riklin, A.; Willner, I. Anal. Chem. 1 9 9 5 , 67, 4118-26.
58) Liaudet, E.; Battaglini, F.; Calvo, E. J. J. Electroanal. Chem. 1 9 9 0 , 293, 55-
were comparable, too, suggesting that the immobilized poly-MPD
in the poly-MPD/ UOx/ Au possesses the electron mediation
68.
Analytical Chemistry, Vol. 71, No. 10, May 15, 1999 1933

UOx/ Au prepared in the present study, the film should also
become dense with an increase in the immobilized amount of poly-
MPD, as indicated by the increase in K
M
app. Presumably, a small
fraction of the loaded polymer would be accommodated into free
spaces which are present between the immobilized bulky UOx
enzymes of a molecular mass of 130 kDa. Because the theoretical
prediction is made for a model enzyme electrode with use of
dissolved electron mediator in solution and under the assumption
that the diffusion and partition coefficients of reaction substrates
are constant, the results shown in the figure might not completely
be explained by the prediction. An alternative speculation is that
an increase of poly-MPD causes an increase in the number of
UOx molecules adjacent to the poly-MPD, resulting in the increase
of Imax up to the optimum amount of poly-MPD. Beyond the
optimum amount of poly-MPD, however, the film should become
dense and the diffusion of uric acid through the film would be
retarded to suppress the Imax value obtained at the electrode. Both
of these effects, i.e., increase in thickness and density of the film,
seems to be related to the observed variation in the current
response depending on the amount of loaded poly-MPD.
Fig u re 6 . Maximum current response (Imax) and apparent Michaelis
constant (KM ) of the poly-MPD/UOx/Au as a function of amount of
app
poly-MPD contained in the cast solution used for preparation of poly-
app
MPD/UOx/Au. The values of Imax and KM
were obtained from
Lineweaver-Burk plots for the results shown in Figure 5. The curves
are aids to the eye.
amount of poly-MPD. It is thought that permeation of uric acid
into the film of the poly-MPD/ UOx/ Au influences the kinetics in
the electrochemical oxidation of uric acid at this electrode;44,59,60
the permeation is reduced as the amount of poly-MPD increases
and the film becomes dense. As shown in the figure, Imax also
increased with increasing amount of poly-MPD up to ∼1.8 mg
CONCLUSIONS
In the present study, UOx-catalyzed oxidation of uric acid with
use of an artificial electron acceptor was achieved for the first
time to our best knowledge. Since the electron acceptor, e.g., MP
and MMP, has the redox potential as negative as ∼-0.2 V vs SCE
(at pH 8.5), the electrochemical oxidation of uric acid catalyzed
by UOx was accomplished in potentials where direct anodic
oxidation of uric acid was not simultaneously induced. These
findings lead us to attempt to fabricate an amperometric uric acid
sensor, and a redox ladder polymer of poly-MPD was found to be
suitable for an electron mediator, which can be immobilized on
an Au electrode substrate. However, in order for this novel
polymer mediator to be applied to other biosensor systems, more
detailed investigations of its electrical and electrochemical proper-
ties have to be made. The amperometric uric acid sensor (poly-
MPD/ UOx/ Au) which does not require the use of special
reagents in test solutions was successfully constructed in the
present study, but further characterizations of the poly-MPD/
UOx/ Au are also needed to obtain information on the stability of
the electrodes and influences of the possible interfering com-
-
3
dm , above which a gradual depletion of Imax was observed. Imax
is the current response obtained at an excess of the analyte and
is usually larger for a higher concentration of immobilized
enzyme.4
4-46,61
However, since the same amount of UOx was
immobilized on all poly-MPD/ UOx/ Au electrodes prepared in the
present study, Imax values might have been determined by another
cause.
It was reported that Imax obtained at the glucose oxidase-
immobilized electrodes for glucose detection varied with the
thickness of the enzyme film, even if the same amount of the
enzyme was immobilized on the electrode.6
2-64
According to the
and some other
theoretical predictions made by Hall et al.6
3-65
groups,66,67 the sensor response should increase with an increase
in the thickness of the enzyme layer up to an optimum thickness,
beyond which it decreases, because in such thick films the analyte
is entirely consumed before reaching the electrode substrate and
enzymatic reaction occurs only near the film/ solution interface.
Such prediction seems valid for the results shown in Figure 6,
since the film thickness would be increased with an increase in
the amount of poly-MPD in the film. However, for the poly-MPD/
2
pounds which are present in blood or urine such as O and
ascorbate. For example, the working potential of this sensor of 0
V vs SCE may still cause oxidation of ascorbate more or less.
The investigations concerning these subjects are now under way
and will be reported in future.
ACKNOWLEDGMENT
(
59) Sundaram, P. V.; Tweedale, A.; Laidler, K. J. Can. J. Chem. 1 9 7 0 , 48, 1498-
04.
The present work was supported by the Asahi Glass Founda-
tion and by Grant-in-Aids for Scientific Research (10555305) from
the Ministry of Education, Science, Culture, and Sports, Japan.
5
(
(
(
(
(
(
(
(
60) Horvath, C.; Engasser, J.-M. Biotechnol. Bioeng. 1 9 7 4 , 16, 909-23.
61) Bartlett, P. N.; Pratt, K. F. E. J. Electroanal. Chem. 1 9 9 7 , 397, 61-78.
62) Bartlett, P. N.; Whitaker, R. G. J. Electroanal. Chem. 1 9 8 7 , 224, 37-48.
63) Gooding, J. J.; Hall, E. A. H. Electroanalysis 1 9 9 6 , 8, 407-13.
64) Gooding, J. J.; Hall, E. A. H. J. Electroanal. Chem. 1 9 9 6 , 417, 25-33.
65) Martens, N.; Hall, E. A. H. Anal. Chem. 1 9 9 4 , 66, 2763-70.
Received for review October 27, 1998. Accepted February
26, 1999.
66) Bartlett, P. N.; Whitaker, R. G. J. Electroanal. Chem. 1 9 8 7 , 224, 27-35.
67) Sorochinskii, V. V.; Kurganov, B. I. Biosens. Bioelectron. 1 9 9 4 , 9, 481-9.
AC981168U
1934 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999