Analytica Chimica Acta 480 (2003) 109–117

Solid-state pH ultramicrosensor based on a tungstic oxide

film fabricated on a tungsten nanoelectrode and its
application to the study of endothelial cells
Katsunobu Yamamoto a , Guoyue Shi b,∗ , Tianshu Zhou c , Fan Xu a , Min Zhu b ,
Min Liu b , Takeshi Kato a , Ji-Ye Jin d , Litong Jin b
a Laboratory of Research and Development, Bioelectroanalytical System Inc., 1-36-6 Oshiage, Sumida-Ku, Tokyo, Japan
b Department of Chemistry, East China Normal University, Shanghai 200062, China
c Department of Environmental Science, East China Normal University, Shanghai 200062, China
d Instrumental Analysis Center, Gifu University, Yanagido, Gifu 501-1193, Japan

Received 6 June 2002; received in revised form 5 December 2002; accepted 11 December 2002

Abstract
In this paper, preparation of a novel pH ultramicrosensor and its physiological application has been discussed. A tungsten
nanoelectrode was produced by an etching method in 0.1 mol/l NaOH solution at the potential of +0.4 V (versus Ag/AgCl
reference electrode) for about 100 s and the diameters ranged from 500 to 800 nm. The pH ultramicrosensor was fabricated by
producing WO3 at W nanoelectrode surface by electrooxidation in 2.0 mol/l H2 SO4 solution between 1.0 and 2.0 V. At last,
Nafion was coated on the surface of WO3 to protect the pH ultramicrosensor. The W/WO3 pH ultramicrosensor exhibited
a good pH linear region from 2.0 to 12.0 with a super-Nernstian slope of −53.5 ± 0.5 mV/pH unit. Response times ranged
from 3 s at about pH 6.0–7.0 up to 15 s at high pH. An interference of various ions to the pH measurement was also studied in
this paper. We also studied the lifetime, stability and reproducibility of the W/WO3 pH ultramicrosensor. In order to testing
the performance of W/WO3 ultramicrosensor, we applied it to measure the extracellular pH values and a pH variation was
also given about the normal, damaged and recovery endothelial cells.
© 2002 Elsevier Science B.V. All rights reserved.
Keywords: W/WO3 pH ultramicrosensor; Endothelial cells

1. Introduction amperometric electrode [1] and potentiometric elec-

The pH sensor is a kind of chemical sensor, which
has been used widely in many fields, such as sci-
entific, industrial, agriculture, and environmental,
clinical and other concern. Scientists have developed
many methods for the pH determination, including
∗ Corresponding author. Tel.: +86-21-6223-2627;
fax: +86-21-62232627.
E-mail address: iamshgy@hotmail.com (G. Shi).
trode [2]. Among them, the most commonly used
pH electrode is the glass electrode because of its
high selectivity, stability and wide pH range. But the
conventional glass electrode also has its obvious lim-
itations, such as the fragility of the glass, difficulty to
remodel its shape and a long time needed to reform
a hydrate layer before use. It is necessary to develop
many suitable pH sensors to use in both aqueous and
non-aqueous medium. Miniaturized pH sensor is also
required in cell determination.

0003-2670/03/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0003-2670(02)01602-1
110 K. Yamamoto et al. / Analytica Chimica Acta 480 (2003) 109–117
During the last decade, efficient efforts have been
given on the designing non-glass pH sensors to over-
come the above-mentioned limitations of the glass
electrode. Many related papers have been published
for pH determination since the earliest example of
the use of a polymer film modified electrode as a pH
sensor reported by Heineman et al. [3]. The modified
polymers include polyaniline, poly(vinyl chloride),
poly(3-cyclohexyl thiophene) and other organic mate-
rials [4–7]. Excellent properties in chemistry, optics,
thermodynamics and mechanics make the sol–gel
technology be used in the fabrication of pH sensor
[8–12]. Many other efficient methods for pH deter-
mination in various samples were also developed in
recent years [13–15].
Furthermore, metal/metal oxide pH electrode re-
ceived more and more attention with its excellent
characteristics of easy miniaturization, a long lifetime
and a strong tip. Several methods have been used to
produce metal oxide for pH sensing. They can be
prepared by thermal decomposition [16], by sputter
deposition [17,18] or by electrochemical growth [19].
Yao and co-workers [20] used iridium oxide electrode
for pH measurement. Honda et al. applied Sb/Sb2 O3
as the pH sensor. Wipf et al. [21] also used iridium
oxide electrode as the tip of scanning electrochemical
microscope (SECM). Antimony microdisk electrodes
for the measurement of pH near electrode surface
with SECM were prepared from antimony shot by
Bard and co-workers [22]. The electrochemical be-
havior of tungsten and tungsten oxide was discussed
by previous work [23–25]. Wrighton and co-workers
also coated a 0.15 ␮m thick layer of polycrystalline
WO3 on Au or Pt microelectrode and studied the pH
characteristics of WO3 [17].
Along with the developing of the field of life sci-
ence, the need for miniaturization of pH sensor has
been recognized. Intracellular and extracellular pH
contribute importantly to the control of many critical
physiological processes. The sensor should be of very
small size to allow spatial resolution and to minimize
distortion of the tissue, which is necessary for reliable
physiological measurements. The pH ultramicrosen-
sors as small as a few micrometers in tip diameter have
been constructed [5,26–29] for in vivo studying, cells
research or ultramicroenvironmental analysis, etc. In
this paper, a novel W/WO3 ultramicrosensor for pH
determination was prepared. Firstly we fabricated a W
nanoelectrode with a tip of about 500–800 nm. Then
WO3 film was produced on the surface of W na-
noelectrode by cyclic voltammetry (CV) in 2.0 mol/l
H2 SO4 solution between 1.0 and 2.0 V at a scan rate
of 20 mV/s. The importance of endothelial cells in the
regulation of vascular biology is being increasingly
recognized. But up to now, there are few papers to
study the relationship between pH variation and dam-
aged endothelial cells directly with miniaturized pH
sensor. Therefore, the main goal of the present study is
the development of pH ultramicrosensor for the mon-
itoring of pH value from cultured endothelial cells
and observes the pH variation generated from normal,
damaged and recovery endothelial cells. A flavonoid
Chinese traditional medicine was also given to the
damaged endothelial cells and then the extracellular
pH value was recovered to normal level.
2. Experimental
2.1. Chemicals and apparatus
Hanks balanced salt solution (HBSS) was prepared
as following: NaCl 8.0 g/l, Na2 HPO4 ·2H2 O 0.06 g/l,
KCl 0.4 g/l, KH2 PO4 0.06 g/l, MgSO4 ·7H2 O 0.2 g/l,
glucose 1.0 g/l, and CaCl2 ·2H2 O 0.19 g/l, pH = 7.4.
Nafion (1 wt.%) was purchased from Aldrich Chem-
ical Company. Deoxygenated with nitrogen (above
99.99%), HBSS was used as buffer solution in all ex-
periments except real sample analysis. All aqueous
solutions were prepared with doubly distilled water.
All electrochemical measurements were performed
with an ALS/CHI 900Electrochemical Workstation
(BASJ, Tokyo, Japan). W wire (10 ␮m diameter)
was purchased from Nilaco company of Japan.
Three-electrodes system included a Pt wire (0.5 mm
diameter) as counter electrode, a modified Ag/AgCl
wire (0.2 mm diameter) as reference electrode, and a
W/WO3 pH ultramicrosensor as working electrode.
Scanning electron micro-analyzer (JSM-5610LV,
Japan) was used to characterize the etching W nano-
electrode. For real sample analysis, an XDS-1 model
stereomicroscope (Chongqing Optical and Electrical
Instrument Company, China) equipped with video
and a WK-2 model micromanipulator (Xi’an North-
west Optical & Electrical Company, China) was used
as instruments for guiding micromanipulation.
 K. Yamamoto et al. / Analytica Chimica Acta 480 (2003) 109–117
2.2. Preparation of the W/WO3 ultramicrosensor
2.2.1. Preparation of W nanoelectrode
A 10 ␮m W wire was connected to a copper wire
with silver conductive paint. Then it was treated in
an etching solution (0.1 mol/l NaOH solution) with a
constant potential of +0.4 V. Every 10 sec, the W wire
was viewed under the stereomicroscope until the diam-
eter we need was received. Fig. 1A is the photograph
of scanning electron microscopy of W nanoelectrode.
At last, the etched W wire connected to a copper wire
was aspirated into a standard glass capillary (Esselte
Leitz, Germany), which was pulled in advance into
pipette with a tip of about 20␮m. The extremity of the
pipette was fixed with ethanol flame, and another ex-
Fig. 1. (A) The photograph of scanning electron microscopy of
tungsten nanoelectrode. (B) The schematic presentation of the
W/WO3 ultramicrosensor.
111
tremity was filled with epoxy resin in order to assure
tightness. The redundant W wire of the tip was cut off
carefully under the microscopy.
2.2.2. Construction of W/WO3 ultramicrosensor
Prior to oxidation, W nanoelectrode was ultra-
sonically cleaned with acetone, 1.0 mol/l NaOH, 1:1
HNO3 and double distilled water, respectively. A Pt
wire as counter electrode, a modified Ag/AgCl wire
as reference electrode, the W nanoelectrode was cy-
cled in the potential range between +1.0 and +2.0 V
in 2.0 mol/l H2 SO4 at the scan rate of 20 mV/s for 20
cycles. The WO3 film was produced on the surface of
W nanoelectrode. Then the W/WO3 ultramicrosensor
was immersed in 2.0 mol/l H2 SO4 solution for about
12 h.
Many substances, such as protein and enzyme exist
in cell samples. They can be absorbed on the surface of
W/WO3 pH ultramicrosensor and effect its sensitivity
and response time. So 2 ␮l 1% Nafion was drop on
the W/WO3 ultramicrosensor surface and air-dried for
10 min. Fig. 1B is the schematic presentation of the
preparation of a W/WO3 pH ultramicrosensor.
In vivo experiments were performed in HBSS un-
der constant temperature (37 ± 0.5 ◦ C) condition.
The three-electrode system (containing a Pt counter
electrode, a Ag/AgCl reference electrode and the
W/WO3 pH ultramicrosensor) was fixed together to
keep the distance of each electrode unchangeable
before it was placed as near as possible close to the
cells.
2.3. pH response measurement
The electromotive force (emf) of the W/WO3 ul-
tramicrosensor was measured as a function of pH of
the test solution. The base solution was HBSS just
as ‘Section 2.1’ mentioned to which either 1.0 mol/l
H3 PO4 or 1.0 mol/l NaOH was added dropwise to
obtain the desired pH value. The dynamic range of
this method is about pH 2.0–12.0. The emf results of
sample solutions were recorded till potential values
became stable. A commercial pH glass electrode was
also used to calibrate the W/WO3 pH ultramicroe-
sensor. A thermostat controlled the temperature of all
the test solutions at 37.0 ± 0.5 ◦ C. The whole appara-
tus was housed in a Faradic cage for eliminating the
interferences.
112 K. Yamamoto et al. / Analytica Chimica Acta 480 (2003) 109–117
2.4. pH ultramicrosensor selectivity
The selectivity of the W/WO3 pH ultramicrosensor
was test under conditions relevant to the physiologi-
cal applications. The W/WO3 ultramicrosensor, along
with the reference electrode and counter electrode,
Fig. 2. The photos of (A) normal group (100 times); (B) damaged
group (250 times) and (C) recovery group (100 times).
was immersed in the pH 7.4 buffer solution. After po-
tential stabilization, different solution of LiCl, NaCl,
KCl, NH4 Cl, CaCl2 or CuCl2 prepared with pH 7.4
buffer solution, was added. The changes of emf were
recorded.
2.5. Endothelial cells culture and pH
measurement of endothelial cells
The endothelial cells were prepared from the sepa-
rated cells of bovine aortic by grown in gelatin-coated
culture plates at 37 ± 0.5 ◦ C. According to the method
of reference [30], cells were fed every 2 days with
RPMI 1640 medium supplemented with 15% bovine
calf serum, 100 U/l penicillin and 100 U/l strepto-
mycin. The cells were divided into two parts, one
was a normal group and the other one was a damaged
group, which was done by ischemic treatment for 15
and 30 min. Fig. 2A and B are the photos of normal
and damaged taken with a stereomicroscope (XDS-1,
Chongqing optical & electrical instrument company).
Then they were all given a flavonoid chinese tradi-
tional medicine. Fig. 2C is the photo of recovery cells
taken with the stereomicroscope.
Before electroanalytical monitoring of pH values
of endothelial cells, the slide used for cell growth on
was washed and filled with HBSS. Endothelial cells
attaching on the slide were counted under the in-
verted stereomicroscope, then placed in 4 cm diameter
petri-dishes, and kept in an incubator at 37.0 ± 0.5 ◦ C
for at least 10 min. Counted under a stereomicroscope,
the amount of the cells was about 1.0 × 106 /ml. Then
the cells were grown statically at 37 ◦ C in a humidi-
fied atmosphere of 95% air and 5% CO2 . After 6–8 h,
the cells settled to the bottom of the culture well. pH
values were recorded from the normal, damaged and
recovery endothelial cells groups.
3. Results and discussion
3.1. Characteristics of the W/WO3 pH
ultramicrosensor
Fig. 1A is a photograph of scanning electron mi-
croscopy (SEM) of tungsten nanoelectrode, which has
etched in 0.1 mol/l NaOH solution at a constant po-
tential of +0.4 V. From Fig. 1A, we can see that the
 K. Yamamoto et al. / Analytica Chimica Acta 480 (2003) 109–117
detailed diameter is about 500–800 nm. It is because
that tungsten is oxidized to dissolved WO4 2− ions un-
der diffusion control by mass transport of OH− ions
as the overall reaction [23].
The electroformed species dissolve chemically in
the electrolysis media. Therefore, we can get differ-
ent size of W electrodes through controlling different
etching time in NaOH solution.
Many scientists have studied the electrochemical
characteristics of tungsten and tungsten oxide in al-
kaline and acidic media [17,23,31–33]. We have also
produced WO3 by electrooxidation in acidic solution.
According to Section 2.2.2, we prepared the W/WO3
pH ultramicrosensor by cycling the W nanoelectrode
in the potential range between +1.0 and +2.0 V at the
scan rate of 20 mV/s for 20 cycles in 2.0 mol/l H2 SO4 .
It is obvious that the thickness of the WO3 film
covered on the W nanoelectrode is very important for
the pH determination. Fig. 3 is the effect of different
cycle number of CV to the response time of W/WO3
ultramicrosensor at different pH solution. Fig. 3 dis-
plays the W/WO3 ultramicrosensor has excellent
response time at 10 or 20 cycles, and the pH ultra-
microsensor exhibits prolonged response time when
the cycle numbers exceed 30 cycles. Furthermore, we
Fig. 3. Effect of different cycle numbers of CV on the response time
of W/WO3 ultramicrosensor at different pH solution A: 10 cycles;
B: 20 cycles; C: 30 cycles; D: 40 cycles.
113
also found that the pH ultramicrosensor had poor se-
lectivity when a 10 cycle number of CV was selected.
So the optimum condition was achieved at the thick-
ness of 20 cycle number of CV ranging from +1.0 to
+2.0 V at the scan rate of 20 mV/s. We can see the
response time is as short as 3 s at pH 6–7 and the
response time is more than about 15 s at extreme pH
of 2 or 12 as shown in Fig. 3. It is beneficial for the
pH determination under the physiological condition.
It is because that WO3 undergoes the following
reversible, proton-dependent redox reaction [17] in
buffer solution:
WO3 + nH+ + ne− ⇔ Hn WO3
The reversible reaction changes the ratio of the
oxide in the film, the membrane potential will also
change subsequently.
3.2. Emf of the W/WO3 pH ultramicrosensor
The pH sensing performance of the W/WO3 ultra-
microsensor was valuated in pH buffer solution. Fig. 4
shows the emf versus Ag/AgCl reference electrode
of the pH ultramicrosensor and commercial glass
electrode at different pH test solutions. It proves that
the W/WO3 ultramicrosensor has a super-Nernstian
response with slope of −53.5 mV/pH unit. Linear pH
Fig. 4. An emf responses of W/WO3 ultramicrosensor and com-
mercial glass pH sensor at different pHs in HBSS with either
1.0 mol/l H3 PO4 or 1 mol/l NaOH added for adjustment to the
desired pH thermostated at 37.0 ± 0.5 ◦ C. (a) glass pH electrode;
(b) W/WO3 pH ultramicrosensor.
114 K. Yamamoto et al. / Analytica Chimica Acta 480 (2003) 109–117

Table 1
Calibration of different W/WO3 pH ultramicrosensors.
Electrode number pH range Slope (mV) R2

1 2.0–12.0 −53.5 0.9990

2 2.0–12.0 −53.8 0.9972
3 2.0–12.0 −53.4 0.9982
4 2.0–12.0 −53.0 0.9979

responses are obtained over a wide pH range of

2.0–12.0 (γ = 0.9990). It is clearly evident that the
characteristics of the W/WO3 pH ultramicrosensor
are very good and well equivalent to the performance
of a commercial glass electrode. Table 1 summarizes
the results of four different W/WO3 ultramicrosen-
sors prepared with the same method. These slopes
are within the range of −53.0 to −53.8 mV. The
following experiments were all done by the no. 1
ultramicrosensor.
The variation of the W/WO3 ultramicrosensor’s re-
sponse was determined from step changes of pH buffer
solution after injection of H3 PO4 or NaOH, as shown
in Fig. 5A and B. As can be seen, the response time
is about 4–6 s in spite of pH from 7.4 to 6.0 or from
7.4 to 9.8.

3.3. Selectivity of the W/WO3 pH ultramicrosensor

Selectivity is very important for pH determination

in biological samples. So the potentiometric selectivity
of the ultramicrosensor for hydrogen ion against ma- Fig. 5. The dependence of the emf response of the W/WO3 pH
jor interfering ions was studied using the mix-solution ultramicrosensor to an injection of (A) 1 mol/l H3 PO4 and 1 mol/l
method in this paper. Table 2 is the results of the ion NaOH into pH 7.4 buffer solution.
selectivity coefficients expressed as log KM,H . In addi-
tion, many electroactive substances, such as ascorbic
acid, urate and neurotransmitters also do not interfere ultramicrosensor is selective for pH determination
with the measurement at the level near physiological and is suitable for the following research work.
concentration. The data prove that the W/WO3 pH Some papers have reported that the Nafion film also
responds to many cations. Anson group [36] found
Table 2 that the electrode modified with redox couples incor-
Ion selectivity coefficients of no. 1 ultramicrosensor porated in Nafion coating has good responses to Li+ ,
Ions KM,H H+ , Na+ and K+ , Zn2+ , Mg2+ . Ugo et al. [37] also
Li+ 8.2
employed the Nafion coated glass carbon electrode for
Na+ 9.6 preconcentrating and detecting Fe2+ and Fe3+ cations
K+ 9.2 from aqueous solutions. So the Nafion modified elec-
NH4 + 7.4 trode is not a selective pH sensor. In this paper, we
Ca2+ 11.0 modified Nafion film on the surface of WO3 . The
Cu2+ 10.7
thickness of the Nafion film was estimated to be only
 K. Yamamoto et al. / Analytica Chimica Acta 480 (2003) 109–117
about 0.05–0.1 ␮m. It has poor response for H+ , espe-
cially in alkali solution and strong acid. But the WO3
film that we prepared on the surface of W wire has ex-
cellent selectivity for H+ determination and response
in the range of pH 2.0–12.0. The similar applications
of Nafion/metal oxide pH sensors have also been re-
ported in recent years [28,38].
3.4. Stability, reproducibility and lifetime of the
W/WO3 pH ultramicrosensor
The W/WO3 ultramicrosensor was test over a pe-
riod of 7 days after fabrication. The particular data
are shown as Fig. 6. Fig. 6 displays that the emf has
slightly variation during the period of 7 days. Further-
more, the value only varied about 10% in 2.0 mol/l
H2 SO4 solution after 1 month of storage. Emf re-
sponses have been determined in HBSS by the same
electrode and the relative standard deviation is 1.5%
(n = 8). This indicates that the pH ultromicrosensor
is stable.
3.5. pH Measurement of normal, damaged and
recovery endothelial cells
The cells were cultured as Section 2.4. In terms of
the culture medium, the experimental samples divided
Fig. 6. The emf responses of W/WO3 pH ultramicrosensor in pH
6.9 buffer solution.
115
into 12 groups, including 6 normal groups, 6 ischemia
groups that were cultured for ischemic treatment for
15 and 30 min, respectively. The above-described
W/WO3 pH ultramicrosensor was used to determine
pH of all the samples. Then a flavonoid medicine
was injected into the normal and damaged groups,
which are the recovery groups. All the experiments
to monitor pH of endothelial cells were performed in
Faraday cage incubated at 37.0 ± 0.5 ◦ C.
Fig. 7 is the pH data recorded with all samples of
endothelial cells. From Fig. 7, we can see the pH of
normal group is about 7.4. When the cells are treated
of ischemia for 15 min, pH value changes from 7.4
to 6.9. The W/WO3 pH ultramicrosensor shows a
variation of 0.5 pH. Along with the treatment of is-
chemia for another 15 min, the pH value of the dam-
aged endothelial cells decreases continuously and at
last reached pH 6.2. This phenomenon is because is-
chemia can result in the excess production of lactic
acid and accumulation of protons, namely acidosis in
biology. This result is similar to the previous studies
[28,34,35].
Flavonoids are a ubiquitous group of polyphenolic
substances, which are present in most plants, concen-
trating in seeds, fruit skin or peel, bark, and flowers.
Fig. 7. pH measurements of (A) normal group; (B) damaged group
(ischemia for 15 min); (C) damaged group (ischemia for 30 min);
(D) normal group after giving medicine; (E) damaged group after
giving medicine.
116 K. Yamamoto et al. / Analytica Chimica Acta 480 (2003) 109–117
A great number of plant medicines contain flavonoids,
which have been reported by many authors as having
antibacterial, anti-inflammatory, antiallergic, antimu-
tagenic, antiviral, antineoplastic, anti-thrombotic, and
vasodilatory actions [39].
In this work, the flavonoid medicine was added
to the normal and damaged groups and then pH
was also determined after about 10 min. We find the
pH has not obvious variation of the normal groups,
but the pH of damaged groups is recovered to the
normal level, from 6.2 to 7.3. It may be because
the flavonoid medicine has the function of resist-
ing free radicals, which play a central role in many
pathological conditions and chronic diseases. At the
same time, the flavonoid medicine is effective to ex-
panding blood vessel, reducing blood viscosity and
improving anoxia endurance. The further research
of the drug and its mechanism are being studied in
our lab.
4. Conclusion
In this paper, a novel pH ultramicrosensor was de-
veloped based on a WO3 film electrodeposited onto
the surface of W nanoelectrode with tip dimensions
ranging from 500 to 800 nm in diameter. The prepared
W/WO3 pH ultramicrosensor exhibits a wide pH range
of 2.0–12.0 and fast respond time. Comparing with
conventional glass pH electrode, the W/WO3 ultrami-
crosensor has a good mechanical strength, nano level
dimension of the tip, simple fabrication and conve-
nient for mass production. The W/WO3 pH ultrami-
crosensor has been applied to the pH measurement of
endothelial cells in this work. As a simple and quick
method, the pH ultramicrosensor is hopeful to be ap-
plied in single cell studying.
Acknowledgements
This work is supported by the Phosphor Project
of Science & Technology of Shanghai for excellent
young people. And we also thank the offer grant by
Opening fund of State key lab of Electroanalytical
Chemistry, Changchun Institute of Applied Chemistry,
Academy of Sciences of China and Bioelectroanalyt-
ical System of Japan.
References
[1] M.S. Ansky, A. Pizzariello, S.S. Anska, S. Miertus, Anal.
Chim. Acta 415 (2000) 151.
[2] A.K. Covington (Ed.), Ion-Selective Electrode Methodology,
vol. 1, CRC Press, Boca Raton, 1979.
[3] W.R. Heineman, H.J. Wieck, A.M. Yacynych, Anal. Chem.
52 (1980) 345.
[4] O. Dinten, U.E. Spichiger, N. Chaniotakis, P. Gehrig, B.
Rusterholz, W.E. Morf, W. Simon, Anal. Chem. 63 (1991)
596.
[5] X.J. Zhang, B. Ogorevc, J. Wang, Anal. Chim. Acta 452
(2002) 1.
[6] D. De Beer, A. Glud, E. Epping, M. Kuhl, Liminol. Oceanogr.
42 (1997) 1590.
[7] R. Yuan, Y.Q. Chai, G.L. Shen, R.Q. Yu, Talanta 40 (1993)
1255.
[8] J.P. Li, T.Z. Peng, C. Fang, Anal. Chim. Acta 455 (2002) 53.
[9] R. Zusman, C. Rottman, M. Ottolenghi, D. Avnir, J. Noncryst.
Solids 122 (1990) 107.
[10] R. Makote, M.M. Collinson, Anal. Chim. Acta 394 (1999)
195.
[11] T.M. Butler, B.D. MacCraith, C. McDonagh, J. Noncryst.
Solids 224 (1998) 249.
[12] J. Brinker, G. Scherer, Sol–Gel Science, Academic Press,
New York, 1989.
[13] P.C. Pandey, G. Singh, Talanta 55 (2001) 773.
[14] T. Sakurai, Y. Husimi, Anal. Chem. 64 (1992) 1996.
[15] P.S. Zhao, W.J. Cai, Anal. Chim. Acta 395 (1999) 285.
[16] S.M. Lin, T.C. Wen, Electrochim. Acta 39 (1994) 393.
[17] M.J. Natan, T.E. Mallouk, M.S. Wrighton, J. Phys. Chem. 91
(1987) 648.
[18] K.G. Kreider, M.J. Tarlov, J.P. Cline Sens. Actuators B B28
(1995) 167.
[19] C. Colombo, T. Kappes, P.C. Hauser, Anal. Chim. Acta 412
(2000) 69.
[20] M. Wang, S. Yao, M. Madou, Sens. Actuators B B81 (2002)
313.
[21] D.O. Wipf, F.Y. Ge, T.W. Spaine, J.E. Baur, Anal. Chem. 72
(2000) 4921.
[22] B.J.R. Horrocks, D. Schmidtke, A. Heller, A.J. Bard, Anal.
Chem. 65 (1993) 3605.
[23] P.I. Ortiz, M.L. Teijelo, M.C. Giordano, J. Electroanal. Chem.
243 (1988) 379.
[24] B. Reichman, A.J. Bard, J. Electrochem. Soc. 126 (1979) 583.
[25] A.T. Vas’ko, in: A.J. Bard (Ed.), Encyclopedia of
Electrochemistry of the Elements, vol. XI, Marcel Dekker,
New York, 1978, p. 69.
[26] C. Nicholson, J. Neurosci. Meth. 48 (1993) 199.
[27] M. Saito, H. Matsuoka, Anal. Chim. Acta 404 (2000) 223.
[28] S.A.M. Marzouk, S. Ufer, R.D. Buck, T.A. Johnson, L.A.
Dunlap, W.E. Cascio, Anal. Chem. 70 (1998) 5054.
[29] V.V. Cosofret, M. Erodosy, T.A. Johnson, R.P. Buck, R.B.
Ash, M.R. Neuman, Anal. Chem. 67 (1995) 1647.
[30] S.L. Ongil, L.G. Santiago, M. Griera, J. Molpeceres, M.R.
Puyol, D.R. Puyol, Life Sci. 70 (2001) 699.
[31] L.D. Burke, D.P. Whelan, J. Electroanal. Chem. 135 (1982)
55.
 K. Yamamoto et al. / Analytica Chimica Acta 480 (2003) 109–117 117

[32] L.D. Burke, T.A.M. Twomey, D.P. Whelan, J. Electroanal. [36] R. Naegeli, J. Redepenning, F.C. Anson, J. Phys. Chem. 90
Chem. 107 (1980) 201. (1986) 6227.
[33] B. Reichman, A.J. Bard, D. Laser, J. Electrochem. Soc. 127 [37] P. Ugo, L.M. Moretto, D. Rudello, E. Birriel, J. Chevalet,
(1980) 647. Electroanalysis 13 (2001) 661.
[34] A.A.M. Wide, G. Aksnes, Cardiovasc. Res. 29 (1995) 1. [38] P.J. Kinlen, J.E. Heider, D.E. Hubbard, Sens. Actuators B
[35] J.N. Weiss, R.C. Shein, Cardiovasc. Res. 28 (1994) B22 (1994) 13.
1125. [39] L. Alan, N.D. Miller, Alt. Med. Rev. 1 (2) (1996) 103.