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Voltammetric Determination of Uric Acid at a Fullerene-C60-
Modified Glassy Carbon Electrode
Rajendra N. Goyal,* Vinod K. Gupta, Aditi Sangal, Neeta Bachheti
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee-247 667, India
*e-mail: rngcyfcy@iitr.ernet.in

Received: June 2, 2005
Accepted: August 3, 2005

Abstract
Glassy carbon electrode modified by microcrystals of fullerene-C60 mediates the voltammetric determination of uric
acid (UA) in the presence of ascorbic acid (AA). Interference of AA was overcome owing to the ability of pretreated
fullerene-C60-modified glassy carbon electrode. Based on its strong catalytic function towards the oxidation of UA and
AA, the overlapping voltammetric response of uric acid and ascorbic acid is resolved into two well-defined
voltammetric peaks with lowered oxidation potential and enhanced oxidation currents under conditions of both linear
sweep voltammetry (LSV) and Osteryoung square-wave voltammetry (OSWV). At pH 7.2, a linear calibration graph
is obtained for UA in linear sweep voltammetry over the range from 0.5 mM to 700 mM with a correlation coefficient
of 0.9904 and a sensitivity of 0.0215 mA mM1 . The detection limit (3s) is 0.2 mM for standard solution. AA in less than
four fold excess does not interfere. The sensitivity and detection limit in OSWV were found as 0.0255 mA mM1 and
0.12 mM, for standard solution respectively. The presence of physiologically common interferents (i.e. adenine,
hypoxanthine and xanthine) negligibly affects the response of UA. The fullerene-C60-modified electrode exhibited a
stable, selective and sensitive response to uric acid in the presence of interferents.

Keywords: Uric acid, Ascorbic acid, C60, Modified electrode, Interferents

DOI: 10.1002/elan.200503353

1. Introduction cardiovascular disease [10, 11]. In patients having conges-
tive heart failure, serum UA is the most powerful predictor
Uric acid (2,6,8-trihydroxypurine) (UA) is a relatively water of survival as well as hospitalization [12]. UA is also
insoluble nitrogenous end product of purine nucleotide employed as a clinical marker for renal dysfunction. UA
catabolism in human system [1]. It is found distributed in level is higher in people at high risk of diabetes mellitus with
biological fluids of human, mainly in serum, blood and urine abnormal glucose tolerance [13]. Besides such detrimental
and is largely excreted from the human system by kidneys [2]. actions, UA also has beneficial functions. It is an important
In a healthy human being, the normal concentration ranges of antioxidant in human adult plasma and is involved in many
UA in human serum, human blood and urine are 240 – pathological changes [14, 15]. Thus, UA concentration
520 mM, 120 –450 mM and 1.2 –4.4 mM, respectively [3, 4]. determination is of paramount importance, due to its crucial
Change in UA concentration leads to numerous diseases and role in health assessment and monitoring.
physiological disorders. An overproduction of UA occurs Various methods such as enzymatic, colorimetric, electro-
when there is excessive breakdown of cells that contain chemical, spectrophotometric and HPLC have been report-
purines or an inability of the kidneys to excrete UA. Greater ed for the analysis of UA in the human body fluids [16 – 19].
than normal levels of UA may be indicated in the diseases such The electrochemical techniques have been found more
as diabetes, gout, hyperuricemia, Lesch-Nyhan syndrome, and selective, less costly and less time-consuming and being
renal failure [5, 6]. Low uric acid levels may be associated with claimed useful over other methods for the determination of
a molybdenum deficiency, copper toxicity, and a worsening of uric acid. However, a major problem has been found due to
multiple sclerosis. An association between low serum uric acid the presence of high concentration of AA in human body
and hypersensitivity problems has been noted. Abnormally fluids, which oxidizes at a similar potential as UA and higher
low uric acid levels may indicate that the patient suffers from potentials are required for their direct electrooxidation at
FanconiBs disease and WilsonBs disease [7]. carbon-based electrodes. The ability to determine UA and
Plasma UA level has been considered as one of the AA selectively has been a major goal of electroanalytical
predictors of blood pressure elevation and obesity [8]. UA research. Voltammetric methods based on Nafion-coated
can independently predict mortality in patients with coro- carbon paste electrode, Nafion coated GCE and b-cyclo-
nary artery disease [9]. Many prospective studies in hyper- dextrin modified electrode incorporating carbon nanotubes
tensive patients show a relationship between serum UA and have been reported for the determination of uric acid that

Electroanalysis 17, 2005, No. 24, 2217 – 2223 G 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2218 R. N. Goyal et al.

offered good selectivity with a low detection limit (0.2 mM) Voltammetric analyzer. The electrochemical measurements
[20 – 22]. However, these modified electrodes involved a were carried out in a single-compartment three-electrode
complex preparation procedure of the electrode and the glass cell with a 3 mm diameter glassy carbon electrode
surface of the electrode is to be renewed each time. The (GCE) as the working electrode, a platinum wire as counter
extent of renewal depends on applied potential and pH of electrode and Ag/AgCl electrode as reference (Model MF-
the cleaning solution. Few other modified electrodes [23, 24, 2052 RB-5B). All experiments were carried out at an
32] reported in literature for the determination of uric acid ambient temperature of 25  2 8C.
also had the same complication.
Cai and co-workers reported an improved voltammetric
method using electrochemically pretreated carbon-paste
2.3. Procedures
electrode in basic electrolyte media [25]. However, the
electrode material was found to swell after 30 measurements. For recording voltammograms, stock solutions of UA
At several other electrodes modified with films of (2 mM) and AA (2 mM) were prepared in doubly distilled
complexes, nanodeposites the lowest detection limit report- water. 2 mL of the stock solution was added to 2 mL of PBS
ed for uric acid is 0.2 mM [26 – 28]. (0.1 M, pH 7.2). Osteryoung square-wave voltammetry
Since the discovery of the new allotrope of carbon, the employed the following parameters: step height, 4 mV;
fullerene, investigations have been initiated to explore square-wave amplitude, 25 mV; frequency, 5 Hz; quiet time,
several possible fields of their applications. The reductive 2s and sensitivity, 100 mA.
electrochemistry of fullerene-C60-films has been studied
extensively. Szucs et al. have established that fullerene films
become conducting upon reduction which implies that it can
2.4. Preparation of Fullerene-C60-Modified Electrode
be used as an electrode material [29]. It was observed that
reduced fullerene films could be used as electrocatalysts. The Prior to electrode modification, the GCE surface was
electrocatalytic behavior of fullerene film electrodes is quite cleaned by polishing with alumina on microcloth pads
different from other type of electrodes since reduced full- (BAS, USA) and then zinc oxide (Aldrich) to a mirror-like
erene film on glassy carbon substrates show microelectrode finish. It was then dipped in a beaker containing 0.2 M H3
characteristics. Fullerene modified electrodes have recently PO4 solution to remove the adhered powder, rinsed with
been widely used for catalytic and sensor applications, and to distilled water and dried in an oven at temperature 45 8C for
study the electrochemistry of biomolecules. The present 2 – 3 minutes. Stock solution of C60 was prepared by
paper describes simple voltammetric techniques for the dissolving in CH2Cl2 (150 mM). A known volume (40 mL)
sensitive and selective determination of UA at fullerene-C60 of this solution was adsorbed onto the surface of the clean
modified glassy carbon electrode at physiological pH i.e. at and dried GCE using a microsyringe and dried in a stream of
pH 7.2. The electrode not only has a strong catalytic function hot air (45 8C). The most important point is the rapid
towards the oxidation of UA and AA, but also resolves the evaporation of dichloromethane providing a fine and even
overlapping voltammetric response of the two compounds dispersion of the deposited particles, thus, avoiding the
into two well-defined voltammetric peaks. The voltammetric formation of large crystalline particles. The C60 film formed
response of UA in the presence of other biologically common was then reduced in 1 M KOH in the potential range 0.0 V to
interferents has also been studied.  1.5 V at 10 mV/s. PBS (50 mM) of pH 7.2 was taken in
another cell and the electrode surface was equilibrated in it
by cyclic scanning in the potential range of 550 mV to
 50 mV (vs. Ag/AgCl) at a scan rate of 20 mV/s for
2. Experimental
20 minutes under a nitrogen atmosphere [31]. The film
changed color upon reduction i.e. the initially brownish
2.1. Reagents
yellow film appears to become brownish red. This very easy
C60 was obtained from Aldrich, USA (purity 98%). UA, technique allowed us to form a compact layer of reduced C60
AA, adenine, xanthine and hypoxanthine were purchased on glassy carbon substrate. The fullerene-C60-modified
from Sigma, USA. All these compounds were used without electrode was then ready for use.
further purification. Phosphate buffer solution (PBS) (m ¼
0.1 M) prepared according to the method of Christian and
Purdy [30] was used as supporting electrolyte. All other
reagents used were of analytical grade. All solutions were
3. Results and Discussion
prepared in double distilled water.
3.1. Electrochemical Behavior of Uric Acid
It has been established that fullerene-C60 becomes conduc-
2.2. Instrumentation
tive upon reduction and doping with alkali metal ions, the
The voltammetric experiments were performed on BAS most active surface being produced with Kþ. These reduced
(Bioanalytical Systems, West Lafayette, IN, USA) CV-50W fullerene-C60 films have been suggested to have special

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Voltammetric Determination of Uric Acid 2219

Fig. 1. Linear sweep voltammograms for (a) 0.5 mM Uric acid at pH 7.2 at bare (- - -) and fullerene-C60-modified (–) glassy carbon
electrode (b) PBS (pH 7.2) at fullerene-C60-modified glassy carbon electrode ( ·· · ·). Sweep rate 100 mV/s.

The dependence of peak current function (with correction
of background current) on the concentration of UA at
pH 7.2 was linear in the range 0.5 mM to 0.7 mM (Fig. 2)
having a correlation coefficient of 0.9904 and can be
expressed by the equation

ip (mA) ¼ 0.0215 C where C is in mM.

The detection limit (3s) is 0.2 mM. The error in the
calculation of detection limit was estimated to be 1.45%.
At concentrations greater than 0.7 mM of UA the peak
current became constant which indicates adsorption of UA
Fig. 2. Dependence of peak current on Uric acid concentration at the surface of the modified electrode at higher concen-
at pH 7.2.Sweep rate 100 mV/s. (~) for linear sweep and (&) for trations. Thus, by using fullerene-C60-modified GCE, UA
OSWV. can be detected in a solution as low as 0.5 mM; however, it is
not possible to detect concentrations greater than 0.7 mM.

electrocatalytic properties [29]. It is also likely that the more
3.2. Oxidation of Ascorbic Acid
conducting KxC60 species, which is formed on reduction,
helps in the electron transfer at the interface. One of the major problems frequently encountered in the
It was observed that modification of electrode surface by a electrochemical detection of biomolecules is the serious
thin film of fullerene-C60 remarkably improves the reactivity interference due to ascorbic acid. The oxidation of AA in
of GCE towards UA and AA oxidation. Figure 1 illustrates linear sweep voltammetry at bare GCE occurred at 440 mV
the voltammograms of UA at a bare GCE and fullerene-C60- and the peak was broad. In contrast, at the fullerene-C60-
modified electrode at pH 7.2. At the bare GCE, UA modified electrode, the oxidation current increased greatly
oxidation takes place at 490 mV, which is in close agreement and the peak potential shifted negatively to 200 mV. Thus,
with a previous report [32]. While at fullerene-C60-modified an increment in the current response with a negative shift
electrode, the electrooxidation of UA occurred at 350 mV (ca.240 mV) of the oxidation potential at the fullerene-C60-
with an enhancement in peak current. This shift of peak modified electrode was obtained for AA. The enhanced
potential (ca.140 mV) to less positive potentials with current responses and lowered oxidation peak potentials for
increased peak current indicates that the exchange current AA clearly indicates the strong catalytic action of fullerene-
increases as a result of a lower overpotential for the reaction C60-modified electrode. Since AA is more hydrophilic and
in the presence of fullerene-C60. more soluble in water than UA, this may account for higher

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2220 R. N. Goyal et al.

catalytic action of the modified electrode towards AA The response of UA and AA, coexisting in a solution, was
oxidation. further studied by Osteryoung square-wave voltammetry
(OSWV), which has been claimed as a more sensitive
technique. Figure 4 shows the typical OSWVs obtained at
fullerene-C60-modified GCE at pH 7.2 for varying concen-
3.3. Simultaneous Detection of Uric acid and Ascorbic
trations of UA keeping the concentration of AA constant.
Acid
Two well-defined oxidation peaks for UA and AA were
It was observed in the linear sweep voltammetric experi- observed at 350 mV and 200 mV, respectively. Figure 2
ments that oxidation potential of AA showed a large represents the relation between the oxidation peak current
negative shift (ca.240 mV) with an enhanced current and the concentration of UA (0.5 mM to 0.8 mM) obtained
response at fullerene-C60-modified electrode. This observa- for OSWV technique. The peak current (ip) increased
tion is explored to detect simultaneously AA and UA using linearly with increase in UA concentration (C) having a
fullerene-C60-modified electrode in the present studies. correlation coefficient of 0.9958. The linear dependence can
Figure 3 shows the linear sweep voltammograms of AA be expressed by the equation
(1.0 mM) and UA (1.0 mM) both coexisting at pH 7.2, at the
bare and fullerene-C60-modified electrode. The bare elec- ip (mA) ¼ 0.0255 C
trode could not separate the voltammetric signals of UA and
AA, and gave a single broad anodic peak at a potential where C is in mM and a sensitivity of 0.0255 mA mM1 is
different from the oxidation potentials of AA and UA. Thus, observed. The detection limit (3s) in OSWV was found as
the peak potentials for UA and AA were indistinguishable 0.12 mM. The error in the calculation of detection limit is
and it was impossible to deduce any conclusive information 1.65%. Ascorbic acid oxidized at  150 mV less positive
from the broad voltammetric peak. On the other hand, the potential than UA and the voltammetric peak of AA
presence of the fullerene-C60-film at the electrode surface remained almost unchanged during the oxidation of UA.
resolved the voltammetric response into two well-defined Thus, using fullerene-C60-modified GCE it is possible to
peaks corresponding to almost the same potential as those determine UA in the presence of AA with detection limit as
obtained for the individual oxidation of UA and AA. The low as 0.12 mM. This method is superior to other methods
separation between the two peak potentials was  150 mV, reported earlier because no prior treatment of the electrode
which was large enough for the simultaneous determination is needed. Also cleaning of the electrode surface after each
of UA and AA in a mixture. run is not required which is a prerequisite to many methods

Fig. 3. Linear sweep voltammograms for solutions containing both uric acid (1.00 mM) and ascorbic acid (1.00 mM) at pH 7.2 at bare
(- - -) and fullerene-C60-modified (–) glassy carbon electrode. Sweep rate 100 mV/s.

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Voltammetric Determination of Uric Acid 2221

surface after each run and studies are carried out at
physiological pH. The ease of preparation of the modified
electrode and its stability is also an advantage over the
reported methods.

3.4. Effect of Interferents
The voltammetric response to UA generally suffers from the
interference of ascorbic acid as well as xanthine, hypoxan-
thine and adenine existing in biological samples. Thus, prior
to the application of a fullerene-C60-modified electrode to
biological samples, effect of interferents on the response of
the specific analyte is checked. The specificity of the
fullerene-C60-modified electrode to 0.05 mM of UA in the
presence of xanthine, hypoxanthine and adenine was carried
out by carrying out OSWV experiments. The OSWV
response for the oxidation of UA (0.05 mM) was recorded
after addition of varying concentration of each interferent
(0.001 – 0.4 mM). The peak current response obtained in
case of adenine, hypoxanthine and xanthine is compiled in
Table 1. Xanthine and hypoxanthine affected the peak
current of UA at 6 and 8 fold-excess respectively. However,
adenine did not show any interference up to 8 fold-excess.
The interference effect of AA was investigated in further
detail. Table 2 shows the effect of varying concentrations of
Fig. 4. Osteryoung square-wave voltammograms of a mixture of AA on the OSWV response of 0.10 mM UA at the fullerene-
AA and UA at the fullerene-C60 modified glassy carbon electrode C60-modified electrode. The peak current for AA increased
at pH 7.2. Concentration of UA was changed keeping the
with increasing AA concentration. The peak potential and
concentration of AA constant, i.e., [AA] ¼ 0.10 mM, [UA]: a)
0.00, b) 0.10, c) 0.25, d) 0.30, e) 0.40, f) 0.50, g) 0.60, h) 0.70, i) peak current of UA remained almost constant in less than 4
0.80 mM. fold-excess of AA as shown in Figure 5. At and above 4-fold
excess of AA, a significant increase in peak current response
Table 1. Effect of interferents on the square-wave voltammetric with a shift in peak potential of UA to more positive
response of 0.05 mM UA at the fullerene-C60-modified glassy potential is observed which indicated that the four-fold
carbon electrode. excess of AA interferes in the determination of UA.
Interferents Concentration Change in
(mM ) peak current (mA )
Adenine 0.40 0.000 3.5. Determination of Uric Acid in Real Samples
Hypoxanthine 0.40 0.348
The linear sweep voltammetric method was applied to the
Xanthine 0.30 0.348
direct analysis of two human urine samples. To fit within the
linear range, the urine samples were diluted 500 times. The
dilution process greatly reduces matrix effect of real
[20 – 22, 33] where low detection limit (0.05 mM) is reported. samples. The results obtained are shown in Table 3. To
The regeneration of the electrode surface is dependent on ascertain the correctness of the results, the samples were
the potential scan and pH of the solution used. The present spiked with certain amounts of UA in about the same
method does not require renewal/regeneration of electrode concentration as found in the samples themselves. The

Table 2. Influence of AA addition on the OSWV response of 0.10 mM UA at the fullerene-C60-modified electrode.
Concentration of AA (mM ) Peak potential of UA (mV ) Peak current of UA (mA ) Peak current of AA (mA )
0.10 351.2 2.526 1.758
0.15 353.7 2.524 1.882
0.20 351.0 2.523 1.961
0.25 353.7 2.515 2.067
0.30 352.9 2.522 2.173
0.35 352.1 2.525 2.278
0.40 381.4 3.229 2.489

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2222 R. N. Goyal et al.

Fig. 5. Osteryoung square-wave voltammograms of a mixture of AA and UA at the fullerene-C60-modified glassy carbon electrode at
pH 7.2. Concentration of AA was changed keeping the concentration of UA constant, i.e., [UA] ¼ 0.10 mM, [AA]: a) 0.10, b) 0.15, c)
0.20, d) 0.25, e) 0.30, f) 0.35, g) 0.40 mM.

Table 3. Determination of UA in urine samples with fullerene- time, the result indicates good stability of the electrode. The
C60-modified electrode. response time of the electrode was very fast, hence, all the
Urine1 Urine2 measurements were done immediately after the fullerene-
C60-modified electrode was immersed into the test samples.
Original value (mM ) 2.968 2.381 The reproducibility of fullerene-C60-modified electrode
Spike (mM ) 3.000 2.400
was also investigated. Repetitive measurements were car-
Recovery (%) 95.74 101.26
Total value [a] (mM ) 1.484 1.191 ried out in 0.01 mM UA solution. The results of five
successive measurements show a relative standard deviation
[a] The total value was obtained by multiplying the detected value and the of 1.76%. This also demonstrates the intraday stability of
dilution factor.
fullerene-C60-modified electrode. To check the reproduci-
bility of the results further, three different glassy carbon
electrodes of almost the same area were modified with same
recovery rates of the spiked samples varied between 95.7% volume of fullerene-C60 solution in dichloromethane and
and 101.2%. their response towards the oxidation of 0.01 mM UA was
tested. The current obtained in five repeated measurements
of the three independent electrodes showed a relative
standard deviation of  1.43 %, confirming that the results
3.6. Stability and Reproducibility of the Fullerene-C60-
are reproducible. Thus, the electrode is very stable and good
Modified GCE
reproducibility is observed.
The procedure of electrode preparation being easy and rapid,
it is not so important for the electrode to be stable for a
prolonged time. However, the day-to-day stability of the 4. Conclusions
electrode was evaluated. As an indicator of its stability, the
performance of a particular fullerene-C60-modified electrode The simultaneous oxidation of UA and AA poses a very
over a period of 7 days with measurements of the oxidation of strong challenge because of their very similar oxidation
peak current for 0.01 mM UA in 0.1 M phosphate buffer peak potentials. The present study demonstrates the use of
solution (pH 7.2) was tracked each consecutive day. Our fullerene-C60-modified electrode, which successfully sepa-
linear sweep voltammetric experiment has demonstrated that rated the peaks of UA and AA present in the mixture,
no apparent decrease in current responses occurred during indistinguishable at the bare GCE. A linear relationship
the first day, and 3% decrease was noted after 3 days. Since between UA concentration and current response was
the activity remains virtually constant during this period of obtained in the concentration range of 0.5 – 700 mM with

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Voltammetric Determination of Uric Acid 2223

excellent reproducibility of the current. Since a better peak [9] C. Bickel, H. J. Rupprecht, S. Blankenberg, G. Rippin, G.
current response is obtained, for UA and AA coexisting in a Hafner, A. Daunhauer, K.-P. Hofmann, J. Meyer, Am. J. Car.
solution, on application of OSWV method, it offers an 2002, 89, 12.
[10] M. H. Alderman, H. Cohen, S. Madhavan, S. Kivlighn,
improved sensitivity in electrochemical signal and thus is Hypertension 1999, 34, 144.
better used in the determination. The fullerene-C60-modi- [11] L. V. Franse, M. Pahor, M. Di Bari, R. I. Shorr, J. Y. Wan,
fied electrode shows a stable and reproducible response G. W. Somes, W. B. Applegate, J. Hypertens. 2000, 34, 1149.
without any influence of interferents commonly existing in [12] S. D. Anker, F. Leyva, P. A. Poole-Wilson, A. J. S. Coats, J
biological fluids. Hence, an excellent approach towards the Am Coll. Cardiol. 1998, 31, A154.
development of a novel fullerene-C60-modified electrode [13] A. Costa, I. Igual, J. Bedini, L. Quint, I. Conget, Metabolism
2002, 51, 372.
has been presented, offering a possibility for extending this
[14] S. Burkhardt, R. J. Reiter, D. X. Tan, R. Hardeland, J.
technique to the routine analysis of UA in clinical samples. Cabrera, M. Karbownik, Int. J. Biochem. Cell Biol. 2001, 33,
The result shows that the method may be especially valuable 775.
in sensor fabrication. [15] F. J. Nieto, C. Iribarren, M. D. Gross, G. W. Comstock, R. G.
Cutler, Atherosclerosis 2000, 148, 131.
[16] T. H. Steel, Tech. Bull. Regist. Med. Technol. 1969, 39, 270.
[17] H. Y. Chang, W. H. Pan, W. T. Yeh, K. S. Tsai, J. Rheumatol.
5. Acknowledgements 2001, 28, 1640.
[18] F. Palmisano, E. Desimoni, P. G. Zambonin, J. Chromatogr.
The authors (A. S.) and (N. B.) are thankful to the Council of 1984, 306, 205.
Scientific and Industrial Research, Delhi for awarding [19] P. Li, S. Wu, H. Zhang, C. Ma, Fenxi Huaxue 2005, 33, 77.
Senior and Junior Research Fellowship (Grant No. 8810 – [20] J. Zen, C. Hsu, Talanta 1998, 46, 1363.
13 – 414), respectively. [21] J. Zen, P. J.Chen, Anal. Chem. 1997, 69, 5087.
[22] Z.Wang, Y.Wang, G.Luo, Analyst 2002, 127, 1353.
[23] L.Lu, X.Lin, Anal. Sciences 2004, 20, 527.
[24] S. B.Khoo, F.Chen, Anal. Chem. 2002, 74, 5734.
6. References [25] X. H. Cai, K. Kalcher, C. Neuhold, B. Ogorevc Talanta 1994,
41, 407.
[1] G. Dryhurst, Electrochemistry of Biological Molecules, Aca- [26] S. Wang, L. Lu, X. Lin, Electroanalysis 2004, 16, 1734.
demic Press, New York 1977. [27] J. Premkumar, S. B. Khoo, J. Electroanal. Chem. 2005, 576,
[2] J. Richards, E. J. Weinman, J. Nephrol. 1996, 9, 160. 105.
[3] P. T. Kissinger, L. A. Pachla, L. D. Reynolds, S. Wright, J. [28] P. R. Roy, T. Okajima, T. Ohsaka, J. Electroanal. Chem. 2004,
Assoc. Anal. Chem. 1987, 70, 1. 561, 75.
[4] J. M. Zen, J. S. Tang, Anal. Chem. 1995, 67, 1892. [29] A. Szucs, A. Loix, J. B. Nagy, L. Lamberts, J. Electroanal.
[5] H. Yamanaka, R. Togashi, M. Hakoda, C. Terai, S. Kashi- Chem. 1995, 397, 191.
wazaki, T. Dan, N. Kamatani, Adv. Exp. Med. Biol. 1998, 431, [30] G. D. Christian, W. C. Purdy, J. Electroanal. Chem. 1962, 3,
13. 363.
[6] I. H. Fox, Metabolism 1981, 30, 616. [31] M. Csiszar, A. Szucs, M. Tolgyesi, A. Mechler, J. B. Nagy, M.
[7] Mosby,s Manual of Diagnostic and Laboratory Tests (Eds: Novak, J. Electroanal. Chem. 2001, 497, 69.
Pagana, K. Deska), St.Louis, Mosby 1998. [32] X. Q. Lin, G. P. Jin, Electrochim. Acta 2005, 50, 3210.
[8] K. Masuo, H. Mikami, T. Ogihara, Michael L. Tuck, Am. J [33] J. W.Luo, M.Zhang, D. W. Pang, Sensors and Actuators B
Hyper 2002, 15, A189. 2005, 106, 358.

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