Microchim Acta 158, 73–78 (2007)

DOI 10.1007/s00604-006-0656-0

Printed in the Netherlands

Original Paper

Selective determination of uric acid in the presence of ascorbic acidusing a penicillamine self-assembled gold electrode

Liang Wang1;�, Jun Yue Bai1;2, Peng Fei Huang1;2, Hong Jing Wang1,

Xiao Wei Wu1, and Yu Qing Zhao1

1 College of Life Science, Dalian Nationalities University, Dalian 116600, China2 School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, China

Received April 5, 2006; accepted June 20, 2006; published online August 30, 2006

# Springer-Verlag 2006

Abstract. The electrochemical behaviors of uric acid

(UA) at the penicillamine (Pen) self-assembled mono-

layers modified gold electrode (Pen=Au) have been

studied. The Pen=Au electrode is demonstrated to pro-

mote the electrochemical response of UA by cyclic

voltammetry (CV). The diffusion coefficient D of UA

is 6.97�10�6 cm2 s�1. In differential pulse voltammet-

ric (DPV) measurements, the Pen=Au electrode can

separate the UA and ascorbic acid (AA) oxidation po-

tentials by about 120 mV and can be used for the se-

lective determination of UA in the presence of AA. The

detection limit was 1�10�6 mol L�1. The modified

electrode shows excellent sensitivity, good selectivity

and antifouling properties.

Key words: Uric acid; ascorbic acid; self-assembled monolayers;

penicillamine.

Uric acid (UA) is a primary end-product of purine

metabolism and abnormal levels of UA are symptoms

of several diseases like gout, hyperuricemia and Lesch-

Nyhan syndrome [1]. Thus the determination of the

concentration of UA in human blood or urine is a pow-

erful indicator in diagnosing diseases. The development

of a simple and rapid methodology for the determina-

tion of UA has therefore attracted attention in recent

years [2–6]. Among various methods, electrochemical

determination of UA shows a higher selectivity than

other methods and it is less costly and less time

consuming [7–15]. Although earlier electrochemical

procedures based on the oxidation of UA at polymer

modified electrode and pretreated carbon electrode

showed good selectivity and sensitivity [16, 17], they

face some drawbacks. For instance, they are mainly

based on adsorption phenomena and thus preconcentra-

tion of UA needs to be done before each measurement

or the electrodes need to be renewed after each mea-

surement; they suffer from the interference of other

electroactive compounds and the oxidation requires a

high overpotential [18]. Moreover, at the bare electrode

the oxidation of ascorbic acid (AA) occurs at a poten-

tial close to that of UA and the bare electrode very

often suffers from fouling effects. Therefore it is neces-

sary to develop an electrochemical sensor, which is free

from the above-mentioned problems [1].

Self-assembled monolayers (SAMs) of organosul-

phur compounds on metal surfaces comprise a wide

field of potential applications due to their versatility in

modifying surfaces in a controllable manner. It has

been shown that organothiol molecules upon adsorp-

tion at gold lose the hydrogen from the thiol group

and that an S–Au bond is formed [19, 20]. The well-

characterized self-assembled monolayers on metal� Author for correspondence. E-mail: qier102@hotmail.com

electrodes have been widely used as a new strategy for

the immobilization, orientation and molecular organi-

zation of biomolecules at interfaces. The stability on

the bond between the specific functional group of a

reagent and the electrode surface over a wide range of

applied potentials and the well-defined microenviron-

ment mimicking biological membranes make such a

system facilitate the electron transfer of biomolecules

and lead to important applications in the development

of biosensors. Such chemically modified electrodes to

improve the selectivity and sensitivity of the electro-

chemical behavior of some biomolecules have been

widely studied [21–23].

In an effort to develop a voltammetric method for

the selective and sensitive detection UA, the present

investigation employed a gold electrode which was

modified with the penicillamine (Pen).

As Pen involves the terminal SH groups, it can be

self-assembled on the gold electrode surface as a new

chemically modified electrode to study electrochem-

istry properties of UA. Herein we describe the utiliza-

tion of the self-assembly of Pen for the selective

detection of UA in the presence of AA by successful

elimination of the fouling effect caused by the oxida-

tion product of AA. This method is very simple and

does not require any mediator or enzymes.

Experimental

Reagents

Penicillamine and uric acid were purchased from Sigma (www.

sigmaaldrich.com) and they were used as received. Ascorbic acid

was from Beijing Chemical Factory (Beijing, China). All other chem-

icals were of analytical grade and were used without further purifica-

tion. A 0.1 mol L�1 phosphate buffer solution was used to control the

pH. All solutions were prepared with deionized water treated in a

Millipore water purification system (Millipore Corp.). All experi-

ments were carried out at room temperature (approx. 20 � 1 �C).

Apparatus

Voltammetric measurements were performed with a CHI 440 elec-

trochemical analyzer (CH Instruments, Chenhua Co. Shanghai,

China). A conventional three-electrode cell was used, including

a saturated calomel electrode (SCE) as reference electrode, a plati-

num wire counter electrode and a bare or modified gold working

electrode. The pH values were measured with a PB-10 pH meter

(Satorius). Unless otherwise stated, the electrolyte solutions were

thoroughly degassed with N2 and kept under a N2 blanket.

Preparation of the Pen=Au electrode

Monolayer was formed by the self-assembling technique on gold

substrates (Scheme 1). The working electrode was a Au disk elec-

trode with a diameter of 2 mm. Prior to each measurement, the

electrode was polished with diamond pastes and an alumina slurry

down to 0.05mm on a polishing cloth (Buehler, Lake Bluff, IL),

followed by sonicating in water and ethanol. Then, the Au electrode

was electrochemically cleaned by cycling the electrodes potential

between 1.6 and �0.4 V (vs. SCE) in 0.5 mol L�1 H2SO4 until a

stable voltammogram was obtained. After it was washed with soni-

cation and dried with a stream of high purity nitrogen, the electrode

was immersed in an aqueous solution of 20 mmol L�1 Pen for about

36 h at 4 �C. Upon removal from the deposition solution, the sub-

strate was thoroughly rinsed with water to remove the physically

adsorbed species. The advancing contact angle was of Pen SAM is

14� [21]. Hereafter the Pen self-assembled gold electrode will be

referred as Pen=Au electrode. The scheme of the resulting self-

assembling configuration at the gold electrode is shown in Scheme 1.

Results and discussion

Characterization of Pen=Au electrode

with cyclic voltammetry

The redox behavior of a reversible couple can be used

to probe the packing structure of the monolayer [24].

Figure 1 shows the cyclic voltammograms of the bare

gold electrode (Fig. 1a) and the Pen=Au electrode

(Fig. 1b) in 1 mmol L�1 FeðCNÞ63�

solution contain-

ing 0.1 mol L�1 KCl. For a bare gold electrode, a cou-

ple of well-defined waves of FeðCNÞ63�=FeðCNÞ6

4�

should appear, and the peak-to-peak separation (�Ep)

should be 60 mV. However, it can be seen that the

Scheme 1. Organization of Pen-SAMs

Fig. 1. Cyclic voltammograms of 1.0 mmol L�1 FeðCNÞ63�=

FeðCNÞ64�

at bare gold electrode (a) and Pen=Au electrode (b).

0.1 mol L�1 KCl; scan rate, 100 mV s�1

74 L. Wang et al.

peak current decreased and �Ep increased for the

Pen=Au electrode. Because Pen is a short mercaptan

molecule, there are many pinhole defects and col-

lapsed sites in the Pen monolayer, and the electron

transfer rate constant at pinhole defects is the same

as that at the bare gold electrode. So the redox couples

can reach the gold surface through pinhole defects in

the Pen monolayer.

From the reductive desorption of Pen monolayer

from the Au electrode, the surface coverage of Pen at

the Au electrode was calculated. First, the charge at-

tributed to the desorption of sulfur atoms of Pen form

the Au surface [25] was 1.386mC which was obtained

by integration of the cathodic peak in the cyclic vol-

tammogram of the Pen=Au electrode in 0.5 mol L�1

KOH. The effective area of the electrode was calcu-

lated to be 0.085 cm2 according to the cyclic voltam-

mogram of 0.5 mol L�1 sulfuric acid at the bare

electrode [26]. Then the surface coverage of Pen at

Au electrode was found to be 1.69�10�10 mol cm�2.

Electrochemical oxidation of UA

at the Pen=Au electrode

Figure 2 shows the cyclic voltammograms at the

bare gold electrode (Fig. 2a) and the Pen=Au elec-

trode (Fig. 2b) in presence of 0.10 mmol L�1 UA in

phosphate buffer of pH 7.0. At the bare electrode,

the electrooxidation of UA occurs at approximately

0.46 V and the voltammetric peak is rather broad, sug-

gesting slow electron transfer kinetics, presumably

due to the fouling of the electrode surface by the

oxidation product. It is reported that the oxidation of

UA is irreversible at GC and metal electrodes and is

quasi-reversible at a graphite electrode [27]. The elec-

trochemical oxidation of UA proceeds in a 2e�, 2Hþ

process lead to an unstable diimine species which is

then attacked by water molecules in a step-wise fash-

ion to be converted into an imine-alcohol and then

uric acid-4,5 diol. The uric acid-4,5 diol compound

produced is unstable and decomposes to various pro-

ducts depending on the solution pH [2]. However, a

well-defined redox wave of UA was obtained at the

Pen=Au electrode. The oxidation peak potential shifts

negatively to 0.34 V and the peak current increases

significantly. The above results suggested that the

Pen=Au electrode promoted the electrochemical reac-

tion of UA. The reason for this is that the Pen-SAMs

can act as a promoter to increase the rate of electron

transfer [21, 23], lower the overpotential of UA at the

bare electrode, and the anodic peak shifts negatively.

The influence of the scan rate on the electrochemi-

cal response of UA at the Pen=Au electrode was

investigated by cyclic voltammetry. The oxidation

peak potential was observed to shift positively with

the increase in scan rate, and in addition, the oxidation

peak current exhibited a linear relation to the square

root of the scan rate in the range from 20 mV s�1 to

300 mV s�1 (Fig. 3). The result indicates that the oxi-

dation of UA at the Pen=Au electrode is a diffusion-

controlled process. The Pen=Au electrode used for the

oxidation of UA did not show any voltammetric signal

for UA after it was transferred to pure supporting elec-

trolyte, confirming that the oxidation process is free

from the adsorption of UA.

The diffusion coefficient D of UA was determined

at the Pen=Au electrode using chronocoulometric

method based on the following equation [28]:

Q ¼ 2nFACðDtÞ1=2

�1=2þ Qdl

The potential step is from 0.0–0.6 V. The concentra-

tion of UA is 0.1 mmol L�1. The electrochemical oxi-

dation of UA involves in a 2e�, 2Hþ process, so the

Fig. 2. Cyclic voltammograms for 0.1 mmol L�1 UA in

0.1 mol L�1 phosphate buffer solution (pH 7.0) at bare Au elec-

trode (a) and Pen=Au electrode (b). Scan rate: 100 mV s�1

Fig. 3. The relationship between the oxidation peak current and

the square root of scan rate for 0.1 mmol L�1 UA in 0.1 mol L�1

phosphate buffer solution (pH 7.0) at Pen=Au electrode

Determination of uric acid in the presence of ascorbic acid using a penicillamine self-assembled gold electrode 75

electron transfer number, n¼ 2 [29]. Based on the

slope of the curve of Q vs. t1=2, 4.89�10�6 C=s1=2,

the diffusion coefficient of UA was calculated as

6.97�10�6 cm2 s�1.

Electrochemical oxidation of AA

at the Pen=Au electrode

Figure 4 shows the cyclic voltammograms of AA

at the bare Au (Fig. 4a) and the Pen=Au electrode

(Fig. 4b). At the bare Au electrode, the oxidation

occurs at around 400 mV and the oxidation peak is

rather broad. Oxidation of AA at bare electrode is

generally believed to be totally irreversible and re-

quires high overpotential and also, no reproducible

electrode response is obtained due to fouling of the

electrode surface by the adsorption of the oxidized

product of AA [30]. However, the oxidation peak is

shifted to less positive potential (140 � 3 mV) at the

Pen=Au electrode, indicating that the Pen SAMs on

the electrode surface favors the oxidation process.

Since Pen molecules form a ‘thin’ monolayer and

this prevents the fouling of the electrode surface, the

electron transfer kinetics for the oxidation of AA are

faster at the Pen=Au electrode. So the possible expla-

nation for the negative shift observed in the oxidation

peak potential of AA could be due to the prevention of

the electrode surface fouling by the oxidation product.

Moreover, the formal potential for the oxidation of

AA is �200 mV [31], which is more negative than a

potential at which the oxidation actually occurs at the

bare electrode and therefore it is reasonable to expect

a negative shift in the oxidation potential at the

Pen=Au electrode [18]. Because the oxidation peak

of AA is shifted to less positive potential it would

not interfere with the measurement of UA. The oxida-

tion peak potential was observed to shift positively

with the increase in scan rate and the oxidation peak

current showed a linear relationship with the square

root of scan rate, indicating a diffusion-controlled

irreversible oxidation process of AA at the Pen=Au

electrode.

Determination of UA in the presence of AA

AA is a main interferent in the voltammetric determi-

nation of UA. The main objective of this study is to

selective detection of UA in the presence of ascorbic

acid. Fig. 5 shows the cyclic voltammograms of UA

and AA (0.1 mM each) coexisting in 0.1 M phosphate

buffer solution at the bare electrode (Fig. 5a) and the

Pen=Au electrode (Fig. 5b). The bare electrode could

not separate the responses of UA and AA and the

voltammetric peak was ill defined. The fouling of the

electrode surface by the oxidation products results in

the single voltammetric peak for both UA and AA.

Therefore it is impossible to use the bare electrode

for the voltammetric determination of UA in the pre-

sence of AA. On the other hand, at the Pen=Au elec-

trode, two oxidation peaks were found at almost the

same potential as those obtained for the individual

oxidations of UA and AA.

For clear confirmation, the response of UA and AA

coexisting in a solution was investigated by the more

sensitive method, differential pulse voltammetry

(DPV). Fig. 6 shows the DPV recordings obtained at

the bare electrode (Fig. 6a) and the Pen=Au electrode

(Fig. 6b) for UA and AA (0.1 mM each) coexisting in

0.1 M phosphate buffer solution. At the bare electrode

a rather broad oxidation peak at about 0.41 V was

obtained and the oxidation peak potentials of UA

and AA were indistinguishable. On the other hand,

in the case of the Pen=Au electrode, two well-defined

Fig. 4. Cyclic voltammograms for 0.1 mmol L�1 AA in 0.1 mol L�1

phosphate buffer solution (pH 7.0) at bare Au electrode (a) and

Pen=Au electrode (b). Scan rate: 100 mV s�1

Fig. 5. Cyclic Viltammograms for 0.1 mmol L�1 UA and

0.1 mmol L�1 AA in 0.1 mol L�1 phosphate buffer solution (pH

7.0) at bare Au electrode (a) and Pen=Au electrode (b). Scan rate:

100 mV s�1

76 L. Wang et al.

oxidation peaks for UA and AA were observed at

0.33 V and 0.10 V, respectively. As the oxidation of

AA is readily oxidized well before the oxidation po-

tential of UA is reached, thus the catalytic oxidation

AA by the oxidized UA is completely eliminated and

the precise determination of UA in the presence of AA

is possible at the Pen=Au electrode. The votammetric

signals of UA and AA remained unchanged in the

subsequent sweeps, indicating that the Pen=Au elec-

trode does not undergo surface fouling. In this case,

the peak potential separation (ca. 120 mV) was large

enough to determine UA and AA individually and

simultaneously.

When DPV was used to investigate the oxidation of

UA at the Pen=Au electrode in the absence of AA, the

voltammetric peak linearly increases with the concen-

tration of UA in the range of 10–160mmol L�1. The

linear regression equation was ipa=mA¼ 0.0760þ0.0090 C=mmol L�1, with correlation coefficients of

0.9955. The detection limit was 1�10�6 mol L�1

based on the signal-to-noise ratio of 3.

Figure 7 shows the DPV recordings obtained while

simultaneously changing the concentration of both

analytes. The calibration curves for both UA and AA

were linear for a wide range of concentrations (10–

160 mmol L�1 for UA and 50–300 mmol L�1 for AA),

with correlation coefficients 0.9981 and 0.9977, re-

spectively. The detection limits for UA and AA were

found to be 1.0 and 12mmol L�1, respectively. The

slopes (�I=�C) of the linear calibration curves were

estimated to be 0.0095 and 0.0018mA=mmol L�1 for

UA and AA, respectively. This suggests that the oxi-

dation of AA mediated by the oxidized UA cannot

occur at the Pen=Au electrode. Thus, the simulta-

neously selective and sensitive detection of UA and

AA was achieved at the Pen=Au electrode. To ascer-

tain further the reproducibility of the results, three

different Au electrodes were modified with Pen SAMs

and their responses towards the oxidation of UA

and AA were tested. The separation between the vol-

tammetric signals of UA and AA and the sensitivities

remained the same at all three modified electrode,

confirming that the results are reproducible. It is in-

teresting to note that the sensitivity of the Pen=Au

electrode towards UA in the absence and presence

of AA remained the same, which demonstrates that

AA does not influence the voltammetric measurement

of UA.

Analytical utility of the Pen=Au electrode has been

examined using human urine samples. Human urine

is diluted 10 times in phosphate buffer of pH 7.0 and

subjected to electrochemical analysis. The amount

of uric acid present in the urine is estimated to be

0.55 � 0.08 g L�1. This value is comparable to the

reported values in the literature [32].

Conclusion

The present study demonstrates an excellent approach

for the development of a novel voltammetric UA sen-

sor based on Pen SAMs. Fast electron transfer, high

selectivity and excellent sensitivity for the oxidation

of UA are achieved at the Pen=Au electrode. The

present monolayer-electrode showed excellent sensi-

tivity, selectivity, reproducibility and antifouling prop-

erty and can separated oxidation peaks towards UA

and AA, which are indistinguishable at the bare elec-

trode. As the voltammetric signals of UA and AA are

well separated at the Pen=Au electrode, the sensitive

Fig. 6. DPVs for 0.1 mmol L�1 UA and 0.1 mmol L�1 AA in

0.1 mol L�1 phosphate buffer solution (pH 7.0) at bare Au elec-

trode (a) and Pen=Au electrode (b). Scan rate: 4 mV s�1; pulse

amplitude: 50 mV; pulse width: 60 ms; pulse time: 200 ms

Fig. 7. DPVs for UA and AA at Pen=Au electrode in 0.1 mol L�1

phosphate buffer solution (pH 7.0) while simultaneously changing

their concentration (i.e., [UA]¼ (a) 10, (b) 60, (c) 100, (d) 130, (e)

160 mmol L�1; [AA]¼ (a) 50, (b) 120, (c) 200, (d) 250, (e)

300 mmol L�1). Scan rate: 4 mV s�1; pulse amplitude: 50 mV; pulse

width: 60 ms; pulse time: 200 ms

Determination of uric acid in the presence of ascorbic acid using a penicillamine self-assembled gold electrode 77

detection UA in the presence of AA or the simulta-

neous detection of UA and AA is possible. The elec-

trode is stable and does not undergo surface fouling

during the measurements.

Acknowledgements. This project was supported by the Doctor

Foundation of Dalian nationalities University (20056101).

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78 Determination of uric acid in the presence of ascorbic acid using a penicillamine self-assembled gold electrode