Renewable sol�/gel carbon ceramic electrodes modified with aRu-complex for the amperometric detection of L-cysteine and

glutathione

Abdollah Salimi *, Sima Pourbeyram

Department of Chemistry, Faculty of Science, Kurdistan University, P.O. Box 416, Sanandaj, Iran

Received 19 January 2003; received in revised form 19 January 2003; accepted 18 February 2003

Abstract

A renewable three-dimensional chemically modified carbon ceramic electrode containing Ru [(tpy)(bpy)Cl] PF6 was

constructed by sol�/gel technique. It exhibits an excellent electro-catalytic activity for oxidation of L-cysteine and

glutathione at pH range 2�/8. Cyclic voltammetry was employed to characterize the electrochemical behavior of the

chemically modified electrode. The electrocatalytic behavior is further exploited as a sensitive detection scheme for L-

cysteine and glutathione by hydrodynamic amperometry. Optimum pH value for detection is 2 for both L-cysteine and

glutathione. The catalytic rate constants for L-cysteine and glutathione were determined, which were about 2.1�/103

and 2.5�/103 M�1 s�1, respectively. Under the optimized condition the calibration curves are linear in the

concentration range 5�/685 and 5�/700 mM for L-cysteine and glutathione determination, respectively. The detection

limit (S/N�/3) and sensitivity is 1 mM, 5 nA/mM for L-cysteine and 1 mM, 7.8 nA/mM for glutathione. The relative

standard deviation (RSD) for the amperogram’s currents with five injections of L-cysteine or glutathione at

concentration range of linear calibration is B/1.5%. The advantages of this amperometric detector are: high sensitivity,

good catalytic effect, short response time (t B/3 s), remarkable long-term stability, simplicity of preparation and

reproducibility of surface fouling (RSD for six successive polishing is 3.31%). This sensor can be used as a

chromatographic detector for analysis of L-cysteine and glutathione.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Modified electrode; Carbon ceramic composite electrode; L-Cysteine; Glutathione; Electrocatalytic oxidation; Sol�/gel;

Ruthenium complex

1. Introduction

Thiols are important in biological systems due

to their widespread occurrence in much proteins

and in natural compounds such as glutathione,

cysteine, coenzyme A and lipoic acid. L-cysteine a

sulfur containing amino acid (CySH) as well as its

oxidative version cystine (CyS�/SCy) play very

important role in living systems [1]. Glutathione

exists in nature in the oxidized (GSSG) and

reduced (GSH) forms, which is an essential

cofactor in much biological process such as* Corresponding author. Fax: �/98-871-6624002.

E-mail address: absalimi@yahoo.com (A. Salimi).

Talanta 60 (2003) 205�/214

www.elsevier.com/locate/talanta

0039-9140/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0039-9140(03)00125-5

catabolism and transportation [2]. The reducedform of glutathione (GSH) is required for main-

taining the iron (II) form of hemoglobin in their

reduced state and for maintaining the structure of

red blood cells [3]. In particular the reversible

redox reactions between thiols and the corre-

sponding disulfides (Eq. (1)) are essential process

in many biological and chemical systems.

2RSH 0 R�S�S�R�2H��2e� (1)

Given the widespread involvement of thiols and

the corresponding disulfides in many essential

biological functions, much effort has been made

to develop sensitive and selective methods for their

detection. Numerous chemical and instrumental

techniques for the determination of glutathione

and L-cysteine have been reported. However most

of them suffer from difficulty with sample pre-paration, the need for derivatization or the lack of

sufficient sensitivity, all of which limit their utility

[4]. Compared to other options, electroanalysis has

the advantages of simplicity and high sensitivity.

Since the direct oxidation of thiols at solid

electrodes is slow and requires a potential of at

least 1.0 V [5,6], the study of electrocatalytic

reactions of L-cysteine and glutathione are impor-tant in the electroanalysis of these thiols. The use

of bare electrodes for electrochemical detection of

glutathione and L-cysteine have a number of

limitations, such as low sensitivity and reproduci-

bility, the slow electron transfer reaction, low

stability over a wide range of solution composition

and high overpotential at which the electron

transfer process occurs. The chemical modifica-tions of inert substrate electrodes with redox active

thin films offer significant advantages in the design

and development of electrochemical sensors. In

operation the redox active sites shuttle electrons

between solution analyte and the substrate elec-

trodes often with significant reduction in activa-

tion overpotential. A further advantage of the

chemically modified electrodes is their less proneto surface fouling and oxide formation compared

to inert substrate electrodes. A wide variety of

compounds have been used as electron transfer

mediators for electrooxidation of L-cysteine and

glutathione. Compton and co-workers reviewed

the electrochemical methods for thiols determina-

tion recently [7]. Alternatively, to detect thiolsseveral recent studies have used electrodes mod-

ified with inorganic catalysts, such as cobalt

phthalocyanine [8], Prussian blue [9], ruthenium

cyanide [10], aquocobalamin [11], copper hexacya-

noferrate [12,13], lead ruthenate pyrochlore [14]

and Bi doped in PbO2 [15]. The water-soluble iron

porphyrin, manganese porphyrin and iron (II)

phenanthrolines [16] have been used for electro-catalytic oxidation of L-cysteine. The methyl and

ethyl derivatives of phenothiazine have been used

by Li and co-workers for electrocatalytic oxidation

of glutathione and L-cysteine [17,18]. The glassy

carbon electrodes modified with coenzyme pyrro-

loquinoline quinone have been used for direct

determination of L-cysteine and glutathione and

as an amperometric sensor for these compounds incapillary electrophoresis [19,20]. Unfortunately,

most modified electrodes have certain disadvan-

tages, such as considerable leaching of electron

transfer mediator and poor long-term stability;

furthermore, the methods of preparation are more

expensive and difficult, the electrodes surfaces

cannot be renewed and the irreversible adsorption

behavior renders the routine analysis difficult andsome of them are not sensitive enough for real

sample analysis. Hence, it is pertinent to explore

and develop a simple and reliable method to

fabricate modified electrodes. The sol�/gel method

offers a possibility of preparing glassy materials at

room temperature that can support the immobili-

zation of different reagents [21]. Due to their

excellent physical and chemical stability and theease with which they can be prepared, it’s well

suited for thin film fabrication and electrochemical

studies [21]. The species embedded in the inert and

stable solid matrices usually preserve their func-

tional characteristics and do not leach out or leach

out very slowly when an appropriate entrapment

procedure is used. These properties will contribute

to the long-term operational stability of the sol�/

gel doping materials under storage conditions,

which is expected to be comparable to covalently

bonded matrices and superior to the adsorption

method because of the isolation of reagent from

the surrounding by the network structure of the

pores [22]. An interesting feature of the carbon

ceramic electrodes (CCEs) is that due to the

A. Salimi, S. Pourbeyram / Talanta 60 (2003) 205�/214206

brittleness of the sol�/gel silicate backbone, theactive section of the electrodes is not clogged upon

repeated polishing and thus the active section of

the electrodes can be renewed by a mechanical

polishing after each use or contamination. The

modified CCEs containing ruthenium metalloden-

drimer, manganese hexacyanoferrate and dirho-

dium-substituted polyoxometalate were fabricated

by the sol�/gel techniques and used for voltam-metric and amperometric detection of L-methio-

nine and L-cysteine [23�/25]. These modified

electrodes have major limitation such as low

catalytic activity for oxidation, low sensitivity,

high detection limit, and they have non-ideal

electrochemical behavior. Considering that

Ru(II)/Ru(III) couple is a good electron transfer

mediator, in continues of our studies to preparemodified electrodes [26�/30], the chemically mod-

ified electrode containing Ru [(tpy)(bpy)Cl] PF6

was constructed by sol�/gel technique, have been

used here for amperometric detection of L-cysteine

and glutathione. Since the electrochemical detec-

tors have high sensitivity and wide linear dynamic

range, this modified electrode can be used as

amperometric detector for L-cysteine and glu-tathione after separation by methods such as

HPLC or capillary electrophoresis.

2. Experimental

2.1. Reagents and solutions

High purity graphite powder was obtained fromMerck. Methyltrimethoxysilane (MTMOS) was

obtained from Fluka and used without any further

purification. Glutathione, L-cysteine, acetonitrile

HPLC grade, other reagents were of analytical

grade from Fluka or Merck and used as received.

The [Ru (bpy)(tpy)Cl]PF6 was synthesized, pur-

ified and characterized as reported [31]. Buffer

solutions (0.1 M) were prepared from H2SO4,H3PO4, CH3COONa, Na2HPO4, HCl and KOH

for the pH range 1�/11. Solutions were deaerated

by bubbling high purity (99.999%) nitrogen gas

through them prior to the experiments and the

electrochemical cell was kept under nitrogen atmo-

sphere throughout the experiments.

2.2. Apparatus and procedure

The Autolab modular electrochemical system

(ECO Chemie, Ultrecht, The Netherlands)

equipped with a PSTA 20 module and driven

GEPS software (ECO Chemie) was used for

amperometric and voltammetric measurements.

A three electrode cell, consisting of a CCE,

modified with Ru-complex as a working electrode,an Ag/AgCl (satd.KCl)/3 M KCl reference elec-

trode and a platinum wire served as auxiliary

electrode. All potentials quoted in the text are

versus this reference electrode. All applied electro-

des were from Methrom. A Metrohm drive shaft

for rotating working electrodes was used in

amperometric measurements. The pH measure-

ments were made with a Metrohm 632 pH meterusing a combined glass electrode. The electroche-

mical measurements were carried out at thermo-

stated temperature of 25.09/0.1 8C. A personal

computer was used for data storage and proces-

sing.

2.3. Fabrication of the bare and Ru-complex

modified CCEs

Carbon composite electrodes were prepared by

a procedure based on previous report [32]. The

binder was a sol�/gel material prepared from a

mixture comprising 0.6 ml methanol containing 1

mg Ru-complex, 0.2 ml MTMOS and 20 ml

hydrochloric acid (11 M). This mixture was

magnetically stirred for 5 min after which 0.6 ggraphite powder was added and the resultant

mixture shaken for additional 2 min. Then the

carbon sol�/gel paste was packed into one end of

Teflon tube (with 2 mm inner diameter and 5 cm

length, the surface of the electrode was :/3.14

mm2). The electrode dried under ambient condi-

tions (25 8C) for 48 h. The same procedure was

used for preparation of bare CCE (without Ru-complex). A copper wire was inserted through the

opposite end to establish electrical contact. The

electrodes were polished with polishing paper and

subsequently rinsed with distilled water. In am-

perometric experiments the modified CCEs ro-

tated by contact them with a Methrom drive shaft.

A. Salimi, S. Pourbeyram / Talanta 60 (2003) 205�/214 207

3. Result and discussion

3.1. Electrocatalytic oxidation of glutathione and

L-cysteine on the Ru-complex doped CCEs

The electrochemical response of a Ru-complex

modified CCE was previously reported [33]. The

catalytic oxidation of L-cysteine and glutathione at

the Ru-complex modified CCEs has been exam-

ined to evaluate the feasibility of using the

electrodes in electro-catalysis and electro-analysis.

In order to test the electrocatalytic activity of theRu-complex doped CCEs, the cyclic voltammo-

grams were obtained in the absence and presence

of these compounds. Fig. 1 shows cyclic voltam-

mograms for the electro-catalytic oxidation of L-

cysteine at the bare and modified CCEs in 0.1 M

phosphate buffer (pH 2). Upon the addition of

2.86 mM L-cysteine, there is a dramatic enhance-

ment of the anodic peak current and the cathodic

peak current disappeared (Fig. 1b), which indicate

a strong catalytic effect. The anodic peak potential

for the oxidation of L-cysteine at Ru-complex

modified CCE is about 800 mV while at the bare

electrode the L-cysteine is not oxidized until 1200

mV (Fig. 1d). Thus, a decrease in overpotential

and enhancement peak current for L-cysteine

oxidation is achieved with the modified electrode.

The same behavior was observed for glutathione at

the surface of modified CCE. Fig. 2 shows the

dependence of the voltammetric response of mod-

ified CCE on the L-cysteine concentration with the

addition of L-cysteine (0.9�/2.86 mM). There was

an increase in the anodic peak current and a

decrease in the cathodic peak current. Plot of Icat

vs. L-cysteine concentration was linear in the

concentration range 0.01�/10 mM. The similar

cyclic voltammograms were observed for glu-

Fig. 1. Cyclic voltammograms of a Ru-complex modified CCE in 0.1 M phosphate buffer (pH 2) solution containing (a) 0 and (b) 2.86

mM L-cysteine. ‘c & d’ as ‘a & b’ for bare CCE. Scan rate 10 mV s�1.

A. Salimi, S. Pourbeyram / Talanta 60 (2003) 205�/214208

tathione and the anodic peak currents depend on

glutathione concentration in the range of 0.01�/3

mM. In order to optimize the electrocatalytic

response of the modified CCE to these compounds

oxidation, the effect of pH on the catalytic

oxidation was investigated. The responses of the

Ru-complex modified carbon composite electrode

in 2.86 mM L-cysteine solution at different pH

values (1�/9) were studied. We find that the

electrocatalytic current decreases as pH increases

and in pH�/5 the electrocatalytic current is low.

The same result was observed for electrocatalytic

oxidation of glutathione. Since the modified elec-

trode shows the electrocatalytic activity at pH

range 1�/9 for oxidation L-cysteine and glu-

tathione, but at pH�/5 for both L-cysteine and

glutathione the results are not repeatable and the

activity of modified electrode is decreased. Hence,

the pH value has an effect on the kinetics of

catalytic reaction; optimum pH for both is 2. By

recording cyclic voltammograms of 2.86 mM L-

cysteine solution at different scan rates, the peak

currents for the anodic oxidation of L-cysteine are

proportional to the square root of the scan rate

(data not shown). This result indicates that at

sufficiently positive potential the reaction is con-

Fig. 2. Cyclic voltammograms of a CCE modified in 0.1 M phosphate buffer (pH 2) at scan rate 10 mV s�1 with various concentration

of L-cysteine from inner to outer 0.0, 0.9, 1.66, 2.3 and 2.86 mM. Inset the plot of catalytic peak current vs. L-cysteine concentration.

A. Salimi, S. Pourbeyram / Talanta 60 (2003) 205�/214 209

trolled by diffusion L-cysteine, which is the idealcase for quantitative applications. It can also be

seen that by increasing the sweep rate the peak

potential for the catalytic oxidation of L-cysteine

shifts to more positive and plot of peak current vs.

square root of scan rate deviates from linearity,

suggesting a kinetic limitation in the reaction

between the redox sites of the Ru-complex and

L-cysteine. These results show that the overallelectrochemical oxidation of L-cysteine at the

modified CCE might be controlled by the cross

exchange process between L-cysteine and the redox

site of Ru(II)/Ru(III) couple and diffusion of L-

cysteine. The same results for oxidation of glu-

tathione were observed. Based on the results the

flowing catalytic scheme (EC? catalytic mechan-

ism) showed that [Ru(bpy)(tpy)Cl]�2 specificallycatalyze L-cysteine and glutathione oxidation in

the flowing electrochemical catalytic pathway:

[Ru(bpy)(tpy)Cl]� 0 [Ru(bpy)(tpy)Cl]�2�e-

(at electrode)(2)

2[Ru(bpy)(tpy)Cl]�2

�2CySH 0Kcat

2[Ru(bpy)(tpy)Cl]�

�CyS�SCy� 2H� (3)

3.2. Electrocatalytic characteristics of L-cysteine

and glutathione oxidation at the modified CCEs

In order to get the information about the rate-

determining step a Tafel plot was drawn, using thedata derived from the rising part of the current

voltage curve recorded at scan rate 10 mV s�1. A

slope of 215 mV decade was obtained, indicating a

one electron process was involved in the rate

limiting step, assuming a charge transfer coeffi-

cient of a�/0.72. The Tafel slope (b) was also

obtained from the linear relationship observed for

Ep vs. log v by using the following equation [34].

Ep�(b log n)=2�constant (4)

The resulting b value was obtained 208 mV,

which correlates with the corresponding value

evaluated from polarization measurement (215

mV). For glutathione the same results wereobserved (a�/0.7), and good agreement was

obtained for results that evaluated from two

methods. Under the above conditions for EC?mechanism the Andriex and Saveant theoretical

model [35] can be used to calculation the catalytic

rate constant. Based on this theory a relation

between the peak current and the concentration of

thiol compound for the case of slow scan rates andlarge catalytic rate constant exist.

Ip�0:496nFAD1=2(nF=RT)1=2Cs (5)

where D and Cs are the diffusion coefficient

(cm2 s�1) and the bulk concentration (mol cm�3)

other symbols have their usual meaning. Low

value of Kcat results in values of the coefficientlower than 0.496. For low scan rate (5�/20

mV s�1) the average value of this coefficient was

found to be 0.40 for a Ru-modified electrode with

a coverage 3�/10�9 mole cm�2, a geometric area

(A ) of 0.0314 cm2 in 3.75 mM glutathione at pH 2.

The diffusion coefficient (D ) value calculated by

chronoamperometry method and it was about

4.2�/10�5 cm2 s�1. According to the approachof Andrieux and Saveant and using Fig. 1 Ref.

[35], the average value of Kcat was calculated to be

2.5�/103 M�1 s�1. The values of diffusion coeffi-

cient and catalytic rate constants for L-cysteine are

3�/10�5 cm2 s�1 and 2.1�/103 M�1 s�1, respec-

tively.

3.3. Amperometric determination of L-cysteine and

glutathione at Ru-complex doped CCEs

Since at concentrations suited for cyclic voltam-

metry, the oxidation of L-cysteine or glutathione in

aqueous solution passivated the composite elec-

trode, the amperometry under stirred conditions

or the flow injection analysis with amperometric

detection is employed rather than cyclic voltam-

metry. Then the amperometry with rotated Ru-complex modified CCE was used to estimate the

lower limit of detection of L-cysteine and glu-

tathione at Ru-complex modified CCE. As Fig. 3

shows, a typical hydrodynamic amperometric

response was obtained by successively adding

glutathione to continuously stirred modified elec-

A. Salimi, S. Pourbeyram / Talanta 60 (2003) 205�/214210

trode in phosphate buffer (pH 2). Fig. 3(A, C)

shows a good response chronoamperograms,

which were recorded for rotating, modified CCE

(rotation speed is 3000 rpm) under conditions,

which the potential of Ru-doped modified elec-

trode was kept at 800 mV during the successive

addition of 5 and 100 mM glutathione. A well-

defined response is observed even for addition 2

mM glutathione (not shown). Under optimized

conditions, which the potential of Ru-doped

modified electrode was kept at 800 mV, the

electrode response time was B/3 s. As shown in

Fig. 3(B, D), the measured currents increase by

increasing glutathione concentration in solution

while for a high concentration of substrate, the

plot of I vs. glutathione concentration deviates

from linearity. The calibration plots for glu-

tathione oxidation current were linear for a wide

range of concentration 5�/700 mM. Linear least

squares calibration curve over the range 5�/43 mM

Fig. 3. Amperometric response at rotating modified CCE (the rotation speed is 3000 rpm) hold at 800 mV in 10 ml, 0.1 M phosphate

buffer (pH 2) for (A) successive addition of 100 ml glutathione 0.01 M. (B) successive addition of 50 ml glutathione 0.001 M (C)

variation of chronoamperometric current vs. glutathione concentration, (D) as (C) for lower concentrations of glutathione.

A. Salimi, S. Pourbeyram / Talanta 60 (2003) 205�/214 211

(9 points) had slope of 7.8 nA/mM (sensitivity) and

correlation coefficient of 0.998. The detection limit

was 1 mM when the signal to noise was 3. The

detection limit, sensitivity and linear calibration

range for L-cysteine determination at this modified

CCE are 1 mM, 5 nA/mM and 5�/685 mM,

respectively. These results are comparable and

even better than those obtained by using several

modified electrodes [13,14,19,20,24,25]. The result-

ing current vs. time curve for a series of replicate

concentration of L-cysteine at concentration range

0.9�/4.5 mM is shown in Fig. 4. In test of

reproducibility, it was found that the relative

standard deviation (RSD) of the amperometry

currents of 4.5 mM L-cysteine for nine replicate

determinations was 0.85%. The RSD for five

replicate determinations was B/1.5% at concentra-

tion range of linear calibration. The poisoning

effect was reduced in chronoamperometric mea-

surements under solution stirring condition at

rotating modified CCE, which limited the accu-

mulation of poisoning species on the electrode

surface. Since the modified CCE has several major

advantages such as high sensitivity, low detectionlimit, good and long-term stability at broader pH

range and fast response to oxidation thiol com-

pounds, it can be used as amperometric detector

for L-cysteine and glutathione after separation by

methods such as HPLC or capillary electrophor-

esis.

3.4. Renewal repeatability and long-term stability

response of modified CCEs

Reproducibility of the response at a given

electrode and of the preparation of the composite

are important factors for analytical applications ofCCEs. In comparing with electrodes modified by

conventional methods, the Ru-complex doped

CCE based on sol�/gel process has certain advan-

tages. The hydrophobic MTMOS monomer pro-

duces a controlled wetting of the composite

electrode in aqueous solutions. Hence a bulk-

modified electrode can be polished using an emery

paper and a fresh surface exposed wheneverneeded. This is especially useful for the electro-

Fig. 4. The recorded chronoamperograms for three repetitive addition of 0.9, 1.6, 2.8 and 3.75 mM glutathione.

A. Salimi, S. Pourbeyram / Talanta 60 (2003) 205�/214212

catalytic study since the catalytic activity is known

to decrease when the electrode is fouled. Fig. 5

shows the cyclic voltammograms for six successive

polishing of a Ru-complex doped CCE at a scan

rate of 100 mV s�1 in 0.1 M phosphate buffer

solution (pH 7). As shown the currents and

potential peaks are almost constant after each

polishing. The RSD is 3.31% by measuring the

anodic peak currents, which reflects the repeat-

ability of surface renewal by polishing. In addi-

tion, only a 1% leakage was found when the

electrode was immersed in 0.1 M phosphate buffer

(pH 7) for 48 h. Stability and reproducibility of the

modified electrode were examined by repetitive

cycling at scan rate 100 mV s�1; after 200 cycles of

repetitive cycling no changes were observed at the

peak height and potential separation. Fouling of

the modified CCE fabricated by Ru-complex was

determined using the continuous amperometry

system in response to a 2 mM L-cysteine or

glutathione. The electrode response after 1 h was

very stable and the amperogram currents were �/

97% of the initial values. On the other hand no

changes was observed for the catalytic peak

current and potential of L-cysteine or glutathione

oxidation at the modified electrode after its hold at

800 mV and in 2 mM amino acids for 2 h (a small

shoulder was observed at 400 mV, that disap-

peared after a simple polishing). The sensor was

used for electrocatalytic oxidation of amino acids,

subsequently stored in ambient condition for 6

months, after this period, the catalytic current for

L-cysteine and glutathione was more than 98% of

the initial value. Then neither electrochemical

reactivation of the surface nor polishing of the

electrode was employed during the series of

experimental for long period of time.

4. Conclusion

The modified CCE contains Ru-complex fabri-

cated by the sol�/gel technique. This three dimen-

sional renewable modified electrode mediate the

oxidation L-cysteine and glutathione. The catalyst

and catalytic activity are stable over a wide pH

range. In view of good stability, short response

time, high sensitivity and low detection limit of

this modified electrode, it holds promise for the

quantitative detection of L-cysteine and glu-

tathione in biological samples. This sensor can be

used as an amperometric detector for L-cysteine

and glutathione detection in flow systems, HPLC

and capillary electrophoresis methods.

Acknowledgements

This project was supported by Kurdistan Uni-

versity. The authors acknowledge Dr H. Hadad-

zadeh (Zahedan University) for synthesis,

purification and identification of ruthenium com-

plex.

Fig. 5. The recorded cyclic voltammograms for six successive

polishing of a Ru-complex doped CCE at scan rate 100 mV�1.

A. Salimi, S. Pourbeyram / Talanta 60 (2003) 205�/214 213

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