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Talanta 70 (2006) 823–832

Simultaneous determination of ascorbic acid, uric acid andneurotransmitters with a carbon ceramic electrode

prepared by sol–gel technique

Abdollah Salimi ∗, Hussein MamKhezri, Rahman HallajDepartment of Chemistry, Kurdistan University, P.O. Box 416, Sanandaj, Iran

Received 18 July 2005; received in revised form 4 January 2006; accepted 7 February 2006Available online 24 March 2006

bstract

A sol–gel carbon composite electrode (CCE) has been prepared by mixing a sol–gel precursor (e.g. methyltrimethoxysilane) and carbonowder without adding any electron transfer mediator or specific reagents. It was demonstrated that this sensor can be used for simultaneousetermination ascorbic acid, neurotransmitters (dopamine and adrenaline) and uric acid. Direct electrochemical oxidation of ascorbic acid, uriccid and catecholamines at a carbon composite electrode was investigated. The experimental results were compared with other common carbonased electrodes, specifically, boron doped diamond, glassy carbon, graphite and carbon paste electrodes. It was found that the CCE shows aignificantly higher of reversibility for dopamine. In addition, in comparison to the other electrodes used, for CCE the oxidation peaks of uric acid,

scorbic acid and catecholamines in cyclic and square wave voltammetry were well resolved at the low positive potential with good sensitivity.he advantages of this sensor were high sensitivity, inherent stability and simplicity and ability for simultaneous determination of uric acid,atecholamines and ascorbic acid without using any chromatography or separation systems. The analytical performance of this sensor has beenvaluated for detection of biological molecules in urine and serum as real samples.

2006 Elsevier B.V. All rights reserved.

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eywords: Electroanalysis; Carbon composite electrode; Sol–gel; Dopamine; A

. Introduction

Dopamine (DA) and other catecholamines play importantoles in central nervous system, cardiovascular, renal and hor-onal systems functioning as well as in drug addiction andarkinson disease [1]. Similarly ascorbic acid (AA) has beensed for prevention and treatment of common cold, mental ill-ess, infertility, cancer, and in some clinical manifestations ofIV infections [2]. Uric acid (UA) is the primary product ofurine metabolism in the human body. It has been shown thatxtreme abnormalities of UA levels are symptoms of several dis-ases (e.g. gout, hyperuricaemia and Lesch–Nyhan syndrome)3,4]. Therefore, it is essential to develop simple and rapid

ethods for determination these biological molecules in rou-

ine analysis. The development of voltammetric sensors for theetermination of neurotransmitters, uric and ascorbic acids in

∗ Corresponding author. Fax: +98 871 6660075.E-mail address: absalimi@yahoo.com (A. Salimi).

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039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2006.02.015

aline; Ascorbic acid; Uric acid; Real samples

uman fluid such as urine and serum has received consider-ble interest in recent years. Neurotransmitters, ascorbic andric acids are electrochemically active compounds co-existingn body fluids and can be determined by electrochemical tech-iques. A major problem is that AA, UA and neurotransmit-ers are oxidized at nearly same potential with poor sensitivityt solid (unmodified) electrodes which results in overlappedoltammetric responses making their discriminations highlyifficult [5]. Simultaneous detection of neurotransmitters, espe-ially dopamine, ascorbic and uric acids is a problem of crit-cal importance not only in the field of biomedical chemistrynd neurochemistry but also for diagnostic and pathologicalesearch. Moreover the solid electrodes very often suffer fromhe fouling effect due to the accumulation of oxidized productn the electrode surface, which results in rather poor selectiv-ty and sensitivity. Electrochemical techniques with modified

lectrodes have received great interest for uric and neurotrans-itters determination in the presence of ascorbic acid as they

re more selective and less time consuming than those basedn other colorimetric or spectrophtometric methods. Different

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24 A. Salimi et al. / Tal

odified electrodes such as DNA modified graphite powdericroelectrode [6], layer by layer assembled carbonnanotubes

7], highly oxidized electrodes [8], self-assembled monolay-rs of hetroaromatic thiols [9], exfoliated graphite electrode10], poly-phenosafranine modified electrodes [11], gold elec-rode modified with N-acetylcysteine self-assembled monolayer12], poly-sulfosalicylic acid modified glassy carbon electrode13], choline and acetylcholine modified glassy carbon elec-rode [14], self-assembled bilayer membrane modified glassyarbon electrode [15], cationic self-assembled monolayers [16],lay modified electrode [17], glassy carbon electrode modi-ed with 2,2-bis(3-amino-4-hydroxyphenyl) hexafluoropropane18], tetrabromo-p-benzoquinone modified carbon past elec-rode [19] and carbon paste electrode modified with iron(II)hthalocyanine complexes [20], have been used for simulta-eous detection of catecholamines and ascorbic acid or uriccid and ascorbic acid. Most of these studies demonstrated thendividual determination of both catecholamines and uric acids well as ascorbic acid by eliminating the others. Also, mostodified electrodes used for catecholamines, uric and ascorbic

cids detections have certain disadvantages, such as consider-ble leaching of electron transfer mediators and poor long-termtability. Furthermore the preparation methods are more expen-ive and difficult or the need for renewal of the surface after eacheasurement. In addition the irreversible adsorption behaviour

enders the routine analysis difficult and some of them are notensitive enough for real sample analysis. Finally the oxidationeaks of biomolecules are not well separated.

Sol–gel technology has aroused great interest in the designnd application of electrochemical sensors due to its simplic-ty, physical rigidity, transparency, porosity and permeability,ersatility and flexibility in the fabrication procedure [21–23].ilver and copper dispersed composite electrodes prepared byol–gel derived ceramic graphite containing 3-mercaptopropylrimethoxysilane have been used for simultaneous determinationf ascorbic acid and dopamine [24,25]. A fluorescent sol–geliosensor has been also used for uric acid determination [26].he modified electrodes prepared with sol–gel technique haveome disadvantageous such as low detection sensitivity andonsuming specific and expensive reagents and materials forheir preparation. Therefore, it is necessary to develop an elec-rochemical sensor based on sol–gel technique, which is freerom the above problems. To our best knowledge, none of theodified electrodes prepared by sol–gel technique show better

erformance in peak separation and simultaneous determina-ion of AA, UA and neurotransmitters. In an effort to constructn efficient electrochemical sensor based on sol–gel techniquend their applications in electroanalysis of important analytesuch as insulin, NADH, hydrazine, hydroxylamine, sulphite, l-ysteine, gluthathione and sulphur oxoanions modified carboneramic electrodes were employed in our group [27–36]. Appli-ation of nafion coated glassy carbon electrode modified withatechin-hydrate and basal plane pyrolytic graphite electrode

odified with immobilization of multiwall carbon nanotubes

or voltammetric and amperometric detection of dopamine andpinephrine in the presence of ascorbic acid was reported in ourecent work [32,35]. In this work we present the application of

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70 (2006) 823–832

arbon ceramic electrode prepared by mixing graphite powdernd methyltrimethoxysilane without adding any electron trans-er mediator or specific reagents for simultaneous determinationf catecholamines (dopamine and adrenaline), uric and ascorbiccid. Finally the analytical performance of this sensor for simul-aneous determination of dopamine, ascorbic and uric acids inrine and human serum samples is evaluated by square waveoltammetry.

. Experimental

.1. Chemical and reagents

Methyltrimethoxysilane (MTMS) and nujel oil were pur-hased from Aldrich. Dopamine, adrenaline, ascorbic acid, uriccid, and high purity graphite powder (50 �M diameter) wereeceived from Merck and used without purification. All otherhemicals and reagents were of analytical grade. Double dis-illate water was used to prepare all solutions. Buffer solu-ions (0.1 M) were prepared from phosphoric acid (H3PO4),odium acetate (CH3COONa), di-sodium hydrogen phosphateNa2HPO4), hydrogen chloride (HCl) and sodium hydroxideNaOH) for the pH range 1–10. Solutions were deaerated byubbling high purity (99.99%) of argon gas through them prioro the experiments. All electrochemical experiments were car-ied out at room temperature 25 ± 0.1 ◦C.

.2. Apparatus and procedures

The Metrohm electrochemical system (757 VA Computraceetrohm, The Netherlands) equipped with a (757 VA Compu-

race software, Metrohm, The Netherlands) was used for voltam-etric and amperometric measurements. A three-electrode cell,

ncluding glassy carbon 0.0314 cm2, carbon paste 0.0314 cm2,oron doped diamond 0.25 cm2, graphite 0.0314 cm2 and car-on ceramic 0.0314 cm2, electrodes as working electrode, ang/AgCl (saturated KCl) and a platinum wire were used as ref-

rence and counter electrode respectively. Glassy carbon (GC),raphite, Pt wire and reference electrodes were from Metrohm

nd boron doped diamond (BDD) was from (BDD, DeBeersndustry Diamond Division Ascot, supplied via Windsor Scien-ific, Slough, UK). The carbon ceramic electrode (CCE) wasrepared by sol–gel technique. The preparation procedure isxplained in the following section. A Metrohm drive shaft forotating working electrode was used in amperometric measure-ents. A personal computer was used for data storage and

rocessing. The following instruments parameters were usedo record the square wave voltammograms: square wave ampli-ude: 35 mV, frequency 25 Hz and step potential 4 mV.

.3. Preparation of the carbon ceramic electrode

The carbon ceramic electrode was prepared through the con-

entional method described by the Lev and co-workers [37], byixing 0.2 ml (MTMS), 0.3 ml methanol and 10 �l hydrochlo-

ic acid (11 M). This mixture was magnetically stirred for 5 minfter which 0.3 g graphite powder was added and the resultant

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ixture shaken for additional 3 min. Then, the carbon sol–gelaste was packed into one end of a Teflon tube (with 2 mm inneriameter and 5 cm length, and the length of composite materialn the tube was about 0.5 cm) and dried under ambient condi-ions (25 ◦C) for 48 h. A copper wire was inserted through theomposite end to establish electrical contact. The electrodes wasolished with polishing paper and subsequently rinsed with dis-illed water. In amperometric measurements the CCEs rotatedy contact them with a Metrohm drive shaft.

. Results and discussions

.1. Electrochemical oxidation of ascorbic acid, dopaminend uric acid

The cyclic voltammograms recorded for ascorbic acid,opamine and uric acid at glassy carbon, carbon paste, basallain pyrolytic graphite electrode, boron doped diamond andarbon ceramic electrodes, are shown in Fig. 1. As can be seent the surface of all electrodes, the oxidation of uric and ascorbiccids are irreversible with large overpotentials although peakotentials for both molecules at less positive values at BPPGEnd CCE in compared to other electrode materials. These

esults indicate the slow electron transfer rate for the oxidationf these biomolecules at utilized electrodes. Such sluggishlectron transfer kinetics is related to the electrode foulingased by the deposition of AA, UA and their oxidation products.

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ig. 1. Cyclic voltammograms of 5 mM AA, 1 mM UA and 1 mM DA at the surface0 mV s−1.

0 (2006) 823–832 825

or dopamine, the cathodic peak was observed at the surfacell electrodes but peak potentials separation are 0.35, 0.45nd 0.6 V at GC, CP and BDD electrodes, respectively. At theurface pyrolytic graphite electrode a quasireversible behaviourEp (150 mV) was observed for oxidation of dopamine. In

omparison to above electrodes at CCE the dopamine oxidations reversible with peak separation 30 mV and cathodic to anodiceak current ratio unity. To examine the ability of different elec-rodes to oxidation of other neurotransmitter derivatives, cyclicoltammogram for adrenaline oxidation at above electrodesere recorded (not shown). The irreversible oxidation peak

or adrenaline was observed at the same dopamine oxidationeak potential for all electrodes. Similar electrochemicalehaviour was observed for oxidation of dopamine and otheratecholamine at glassy carbon electrode modified with singleall carbon nanotubes [38] and microcylander polymer mod-

fied electrodes [39]. The effect of potential scan rate on thexidation response of dopamine and adrenaline were examinedn the 20–200 mV s−1 range of scan rates (not shown). Foropamine the ratio of cathodic to anodic peak currents is aboutne, but for adrenaline only at scan rate above 50 mV s−1 theathodic peak is observed. The oxidation currents for bothatecholamines, uric and ascorbic acids are increasing with

he square root of scan rate, indicating that the oxidationurrents were diffusion controlled, which is the ideal case foruantitative measurements. Fig. 2 shows the dependence ofhe voltammetric response of the CCE on the dopamine and

different modified electrodes in phosphate buffer solution (pH 5) at scan rate

826 A. Salimi et al. / Talanta 70 (2006) 823–832

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ig. 2. Cyclic voltammograms of a CCE in 0.1 M phosphate buffer solution (pHo outer 0.1,0.2,0.3,0.4,0.5, 0.6,0.7 and 0.8 mM; (B) adrenaline from inner to ou

drenaline concentration with addition of these biomolecules.sing a constant scan rate of 20 mV s−1 the variation in the peak

urrents were analysed as a function of added dopamine anddrenaline concentration. The response was found to produce ainear response over the entire concentration range of 0.1–1 mMIp/�A = 0.0056[adrenaline]/mM + 0.728 �A, R2 = 0.9991)nd 0.1–0.80 mM (Ip/�A = 0.009 [dopamine]/mM + 0.306 �A,2 = 0.998) with a limit of detections (signal to noise = 3)

o the 30 and 20 �M for adrenaline and dopamine, respec-

ively. For uric and ascorbic acids the cyclic voltammogramsere recorded at different concentration (not shown). The

elationships between concentration and peak currentsere linear over the concentration range, 0.2–0.8 mM

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ig. 3. Cyclic voltammograms of 2.0 mM dopamine recorded at different pH values frnd peak currents vs. pH.

t scan rate 20 mV s−1 with various concentration of: (A) dopamine from inner1 to 1 mM. Insets: the plots of peak currents vs. biomolecules concentration.

Ip/�A = 0.0108[uric acid]/mM + 4.47 �A, R2 = 0.9996) and.1–1 mM (Ip/�A = 0.0014[ascorbic acid]/mM + 1.31 �A,2 = 0.9997) with a limit of detections (signal to noise = 3) to

he 15 and 25 �M for uric acid and ascorbic acid respectively.Since the electrochemical behaviour of above compounds

re pH dependent, we investigated the oxidation activity ofopamine, adrenaline, uric and ascorbic acids on the CCE inuffers with pH values 1–10. Fig. 3 shows the recorded cyclicoltammograms of 2.0 mM dopamine in solution with pH val-

es ranging from 1 to 10. As can be seen in the inset of Fig. 3,p decreased by increasing the pH value, and current response

or dopamine oxidation is high at pH range 4–7, clearly show-ng that protons are involved in the oxidation process. The slope

om right to left 1 to 10, scan rate 20 mV s−1, Insets: the plots of peak potentials

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f Ep versus pH is 55 mV/pH unit, which is very close to thenticipated Nernstian value for a two-electron two-proton pro-ess. A similar behaviour was observed for other compounds,hereas for adrenaline a small current changes were produced

n the entire pH range. Based on both, high peak current andow possible peak potential, pH 5 was selected as the workingH value to carry out the square wave voltammetry and amper-metric determination of these compounds.

.2. Cyclic voltammetry of a mixture of ascorbic acid, uriccid, dopamine or adrenaline

Since uric acid, dopamine, adrenaline and ascorbic acidave similar oxidation potential at most solid electrodes andany modified electrodes, separate determination of these

iomolecules is a great problem due to overlapped signals.he main objective of the present study is to simultaneouslyetermine AA, UA, DA or adrenaline using carbon ceramic elec-rode prepared by sol–gel technique. To evaluate the sensitivitynd selectivity of this sensor for simultaneous determination ofbove biomolecules, cyclic voltammograms mixture of thesepecies are recorded at the surface CCE and compared withther electrode materials (Fig. 4). We can see the peak potentialsor dopamine, uric and ascorbic acids are indistinguishable at a

C, BDD, and carbon paste electrodes. At the surface pyrolyticraphite electrode separate peaks for oxidation dopamine andric acid was observed, however, the broad overlapped voltam-etric peak for ascorbic acid oxidation is a serious obstacle

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ig. 4. Cyclic voltammograms a mixture of AA (1 mM), DA (1 mM) and UA (2 mM

0 (2006) 823–832 827

n mixture determination. Fortunately at CCE the overlappedoltammetric peaks is resolved into three well defined CV peaksFig. 4) at about 0.1, 0.3 and 0.45 V corresponding to the oxida-ion of AA, DA, and UA, respectively.

.3. Simultaneous determination of AA, UA, DA ordrenaline by square wave voltammetry technique

Since square wave voltammetry has much higher current sen-itivity and ability for peak separations than cyclic voltammetryt was employed to estimate the lower limit of detection andimultaneous determination AA, UA and DA or adrenaline. It isell known that AA is a major interference in the voltammetricetection of UA and neurotransmitters (vice-versa) as they arexidized at about the same potentials. Also the electrochemi-al sensing of the other biomolecules using solid unmodifiedlectrodes suffers from the interference of AA that is present atilimolar level in biological fluids. In addition the fouling effect

f the AA and its oxidation products decreases the reproducibil-ty of analytical method for biomolecules detection. One of the

ain objectives of this study is to detect AA, UA and DA ordrenaline simultaneously. Fig. 5A shows the SQWV of a mix-ure of ascorbic acid, dopamine and uric acid. Fig. 5B shows theQW voltammograms obtained at CCE when the concentration

A, UA and DA were simultaneously changed. The calibration

urves and correlation coefficients for these biomolecules arehown in inset of Fig. 5B. The detection limit, linear calibra-ion range and sensitivity of AA, UA, DA and adrenaline were

) at the surface different electrode materials at (pH 5), scan rate 20 mV s−1.

828 A. Salimi et al. / Talanta 70 (2006) 823–832

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ig. 5. (A) Square wave voltammograms of 10 �M of AA, 1 �M DA, and 2 �

f AA, DA, and UA are simultaneously changed: 10, 20, 30, 40, 50 �M of AAiomolecules concentrations.

eported in Table 1. The analytical parameters are better or com-arable with results reported for simultaneously determinationf these biomolecules at the surface different modified electrodes

Table 1 results and Refs. [44–50]). The electrode response wasery stable and showed excellent antifouling properties againsthe oxidation products. For evaluation of the ability of CCE toetermination UA or catecholamines in the presence of ascorbic

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at CCE in 0.1 M phosphate buffer solution (pH 5). (B) as (A) concentration1, 2, 3, 4, 5 and 6 �M DA and UA. Insets: the variation of peak currents vs.

cid, the SQW voltammograms was recorded for the oxidationf UA and dopamine in the presence of AA (Fig. 6). A linearncrease in the peak current of neurotransmitters and uric acid

ere observed while increasing the concentration of dopamine

nd UA and the value of voltammetric peak current of AA isemained almost unchanged during the addition of UA and neu-otransmitters (only the background current is changed). These

A. Salimi et al. / Talanta 70 (2006) 823–832 829

Table 1Analytical parameters for simultaneous detection UA, AA and neurotransmitters at several modified electrodes

Electrode Method Analyte Dynamicrange(�M)

Limit ofdetection(�M)

Sensitivity(�A �M−1)

Reference

Sol–gel composite containing copper and MPSa Amperometry AA 16–5000 8.6 – [24]DA 7.3–2200 5.8 –

Glassy carbon modified by propionylcholine DPVb UA 0.07–70 0.02 – [41]AA – –

Novel choline and acetylcholine modified glassy carbon DPV DA 0.7–5 0.3 – [14]AA 7–90 0.9 –5-HTc 1–30 0.5 –

Sol–gel composite containing silver and MPS Amperometry DA in the presence ofAA

6.6–1200 1.4 0.026 [25]

Self-assembled lipid bilayer (DTDB-BLM)d modifiedglassy carbon electrode

DPV DA 0.01–100 0.01 – [15]

FIAe In the presence of AA 5 × 10−4–500 5 × 10−5 –Carbon paste electrode containing iron II

phethalocyanin and iron(II) tetrasulfo phethalocyaninOSWVf DA 1–15 1 – [20]

5-HT 1GC modified with (BAHHFP)g DPV DA 0.05–3 0.005 138 [18]

UA 0.5–100 0.1 4.67Gold modified electrode cationic self-assembled

monolayerSWVh DA 0.5–100 0.5 0.021 [16]

AA 0.3–100 0.3 0.036Modified carbon paste electrode by

tetrabromo-p-benzoquinoneDPV AA 10–600 0.62 0.005 [19]

DA 10–100 – 0.0074UA 10–100 – 0.0022

Highly oxidized electrode LSVi AA 100–7000 100 – [8]UA 0.2–60 0.2 –

Clay modified electrode SWV DA 0–6 0.0027 – [17]UA 0.1–10 0.2 17

Gold electrode self-assembled monolayer ofhetroaromatic thiol

OSWV AA – – 6.47 [9]UA 1–300 1 14.9

Oxidized diamond film electrode Amperometry UA in the presence ofAA

Up to 1 0.015 – [40]

2,4,6-Triphenylpyrylum ion encapsulated into zeolyte Ysol–gel ceramic film incorporating methylene-blue onGC electrode

DPV DA in the presence ofAA

1–250 0.2 0.062 [42]

Sol–gel ceramic film incorporating methylene-blue onGC electrode

UA – 0.001 – [43]AA – 0.005 –

Nafion coated glassy carbon electrode modified withcatechin hydrate

Amperometry DA in the presence ofAA

0.1–100 0.011 0.016 [33]

Bppgj electrode modified with MWCNTsk Amperometry Epinephrine in thepresence of AA

0.1–100 0.02 0.42 [35]

Amperometry DA 0.5–50 0.07 0.0414 This workUA 1–50 0.4 0.0303AA 5–1000 1.7 8.5AD 0.5–100 0.07 0.044

Sol–gel carbon ceramic composite electrode SWV DA 0.5–20 0.1 0.75UA 0.5–20 0.1 0.754AA 10–200 5 0.36AD 5–100 1 0.83

a (3-Mercaptopropyl)trimethoysilane.b Differential pulse voltammetry.c 5-HT serotonin.d 5,5-Ditetradecyl-2-(2-trimethyl-ammonioethyl)-1,3-dioxane bromide.e Flow injection analysis.f Osteryoung square wave voltammetry.g 2,2-Bis(3-amino-4-hydroxyphenyl)hexafluoropropane modified GC.h Square wave voltammetry.i Linear sweep voltammetry.j Basal plane pyrolytic graphite.k Multi wall carbon nanotube.

830 A. Salimi et al. / Talanta 70 (2006) 823–832

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DfomaFsr(Since fabrication procedure of CCE is easy without using anyelectron transfer or specific reagents, the long-term stability ofthe electrode is not a big issue. Electrode can be renewed sim-ply by polishing its surface. One of the big advantages of this

ig. 6. Square wave voltammograms of simultaneously concentration of DAnd UA changed from 1 to 5 �M in 0.1 M phosphate buffer solution (pH 5) inhe presence 0.1 mM ascorbic acid.

esults indicate that the homogeneous catalytic oxidation has noteen found and the voltammetric peak of AA was unaltered byhe oxidation of UA and neurotransmitters.

.4. Amperometric determination of AA, UA, DA anddrenaline

For compounds such as dopamine, adrenaline and other neu-otransmitters derivatives, a separation step is needed whenixture of these compounds want to be analysed by using an

mperometric detection because of they have the very similarxidation potentials. The CCE is suitable amperometric detectoror catecholamines determination because it has high sensitivitynd antifouling properties. Fig. 7 shows amperogram recordedrom a rotating disk carbon ceramic electrode. In this experimenthe potential of electrode was controlled at the constant valuef 0.4 V during the successive addition of 0.5 �M dopamine. Aell-defined response is observed for addition of dopamine up

o 100 nM. As shown in inset of Fig. 7, the measured currentsncrease by increasing the dopamine concentration in solution.owever, for a high concentration of dopamine the I versus DA

oncentration curve deviate from linearity. The detection lim-ts, linear calibration range and sensitivity for DA, UA, AA anddrenaline are presented in Table 1. These analytical parame-ers are comparable or even better than those obtained whensing several modified carbon ceramic or other kind modifiedlectrodes (see results of Table 1 and Refs. [44–50]). In addi-ion, small variation (2–4%) in the sensitivity was observedhen the concentration of above molecules was changed in

he concentration calibration range. Fig. 8 shows the steady-tate response of carbon ceramic electrode obtained for thexidation of DA, AD, UA and ascorbic acid. The steady-state

urrent for oxidation of these biomolecules was examined atonstant potential of electrode at 0.5 V in phosphate buffer solu-ion (pH 5) and individually injection of DA, AD, UA and AAnto the solution. The current response for adrenaline, UA and

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ig. 7. Chronoamperometric response of the CCE for the successive addition of.5 �M dopamine to the stirring 0.1 M phosphate buffer solution; pH 5; stirringate: 1000 rpm and electrode potential was kept at 0.4 V. Inset: calibration plot.

A before and after the injection of AA is very similar. Thisact confirms the elimination of the above mentioned catalyticxidation at the CCE, and these results indicate SQW voltam-etric measurements. The selective determination of AA, DA

nd UA can be made by controlling the electrode potential.or instance, by holding the electrode potential at 0.15 V, theelective determination of ascorbic acid can be done. The cur-ent responses for AA, DA, UA and adrenaline were very fast<1 s) and stable upon repeated injection of these biomolecules.

ig. 8. Steady-state current response obtained for the oxidation of DA, AD, UAnd AA at CCE upon the injection of (A), (B), (C) 1 �M of DA, AD, UA and (D)0 �M of AA, respectively. Electrode potential and rotation rate were 500 mVnd 1000 rpm.

A. Salimi et al. / Talanta 70 (2006) 823–832 831

Table 2Determination of UA, AD and DA in real samples with carbon ceramic electrode

Sample Original (�M) Added (�M) Found (�M)a Recovery (%) Totalb (mg/l)

Serum Uric acid, 23.34 10 33 (±1) 100(±3)20 44 (±1) 100 (±3) 41 (±2)30 55 (±1) 103 (±3)

Serum Adrenaline 2 2.1(±0.05) 105(±2.3)3 3.2(±0.1) 106(±3)4 4 (±0.1) 95(±4)

Urine Uric acid, 39.13 10 50 (±01) 103(±3)20 58 (±1) 99 (±2) 548 (±3)30 68(±1) 98 (±2)

Urine Dopamine 1 1.02(±0.02) 102(±1)2 1.85(±0.05) 92 (±2)3 3.3 (±0.1) 110(±3)

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CE for amperometric detection of above biomolecules is itsigh stability of amperometric response during the long periodf time towards AA, DA, UA and adrenaline oxidation. Weave recorded the amperometric response of AA, DA, UA anddrenaline for the period 20 min (not shown). It was interest-ngly observed that oxidation current only decreased less than% after 20 min.

.5. Analytical performance of carbon ceramic electrodeor biomolecules determination in real samples

We examined the applicability of our carbon ceramic elec-rode for determination of uric acid, dopamine and adrenaline ineal samples by measuring UA, DA and adrenaline in urine and

ig. 9. Square wave voltammogram obtained for the oxidation of real urineample (83 times diluted with pH 5 phosphate buffer) (a) at CCE surface: serumn the presence different concentration of uric acid, (b) 10 �M, (c) 20 �M, (d)0 �M and (e) 40 �M.

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etected value by the appropriate dilution factor (83 for urine and 10 for serum

erum samples. The urine and serum samples were diluted 83nd 10 times with phosphate buffer solution before the measure-ents to prevent the matrix effect of real samples. The squareave voltammograms recorded for urine sample were shown inig. 9. As can be seen a voltammetric peaks at 0.11 and 0.4 Vere observed, corresponding to the oxidation ascorbic and uric

cid respectively. However no detectable peaks observed for oxi-ation DA and adrenaline in potential range of 0.15–0.3 V. Theesults obtained for UA in serum and urine samples are listedn Table 2. To ascertain the correctness of the results the urinend serum samples were spiked with certain amount of UA, DAnd adrenaline in about the same concentration as found in theamples themselves or linear concentration range of method.he recovery rates of the spiked samples were determined and

anged between 97 and 105% for, UA, DA and adrenaline inerum and urine samples. The total value concentration of UAn the serum and urine sample are in close agreement with thealue reported by other research groups [16,17,25].

. Conclusion

In this paper we presented a method for preparation of car-on ceramic electrode by sol–gel technique without using anylectron transfer mediator or specific reagents. The results indi-ated that the CCE facilitates the simultaneous determinationf uric acid, ascorbic acid, dopamine or adrenaline with goodensitivity and high selectivity compared to the similar basedlectrodes. This sensor can be used for square wave voltammet-ic or amperometric determination of UA, and neurotransmitterss low as 0.5 �M in the presence of 0.1 mM ascorbic acid withoutnterfering and surface fouling. The CCE is sensitive, stable andast response for biomolecules determination and was shownhat it can be successfully used for determination of UA and

A in diluted real samples and neurotransmitters detection in

eal samples. Simple fabrication procedure, wide linear range,ow detection limit, high stability and good reproducibility forepeated determination, suggest this electrode as a good andttractive candidate for practical applications.

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32 A. Salimi et al. / Tal

cknowledgments

This research was supported by research office of Kurdistanniversity. The authors are grateful to Dr. Saied Soltanian, Dr.abibollah Danyali and Dr. Gholam Hossain Karimi Dostan for

heir valuable discussion and Dr. Mehrdad Parki (Shafa Medicalaboratory of Sanandaj) for serum samples.

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