Journal of Electroanalytical Chemistry 705 (2013) 86–90

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Journal of Electroanalytical Chemistry

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Bismuth film random array carbon fiber microelectrodesfor determination of cysteine and N-acetyl cysteine

1572-6657/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.jelechem.2013.07.031

⇑ Corresponding author. Tel.: +385 21329475; fax: +385 21329461.E-mail address: brinic@ktf-split.hr (S. Brinic).

Slobodan Brinic ⇑, Nives Vladislavic, Marijo Buzuk, Marija Bralic, Mislav ŠolicDepartment of Chemistry, Faculty of Chemistry and Technology, University of Split, Teslina 10/V, 21000 Split, Croatia

a r t i c l e i n f o

Article history:Received 3 May 2013Received in revised form 22 July 2013Accepted 24 July 2013Available online 3 August 2013

Keywords:Bismuth filmMicroelectrodesCarbon microfiberStripping voltammetryCysteineN-acetyl cysteine

a b s t r a c t

The electrochemical behaviors of bismuth at random array carbon fiber microelectrodes (RACFMEs) arestudied by cyclic voltammetry. Obtained results indicate that dissolution of bismuth is facilitated in thepresence of cysteine, by formation of bismuth cysteinate on electrode surface. Ex situ prepared bismuthfilm random array carbon fiber microelectrodes (BiF-RACFMEs) were applied for determination of cys-teine and N-acetyl cysteine (NAC) by using square wave cathodic stripping voltammetry (SWCSV). For-mation of bismuth cysteinate was performed at �0.4 V, followed by bismuth reduction duringcathodic sweep.

The electrodes presented a linear response range toward cysteine and NAC, with estimated detectionlimits of 0.028 lmol L�1 and 0.069 lmol L�1, respectively. In the case of NAC two different linear concen-tration ranges of 0.1 lmol L�1 to 1.0 lmol L�1 and of 1.0 lmol L�1 to 10.0 lmol L�1 were obtained. Theprepared BiF-RACFMEs have been successfully applied for determination of NAC in pharmaceutical sam-ples (food supplement) with excellent recoveries.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Cysteine plays an important role in biochemical processes andenvironmental systems [1,2]. It is also presented in food (as anantioxidant) and in various cosmetic and pharmaceutical prepara-tions [3]. Analysis of cysteine and its derivates in biological fluids(urine, blood, etc.), can provide information about a number of dis-eases, such as growth disorder in children, depigmentation of hair,liver damage and some pathological condition such as Alzheimer’sand Parkinson disease [4–6].

Thus, determination of cysteine has attracted considerableattention up to nowadays. In this sense, electrochemical methodspresent advantages of simplicity and high sensitivity. Severalpotentiometric determination of cysteine were reported: determi-nation with electrodes based on incorporated carrier in PVC [7],determinations with Ag2S [8] and AgI [9,10] electrodes, and deter-minations with Trichosporon jirovecii yeast cells coupled with sul-fide electrode [11]. On the other hand, one of the main problemsthat occur during amperometric or voltammetric determinationof cysteine (and other thiols) is high positive overpotential for theiroxidation at most conventional electrodes. Oxidation of thiols in-cludes electron transfer between electrode surface and electroac-tive species in solution, which require appropriate mediators,thus construction of these electrodes become too complicated.

Detail information about modifiers, electrode modification, meth-ods of determination and analytical characteristics were summa-rized in the works of Lima et al. [3], Santhiago et al. [12], Liuet al. [13] and Hallaj et al. [14].

The high affinity of reduced sulfur for mercury was used indetermination of cysteine by cathodic stripping voltammetry(CSV) using mercury electrodes [15]. Interestingly, investigationsof sulfur containing organic molecules with electrodes based onbismuth or bismuth compounds were reported by only a few con-tributors. Baldrianova et al. [16] reported a Bi–powder carbonpaste electrode (Bi–CPE) for determination of cysteine by cathodicstriping voltammetry, while Nosal-Wiercinska [17,18] studiedelectroreduction of Bi3+ in the presence of cysteine at the drop-ping mercury electrode. This is surprising, bearing on mindresemblance in behavior between Bi and Hg toward cysteine(both, Bi3+ and Hg2+ form stabile cysteinate complex), advantageof the Bi as environmental friendly element over Hg and the factthat conventional and microbismuth film electrodes have beenwidely used in electrochemical analysis of various organic com-pounds [19–26].

To the best of our knowledge, no study has been reported on thedetermination of biological thiols using bismuth film electrode(BiFE). As supporting material, for bismuth film, carbon fiber elec-trodes were chosen due to their advantages over other carbonbased materials, such as decreasing of capacitive current, increas-ing of mass transfer rate, faster equilibrium time and negligiblyohmic drop [27].

-1.0 -0.5 0.0 0.5 1.0

-20

0

20

40 1 × 10 mol L cysteine-3 -11 × 10 mol

Start

Without cysteine

E / V

A

C

S

Fig. 1. Cyclic voltammograms obtained at RACFMEs containing 1 � 10-3 mol L�1

Bi(III) in acetate buffer solution (pH 4.5) in absence and presence of 1 � 10�4 mol L�1

cysteine; scan rate 20 mV/s.

-0.70 -0.65 -0.60 -0.55 -0.50 -0.45 -0.40

BiF-RACFMEs in buffer solution with cysteine: 8, 6, 4 mol L-1

Start

-0.7

-0.6

-0.5

-0.4BiF-RACFMEs in buffer solution

-0.3

E / V

Fig. 2. Cyclic voltammograms recorded at BiF-RACFMEs in acetate buffer solution(pH 4.5), in absence and presence of 4, 6 and 8 lmol L�1 cysteine; scan rate20 mV s�1. 0 20 40 60 80 100

-1.0

-0.5

0.0

Es = 2 mV= 2 mV

Es = 5 mV= 5 mVEs = 8 mV= 8 mV

f / Hz

(A)

I p/

-2.0

-1.5

-1.0

I p/

S. Brinic et al. / Journal of Electroanalytical Chemistry 705 (2013) 86–90 87

Contrary to the methods based on oxidation of cysteine onmodified carbon electrodes, which are mostly explored, methodsbased on reduction of Bi-cysteine complex, have been poorly stud-ied and reported.

In this sense, the present paper offers a novel approach to thedetermination of the cysteine and NAC, based on suitability ofthe ex situ prepared bismuth film random array carbon fibermicroelectrodes (BiF-RACFMEs), in conjugation with square wavecathodic stripping voltammetry (SWCSV). Remarkable analyticalcharacteristics, together with simplicity and reliability, comparedto other reported methods were achieved. Also, reliability of NACdetermination in pharmaceutical sample was performed andreported.

E /p mV

0 50 100 150

-0.5

0.0

(B)

Fig. 3. Effect of the electrochemical parameters on peak current for SWCSV on BiF-RACFMEs in acetate buffer solution (pH 4.5), containing 3 lmol L�1 cysteine: (A)frequency (f) with different potential increment (DEs); (B) pulse height (DEp).

2. Experimental

2.1. Chemicals and solutions

Carbon microfibers were obtained from Good-fellow (LS249697; filament diameter 7 lm; 12,000 filaments). Nitric acid, so-dium acetate, sodium chloride and acetic acid, all purchased fromKemika (Croatia), were prepared by dissolution in redistilled water.Stock solution of the bismuth nitrate (1 � 10�3 mol L�1 Bi3+) wasprepared by dissolution of 99.99% Bi(NO3)3 � 5H2O (Sigma–Aldrich, Inc.) in acetate buffer solution (0.1 mol L�1; pH 4.5).

Solution of cysteine and NAC (both from Merck), histamine, lauricacid, penicillamine and glutathione (all from Sigma Aldrich) wereprepared daily by dissolution of appropriate amount of the aminoacid in redistilled water, previously deaerated with N2. The foodsupplement (NAC-Twinlab� dietary supplement) was purchasedfrom local drug store and prepared for analysis in same manneras solution of the cysteine.

2.2. Apparatus

Electrochemical measurements were carried out with potentio-stat (Autolab PGSTAT 302N), connected to PC and driven by GPES4.9 Software (Eco Chemie). An electrochemical cell with threeelectrodes was used with saturated calomel electrode (SCE) asreference, Pt plate as auxiliary and RACFMEs or BiF-RACFMEs asworking electrodes were used. All experiments were carried outat 25 �C.

pH

3 4 5 6 7 8

-1.5

-1.2

-0.9

-0.6

-0.3

0.0

I p/

Fig. 4. Influence of the pH on peak current for SWCSV on BiF-RACFMEs in acetatesolution containing 3 lmol L�1 cysteine with f = 50 Hz, DEs = 5 mV andDEp = 50 mV.

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

I c= 0.256 + 0.405=

R = 0.998=2

1.5

2.0

2.5

3.0

I p/

A

E / V

I c= 0.293 + 0.458=

-0.8 -0.7 -0.6 -0.5 -0.4-0.8

-0.6

-0.4

-0.2

0.0

c / mol L-1

0 0.2 0.4 0.6 0.8 1.0 1.2

0.50

0.55

0.60

0.65

0.70

0.75

R = 0.990=2

0

(A)

I p/

A

88 S. Brinic et al. / Journal of Electroanalytical Chemistry 705 (2013) 86–90

2.3. Electrodes fabrication

RACFMEs were fabricated by dip-coating of carbon microfibersin epoxy resin. Prior to bismuth deposition, electrode was mechan-ically polished with emery paper (2000 grit), followed by polishingwith alumina powder down to 0.05 lm. After polishing, the elec-trode was repetitive cyclized in 0.5 mol L�1 HNO3 solution, be-tween �1 V and 1 V (scan rate of 100 mV s�1). Formation ofbismuth film on such prepared RACFMEs was performed from ace-tate buffer solution (0.1 mol L�1; pH 4.5) containing 1 � 10�3

mol L�1 Bi3+ at �0.7 V for 600 s.

E / V

-0.8 -0.7 -0.6 -0.5 -0.4

-3.5

-3.0

0 2 4 6 8 100

0.5

1.0

(B)c / mol L

-1

Fig. 5. The SWCSVs, with baseline correction, recorded at BiF-RACFMEs in acetatebuffer solution (pH 4.5) with successive addition of NAC in: (A) 0.1 lmol L�1 stepsfrom 0.1 lmol L�1 to 1.0 lmol L�1 and (B) 1 lmol L�1 steps from 1 lmol L�1 to10 lmol L�1, together with background response. Experimental conditions: accu-mulation potential �0.4 V, accumulation time 540 s, equilibration time 60 s,f = 50 Hz, DEs = 5 mV and DEp = 50 mV. The insets show the corresponding calibra-tion plots.

2.4. Measurements procedure

Appropriate amount of cysteine or NAC was added in acetatebuffer solution (0.1 mol L�1; pH 4.5). Accumulation was performedat cathodic potential (�0.4 V) during 540 s in stirred solution and60 s in quiescent solution. After accumulation, SWCSV was per-formed in quiescent solutions from �0.4 V toward �0.8 V, with po-tential scan frequency of the 50 Hz, pulse height of the 50 mV andpotential increment of the 5 mV. Before determination, a back-ground response was recorded at same condition as mentionedabove. These measurements were carried out in deaerated solu-tions and nitrogen blanket was kept above solution during cysteinedetermination.

3. Results and discussion

3.1. Electrochemical behavior of bismuth

Electrochemical behavior of bismuth film deposition and disso-lution in acetate buffer (pH 4.5) Bi(III) solution, in the absence andpresence of cysteine, are examined by cyclic voltammetry at RACF-MEs. Typical cyclic voltammograms recorded in the absence andpresence of cysteine, between �1.0 V and 1.0 V, are shown inFig. 1. The voltammogram, obtained in the absence of cysteineshows well defined cathodic (C) and anodic (A) peaks, which corre-sponds to bismuth deposition and dissolution, respectively. In the

presence of cysteine these peaks are higher and bismuth reductionpeak is slightly shifted toward positive potential. This implies thatelectroreduction of bismuth species is facilitated in the presence ofcysteine. Also, in the presence of cysteine during anodic cycle, cur-rent started to increase at more negative value and clearly pro-nounced shoulder (S), before current due to massive bismuthoxidation, can be seen. Similar behavior, noticed on Hg electrode[18], was attributed to mercury cysteine complexes formation atthe electrode surface, which can mediate in electron transfer. Atbismuth carbon paste electrode, such phenomenon, Baldrianovaet al. [16] associated to

BiðsÞ þ 3cysteine! BiðCySÞ3ðadsÞ þ 3Hþ þ 3e� ð1Þ

Table 1Comparison of the analytical performances, reported for the electrochemical determination of cysteine and NAC, at modified carbon materials with our work.

Electrode Method Ep (V) vs Ag/AgCl

LOD(lmol L�1)

Linear range(lmol L�1)

Sensitivity(nA lmol�1 L)

Ref.

Carbon paste electrode modified with 4-nitrophthalonitrile Cysteine CA 0.33 0.25 0.8–13.2 37 [3]Cobalt(II)-4-methylsalophen into carbon paste Cysteine CV, DPV 0.50 0.2 0.5–100 0.036 [33]Microcrystals fullerene-C60 adhered on GCE Cysteine CV 0.58 Not

reported200–1200 15.5 [34]

Bulk carbon electrodes modified with cobalt phthalocyanine Cysteine CV, CA 0.40 0.2 1–12 8.89 [35]Aquocobalamin adsorbed on glassy carbon Cysteine FIA 0.80 1.7 Not reported 9.28 [36]Glassy carbon modified with Nile blue Cysteine CA �0.45 1.3 10–250 Not reported [37]Glassy carbon electrode modified with MWCNTs Cysteine Amperometry 0.18 5.4 10–500 3 [38]Oxidation product of guanine at ZnOx nanoparticles modified GCE Cysteine Amperometry 0.50 0.05 0.3–20 28.5 [14]Boron-doped carbon nanotube modified GCE Cysteine CA 0.47 vs SCE 0.26 0.78–200 0.025 [39]Pt nanoparticles/poly(o-aminophenol) film on GCE Cysteine Amperometry 0.41 0.08 0.4–6300 Not reported [13]Positively charged poly(diallyldimethylammonium chloride) and

negatively charged MWNTs on glassy carbonCysteine Amperometry 0.80 0.3 20–1300 Not reported [40]

Catechol on glassy carbon Cysteine CV 0.22 6.0 15–50 Not reported [41]Bi–powder carbon paste electrodes Cysteine SWCSV �0.60 0.3 1–50 203 [16]Carbon fiber clustered electrode modified with Ag nanoparticles Cysteine CV 0.25 1 � 10�7 1 � 10�7 to

1 � 1049.612 and0.522

[42]

Conducting polymers/gold nanoparticles on glassy carbon Cysteine CV,Amperometry

0.55 0.05 0.5–200 115 [43]a

1-Benzyl-4-ferrocenyl-1H-[1,2,3]-triazole (BFT)/carbon nanotubemodified GCE for determination of NAC

NAC CV, CA, SWV 0.30 0.062 0.1–600 Not reported [44]

Carbon-paste electrode modified with 2,7-bis(ferrocenylethyl)fluoren-9-one (2,7-BF) and carbon nanotubes (CNTs)

NAC DPV 0.40 0.052 0.07–300 421 and 61 [45]

N-(3,4-dihydroxyphenethyl)-3,5-dinitrobenzamidemodifiedmultiwall carbon nanotubes paste electrode

NAC DPV 0.55 0.2 0.5–200 103 [46]

Carbon-paste electrode modified with catehol NAC CV 0.40 10 30–2000 Not reported [47]Carbon-paste electrode modified with cobalt salophen NAC DPV 0.05 0.1–100 64.6 [48]BiF-RACFMEs Cysteine SWCSV �0.40 0.028 1.0–10 398 Our

workNAC 0.069 0.1–10 293

CA: chronoamperometry; CV: cyclic voltammetry; DPV: differential pulse voltammetry; DNP: differential normal pulse; FIA: flow injection amperometry; SWCSV: squarewave cathodic stripping voltammetry; SWV: square wave voltammetry.

a Contains excellent lists of the carbon based modified electrode for cysteine detection between 2008 and 2011.

Table 2The application of BiF-RACFMEs for determination of NAC (n = 3) in NAC-Twinlab� dietary supplement.

Sample no. Original content (lmol L�1) Added (lmol L�1) Peak current (lA) Found (lmol L�1) Recovery (%)

1 1 – 0.67 – –2 1 2 1.16 2.95 97.53 1 4 1.65 4.93 99.04 1 6 2.19 7.03 100.55 1 8 2.69 9.01 100.1

S. Brinic et al. / Journal of Electroanalytical Chemistry 705 (2013) 86–90 89

According to above, for determination of cysteine, we appliedSWCSV on previously formed bismuth(III) cysteinate (Bi(CyS)3 atBiF-RACFMEs, due to

BiðCySÞ3ðadsÞ þ 3e� ! BiðsÞ þ 3CyS� ð2Þ

3.2. Electroanalytical determination of cysteine and NAC

Cyclic voltammograms obtained at BiF-RACFMEs in acetate buf-fer (pH 4.5), in the absence and presence of different cysteine con-centration, are presented in Fig. 2. As can be seen, successiveaddition of cysteine increases anodic current, which in this poten-tial range probably represent surface film formation of bismuth(III)cysteinate, according to Eq. (1). Consequently, at higher cysteineconcentration, cathodic peak of Bi(Cys)3 reduction can be noticed.Thus, for SWCSV determination of cysteine, accumulation potentialof �0.4 V was chosen. This potential ensures formation of bis-muth(III) cysteinate at electrodes surface and, in the same time,avoids active dissolution of Bi which may distort adsorbed Bi(Cys)3

layer.The SWCSV procedure, used in this work, has been established

by monitoring the influence of applied potential increment (DEs),frequency (f), and pulse height (DEp) on peak currents (Ip) and

obtained results are presented in Fig. 3A and B. For all examinedvalues of DEs, an increase of the frequency up to 50 Hz results inan increase of Ip. At frequencies higher than 50 Hz, the currentpeaks decrease, which can be attributed to kinetic limitation[28]. As it can be seen from Fig. 3B the Ip has a practically linearvariation with the DEp for values lower than 80 mV. Thus, allsubsequent SWCSV were carried out with: f = 50 Hz, DEs = 5 mVand DEp = 50 mV.

Effect of the pH on the analytical signal was investigated be-tween pH 3 and pH 7 in acetate solutions containing 3 lmol�1 Lcysteine. As it is shown in Fig. 4, maximum value of the currentpeak was reached at pH 4.5. Decreasing of current peak at lowerpH values (pH = 3.5) can be attributed to weak dissociation of thecysteine –SH group. By increasing of the pH values, distributionof the ionic fractions of the cysteine in solution increase and forma-tion of Bi-cysteinate complex at electrode surface is enhanced.However, at higher pH value, not only Bi-cysteinate complex, butalso Bi hydroxide species at the electrode surface could be formed.As a consequence of the formation of Bi hydroxide species, yield ofthe Bi-cysteinate will be decreased.

When cysteine was replaced with NAC, no discrepancy in ana-lytical performance was noticed. The results of SWCSV in the pres-ence of NAC, are shown in Fig. 5A and B, with corresponding

90 S. Brinic et al. / Journal of Electroanalytical Chemistry 705 (2013) 86–90

calibration graphs as insets. The voltammograms were obtained bystandard addition method at BiF-RACFMEs and presented curvesare background-subtracted reduction currents.

In determination of NAC two different linear concentrationranges of 0.1 lmol L�1 to 1.0 lmol L�1 and of 1.0 lmol L�1 to10.0 lmol L�1 were obtained. These two ranges are characterizedwith good linearity (R2 = 0.990 and 0.998) and with sensitivity of293 nA lmol�1 L and 254 nA lmol�1 L, respectively. The calculateddetection limit, based on the 3r criterion [29], was 0.069 lmol L�1.The change in slope, with linearity preserving, can be attributed tothe increased amount of Bi(CyS)3 accumulated at electrode surface,resulting in saturation of electrode surface, as it was reported forthe determination of cysteine on Hg [30] or to the change in sur-face layer morphology upon thickening [31]. However, this willnot take affect on analytical capabilities since these ranges are welldefined and reproducible, although some authors [16] suggestemploying of shorter accumulation time at higher analyteconcentrations.

In the case of cysteine concentrations between 1 lmol�1 L and10 lmol�1 L, the sensitivity of 398 nA lmol�1 L was obtained andcalculated detection limit was 0.028 lmol L�1.

Slightly lower sensitivity in the case of NAC (254 nA lmol�1 dm3)over cysteine (398 nA lmol�1 L), in a given data range, can beattributed to the presence of the acetyl moiety (in NAC), which canreduce reactivity of the thiol group [32].

The possible interference was studied by addition of appropri-ate amount of histamine, lauric acid, penicillamine and glutathioneinto solution containing 3 lmol�1 L cysteine. The criterion forinterference was a ±5% error in the peak current. No significantinterferences from histamine and lauric acid were noticed. Ob-tained tolerance level, in the presence of penicillamine, was5 lmol�1 L while determination of cysteine in the presence of glu-tathione is hampered. Even a small quantity of glutathione(0.5 lmol�1 L) results with relative error of the 5.0%.

The intra-day repeatability of the proposed method using BiF-RACFMEs was examined by five successive calibration measure-ments of 3 lmol�1 L cysteine solution at the same film. Obtainedrelative standard deviation (RSD) of the 4.8% indicates good repeat-ability. The reproducibility (RSD = 4.9%) was evaluated using sevenfreshly prepared bismuth film (as explained in Section 2.3.) over7 days. Compared to the previously studies, reported for modifiedcarbon materials, this method exhibited remarkable analytical per-formances (high sensitivity, low detection limit, broad linearrange), together with simplicity over more complex electrodeassembly presented in Table 1.

3.3. Analytical application of BiF-RACFMEs in pharmaceutical sample

In order to evaluate the analytical applicability of the proposedmethod, it was applied for the detection of NAC in NAC-Twinlab�

dietary supplement and results are given in Table 2. The concentra-tions of NAC were determined using the standard addition method.Recoveries of 97.5–100.1% from food supplement suggest that thismethod is very reliable and sensitive.

4. Conclusions

Electrochemical study on RACFMEs revealed that oxidation ofbismuth is facilitated in the presence of cysteine with simulta-neously formation of bismuth cysteinate at the electrode surface.

Reduction current of bismuth cysteinate, formed on BiF-RACF-MEs at pH 4.5, was used as analytical signal for determination ofcysteine and NAC. For this purpose SWCSV was used. Estimateddetection limits for cysteine and NAC were 0.028 lmol L�1 and0.069 lmol L�1, respectively.

The determination of cysteine was not significantly influencedin the presence of the histamine, lauric acid and penicillamine,while determination is hampered in the presence of glutathione.

Due to a non-toxic character in comparison with commonlyused mercury analogues, simple procedure and analytical perfor-mances, make this method advantageous over other reported stud-ies. Practical applicability was proven by determination of NAC inpharmaceutical sample with obtained recoveries of 97.5–100.1%.

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