Electrocatalytic Oxidation and Determination of Cysteine at Oxovanadium(IV) Salen Coated Electrodes

A transition metal complex, oxovanadium(IV) salen (where salen represents N,N-bis(salicylidene)ethylenediamine) is immobilized on glassy carbon (GC) electrodes and utilized for electrocatalytic oxidation of cysteine. In presence of oxovanadium(IV) salen, increased oxidation current is observed due to the effective oxidation of cysteine by the electrogenerated oxovanadium(V) salen species. The oxidation current linearly varies with the concentration of cysteine from 0.1 to 1.0mM. The modified electrode has good sensitivity and low limit of detection.These properties make the oxovanadium(IV) salen as an effective electrocatalyst for the determination of cysteine.


Introduction
Transition metal complexes of N,N  -bis(salicylidene)ethylenediamine (salen) are used as effective catalysts for several reactions including some electrochemical reactions [1,2].A variety of catalytic reactions are reported by metal salen complexes.For example, oxovanadium(IV) salen (VO 2+ -S) complex exhibits effective and selective catalysis for the conversion of aromatic and aliphatic aldehydes to cyanohydrin [3,4].Similarly, a number of metal complexes of vanadium(IV) which possess VO 2+ unit have been synthesized and studied for their catalytic properties [5].The catalytic efficiency and other characteristics of oxovanadium(IV) compounds depend on the nature of ligand, coordination number, and stereochemistry [6].VO 2+ -S is already used for the electrocatalytic oxidation and subsequent determination of pyridoxine [7], ascorbic acid [8], sulphide [9], cysteine [6], and other analytes [10][11][12].
Recently, accurate determination of cysteine has attracted many researchers due to its important role in structural and functional modification of proteins [6].Oxidation of cysteine gives information about structural and functional modification of proteins and it could be used as a destined radiation protector and cancer indicator [13,14].Thus, quantitative determination of cysteine and elucidating the cysteine oxidation mechanism are important in the protein chemistry [15,16].Cysteine oxidation and subsequent determination at bare electrodes suffer because of electrode fouling, low sensitivity, and high oxidation overpotential [5,6].These problems can be solved partially by the utilization of modified electrodes which normally contain a suitable electrocatalyst immobilized on the electrode surface [17,18].Electrocatalysts can be immobilized on the electrode surface in various ways using polymers containing covalently attached electrocatalyst, electrocatalyst immobilized composite/nanocomposite materials, porous inorganic substances possessing electrocatalysts, and so forth [2][3][4][5].Alternatively, conducting polymers or self-assembled monolayers can also be used for the electrocatalytic reactions [19,20].Success of such modified electrodes mainly depends on diffusion of the target analyte through the chemical film to the electrode or to the immobilized electrocatalyst where an electron transfer reaction can occur [21][22][23][24][25].In the present work, VO 2+ -S is synthesized, characterized, and immobilized on a glassy carbon electrode for the electrocatalytic oxidation and subsequent determination of cysteine.

Experimental
2.1.Chemicals and Reagents.Vanadium(IV) oxide sulphate was purchased from Sigma-Aldrich (India).Salicylaldehyde, ethylenediamine, dichloromethane, and cysteine were purchased from SD Fine Chemicals (India).Triple distilled water was used in all experiments.All other chemicals were of analytical grade.The ligand, salen, and the metal complex, VO 2+ -S are synthesized according to a reported procedure [6].

Instrumentations.
FT-IR spectroscopic analysis is performed on PerkinElmer spectrometer (Spectrum 2, UK) and KBr disk method is employed over the range of 400-4000 cm −1 .UV-vis spectra were recorded with a 2802 PC UV-vis absorption spectrophotometer (Unico, USA).All electrochemical experiments were performed with CHI-660C electrochemical workstation (CH instruments, USA) using three-electrode system.Glassy carbon (GC) as working electrode (area = 0.07 cm 2 ), calomel electrode saturated with potassium chloride (SCE) as reference electrode, and Pt wire as auxiliary electrode are employed.To remove dissolved O 2 present in the electrochemical solution, N 2 gas was bubbled through the solution for 20-30 min prior to the electrochemical experiments.

Preparation of Modified
Electrode.VO 2+ -S was immobilized on polished GC electrodes as follows.Typically, 20 L solutions of 0.2% VO 2+ -S and 0.05% polystyrene (as a binder) in dichloromethane were drop coated on a GC electrode and allowed to dry (represented as GC/VO 2+ -S).2+ -S.UV-vis absorption spectra of VO 2+ -S and free salen ligand are shown in Figure 1.The absorption spectra of free salen ligand in N,N-dimethylformamide show one band (curve (f)) at 316 nm.After complexation, it shows peak at 362 nm indicating the successful synthesis of VO 2+ -S.Inset of Figure 1 shows plot of VO 2+ -S absorbance at 362 nm against various concentrations of VO 2+ -S.The calculated molar extinction coefficient,  (9.44 × 10 3 L mol −1 cm −1 ), is comparable with the literature value (370 nm, 8.35 × 10 3 L mol −1 cm −1 ) [1,26].

Characterization of VO
FT-IR spectrum of salen ligand shows bands at 1632, 1949, and 1295 cm −1 due to C=N, C-N, and C-O groups, respectively (Figure 2).In the metal complex, C=N and C-N peaks shifted to 1622 cm −1 and 1939 cm −1 because of the interaction of the vanadium with N atom present in the salen ligand.Free -OH group present in the salen ligand also binds with vanadium metal ion, which results in the shift of C-O group from 1295 to 1305 cm −1 .Occurrence of these bands in the IR spectrum of metal complex provides the evidence for the formation of VO 2+ -S [5,17,18].at room temperature (Figure 3).Anodic peak potential is observed at 0.55 V and cathodic peak potential is observed at 0.36 V. Anodic peak current is observed due to the oxidation of VO 2+ to VO 3+ , where vanadium changes its oxidation state from IV to V. Cathodic peak current is observed due to reduction of VO 3+ to VO 2+ where it reduces back from V to IV [17,19].  /  ratio and Δ  are found to be 0.83 and 190 mV indicating a quasi-reversible reaction [6].

Electrochemical Characteristics of the VO
Inset of Figure 3 shows the plot of anodic and cathodic peak currents against the square root of scan rate.Such linearity over a long range of scan rates specifies the diffusion controlled process at the GC/VO 2+ -S electrodes.electrodes further indicate that the VO 2+ -S film is strongly seized on the GC electrode surface and can be availed for the electrocatalytic applications.

Electrochemical Oxidation of Cysteine.
Electrochemical oxidation of cysteine at bare GC electrodes causes electrode poisoning since the oxidized product, cystine, forms an inert polymeric film on the electrode surface which hinders the electron transfer activities.To avoid this type of problem, generally modified electrodes with immobilized electrocatalysts are used.For efficient immobilization of electrocatalysts, electrode modifiers such as Nafion, organic polymers, clays, zeolites, and sol-gel silica materials are largely used [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42].Coating of such electrode modifiers not only avoids the electrode poisoning but also disperses the electrocatalyst uniformly on the electrode surface and increases the active surface area [43][44][45].Therefore, we tried to immobilize VO 2+ -S onto MCM-41 type silica using a procedure reported for the incorporation of metal phthalocyanines, Co(salen) and [Mn(salen)] [46,47] on MCM-41 type silica.However, our attempts failed and VO 2+ -S could not be incorporated/adsorbed on MCM-41 type silica materials due to unknown reasons.Similarly, clay film [48,49] and Nafion film [31,32,38] coated electrodes were also examined to ion-exchange/incorporate VO 2+ -S into the respective films as per our previous procedures [50].Surprisingly, VO 2+ -S could not be ion-exchanged/incorporated on clay and Nafion films also.Therefore, a solution of polystyrene and VO 2+ -S in dichloromethane is used to attach the electrocatalyst, VO 2+ -S, on the GC electrode surface.Though this method is not widely used and the immobilized electrocatalysts are prone to desorb from the GC electrode surface to solution easily, this method is still advantageous over the carbon paste electrodes (CPEs) [7].In CPE, the homogeneous distribution of electrocatalysts is almost impossible and the current generally varies for each new surface of the same CPE.However, polystyrene-VO 2+ -S film displays stable and reproducible results and exhibits electrocatalysis towards the oxidation of cysteine (vide infra).
Figure 4 shows linear sweep voltammograms (LSVs) of GC/VO 2+ -S and bare GC electrodes in 0.1 M KCl in the absence and presence of 0.1 mM cysteine.Bare GC electrode (Figures 4(c) and 4(d)) did not exhibit any characteristic oxidation peak in both the presence and absence of cysteine, indicating that cysteine cannot be oxidized at bare GC electrode in the potential window studied (0.1-0.8 V).At GC/VO 2+ -S electrode, in the absence of cysteine an oxidation peak at 540 mV (Figure 4(a)) is observed due to the oxidation of VO 2+ -S.Upon addition of 0.1 mM cysteine to the supporting electrolyte, the same GC/VO 2+ -S electrode shows large increase in oxidation peak current at the same potential (Figure 4(b)).This increase in anodic peak current clearly shows the electrocatalytic oxidation of cysteine at GC/VO 2+ -S electrode by the electrogenerated oxovanadium(V) salen [2,20].The LSVs at GC/VO 2+ -S electrode with successive addition of cysteine are shown in Figure 5. Upon incremental addition of cysteine, oxidation current increases linearly from 0.1 to 1.0 mM (Figure 6).Further addition of cysteine increases the oxidation current and, after 3.0 mM, the current levels off or slightly decreases (inset of Figure 6).The linear increase in current with increasing concentration of cysteine can be used to quantitatively determine cysteine presence in real samples [20].The deviation from linearity and leveling off of current above 1.0 mM cysteine could be due to possible changes in the catalytic reaction conditions because cysteine oxidation products formed on the surface of the GC/VO 2+ -S electrode.The continuous increase in catalytic current of cysteine oxidation in the range of 0.1 to 1.0 mM (Figure 6) represents the effective and reproducible electrocatalytic oxidation of cysteine.The repetitive use of the same electrode for multiple analysis of cysteine in aqueous solutions does not show any decrease in peak current.Therefore, using this electrode, one can determine the concentration of cysteine present in aqueous solutions of simple matrix.

Mechanism of Cysteine Oxidation.
According to the literature [2] and our present study, the electrocatalytic oxidation of cysteine by VO 2+ -S can be represented as shown in ( 1) and (2).First, the VO 2+ -S is oxidized electrochemically to VO 3+ -S at the modified electrode surface.This electrogenerated VO 3+ -S reacts with cysteine (CySH in (2)) to produce cystine (CyS-SCy in (2)) and therefore converted back to VO 2+ -S metal complex: VO 2+ -S → VO 3+ -S + e − (electrochemical oxidation) (1) The main purpose of this study is to demonstrate the utilization of VO 2+ -S films in the electrochemical cysteine determination which are formed by simple adherence of International Journal of Electrochemistry  polystyrene to the GC electrode surface.Therefore, evaluation of extensive analytical and/or kinetic parameters was not attempted.

Conclusion
VO 2+ -S is immobilized on a GC electrode as thin film.This film electrocatalytically oxidizes cysteine to cystine at low oxidation potentials.This electrocatalytic process leads to an enormous increase in oxidation current which can be utilized for the sensitive determination of cysteine present in the aqueous solution.The oxidation current linearly varies with the concentration of cysteine from 0.1 to 1.0 mM.

Figure 4 :
Figure 4: Linear sweep voltammograms of GC/VO 2+ -S (a, b) and GC (c, d) in absence (a and c) and in presence (b and d) of 0.1 mM cysteine in 0.1 M KCl.Potential scan rate: 20 mVs −1 .

Figure 6 :
Figure 6: A plot showing the variation of oxidation peak current with incremental addition of cysteine.Straight line indicates the linearity of peak current in the 0.1 to 1.0 mM concentration range of cysteine.Potential scan rate is 20 mVs −1 .Inset represents the peak current of cysteine oxidation from 0.1 mM to 5 mM concentration.