Electrochimica Acta 125 (2014) 566–572

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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta

Electrochemically Active L-Cysteine Gold Modified Electrodes

Fernando C.M. de Oliveira, Silvia H.P. Serrano ∗
Institute of Chemistry–University of São Paulo, Avenida Professor Lineu Prestes, 748 - Bloco 2 Superior, Cidade Universitária CEP: 05508-000, São Paulo
(SP), Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Gold electrodes were easily modified using MPA (3- mercaptopropionic acid) to produce a self assem-
Received 12 September 2013 ble monolayer, SAM, in which electrochemically active cysteine molecules were anchored to produce
Received in revised form Au/MPA and Au/MPA/CSH electrodes. The functional groups, present on the modified electrode surfaces,
23 December 2013
were identified using cyclic voltammetry and ferricyanide ions, an electrochemical probe, at variable pH
Accepted 13 January 2014
values. The steps which describe the global electrode process were identified and characterized using
Available online 30 January 2014
cyclic voltammetry. It involves the deprotonation of the R-SH superficial groups; production of R-S free
radicals from R-S- electrochemical oxidation and attack and of R-S free radicals to the RSH adjacent
Keywords:
MPA
molecules on the electrode surface, followed by the second electrochemical oxidation step to produce
L-cysteine RSSR. The Au/MPA/CSH electrode showed electrocatalytic activity toward oxidation of quercetin in the
Gold Modified Electrodes range of 0.50 to 12.2 ␮molL−1 with a detection limit of 0.15 ␮molL−1 .
Cyclic Voltammetry and Quercetin © 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Modified electrodes containing a reversible or quasi reversible
redox couples confined on the electrode surface has been widely
used for manufacturing sensors with electrocatalytic properties
[1,2] and several molecules, such as metalloproteins [3,4], enzymes
[5,6] and redox coordination compounds containing metal ions
have been used for these purposes [7–8]. The third generation of the
electrochemical biosensors, where the electron transfer takes place
directly between the electroactive confined species and the surface
of electrode across tunneling process, simplifies the electrochemi-
cal devices and increases the possibility of the sensor applications
[9–11].
In this context, thiol modified electrodes have a great poten-
tial to study the electron-transfer reactions in peptide, molecules
which act as cellular signal with–SH moieties being the main elec-
troactive groups [12,13]. Low molecular weight peptides such as
cysteine (CSH) and glutathione (GSH), could be used as model com-
pounds to the electron-transfer reactions in peptide, since they
have been used to improve the electron transfer in free-mediated
biosensors [14]. Self Assembled Monolayers (SAM’s) are given spe-
cific properties to recover the metal electrode surfaces [15–17].
L-cysteine molecule, due to its amphoteric characteristics, has been
used to produce SAM’s on gold electrodes providing a positive, neg-
ative or neutral charge electrode surface, according to the pH of
∗ Corresponding author.
E-mail addresses: shps@iq.usp.br, silvia.iqusp@gmail.com (S.H.P. Serrano).
media [18–21] and the 3-mercaptopropionic acid (MPA) was used
to anchoring peptides on gold surface [22].
This paper presents a new alternative to produce thiol gold mod-
ified electrodes, in which MPA was simultaneously used to cover
gold electrode surface and anchor L-cysteine molecules (CSH).
Thiol-cysteine residues showed quasi reversible redox reaction due
the formation and breaking of disulfide bridges involving thiol
groups on CSH adjacent residues on the gold electrode surface.
The steps which describe the global electrode process were iden-
tified and characterized using cyclic voltammetry. It involves the
deprotonation of the R-SH superficial groups; production of R-S•
free radicals from R-S− electrochemical oxidation and attack and
of R-S• free radicals to the RSH adjacent molecules on the electrode
surface followed by the second electrochemical oxidation step to
produce RSSR, which is further reduced to RSH and R-S• .
2. Materials and Methods
2.1. Reagents and Solutions
All solutions were prepared by dissolution of appropri-
ated quantity of the salts in deionized water. Morpholine-
ethanesulfonic acid (MES), from Sigma&Aldrich, Na2 HPO4 e
NaH2 PO4 , from SIGMA and ammonium from QM, were used as
received. MPA solution at concentration of 25 mmol L−1 was pre-
pared by dilution 1: 1 (v/v) of Fluka stock solution 98% (w/v) in
anhydrous ethanol (Merck). Solutions of cysteine (CSH) 0.2 mol L−1
were prepared through the dissolution of appropriate quantity of
cysteine chloridrate (Sigma&Aldrich) in MES buffer solution.

0013-4686/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.electacta.2014.01.079
 F.C.M.d. Oliveira, S.H.P. Serrano / Electrochimica Acta 125 (2014) 566–572
2.2. Equipments
All aqueous solutions were prepared with deionized water
obtained from a reverse osmosis deionizer Gehaka model OS10LX.
A pH-Meter model 654 W and a combined glass electrode, both
from Metrohm, were used to adjust the pH of the solutions. All
voltammetric measurements were performed using a ␮-Autolab
type III potentiostat/galvanostat from EcoChemie (Netherland). Au;
Au/MPA or Au/MPA/CSH were used as working electrodes, while
Pt and Ag/AgCl, KCl(sat) were used as auxiliary and reference elec-
trodes, respectively.
2.3. Cleaning of Au Electrode Surface
The Au electrode with 0.3 mm diameter was polished in a metal-
lographic felt using alumina 1.0 and 0.3 ␮m, washed with deionized
water and submitted to an electrochemical treatment, which con-
sisted of 10 cyclic voltammograms in 0.1 mol L−1 H2 SO4 solution at
100 mV s−1 in the range of + 0.2 to + 1.6 V vs Ag/AgCl, KCl(sat) .
2.4. MPA Self-assembled Monolayer Formation on Gold Electrode
(Au/MPA)
The gold electrode, previously treated as described above, was
immersed in an ethanolic solution of 25 mmol L−1 MPA, without
shaking, during 25 minutes at room temperature, and subsequently
washed with deionized water. This optimized MPA concentration
was chosen as function of the highest peak area of the MPA des-
orption process in a KOH 0.5 molL−1 solution. The assembling time
has not been rigorously studied because the proposed goal was to
modify the electrode in a relatively short time.
2.5. CSH immobilization on Au/MPA electrode
The Au/MPA electrode was immersed for 10 minutes, under stir-
ring, in a 0.1 mol L−1 CSH solution, prepared in 0.2 mol L−1 MES
buffer at pH 6.2. After that, the Au/MPA/CSH electrode was sub-
sequently washed with MES buffer 0.1 mol L−1 at pH 6.2. The CSH
concentration was chosen taking in account the maximum value
obtained from CSH concentration on the electrode surface versus
CSH solution assembling concentration plot. No significant increase
or decrease in the electrochemical responses was observed for
CSH concentrations higher than 0.1 mol L−1 . In this condition, the
immersion time is not so relevant, because the superficial activat-
ing process can help the molecular rearrangement on the electrode
surface, process which may also be obtained using long time for the
SAM’s preparation.
2.6. Activating of Au/MPA/CSH Electrode
The Au/MPA/CSH electrode was transferred to the electrochem-
ical cell containing 0.1 mol L−1 ammonium acetate, at pH 7.2, as
supporting electrolyte and 100 cyclic voltammograms were carried
at 10 mV s−1 in the range of - 0.2 ≤ Eappl ≤ + 0.3 V. When not in use,
the Au/MPA/CSH electrode was immerse in MES buffer 0.1 mol L−1
and its superficial activity was maintained by 3 days.
2.7. Determination of the superficial functional groups on
Au/MPA and Au/MPA/CSH electrodes
The superficial molecular architecture of the gold modified elec-
trodes was evaluated as described on [22]: cyclic voltammograms
at 100 mV s−1 were performed in a 0.1 mol L−1 NaH2 PO4 /Na2 HPO4
buffer solution, at variable pH values, containing 1.0 mmol L−1 fer-
ricyanide ions (used as an electrochemical probe). Since that Ep
(Ep,a–Ep,c) and current levels are function of the superficial charge
567
Fig. 1. Cyclic voltammograms obtained at Au/MPA electrode in 0.1 mol L−1 phos-
phate buffer solution containing 1.0 mmol L−1 de Fe(CN)6 3− , pH 6.0 (a) and pH 2.0
(b); and at Au/MPA/CSH, pH 6.0 (c). Experimental conditions: Einitial = - 0.1, E␭ =
+0.5 V, Efinal = - 0.1 V at 0.100 Vs−1 .
density and, therefore, of the protonation degree of the superficial
groups in contact with the solution, it was easy to identify which
group was available on the electrode surface [24].
3. Results and Discussions
3.1. Identification of the groups with acid-base properties on the
Au/MPA and Au/MPA/CSH surfaces
Pseudo titrations of the acid-base groups, present on the
Au/MPA and Au/MPA/CSH surfaces, were performed by record-
ing cyclic voltammograms in 0.1 mol L−1 phosphate buffer solution
containing 1.0 mmol L−1 of [Fe(CN)6 ]3− ions at variable pH values
[22]. After deprotonation of the superficial acid groups,–COOH at
Au/MPA and–SH at Au/MPA/CSH, the electrode, due its negative
superficial charge, makes it harder for the [Fe(CN)6 ]3− ions to get
closer. In this condition both, distortion and decreasing in the cur-
rent levels were observed in the voltammograms, Fig. 1.
Fig. 1 Ip,c - pH titrations plots for MPA and MPA/CSH were
used to determine the pKa of the acid-base group present on the
electrode surfaces. Using dIp,c/dpH vs pH curves derived from
data presented on Fig. 2a and 2b, the experimental pKa values
were determined as 5.5 and 8.1 for–COOH and–RSH groups at
Au/MPA and Au/MPA/CSH electrodes, respectively, values which
are in agreement with the pKa of 5.41 for the carboxyl group on
MPA [22] and 8.14 for the thiol group on CSH [23].
Fig. 2 These results confirm the presence of–SH free group in
the surface of Au/MPA/CSH modified electrode, indicating that the
previous modification of gold electrode with MPA monolayer was
effective in the maintenance of free SH groups on the electrode
surface which are able to promote electrochemical reaction at the
interface electrode/solution.
3.2. Activating of Au/MPA/CSH Modified Gold Electrodes
Au/MPA/CSH modified electrode presents, in a 0.1 mol L−1 phos-
phate buffer solution at pH 7.2, a redox couple, with anodic
and cathodic peak potentials, Ep,a and Ep,c, shifting from + 0.052
to + 0.075 V and–0.025 at–0.015 V, respectively. Current peaks, Ip,a
and Ip,c, increased during the course of the successive cycles, but
stable values were obtained after the 40th cycle, Fig. 3.
568 F.C.M.d. Oliveira, S.H.P. Serrano / Electrochimica Acta 125 (2014) 566–572

Fig. 4. Cyclic voltammograms obtained in 0.1 mol L−1 phosphate buffer solution at
pH 7.2 at (a) Au/MPA electrode and (b) Au/CSH electrode at 0.100 Vs−1 . The electrode
Fig. 2. Ic,p vs. pH pseudo titration of (a)–COOH at Au/MPA and (b)–SH at Au/MPA/CSH
Au/MPA was prepared as described in section 2.4 and the electrode Au/CSH was
electrodes.
prepared for immersion in 10 mmolL−1 CSH for 1 hour and washed with deionized
water before use. Other experimental conditions as described on Fig. 3.

Fig. 3 These results indicated that the Au/MPA/CSH surface is

modified during the cycling process, which may involve the re-
orientation of the MPA and CSH molecules on the electrode surface,
facilitating the electron-transfer between CSH molecules and the
gold electrode surface via tunneling electron transfer effect. The pka
value of 8.14 allows us to conclude that the redox couple, detected
on Fig. 3, corresponds to the electrochemical signal of the R-S-S-
R/RSH redox system. Additionally, cyclic voltammograms obtained
in supporting electrolyte at Au/MPA and Au/CSH electrodes, Fig. 4,
show no redox process in the same potential range used in Fig. 3.
Fig. 4 Taking in account the data presented on Figs. 3 and 4, the
Scheme 1 was proposed to describe the MPA adsorption on gold
electrode surface, as well as, the CSH molecular binding to the MPA
surface, respectively.
3.3. Electron-transfer at MPA/CSH modified electrode
The peak potentials due the redox couple presented on Fig. 3
shifted linearly as pH function, but low current levels were
observed at extreme pH values, Fig. 5c.
Fig. 5 As can be observed from Fig. 5c, the peak current goes to
zero at low pH values and to half maximum value at high pH values.
Since the oxidation process does not take place in low pH values
or, can occur, but in low extension, it can be concluded that the
electrochemical oxidation of the–SH groups is preceded by a depro-
tonation step. Thus, the first step of the mechanism should involve
the oxidation of RS− specie (instead RSH) to produce R-S• , which
is able to attack another R-SH group from an adjacent adsorbed
molecule, to produce R-S-S-R. In fact, the signal due the disulphide
bridged was detected at 500 cm−1 by using SERS (Surface-Enhanced
Raman Spectroscopy), data not showed. The major levels of peak
currents observed in Fig. 5c take place in the pH interval of 5.0 to 6.0,
range in which these two species, RS− and R-SH, are present in a suf-
ficient concentration to promote the bimolecular reaction between
RS• and R-SH. At pH greater than 8.0, the concentration of R-SH
decreases due the overall deprotonation of thiol group and, in this
condition; the second electron is not transferred. The set of steps
proposed to mechanism reactions are described by the equations
(1) to (4):

Ka
R − SH−→R − S− + H+ (1)

ks,f
1
R − S− ⇄ R − S• + e− (2)
ks,b
2

ks,f
2
R − S• + R − SH ⇄ R − S − S − R + E + + e−
ks,b
1
(3)
2R − SH ⇄ R − S − S − R + 2H+ + 2e−

(Overall Thiol Oxidation Step − Quasi Reversible Process) (4)

The mean value of slopes of Ep,a and Ep,c versus pH was determined
as 0.037 V/pH. Using this value we calculated the protons-to-
electrons ratio as 0.62 according to equation 5.
 
∂Ep,c
n 
H+
−1 = 0, 059 (at 25◦ C) (5)
Fig. 3. Cyclic voltammograms obtained at Au/MPA/CSH electrode in 0.1 mol L ∂pH ne−
phosphate buffer, pH 7.2. Experimental Conditions: Einitial = - 0.2 V, E␭ = + 0.3 V and
Efinal = - 0.2 V at 0.0100 V s−1 .
 F.C.M.d. Oliveira, S.H.P. Serrano / Electrochimica Acta 125 (2014) 566–572 569

Scheme 1. Molecular binding of CSH to Au/MPA electrode surface.

This value indicated that the deprotonation of–SH species is not 3.4. Determination of kinetic parameters involved in the
involved in the first step of the electron transfer reaction (Equation R-S-S-R/RSH redox couple on the Au/MPA/CSH electrode
2) and it can be considered that the deprotonation process is easier
than the R-S- oxidation process to generate the R-S• radical. The charge transfer coefficient (␣), the surface excess (Ŵ0 ) and
Considering the experimental values of (Ep,c-Ep,c/2), 0.045 V rate constants (ks ) associated to the R-S-S-R/RSH redox couple at
and (Ep,a-Ep,a/2), 0.065 V, obtained at pH 7.2, and using the equa- the Au/MPA/CSH electrode were obtained using the voltammo-
tions (6) and (7) [24], ␣ (the charge transfer coefficient) and n grams performed under different scan rates, Fig. 6.
(number of electrons) were determined as 0.59 and 2.3, respec- Fig. 6 The slopes of the curves Ip,a and Ip,c versus ␯, ∂Ip,a/∂␯
tively. and ∂Ip,c/∂␯, obtained from Fig. 6b, were introduced into the equa-
tions (8) and (9) [24] and, the superficial excess (Ŵо ), as well as, the
0, 062 electron transfer coefficient (␣) for the CSSC/CSH charge transfer
Ep,c − Ep/2,c = (6)
˛n reaction were calculated.
   
0, 062 ∂Ip,a F 2A
Ep,a − Ep/2,a = (7) = (1 − ˛) ◦ (8)
(1 − ˛)n ∂ 2, 718RT

Scheme 2. Redox process of CSH molecules anchored on the Au/MPA/CSH electrode at low scan rate values and description of the redox mechanism involved in the
Au/MPA/CSH modified electrode.
570 F.C.M.d. Oliveira, S.H.P. Serrano / Electrochimica Acta 125 (2014) 566–572

Fig. 6. (a) Cyclic voltammograms obtained at MPA/CSH modified gold elec-

trode in phosphate buffer 0.1 mol L−1 , pH 7.2 under different scan rates:
0.0100 ≤ ␯ ≤ 1.50 Vs−1 . Ei = - 0.2 V, E␭ = 0.3 V and Ef = - 0.2 V and (b) Ip versus ␯ plot.
Ia,p = (0.017 ± 0.08) + (0.628 ± 0.011) ␯; Ic,p = (0.029 ± 0.011) - (0.585 ± 0.016) ␯·.

s and dividing the equation (8) by equation (9), ␣ and Ŵ0 were

calculated as 0.52 and 1.23 × 10−11 mol cm−2 , respectively.
The curves of Ep versus ln ␯ at scan rate values higher than
600 mV s−1 , allowed the calculation of the value of ks , using equa-
tions (10) and (11) [13,24].
  k
RT RT
 
′ s,f
Ep,a = E 0 + ln (10)
(1 − ˛)F (1 − ˛)F 

 RT   RT   k 
′ s,b
Ep,c = E 0 + ln (11)
˛F ˛F 

The value of 0.046 V for E0 , was determined by extrapolation of the

linear segments in the curve of Ep versus ln ␯, Fig. 7.
Fig. 7 Two reduction steps were observed at high scan rates
Fig. 5. (a) Cyclic voltammograms recorded at Au/MPA/CSH; (b) Ep–pH plots and (c) (Ep,c1 and Ep,c2 - Fig. 5a) with the second step being attributed
Ip - pH plots. Ec,p = 0.287–0.039 pH and Ep,a = 0.346–0.037 pH. Data obtained from
to the reduction of R-S• to R-S- (ks,b2), Equation 2.
the cyclic voltammograms carried out under identical experimental conditions as
described on Fig. 3. The equations (12) and (13) were obtained rearranging the
equations (10) and (11) by introduction of Ep as function of ln ␯
in the Laviron’s equation.
   
∂Ip,c F 2A  RT   RT   RT 
=˛ ◦ (9) Ep,c = E 0 +
′
ln + ln ks,b (12)
∂ 2, 718RT ˛F ˛F ˛F
RT RT RT
     
Using A= 0.071 cm2 , R= 8.314 J mol−1 K, T = 298 K, Ep,a = E 0 +
′
ln − ln ks,f (13)
(∂Ip,a/∂␯) = 0.585 ± 0.016 and (∂Ip,c/∂␯) —
— − 0.628 ± 0.011 ␮AV−1 (1 − ˛)F (1 − ˛)F (1 − ˛)F
 F.C.M.d. Oliveira, S.H.P. Serrano / Electrochimica Acta 125 (2014) 566–572
Fig. 7. Curves of Ep versus ln ␯ obtained from cyclic voltammograms shown in
Fig. 5a.
Introducing the values of E◦ , ␣ and the values of Ep,a, Ep,c1 and
Ep,c2 obtained from Fig. 6a in the equations (12) and (13), the rate
constants, ks,f1 , ks,b1 and ks,b2 , were calculated as 8.5 s−1 , 14.5 s−1
and 3.2 s−1 respectively.
Although two oxidative processes are involved in the overall
electrode process (equations 2 and 3), only one peak was observed
Fig. 8. Cyclic voltammograms obtained in 0.1 molL−1 phosphate buffer at pH 7.2
containing 5.6 ␮molL−1 quercetin at: (1) Gold bare electrode and (2) Au/MPA/CSH
electrode. Experimental conditions: Ei = - 0.2 V; E␭ = +0.3 V; Ef = - 0.2 V; ␯ = 0,010 mV
s−1 in presence of O2 .
571
in the positive potential region even at high ␯ values. Accordingly
to the equations (2) and (3), the first oxidative process refers to
the radical R-S• formation, while the second oxidative process can
be attributed to the attack of R-S• to the RSH adjacent molecule, a
highly energetic reaction, which is kinetically favored by the prox-
imity between the radical and the RSH, the target molecule. In these
conditions, the first, and single, oxidation process can be consid-
ered as the slow step in the global mechanism, which includes the
second oxidative step.
The second reduction process (Equation 2) is slightly favored
(ks,b2 = 3.2 s−1 ), due to the competition between the reduction pro-
cess and the sulfyl radical attack to the RSH molecule (Equation 3).
The first reduction process is a second order reaction and is pH-
dependent. Thus, the ks,b1 is a pseudo-first order rate constant and,
the second order reaction for to break of the disulfide bridge can be
calculated as:
ks,b1 = ks,b1′ .[H + ] Equation (14)
The ks,b1 calculated value was 2.30 × 108 L mol−1 s−1 at pH 7.6.
Scheme 2 synthesizes the redox mechanism proposed for the
electrode global process.
4. Detection of Quercetin at Au/MPA/CSH Electrode
Fig. 8 show that quercetin, compound with well-known antiox-
idant activity, can be successfully detected at the Au/MPA/CSH
electrode.
Fig. 8 As can be seen from Fig. 8a the anodic current increases
after the addition of quercetin to the electrochemical cell con-
taining the supporting electrolyte and, linear response to this
compound was observed in the range of 0.50 to 12.2 ␮molL−1 with
a detection limit of 0.15 ␮molL−1 , Fig. 8b.
5. Conclusions
Gold electrodes were easily modified using MPA (3- Mercap-
topropionic acid) to produce a self assemble monolayer, SAM, in
which electrochemically active cysteine molecules were anchored.
The S-S/-SH cysteine redox couple is able to exchange electrons
directly at the gold surface by tunneling effect.
The steps which describe the global electrode process were
identified and characterized, showing the deprotonation of the
superficial R-SH groups; R-S− electrochemical oxidation to pro-
duce R-S• free radical; attack of the free radical to the RSH adjacent
molecule (on the electrode surface) together with a second electro-
chemical oxidation process to produce RSSR, which was reduced
to RSH and R-S• . The electroactivity of the modified electrode
increased slowly with time due the molecular reorganization of
the superficial CSH adsorbed molecules in a monolayer of MPA and
the process was accelerated during the cycling in the supporting
electrolyte solution due to the applied potential. The mechanism
of formation (electrochemical oxidation) and breaking (electro-
chemical reduction) of disulfide bridge is pH-dependent and the
oxidation process occurred in two steps, where the slower step was
the formation of sulfyl radical which attacked–SH residues of adja-
cent L-cysteine molecules, favoring the second electron transfer in
a magnitude, which due to the very short time that takes place,
requires other time accurate techniques to the determination of
the rate constant. The electrocatalytic effect of the Au/MPA/CSH
toward oxidation of quercetin was demonstrated.
572 F.C.M.d. Oliveira, S.H.P. Serrano / Electrochimica Acta 125 (2014) 566–572
Acknowledgements
The authors thank to CNPq, by the Doctoral Scholarship of Fer-
nando C. M. de Oliveira and, to FAPESP (Process n◦ 2009/18223-0)
and CNPq (Process n◦ 308787/2012-2), for the financial support.
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