Electrocatalytic oxidation and sensitive detection of cysteine on a lead ruthenate pyrochlore modified electrode

Article abstract of DOI:10.1021/ac0010781

Electrocatalytic oxidation of cysteine (CySH) at Nation/ lead ruthenate pyrochlore (Py) chemically modified electrodes was thoroughly studied. Electrochemical ac impedance spectroscopy analysis indicated the formation of Py microparticles in the interfacial galleries of Nation. Experiments with benchmark systems of Fe(CN)₆^3-/4- and Ru(bpy)^2+/3+ reveal the suppression of Nation's anionic character after the in situ precipitation of Py. Michaleis-Menten-type kinetics with the rate determination step of CyS-Py-Ru^VI → Py-Ru^IV + CyS-SCy was proposed for this catalytic oxidation. The electrocatalytic behavior is further developed as a sensitive detection scheme for CySH by square-wave voltammetry (SWV) and flow injection analysis (FIA). Under the optimized conditions, the calibration curve is linear up to 560 μM with a detection limit (signal/noise 3) of 1.91 μM in SWV. The detection limit can be improved to 1.70 nM (i.e., 24.22 ng in a 20-μL sample loop) in FIA. This is the lowest value ever reported for direct CySH determination without preliminary accumulation.

Full text of DOI:10.1021/ac0010781

Anal. Chem. 2001, 73, 1169-1175
Electrocatalytic Oxidation and Sensitive Detection
of Cysteine on a Lead Ruthenate Pyrochlore
Modified Electrode
Jyh-Myng Zen,* Annamalai Senthil Kumar, and Jyh-Cheng Chen
Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan
disadvantages to extend them into real application. For example,
Electrocatalytic oxidation of cysteine (CySH) at Nafion/
lead ruthenate pyrochlore (P y) chemically modified elec-
trodes was thoroughly studied. Electrochemical ac im-
pedance spectroscopy analysis indicated the formation of
P y microparticles in the interfacial galleries of Nafion.
2+
the Nafion/ Os(bpy)3 electrode showed considerable leaching
2+
of Os(bpy)3 even after it was stabilized in Nafion film.3 The
oxidation process of CySH on polycrystalline gold electrode
displayed complicated kinetics and the irreversible adsorption
behavior rendered the routine analysis difficult.4 Although the Pc
complexes possess substantial catalytic activity, there is a solubility
problem in an acidic environment. The ST-NiTsPc/ carbon paste
electrode was reported recently to overcome these problems
except that the detection range (1-7 mM) is not sensitive enough
for real-sample analysis and the interference from other biological
chemicals is considerably high.10
Surprisingly, metallic oxide electrodes have hardly been used
for this purpose, although they possess redox groups with tunable
oxidation state and large surface area. Ruthenium dioxide (RuO2)
has been reported to have excellent electrocatalytic activity toward
a number of organic compounds through mediation by Ru(VII)/
Ru(VI), Ru(VI)/ Ru(IV), or Ru(IV)/ Ru(III) redox couples.11-21
Nevertheless, the high-temperature pyrolysis (300-700 °C) route
in preparation and large double-layer charging effect make RuO2
less favorable for analytical applications.19-21 To overcome the main
drawbacks of conventional RuO2 electrodes, we disclosed a
Nafion/ lead ruthenate pyrochlore (Py) chemically modified elec-
trode (designated as NPyCME) in electrocatalytic application with
excellent sensitivity.22-34 In this paper, we report the detail and
3 -/ 4 -
Experiments with benchmark systems of Fe(CN)6
and Ru(bpy)2 +/ 3 + reveal the suppression of Nafion’s
anionic character after the in situ precipitation of P y.
Michaleis-Menten-type kinetics with the rate determina-
tion step of CyS-P y-RuVI f P y-RuIV + CyS-SCy was
proposed for this catalytic oxidation. The electrocatalytic
behavior is further developed as a sensitive detection
scheme for CySH by square-wave voltammetry (SWV) and
flow injection analysis (FIA). Under the optimized condi-
tions, the calibration curve is linear up to 5 6 0 µM with a
detection limit (signal/ noise 3 ) of 1 .9 1 µM in SWV. The
detection limit can be improved to 1 .7 0 nM (i.e., 2 4 .2 2
ng in a 2 0 -µL sample loop) in FIA. This is the lowest value
ever reported for direct CySH determination without
preliminary accumulation.
The sulfur-containing molecule cysteine (CySH) plays a crucial
role in biological systems, especially in folding and defolding
mechanisms.1 Because CySH possesses a very low molar extinc-
tion coefficient, a spectroscopic method is suitable for its detection
only with derivatization via the sulfhydryl functionality.2 Compared
to other options, electroanalysis has the advantage of simplicity
and high sensitivity. Several electrochemical systems, such as
Nafion/ Os(bpy)32+, polycrystalline gold, vitamin B12-adsorbed
graphite, phthalocyanine (Pc) complexes of Co and Mo, water-
soluble Fe and Mn porphyrins, and Ni-Pc immobilized silica gel-
modified TiO2 (ST-NiTsPc) electrodes, were reported for CySH
detection.3-10 Unfortunately, most electrodes contain certain
(10) Perez, E. F.; Kubota, L. T.; Tanaka, A. A.; De Oliveira Neto, G. Electrochim.
Acta 1 9 9 8 , 43, 1665-1673.
(11) Burke, L. D.; Murphy, O. J. J. Electroanal. Chem. 1 9 7 9 , 101, 351-361.
(12) Burke, L. D.; Healy, J. F. J. Electroanal. Chem. 1 9 8 1 , 124, 327-332.
(13) O’Sullivan, E. J. M.; White, J. R. J. Electrochem. Soc. 1 9 8 9 , 136, 2576-
2583.
(14) Shieh, D.-T.; Hwang, B.-J. J. Electrochem. Soc. 1 9 9 5 , 142, 816-823.
(15) De Andrade, A. R.; Donate, P. M.; Alves, P. P. D.; Carlos, H. V. F. J.
Electrochem. Soc. 1 9 9 8 , 145, 3839-3843.
(16) Leech, D.; Wang, J.; Smyth, M. K. Analyst 1 9 9 0 , 115, 1447-1450.
(17) Lyons, M. E. G.; Lyons, C. H.; Michas, A.; Bartlett, P. N. Analyst 1 9 9 4 ,
119, 855-861.
* To whom correspondence should be addressed. Fax: 886-4-2862547.
E-mail: jmzen@dragon.nchu.edu.tw.
(1) Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; John Wiley & Sons: New York,
1995; p 1263.
(2) Chwatko, G.; Bald, E. Talanta 2 0 0 0 , 52, 509-515.
(3) Chen, X.; Xia, B.; He, P. J. Electroanal. Chem. 1 9 9 0 , 281, 185-198.
(4) Fawcett, W. R.; Fedurco, M.; Kovacova, Z.; Borkowska, Z. J. Electroanal.
Chem. 1 9 9 4 , 368, 265-274.
(18) Wang, J.; Taha, Z., Anal. Chem. 1 9 9 0 , 62, 1413-1416.
(19) Trasatti, S. In Electrochemistry of Novel Materials; Lipkowski, J., Ross. P.
N., Eds.; VCH Publishers: New York, 1995; p 207.
(20) Senthil Kumar, A. Ph.D. Thesis, University of Madras, Alagappa College of
Technology, Madras, India, 1998.
(21) Senthil Kumar, A.; Chandrasekara Pillai, K. J. Solid State Electrochem. 2 0 0 0 ,
(5) Zagal, J. H.; Aguirre, M. J.; Parodi, C. G. J. Electroanal. Chem. 1 9 9 4 , 374,
4, 408-416.
215-222.
(22) Zen, J.-M.; Wang, C.-B. J. Electrochem. Soc. 1 9 9 4 , 141, L51-L52.
(23) Zen, J.-M.; Wang, C.-B. J. Electroanal. Chem. 1 9 9 4 , 368, 251-256.
(24) Zen, J.-M.; Tang, J.-S. Anal. Chem. 1 9 9 5 , 67, 208-211.
(25) Zen, J.-M.; Tang, J.-S. Anal. Chem. 1 9 9 5 , 67, 1892-1895.
(26) Zen, J.-M.; Ting, Y.-S. Anal. Chim. Acta 1 9 9 7 342, 175-180.
(6) Li, H.; Li, T.; Wang, E. Talanta 1 9 9 5 , 42, 885-890.
(7) Halbert, M. K.; Baldwin, R. P. Anal. Chem. 1 9 8 5 , 57, 591-595.
(8) Mafatle, T. J.; Nyokong, T. J. Electroanal. Chem. 1 9 9 6 , 408, 213-218.
(9) Chen, S.-M. Electrochim. Acta 1 9 9 7 , 42, 1663-1673.
10.1021/ac0010781 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/15/2001
Analytical Chemistry, Vol. 73, No. 6, March 15, 2001 1169

systematic investigation for the catalytic mechanism of CySH
oxidation at the NPyCME. Electrochemical impedance spectros-
copy (EIS) provides insight information about the formation of
Py particles inside the Nafion membrane. The catalytic mechanism
was further applied in the determination of CySH at trace level
by square-wave voltammetry (SWV) and flow injection analysis
(FIA). An extraordinary sensitivity for CySH was obtained at the
NPyCME by FIA.
EXPERIMENTAL SECTION
Chemicals and Reagents. DL-Cysteine (99%), cystine, N-
acetylcysteine, glutathione, glycine, Nafion perfluorinated ion-
exchange powder, 5 wt % solution in a mixture of lower aliphatic
alcohols and 10% water, lead nitrate, ruthenium trichloride, K3-
[FeIII(CN)6], and [RuII(bpy)3]Cl2 were all obtained from Aldrich
(Milwaukee, WI). All the other compounds (ACS-certified reagent
grade) were used without further purification. Aqueous solutions
were prepared with doubly distilled deionized water.
Apparatus. Cyclic voltammetry (CV) and SWV were per-
formed on a BAS 100B electrochemical analyzer and a BAS VC-2
electrochemical cell (West Lafayette, IN). The three-electrode
system consisted of a NPyCME working electrode, an Ag/ AgCl
reference electrode, and a platinum wire auxiliary electrode. Since
oxygen did not interfere with the voltammetric analysis of CySH,
no deaeration was performed for qualitative and quantitative
determinations. The EIS measurements were performed using an
Autolab frequency response analyzer with FRA2 module that was
controlled by an IBM-compatible PC. It was measured at 10
discrete frequencies per decade from 0.01 to 105 Hz with 5-mV
amplitude at an applied bias potential of 0.8 V versus Ag/ AgCl.
An in-house built FRA2 software fitting program was used for
circuit analysis. A wall-jet-type working system was used in FIA.
This consisted of a Cole-Parmer microprocessor pump drive, a
Rheodyne model 7125 sample injection valve (20-µL loop) with
interconnecting Teflon tube, and a BAS electrochemical detector.
A carrier solution of pH 7.4 phosphate-buffer solution (PBS; I )
0.1 M) was used throughout the FIA experiments. UV-visible
spectra were recorded with a Hitachi U-3000 spectrophotometer.
P rocedures. The NPyCME was prepared as described previ-
ously.23 Briefly, the Nafion-coated GCE (designated as Nafion-
GCE) was first formed by spin-coating with 4 µL of 4 wt % Nafion
solution at 3000 rpm. The Nafion-GCE was then ion-exchanged
with Ru3+ and Pb2+ and further reacted in 1.1 M KOH at 53 °C
for 24 h with constant purging of O2. The preparation procedure
resulted in uniform distribution of the catalytically active micro-
particles throughout the Nafion matrix. The formation of Py inside
the Nafion film was confirmed by the X-ray diffraction pattern in
our previous study.23 As to the preparation of the Nafion/ Py
composite electrode (designated as NPyCE), 4 wt % Nafion was
mixed with 1.5 µg/ cm2 Py powder and 4 µL of this mixture was
Figure 1. Cyclic voltammograms with (solid lines) and without
(dotted lines) 1 mM CySH at different electrodes in pH 7.4 PBS at a
scan rate of 50 mV/s.
spin-coated on a clean GCE. The Py powders were prepared
according to the procedure reported earlier.31
SW voltammograms were obtained by scanning the potential
from 0.3 to 1.0 V at a frequency and amplitude of 25 Hz and 55
mV, respectively. At a step height of 4 mV, the effective scan rate
is 100 mV/ s. The CySH quantification was achieved by measuring
the oxidation peak current. Unless otherwise mentioned, 0.1 M,
pH 7.4 PBS was used for all electrochemical measurements.
RESULTS AND DISCUSSION
Voltammetric Study. Figure 1 shows the CV responses of 1
mM CySH on a bare GCE, Nafion-GCE, and NPyCME in pH 7.4
PBS at a scan rate of 50 mV/ s. As can be seen, only at the
NPyCME was an anodic peak corresponding to the oxidation of
CySH to CyS-SCy observed.32 This result clearly demonstrates
the effective catalytic behavior of the NPyCME. The absence of
the corresponding cathodic peak indicates the irreversible nature
of the CySH oxidation process. It is surprising that there is no
obvious faradaic behavior on the NPyCME in bare electrolyte. It
may be due to very low oxide content (i.e., loading) by in situ
precipitation or due to the highly porous and amorphous nature
of Py in the Nafion film. Nevertheless, the Py particles can still
show their specific catalytic activity. Similar behavior was also
noticed on PVC/ Py and Nafion/ Py composite electrodes, which
are not prepared by in situ precipitation procedure like NPyCME.31
The plot of log(ipa) versus log(v) yielded a slope very close to
0.5 on the NPyCME, suggesting that the oxidation follows a
diffusion-controlled charge-transfer mechanism with a current
function, ipa/ (v1/ 2CCySH), of 54.01 A V-1/ 2 s1/ 2 mol-1 cm3. On the
basis of Epa ) [balog(v)]/ 2 + constant, for a totally irreversible
(27) Zen, J.-M.; Ting, Y.-S. Shih, Y. Analyst 1 9 9 8 , 123, 1145-1147.
(28) Zen, J.-M.; Chang, M.-R.; Ilangovan, G. Analyst 1 9 9 9 , 124, 679-684.
(29) Zen, J.-M.; Lai, Y.-Y.; Ilangovan, G.; Senthil Kumar, A. Electroanalysis 2 0 0 0 ,
12, 280-286.
(30) Zen, J.-M.; Senthil Kumar, A.; Chang, M.-R. Electrochim. Acta 2 0 0 0 , 45,
1691-1699.
(31) Zen, J.-M.; Senthil Kumar, A.; Chen, J.-C. J. Mol. Catal. A 2 0 0 1 , 165, 177-
188.
(32) Zen, J.-M.; Senthil Kumar, A.; Chen, J.-C. Chem. Lett. 1 9 9 9 , 8, 743-744.
(33) Zen, J.-M.; Senthil Kumar, A.; Chen, J.-C. Electroanalysis, in press.
(34) Zen, J,-M.; Senthil Kumar, A.; Wang, H.-F. Analyst 2 0 0 0 , 125, 2169-2172.
1170 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001

Scheme 1. Electrocatalytic Mediated Mechaism
for CySH Oxidation at the NPyCMEa
a (A) Illustration of the partcipation of the Py-RuVI/IV redox sites
in the mediated mechanism. (B) Reaction pathway based on the
Michaelis-Menten kinetics.
diffusion-controlled process,35,36 the ba (i.e., Tafel slope) value is
measured as 30.3 mV/ decade. Assuming one-electron transfer in
the rate determination step (i.e., na ) 1), since ba ) 2.303RT/
naFRa, the anodic-transfer coefficient (Ra) can then be calculated
as 0.99. This indicates the effective catalytic performance of the
NPyCME. The ipa versus [CySH] plot at a quasi-steady-state
condition (i.e., at v ) 10 mV/ s) yielded a straight line up to 40
mM and then reached a plateau. This behavior follows surface
saturation kinetics, which is close to the operation of a Michaelis-
Menten-type reaction mechanism.17 The reaction order, m, was
estimated as 0.94 from the log(ipa) versus log[CySH] plot and the
value is close to the Michaelis-Menten prediction.17,29
Figure 2. Electron-transfer reaction with benchmark model systems.
Our preliminary report has demonstrated the Michaelis-
Menten kinetics and its corresponding basic mechanistic param-
eters, such as catalytic (kc), electrochemical (k′ME), and Michaelis-
Menten (Km) rate constants.32 These data were evaluated from
the Lineweaver-Burke (LB) analysis, and some of the results
were compared to a recent report on the ST-NiTsPc electrode.10,32
Scheme 1 shows the electrocatalytic oxidation mechanism of
CySH to CyS-SCy through the Py-RuVI/ RuIV redox couple. In
the electrocatalytic pathway, the reaction of CyS-Py-RuVI f Py-
RuIV + CyS-SCy is considered as the rate determination step
(A) CV responses on different electrodes with 5 mM Fe(CN)63-/4- at
2+/3+
v ) 10 mV/s. (b) CV responses in 1 mM Ru(bpy)3
at v ) 50
mV/s before (dashed line) and after (solid line) transfer to the pure
base electrolyte solution.
Fe(CN)63-/ 4- and cationic Ru(bpy)32+/ 3+ indicate the malfunction
of the ion-exchange ability of Nafion at the NPyCME. Figure 2A
3-/ 4-
shows the typical CV response of 5 mM Fe(CN)6
with the
GCE, NPyCME, and NPyCE at v ) 10 mV/ s. Due to the strong
repulsion effect of Nafion with Fe(CN)63-/ 4-, a poor CV behavior
was observed on the NPyCE. The CV response at the NPyCME,
however, showed as clear and sharp redox signal as that at GCE.
The absence of repulsion effect indicates that the cationic sites
of Nafion are occupied by Py units in the NPyCME. Further
(rds) with a rate constant (kc) of 6.33 s-1 32
. Note that CyS-Py-
RuVI is a high-energy intermediate complex close to the Eryings
activated complex and it is difficult to trap either by electrochemi-
cal and spectroscopic techniques.29 On the other hand, the
physical picture regarding its equilibrium kinetics can be seen
from the Michaelis-Menten rate constant (Km). The UV-visible
spectrum of the electrochemically (ec) oxidized products in pH 4
and 7 confirms the overall basic mechanism as illustrated in
Scheme 1. It is performed by injecting a small volume of 1 mM
CySH at 1.1 V, and a specific peak at λmax ) 283 nm was observed.
This absorption wavelength is identical to the characteristics of
-S-S- link as reported earlier.37,38
2+/ 3+
evidence was provided by studying with cationic Ru(bpy)3
complex at v ) 50 mV/ s as shown in Figure 2B. In all cases,
before transfer to its blank solution, a well-defined anodic peak
of Ru(bpy)32+/ 3+ at 1.10 V was noticed. However, only the NPyCE
still shows a peak response after transfer to blank solution. This
result again proves the absence of cationic exchange ability of
Nafion at the NPyCME.
EIS Study. The purpose of EIS studies is to pursue insight
regarding the Py formation and the film resistivity. Supporting
evidence for the in situ crystallization of Py and its physical
properties in the NPyCME can also be obtained from this study.
Figure 3 shows the complex plane plots for the Nafion-GCE and
NPyCME with/ without 1 mM CySH at a bias potential of 0.8 V in
a wide frequency range of 0.01-105 Hz. Both electrodes were
found to follow a similar trend in blank electrolyte except that
The Py trapped in the interfacial sites of the Nafion galleries
is expected to be responsible for the electrocatalytic oxida-
tion. Experiments with benchmark model systems of anionic
(35) Xia, H.; Li, H.-L., Z. J. Electroanal. Chem. 1 9 9 7 , 430, 183-187.
(36) Golabi, S. M.; Zare, H. R. J. Electroanal. Chem. 1 9 9 9 , 465, 168-176.
(37) Clevland, W. W. Biochemistry 1 9 6 4 , 3, 480-482.
(38) Morioka, Y.; Kobayashi, K. Biol. Pharm. Bull. 1 9 9 7 , 20, 825-827.
Analytical Chemistry, Vol. 73, No. 6, March 15, 2001 1171

Figure 3. Complex plane plots for different electrodes with and
without 1 mM CySH at an applied bias potential of 0.8 V versus Ag/
AgCl in pH 7.4 PBS.
Figure 4. Plots of Epa and ipa versus pH for 1 mM CySH on the
NPyCME using CV in different pHs at v ) 50 mV/s.
(Figure 5A).39 The SH group possesses the strongest oxidizing
tendency among the three functional groups and can form the
disulfide (CyS-SCy) bond easily. To our knowledge, there is no
specific study about the dissociation (i.e., speciation) of CySH so
far. We deconvoluated the different chemical species of CySH
against pH using simple chemical equilibrium analysis as shown
in Figure 5B.40 As can be seen, the dominant species change with
pH and can be used to explain the experimental results. For
example, an increasing trend in the pH range of 6-10.77 was
reported on a vitamin B12-adsorbed OPG electrode toward CySH
oxidation.5 On the basis of the distribution diagram, the respon-
sible species in this case is [HA-], i.e., CyS-. The NPyCME shows
maximum current values at pH <5 and thus the cationic [H3A+]
(i.e., CySH2+) or the neutral [H2A] (i.e., CySH) is responsible for
the disulfide formation and can be explained as follows.
For metallic oxides, the surface hydroxyl groups play an
essential role for the electrocatalytic reactions.19-21,31,41-43 The
reported pHPZC for Py is ∼4.0, and the oxide surface is acidic when
pH is <4 and basic when pH is >4.31,43 It is therefore expected
that the acidic surface sites are responsible for the enhanced
disulfide formation. Indeed, sulfur-containing ligands were re-
ported to lead to an effective catalytic activity on acidic surface
sites of metallic oxides.41 Considering the electronegativity of O
(3.5) > N (3.0) > S (2.5), the oxygen-containing terminals should
directly face the acidic surface sites and either N or S sites are
loosely coordinated to the high-valence metal ions. To check the
expectation, parallel experiments were carried out using glu-
the resistance in Ω was dramatically lowered by ∼3 orders for
the NPyCME. Obviously, it is due to the existence of conducting
crystallite catalysts inside the interfacial sites of the Nafion matrix.
The complex plane plot is dramatically different between the
Nafion-GCE and NPyCME in the presence of CySH. A semicircle
with a frequency maximum at 1.322 Hz was observed at the
Nafion-GCE. Whereas, a broad and distorted semicircle curve at
a comparatively lower Ω value was noticed on the NPyCME.
Simple semicircle analysis, using the Boucamp circuit of Rs(RctCdl),
shows calculated values of Rct ) 1.719 × 105 Ω, Cdl ) 59.06 nF,
and n ) 0.83 for the Nafion-GCE with CySH. In the circuit, Rs,
RCT, and Cdl represent solution resistance, faradaic charge-transfer
resistance, and double-layer capacitor, respectively. Note that, n
) 1 indicates a pure capacitative behavior of the Cdl. These results
show the sluggish faradic electron transfer with high resistance
of CySH on the Nafion-GCE as shown in Figure 1 and Figure 3.
On the other hand, the EIS analysis confirms the formation of Py
microparticles in the interfacial galleries of Nafion. The lowering
in resistivity of the film can in turn mediate the electrocatalytic
oxidation of CySH. The obtained EIS plot for the NPyCME with
CySH is not a Randle-type semicircle behavior and is different
from that of GCE and Nafion-GCE. Thus, we believe the NPyCME
has a complex internal electrical circuit. Detailed EIS analysis on
the above electrode is necessary to solve this problem.
Effect of pH. Figure 4 shows the effect of pH on the CV
response of 1 mM CySH on the NPyCME. As can be seen, no
simple increase in Epa versus pH was observed and relatively
higher ipa was noticed when pH was <5. The behavior is very
different from uric acid and norepinephrine (NE) and other
organic compound oxidation on the NPyCME with a linear
increase in Epa versus pH.25,31,33 The discrepancy has something
to do with CySH since it contains various functional groups of
COOH, SH, and NH2 with pKa of 1.71, 8.36, and 10.77, respectively
(39) The Merk Index, VIth ed.; Merck & Co. Inc.: Rahway, NJ, 1989.
(40) Rubinson, K. A. Chemical Analysis; Little, Brown and Co.: Boston, 1987; p
34.
(41) Ziolek, M.; Kujawa, J.; Saur, O.; Lavalley, J. C. J. Mol. Catal. A 1 9 9 5 , 19,
49-55.
(42) Goodenough, J. B.; Manoharan, R.; Paranthaman, M. J. Am. Chem. Soc.
1 9 9 0 , 112, 2076-2085.
(43) Zen, J.-M.; Manoharan, R.; Goodenough, J. B. J. Appl. Electrochem. 1 9 9 0 ,
22, 140-150.
1172 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001

Figure 5. (A) Various forms of CySH with respect to its pKa value. (B) Distribution curves for various CySH species versus its pH. Distribution
curves were simulated for 1 mM CySH.
Figure 6. (A) SW voltammograms for 1 mM CySH at different electrodes in pH 7.4 PBS. SWV parameters: amplitude, 55 mV; frequency, 25
Hz; step height, 4 mV. (B) Plots of ipa and Epa versus pH on the NPyCME for 1 mM CySH by SWV.
tathione, N-acetylcysteine, cystine, and glycine under similar
conditions. Interestingly, the obtained qualitative results are
similar to that of CySH. On the other hand, no faradaic signals
were noticed with glycine and cystine (RS-SR). These observa-
tions prove the above expectation that only the SH group alone
can get involved in the electrocatalytic oxidation process.
Thus, on the basis of Scheme 1, the irreversible disulfide
formation is considered as the rds. The continuous decreasing of
ipa, when pH is >5, is a direct indication for the decrease in the
overall rate constant (i.e., kc). In other words, the anionic S- group
can somehow lead to a decrease in kc. The appearance of two
oxidation peaks at 1.0 and 0.5 V at pH 6 confirms the transition
of reacting species from SH to S- on the Py-RuVI sites. Therefore,
both the acidic sites of Py trapped in Nafion and the SH group
are key for the electrocatalytic oxidation process. The electro-
catalytic behavior is further utilized as a sensitive detection scheme
for CySH at the NPyCME.
Analytical Application. Both SWV and FIA were studied and
applied for sensitive detection of CySH. Figure 6A shows the
typical SWV response for 1 mM CySH on various electrodes. The
electrocatalytic effect of the NPyCME is qualitatively similar to
those of CV results as shown in Figure 1. The effect of pH on the
SWV response was first investigated and the ipa and Epa versus
pH plots obtained are shown in Figure 6B. As can be seen, the
E
pa versus pH result is similar to that in Figure 1; whereas, there
is an irregular trend at pH 7 for the ipa versus pH result. It may
have something to do with the fact that the SWV response is
proportional to the reversibility of the system. Nevertheless, this
is in effect an advantage considering that the biological reaction
takes part in neutral condition. Moreover, it is believed that, at
pH 7, the basic electrocatalytic mechanism is the same except
with a lower kc value.
To optimize the SWV response for trace detection of CySH,
the corresponding instrumental SWV parameters were studied
Analytical Chemistry, Vol. 73, No. 6, March 15, 2001 1173

Figure 7. (A) FIA responses for different concentration of CySH (10 nM-100 µM) at the NPyCME. Carrier solution was pH 7.4 PBS with a
flow rate of 0.30 mL/min and an applied potential of 1.0 V. (B) Interference effect of 1 mM CySH with cystine (a), glucose (b), and oxalic acid
(c).
next. These parameters are interrelated and have a combined
effect on the response SWV peak, but here only the general trends
will be examined. It was found that these parameters had little
effect on the peak potential. When the SW amplitude was varied
between 10 and 60 mV, the peak currents increased with
increasing amplitude. However, the peak width also increased at
the same time, in particular when the amplitude was higher than
60 mV. The step height together with the frequency defines the
effective scan rate. Hence, an increase with either the frequency
or the step height results in an increase in the effective scan rate.
The response for CySH oxidation peak current increases with SW
frequency up to 25 Hz, above which the peak current was unstable
and obscured by a large residual current, in turn decrease in the
peak current was noticed. By maintaining the frequency as 25
Hz, the effect of step height was studied. As step height is greater
than 4 mV, too few points are sampled, thus affecting the
reproducibility of the detection. Overall, the optimized parameters
are as follows: preconcentration potential, 0 V; preconcentration
time, 20 s; SW frequency, 25 Hz; SW amplitude, 55 mV; step
height, 4 mV.
Linear calibration curve is obtained up to 560 µM (67.8
mg/ L) with a slope and correlation coefficient of 0.0323 µA/ µM
and 0.997, respectively, for CySH detection on the NPyCME. The
detection limit (S/ N ) 3) is 1.91 µM. To characterize the
reproducibility of the NPyCME, seven repetitive measurement-
regeneration cycles were carried out for 40 and 500 µM CySH.
The electrode renewal gives a good reproducible surface since
the relative standard deviation obtained was 1.32 and 1.49%,
respectively.
obtained for different concentrations of CySH in the range of 10
nM to 100 µM. Based on the peak currents in Figure 7A, a linear
calibration plot was obtained with a slope and regression co-
efficient of 0.0788 nA/ nM and 0.9999, respectively. Using 10 nM
CySH as the test solution, the detection limit (S/ N ) 3) was 1.70
nM (i.e., 24.22 ng in a 20-µL sample loop). To our knowledge,
this is the lowest detection limit ever reported for direct CySH
determination without any preliminary accumulation. Thus, by
coupling these electrodes with real protein samples, one can
certainly get information about the SH quantification and in turn
the protein analysis.
The effect of typical organic interferences such as cystine,
glucose, and oxalic acid was also studied (Figure 7B). With 10×
excess in concentration, only the oxalic acid showed ∼20%
alteration in the FIA signal of 1 mM CySH. Nevertheless, the FIA
signal was very stable when the concentration of interferences
was reduced to 5× excess. The sensitivity can certainly be
increased provided that pH is <4 in FIA. Furthermore, since the
process is diffusion-controlled, the response time for the NPyCME
is very fast. All the measurements can be done immediately after
the NPyCME is immersed into the test samples. Finally, no
decrease in CySH response, either peak potential or peak current,
was observed after the same electrode was repeatedly used and
stored in 1.1 M KOH solution for more than 2 months.
CONCLUSIONS
The NPyCME showed excellent electrocatalytic effect toward
the CySH oxidation. The ac impedance measurements give
evidence of the participation of Py in the electrocatalytic oxidation
process. Studies with standard benchmark systems reveal the
unique property of the NPyCME. The Michaelis-Menten-type
kinetics through the participation of the CyS-Py-RuVI intermedi-
ate was well suited for the electrocatalytic oxidation. It is
concluded that the SH groups were oxidized effectively on the
acidic sites of Py. An excellent detection limit was noticed by FIA
in the present system. The reliability and stability of the NPyCME
It is known that FIA can work with high dead volume with
good sensitivity.44 Experiments regarding hydrodynamic flow rate
and applied potential were first systematically optimized as 0.3
mL/ min and 1.0 V, respectively, for 1 µM CySH in pH 7.4 PBS.
Under this optimized FIA condition, Figure 7A shows the results
(44) Wang, J. Analytical Electrochemistry; VCH Publishers: New York, 1994; pp
54-64.
1174 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001

offer a good possibility for extending the technique to routine
analysis of CySH in real samples.
Received for review September 11, 2000. Accepted
January 12, 2001.
ACKNOWLEDGMENT
The authors gratefully acknowledge financial support from the
National Science Council of the Republic of China.
AC0010781
Analytical Chemistry, Vol. 73, No. 6, March 15, 2001 1175