Direct electrochemical oxidation of disulfides at anodically pretreated boron-doped diamond electrodes

Article abstract of DOI:10.1021/ac020583q

Anodically oxidized diamond electrodes have been used to oxidize disulfides, thiols, and methionine in aqueous acidic media and tested for amperometric detection of these compounds after chromatographic separation. Cyclic voltammetric signals for 1 mM glutathione disulfide (GSSG) were observed at 1.39 and 1.84 V vs SCE, the values being less positive than those of its as-deposited counterpart as well as glassy carbon electrode. The voltammetric and chronocoulometric results have indicated the high stability of the electrode with negligible adsorption. A positive shift in the peak potential with increasing pH indicated the attractive electrostatic interaction between the anodically oxidized diamond surface and the positively charged GSSG in acidic media that promoted its analytical performance. The results of the electrolysis experiments of disulfides and thiols showed that the oxidation reaction mechanism of glutathione (GSH) and GSSG involves oxygen transfer. Following separation by liquid chromatography (LC), the determination of both GSH and GSSG in rat whole blood was achieved at a constant potential (1.50 V vs Ag/AgCl), and the limits of detection for GSH and GSSG were found to be 1.4 nM (0.028 pmol) and 1.9 nM (0.037 pmol) with a linear calibration range up to 0.25 mM. These detection limits were much lower than those reported for the amperometry using Bi-PbO₂ electrodes and LC-mass spectrometry, and the LC method using diamond electrodes were comparable with enzymatic assay in real sample analysis. The high response stability and reproducibility together with the possibility of regeneration of the electrode surface by on-line anodic treatment at 3 V for 30 min further support the applicability of anodically pretreated diamond for amperometric detection of disulfides.

Full text of DOI:10.1021/ac020583q

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Anal. Chem. 2003, 75, 1564-1572
Articles
Direct Electrochemical Oxidation of Disulfides at
Anodically Pretreated Boron-Doped Diamond
Electrodes
C. Terashima,† Tata N. Rao,† B. V. Sarada,† Y. Kubota,‡ and A. Fujishima*,†
Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,
Tokyo 113-8656, Japan, and Department of Urology, School of Medicine, Yokohama City University,
3-9 Fukuura, Kanazawa-ku, Yokohama 236-8567, Japan
thione is found in vivo as both glutathione (GSH) and glutathione
disulfide (GSSG). Under oxidative stress, GSH is known to be
oxidized to GSSG in living cells. Glutathione is a potent antioxidant
which is found at high concentrations in many living cells. It forms
GSSG in the presence of oxidants and rapidly converts back to
GSH by the action of the enzyme glutathione reductase. Because
of this rapid turnover, GSSG is usually found in very low
concentrations in comparison to GSH in normal individuals. The
ratio of GSH to GSSG serves as a sensitive indicator of oxidative
stress and is a key marker for the redox status of cells.1 Therefore,
the detection methods developed for the analysis of these
compounds should exhibit high sensitivity because of the low
available concentrations of GSSG in biological samples.
Anodically oxidized diamond electrodes have been used
to oxidize disulfides, thiols, and methionine in aqueous
acidic media and tested for amperometric detection of
these compounds after chromatographic separation. Cy-
clic voltammetric signals for 1 mM glutathione disulfide
(GSSG) were observed at 1 .3 9 and 1 .8 4 V vs SCE, the
values being less positive than those of its as-deposited
counterpart as well as glassy carbon electrode. The
voltammetric and chronocoulometric results have indi-
cated the high stability of the electrode with negligible
adsorption. A positive shift in the peak potential with
increasing pH indicated the attractive electrostatic inter-
action between the anodically oxidized diamond surface
and the positively charged GSSG in acidic media that
promoted its analytical performance. The results of the
electrolysis experiments of disulfides and thiols showed
that the oxidation reaction mechanism of glutathione
(GSH) and GSSG involves oxygen transfer. Following
separation by liquid chromatography (LC), the determi-
nation of both GSH and GSSG in rat whole blood was
achieved at a constant potential (1 .5 0 V vs Ag/ AgCl), and
the limits of detection for GSH and GSSG were found to
be 1 .4 nM (0 .0 2 8 pmol) and 1 .9 nM (0 .0 3 7 pmol) with
a linear calibration range up to 0 .2 5 mM. These detection
limits were much lower than those reported for the
amperometry using Bi-P bO2 electrodes and LC-mass
spectrometry, and the LC method using diamond elec-
trodes were comparable with enzymatic assay in real
sample analysis. The high response stability and repro-
ducibility together with the possibility of regeneration of
the electrode surface by on-line anodic treatment at 3 V
for 3 0 min further support the applicability of anodically
pretreated diamond for amperometric detection of disul-
fides.
A number of analytical methods using liquid chromatography
(LC) have been developed for the analysis of GSH and GSSG on
the basis of different detection techniques. However, most of these
methods are based either on pre- or postcolumn derivatization
followed by fluorimetric detection or on the conversion to their
phenyl or pyridine derivatives followed by UV detection. Although
electrochemical detection is versatile, detection of these com-
pounds, especially disulfides, is very difficult as a result of their
high overpotentials for oxidation. Earlier methods were based on
reduction of disulfides at an upstream electrode aligned in series
against the flow followed by detection of the thiols formed at the
downstream gold/ mercury electrode.2 Platinum and gold elec-
trodes have also been used for anodic detection of sulfur
compounds.3,4 The anodic reaction of disulfides at Au and Pt
electrodes is mediated by surface oxides in that potential region
that complicates analytical applications. Thus, chemically modified
electrodes with inorganic electrocatalysts have been used to detect
thiols (not disulfides), but have been limited by the fact that their
response decreases with time.5,6 More recently, a coulometric-
type electrochemical cell coupled to LC has been shown to be
successful in simultaneously detecting thiols and disulfides with
Thiols and disulfides are widely distributed in living cells and
are involved in many biological functions. The tripeptide gluta-
(1) Morel, Y.; Barouki, R. Biochem. J. 1 9 9 9 , 342, 481-496.
(2) Allison, L. A.; Shoup, R. E. Anal. Chem. 1 9 8 3 , 55, 8-12.
(3) Vandeberg, P.; Johnson, D. C. Anal. Chem. 1 9 9 3 , 65, 2713-2718.
(4) Hoekstra, J. C.; Johnson, D. C. Anal. Chem. 1 9 9 8 , 70, 83-88.
(5) Nalini, B.; Narayanan, S. S. Electroanalysis 1 9 9 8 , 10, 779-783.
(6) Inoue T.; Kirchhoff J. R. Anal. Chem. 2 0 0 0 , 72, 5755-5760.
* Corresponding author. Tel: 81 3 3812 9276. Fax: 81 3 3812 6227. E-mail:
akira-fu@ fchem.chem.t.u-tokyo.ac.jp.
† The University of Tokyo.
‡ Yokohama City University.
1564 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003
10.1021/ac020583q CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/25/2003

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high stability and sensitivity.7,8 However, this method is not
suitable for the analysis of small amounts of samples. For example,
it cannot be coupled with capillary LC and capillary electrophoresis
for the analysis of single cells9 and microdialysis samples10,11 that
involve small amounts of samples. An electrode material that can
simultaneously detect thiols and disulfides amperometrically is
the best choice for such applications.
usefulness of amperometric detection. The electrolysis results of
this study suggest that the oxidation mechanisms at BDD are
different from the electrochemical reactions at the ordinary carbon
electrodes, in which thiols are oxidized to the corresponding
disulfides. We identified the products of the electrolysis by mass
spectrometry and suggested an oxidation mechanism of sulfur
compounds at BDD. Finally, we applied BDD for the analysis.
After LC separation, the levels of both GSH and GSSG in rat whole
blood were determined by amperometric detection and were
compared with the commercial enzymatic assay.
Highly boron-doped diamond electrodes (BDD) have recently
received a great deal of attention,12 particularly for electroanalysis,
owing to their unique electrochemical properties, for example,
(i) wide electrochemical potential window in aqueous solutions,13
(ii) very low background current,14 and (iii) long-term stability of
the response.15,16 Because of their wide potential window, BDD
electrodes have been successfully used for the detection of several
analytes, such as polyamine and histamine, which possess high
overpotentials for oxidation.17-19 Recently, we demonstrated the
outstanding performance of anodically oxidized (AO) diamond
electrodes for the detection of chlorophenols in contrast to the
as-deposited (AD) diamond electrodes.20 These results have
indicated that surface oxygen of the diamond electrode plays a
very important role in electroanalysis. Swain et al. reported the
oxidation and detection of polyamine, based on the oxygen transfer
reactions from reactive OH radicals produced during the initial
stage of O2 evolution,17 even though the oxidation product for
polyamine was not determined. These OH radicals have also
served as the source of the oxygen transfer reactions in the
electroanalysis of polyamine. Foti et al. reported the involvement
of physisorbed OH radicals at BDD in the decomposition of
organic pollutants to fully oxidized reaction products, such as CO2
and H2O.21
EXPERIMENTAL SECTION
Materials. All solutions were prepared from reagent grade
chemicals that were used as received. The reduced form of
glutathione and metaphosphoric acid were obtained from Sigma
(St. Louis, MO). All other chemicals were from Wako (Osaka,
Japan). HPLC-grade acetonitrile was purchased from Nacalai
Tesque (Kyoto, Japan). All solutions were prepared using Milli-Q
water (Millipore).
Electrochemical Measurements. Highly boron-doped dia-
mond electrodes were deposited on Si (100) wafers using a high-
pressure microwave plasma-assisted chemical vapor deposition
(CVD) system (ASTeX Corp., Woburn, MA). The details of the
preparation have been described previously.14 The typical boron
concentration obtained under the present conditions was ∼1.5 ×
1021 at. cm-3, estimated by the nuclear reaction measurements
8
[11B(p, R) Be], which was carried out by means of MeV proton
bombardment and subsequent comparison of the R spectrum in
the 6-8 MeV region with a BN standard.23 Anodic pretreatment
of the diamond electrode was carried out at a constant current
density of +8 mA cm-2 in pH 2 Britton-Robinson buffer for 20
min. The film quality before and after anodic pretreatment was
confirmed by Raman spectroscopy (Renishaw System 2000). The
Raman spectra of diamond films showed them to be of high
quality, as evident from the strong characteristic peak at 1332
cm-1. In addition, a broad peak centered at ∼1200 cm-1 was
observed, which is characteristic of highly boron-doped samples.
No additional peaks due to sp2 carbon were observed around 1500
cm-1, indicating the high quality of these films. The XPS atomic
O/ C ratios obtained before and after anodization for the AD
diamond are in agreement with our previous report on chloro-
phenols.20
In general, carbon electrodes, as represented by glassy carbon,
show no signal corresponding to disulfides and methionine, but
the signals for thiols, for example, GSH and cysteine (CySH), are
observed at the potential of ∼1 V vs SCE. The products of thiols
are known to be the corresponding disulfides.22 In the present
work, we report the possibility of electrochemical detection of GSH
and GSSG at anodically pretreated diamond electrodes and the
(7) Melnyk, S.; Pogribna, M.; Pogribny, I.; Hine, R. J.; James, S. J. J. Nutr.
Biochem. 1 9 9 9 , 10, 490-497.
(8) Manna, L.; Valvo, L.; Betto, P. J. Chromatogr., A 1 9 9 9 , 846, 59-64.
(9) Ewing, A. G. J. Neurosci. Methods 1 9 9 3 , 48, 215-224.
(10) Malone, M. A.; Zuo, H.; Lunte, S. M.; Smyth, M. R. J. Chromatogr., A 1 9 9 5 ,
700, 73-80.
(11) Hadwiger, M. E.; Torchia, S. R.; Park, S.; Biggen, M. E.; Lunte, C. E. J.
Chromatogr., B 1 9 9 6 , 681, 241-249.
Electrochemical measurements were made with a potentiostat/
galvanostat (Hokuto Denko Research, model HZ-3000). Cyclic
voltammograms were obtained at a scan rate of 0.1 V s-1. The
electrochemical studies were performed in a single-compartment
glass cell. The planar working electrode was mounted on the
bottom of the glass cell by use of a silicon O-ring. The geometric
area of the electrode in the cell was estimated to be 0.09 cm2. A
commercial saturated calomel electrode (SCE) was used as the
reference, and a Pt coil was used as the counter electrode. All
measurements were made at room temperature (23 ( 2 °C). The
electrolyte was a Britton-Robinson buffer solution (pH 2.0),
composed of a mixture of boric, acetic, and phosphoric acids (each
0.04 M) and adjusted to the appropriate pH by the addition of 0.2
(12) Rao, T. N.; Fujishima, A. Diam. Relat. Mater 2 0 0 0 , 9 (3-6), 384-389.
(13) Strojek, J. W.; Granger, M. C.; Dallas, T.; Holtz, M. W.; Swain, G. M. Anal.
Chem. 1 9 9 6 , 68, 2031-2037.
(14) Yano, T.; Tryk, D. A.; Hashimoto, K.; Fujishima, A. J. Electrochem. Soc. 1998,
145, 1870-1876.
(15) Rao, T. N.; Yagi, I.; Miwa, T.; Tryk, D. A.; Fujishima, A. Anal. Chem. 1 9 9 9 ,
71, 2506-2511.
(16) Popa, E.; Notsu, H.; Miwa, T.; Tryk, D. A.; Fujishima, A. Electrochem. Solid-
State Lett. 1 9 9 9 , 2 (1), 49-51.
(17) Koppang, M. D.; Witek, M.; Blau, J.; Swain, G. M. Anal. Chem. 1 9 9 9 , 71,
1188-1195.
(18) Witek, M.; Swain, G. M. Anal. Chim. Acta 2 0 0 1 , 440, 119-129.
(19) Sarada, B. V.; Rao, T. N.; Tryk, D. A.; Fujishima, A. Anal. Chem. 2 0 0 0 , 72,
1632-1638.
(20) Terashima, C.; Rao, T. N.; Sarada, B. V.; Tryk D. A.; Fujishimrna, A. Anal.
Chem. 2 0 0 2 , 74, 895-902.
(21) Foti, G.; Gandini, D.; Comninellis, Ch.; Perret, A.; Haenni, W. Electrochem.
Solid-State Lett. 1 9 9 9 , 2 (5), 228-230.
(22) Ralph, T. R.; Hichman, M. L.; Millington, J. P.; Walsh, F. C. J. Electroanal.
(23) Notsu, H.; Yagi, I.; Tatsuma, T.; Tryk, D. A.; Fujishima, A. J. Electroanal.
Chem. 2 0 0 0 , 492, 31-37.
Chem. 1 9 9 4 , 375, 1-15.
Analytical Chemistry, Vol. 75, No. 7, April 1, 2003 1565

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M NaOH.24 The solutions were deoxygenated with nitrogen for
10 min prior to analysis. The glassy carbon electrode (Tokai
Carbon Co., Ltd) was pretreated by polishing with diamond paste,
followed by ultrasonication in high-purity water before the experi-
ment.
(BIOXYTECH GSH/ GSSG-412). GSH can be monitored by a
spectrophotometrically detectable product at 412 nm after reaction
with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). On the other hand,
GSSG can be determined following the reduction of GSSG to GSH
by glutathione reductase and nicotinamide adenine dinucleotide
phosphate, using 1-methyl-2-vinylpyridinium trifluoromethane-
sulfonate (M2VP) as a scavenger of GSH. The details of the
procedure have been described previously.25
Controlled P otential Electrolysis. The controlled potential
electrolysis was carried out using a BDD electrode (A ) 1 cm2).
The applied potential was obtained using HZ-3000 potentiostat.
This electrolysis was carried out in a single compartment cell. A
nitrogen stream and stirring were maintained in the solution
during the electrolysis. The initial concentration of the electrolysis
solution was 1 mM; after chosen intervals, 20 µL of the electro-
lyzed solution was injected into the chromatographic system.
Chromatographic Analyses. LC measurements were carried
out by using a binary pump (GL Sciences, Inc., PU611), an
autosampler (Spark-Holland, Triathlon) for variable injections, a
thin-layer flow cell (GL Sciences, Inc.), an amperometric detector
(GL Sciences, Inc., ED623), and a data acquisition system
(EZChrom Elite, Scientific Software, Inc.). The thin-layer flow cell
consisted of the Ag/ AgCl reference electrode and a stainless steel
(type 316) counter electrode. A 500-µm-thick silicon gasket was
used as a spacer in the cell. The geometric area of the electrode
in the cell was estimated to be 0.4 cm2. As chromatographic
column, an Inertsil ODS-3 C-18 column (4.6-mm i.d. × 75 mm)
was used, and the particle size was 3 µm. During experiments,
the flow cell and the separation column were kept at 25 ( 0.1 °C.
The mobile phase for LC separation consisted of 0.1% trifluoro-
acetic acid (TFA) and acetonitrile in the ratio 98:2 (v/ v), and the
flow rate was set at 0.7 mL min-1. The optimum potential of the
working electrode for the amperometric measurement of GSH
and GSSG was set at 1.50 V vs Ag/ AgCl.
Analysis of the Oxidation P roduct by LC-MS/ MS. The
byproducts of electrolysis were analyzed by LC coupled with UV
detector (210 nm) with the chromatographic conditions the same
as that for electrochemical detection for GSH and GSSG. Mass
spectrometric (MS) analysis coupled to LC-UV was used for the
identification of products. The electrospray ionization (ESI)
tandem MS was carried out on an API 2000 triple stage quadrupole
mass spectrometer (PE-Sciex, Langen, Germany) equipped with
the articulated ion spray ionization source. Mass spectra were
generated in the positive ion mode. Tandem quadrupole mass
spectrometry was performed by subjecting the parent ions to
collision-activated dissociation using nitrogen as the collision gas.
Collision energy was 30 eV. Scans were performed at an ionization
voltage of 4500 V over a mass range of 50-700 amu.
RESULTS AND DISCUSSION
Cyclic Voltammetry of GSH at As-Deposited Diamond.
Direct oxidation of thiols is known to produce the corresponding
disulfides on a carbon surface.
2RSH f RSSR + 2H+ + 2e-
(1)
Spataru et al. reported CySH oxidation at BDD.26 From the pH
dependence of the voltammetric signal, they concluded that the
ionization of CySH precedes the electrochemical step, and the
electrochemically active species are CyS- ions, indicating that the
AD diamond behaves in a way similar to a glassy carbon electrode
in terms of electron transfer, except that the rate-determining step
is different. In the present work, similar voltammetric and pH
dependence experiments were performed on GSH oxidation at
the AD diamond electrode. A well-defined peak for 1 mM GSH
was obtained at 0.54 V vs SCE in Britton-Robinson buffer (pH
9.0). The results for GSH oxidation, similar to CySH, show that
the process is not pH-dependent in alkaline media, whereas in
neutral and acidic media, the peak potential shifts linearly toward
more positive potentials with a slope dEp/ dpH of 78.9 ( 6.4 mV/
pH (n ) 3). Furthermore, the inflection point of the Ep versus pH
plots correlates quite well with the pK value of the dissociation of
the proton from the thiol group in GSH (pKSH ) 9.65). In addition,
no anodic peaks for GSSG were observed within the investigated
potential range (0 to +1.1 V) at the AD diamond electrode. These
results indicate that the oxidation for GSH is quite similar to the
case of CySH.
Cyclic Voltammetry of GSSG and GSH at Anodically
Oxidized Diamond. Conductive diamond electrodes, which are
deposited by a microwave plasma-assisted chemical vapor deposi-
tion (CVD) system, are known to be hydrogen-terminated on the
surface because of the use of high-purity hydrogen as the carrier
gas during the deposition process. Such an AD diamond surface
can be modified to the oxygen-terminated surface by anodic
oxidation or by oxygen plasma treatment.27 In the present
experiments, the oxygen termination was induced by anodic
treatment, as mentioned in the Experimental Section. Figure 1
shows the linear sweep voltammograms for GSH and GSSG at
the AD and AO diamond electrodes in acidic medium (pH 2-2.5).
At the AD diamond in Figure lAa, GSH shows a voltammetric
peak at ∼0.8 V vs SCE, corresponding to the direct oxidation to
GSSG, as mentioned before. However, interestingly, this peak
disappears at the AO diamond, and a pair of new peaks appears
Blood Sample P rocessing. Female rats were anaesthetized
with diethyl ether, and then whole blood was freshly drawn using
Na2EDTA as an anticoagulant. For deproteinization, 200 µL of
whole blood was mixed with 600 µL of ice-cold 5% metaphosphoric
acid, and the mixture was centrifuged for 10 min at 3000g to
separate the precipitated proteins and other nonsoluble matter.
A 50-µL aliquot of supernatant after centrifuging was diluted to 1
mL with the LC mobile phase. The sample was injected into the
LC system without any other treatment.
(25) Richie, J. P.; Skowrouski, L.; Abraham, P.; Leutzinger, Y. Clin. Chem. 1 9 9 6 ,
42, 64-70.
(26) Spataru, N.; Sarada, B. V.; Popa, E.; Tryk, D. A.; Fujishima, A. Anal. Chem.
Enzymatic Assays. An assay kit for GSH and GSSG based
on colorimetric determination was purchased from OxisResearch
2 0 0 1 , 73, 514-519.
(24) Dobos, D. Electrochemical Data: A Handbook for Electrochemists in Industry
and Universities; Akademiai Kiado: Budapest, 1975; p 239.
(27) Yagi, I.; Notsu, H.; Kondo, T.; Tryk, D. A.; Fujishima, A. J. Electroanal. Chem.
1 9 9 9 , 473, 173-178.
1566 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003

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Figure 2. Consecutive cyclic voltammograms for the oxidation of
1 mM GSSG in Britton-Robinson buffer (pH, 2) at (A) AO diamond
and (B) GC electrodes. Bold lines represent first cycle; bold dotted
lines represent second cycle; and thin lines, background voltammo-
grams. The solutions were vigorously stirred after each cycle.
consecutive runs in Figure 2, the bold solid line for the first cycle
and the bold dotted line for the second cycle, were carried out
with vigorous stirring after each measurement. Surface fouling
on the GC electrode due to a buildup of adsorbed reaction
products was observed during GSSG oxidation. The oxidation of
the GC surface itself also caused an increase in the background
current because of the higher applied potential, up to 2.2 V vs
SCE. The results, showing the decrease in the peak current after
one cycle and the disappearance of the peak around 1.8 V, indicate
that the active sites of the GC surface are blocked by adsorption
of reaction products. In contrast, the oxidation peak current and
potential at the AO diamond electrode were essentially unchanged
between the first and second cycles, indicating its inertness to
adsorption of oxidation products.
To understand the adsorption process at AO diamond, a
chronocoulometric study was carried out by applying a potential
step in the presence and absence of low concentrations of GSSG.
A plot of total charge, Qtotal vs t1/ 2 should be a straight line with
an intercept equal to the sum of the charges for a double layer
and adsorption (Qdl + Qads). Since the difference between the
intercepts of Qtotal and Qdl (blank) in this case was approximately
0 (data not shown), there was no adsorption of reactant in the
range of GSSG oxidation. According to the investigation of Koryta
et al. at platinum and gold electrodes,28,29 CySSyC is strongly
adsorbed at Pt and Au electrodes; furthermore, the byproduct of
CySSyC oxidation shows a cathodic signal owing to the adsorbed
product species. At the AO diamond electrode, no cathodic peaks
of oxidation products of GSSG and CySSyC were observed on
the reverse scan within the potential range (2.2 V to -1.0 V).
Furthermore, the anodic peak currents on GSSG concentration
are linear with 0 intercept over the range of 4 µM to 1 mM. The
values of r2 were 0.9986 and 0.9972 for the first and second peaks,
respectively. These results show that the diamond electrode is
free from adsorption of the oxidation products of GSSG and that
the response stability is very good.
Figure 1. Linear sweep voltammograms for (A) GSH and (B) GSSG
in acidic Britton-Robinson buffer at (a and c) AD diamond and (b
and d) AO diamond. The concentration of GSH and GSSG was 1
mM each. The potential sweep rate was 0.1 V s-1. Dashed line
represents background current.
at higher anodic potentials (Figure lAb). In the case of GSSG, a
broad shoulder was observed at 1.51 V for the AD diamond, while
two clear peaks were observed for the AO diamond. It is also
interesting to note that the linear sweep voltammetric i-E curve
for GSH at the AO diamond is similar in shape to that for GSSG,
except that the peak potentials of GSH are shifted to a more
negative potential, and the peak currents of GSH are lower. These
results indicate that the mechanism for the oxidation is probably
similar for both of the molecules at this electrode. The improved
response of the GSSG at the AO diamond with two clear peaks in
comparison to that at AD diamond can be explained on the basis
of the ion-dipole interactions at the electrode surface. As
mentioned in our previous reports,20 we believe that the oxygen
functional groups, such as carbonyl or hydroxyl groups, formed
on the facets of the diamond microcrystals form a negative dipolar
field, which electrostatically attracts the positively charged GSSG
molecule. The ion-dipole interaction between GSSG, an amino
acid that carries a positive charge in the acidic medium, and the
AO diamond surface is believed to facilitate the oxidation reaction.
In contrast, GSH also being positively charged in acidic media,
its voltammetric peaks appear at more positive potentials in
comparison to that at AD diamond. This is probably due to the
change in the oxidation mechanism. Although the mechanism of
thiol oxidation at AD diamond is known to involve the formation
of disulfide,26 the oxidation at the AO diamond is believed to
involve an oxygen transfer reaction, as expected for the GSSG.
The fact that the both compounds produce similar voltammograms
provides support for this hypothesis. The experimental support
for this hypothesis comes from the identification of products of
exhaustive electrolysis, as discussed in the later section.
pH Dependence of GSSG and GSH at Anodically Oxidized
Diamond. Figure 3 shows the influence of pH dependence of
Figure 2 shows cyclic voltammograms of 1 mM GSSG both
for AO diamond (Figure 2A) and for GC (Figure 2B). Two
(28) Pradac, J.; Koryta, J. J. Electroanal. Chem. 1 9 6 8 , 17, 167-175.
(29) Koryta, J.; Pradac, J. J. Electroanal. Chem. 1 9 6 8 , 17, 177-183.
Analytical Chemistry, Vol. 75, No. 7, April 1, 2003 1567