Determination of physiological thiols by electrochemical detection with piazselenole and its application in rat breast cancer cells 4T-1

Article abstract of DOI:10.1021/ja802273p

Glutathione (GSH), the most abundant cellular thiol, has been shown to play an important role in maintaining cellular redox equilibrium that is pivotal for cell growth and function. In the present paper a novel electrochemical probe of piazselenole containing a Se-N bond was well developed for the determination of GSH. The cyclic voltammogram of piazselenole scanned at 100 mV/s displayed an irreversible reduction peak at -0.106 V (vs Ag/AgCl electrode) and a significant peak current decrease could be further provoked with the addition of GSH into piazselenole solution. On the basis of the peak current decrease of piazselenole recorded by differential pulse voltammetry with the increase of GSH concentration, a working curve was constructed for GSH determination in the range of 5.0 × 10^-10～2.2 × 10^-8 M with the linear regression equation as Δi_P (10^-6A) = 0.0952 + 0.4287 × C_GSH (10^-8 M) and the detection limit (3σ) as 83 pM. The proposed method was satisfactorily applied to the extracts of rat breast cancer cells 4T-1 for intracellular thiols detection. Copyright

Full text of DOI:10.1021/ja802273p

Published on Web 07/25/2008
Determination of Physiological Thiols by Electrochemical Detection with
Piazselenole and Its Application in Rat Breast Cancer Cells 4T-1
Wei Wang,† Lin Li,† Shufeng Liu,† Cuiping Ma,‡ and Shusheng Zhang*,†
Key Laboratory of Eco-chemical Engineering of Ministry of Education, College of Chemistry and Molecular
Engineering, Qingdao UniVersity of Science and Technology, Qingdao 266042, P. R. China, and Department of
Molecular Biology, School of Medicine and Pharmacy, Ocean UniVersity of China, Qingdao, 266003, P. R. China
Received March 27, 2008; E-mail: shushzhang@126.com
Scheme 1. Electrochemical Reaction of Piazselenole
The low molecular weight thiols, widely distributed in the tissues
and cells, have been proven to play an important role in metabolism
and cellar homeostasis.1,2 Herein, glutathione is found to be the
most abundant cellar thiol and exists in redox equilibrium between
sulfhydryl (reduced form, GSH) and disulfide (oxidized form,
GSSG) forms.3 It has been extensively revealed to play a central
role in combating oxidative stress and maintaining redox homeo-
stasis that is pivotal for cell growth and function,4 and its level has
been directly linked to some diseases and cancers.5-8
Scheme 2. Reaction of Piazselenole with GSH
Various conventional techniques for the determination of GSH
and related thiols such as high performance liquid chromatography
coupled with different detection methods usually suffer from several
substantial difficulties or drawbacks in terms of equipment cost,
complexity, sample processing, and applicable feasibility in in vivo
analysis.9,10 In contrast, electrochemical detection poses an attractive
solution because of the advantages of simplicity, sensitivity, and
low instrumental cost, etc.11 Direct electro-oxidation of thiol
compounds on common solid electrodes limits its selectivity because
of the similar oxidation potential of most biological reducing
substances with that of thiol.12 Alternatively, the mercury or
mercury amalgam electrodes are employed,13 but the toxicity with
the involvement of mercury hinders further use for in vivo analysis.
Recently, considerable interest has turned to the use of some organic
or inorganic electroactive indicators as electron mediators for thiol
compounds detection, yet the requirement of delicate and finely
tuned experimental conditions for the success of the electrocatalytic
reaction between the thiol compounds and the indicator limits their
wide applications. Thereby, additional efforts are needed to create
a more broadly applicable electrochemical approach that would
allow accurate, sensitive, rapid, and low-cost determination of GSH
and related thiols, especially suitable for in vivo analysis.
The electrochemical behavior of piazselenole was studied by
measuring the cyclic voltammogram on a Au electrode in pH 2.0
Britton-Robinson buffer (BR buffer) solution. As shown in the
cyclic voltammogram of piazselenole (see Supporting Information,
SI), only a cathodic signal (at -0.106 V) was observed. It was
suggested that piazselenole followed a six-electron electrochemical
reduction pathway on the Au electrode with the equation shown as
follows (Scheme 1). The subsequent addition of GSH to the solution
of piazselenole caused a decrease in the peak current, which could
be ascribed to the consumption reaction of electrochemically active
piazselenole with GSH, yielding the nonelectroactive species in the
studied potential region according to Scheme 2.
The peak current decrease of piazselenole recorded by differential
pulse voltammetry could be exploited as an assay for GSH
concentration. With the GSH concentration changed in the range
of 5.0 × 10-10∼2.2 × 10-8 M (Figure 1), a good linearity was
plotted with the regression equation as ∆iP (×10-6 A) ) 0.0952
+0.4287 × CGSH (×10-8 M) (γ ) 0.9940), and a detection limit
(3σ) of 83 pM was achieved with the proposed approach.
The effect of interference of a variety of biorelevant analytes on
monitoring GSH was also studied for control experiments. The
various bioanalytes employed mainly included metal ions, bioam-
ines, and biological antioxidants listed below (tolerance ratio with
5.0 nM GSH and final concentration): Fe3+, Al3+, Mg2+, Na+,
Zn2+, Cu2+, K+, dopamine, histamine, L-adrenaline (1000, 5.0
Recently, Tang et al. devised a novel fluorescence probe
containing a Se-N bond and successfully utilized it for thiol
determination.14 Inspired by this strategy, we envisioned that an
electrochemical probe containing a Se-N bond should be reason-
ably conceivable for thiol compound determination. As a proof of
the notion, in present study, an electroactive piazselenole containing
the Se-N bond was synthesized. The decreased current signal of
piazselenole was observed with the increase of GSH concentration
due to the consumption of electroactive piazselenole by the
nucleophilic reaction of GSH. Thus, a novel electrochemical
approach for thiol determination was developed. It was also further
used in the application in cell extracts. As far as we know, this is
the first case of electrochemical detection of GSH and application
in a cell using electroactive species such as piazselenole containing
the Se-N bond for the nucleophilic reaction of sulfhydryl.
Figure 1. Differential pulse voltammograms of piazselenole (0.25 µM)
toward different concentrations of GSH (final concentration: 0, 0.5, 2.5,
5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5 nM) after incubation at 25 °C for
20 min in 0.2 M BR buffer (pH 2.0).
† Qingdao University of Science and Technology.
‡ Ocean University of China.
9
10846 J. AM. CHEM. SOC. 2008, 130, 10846–10847
10.1021/ja802273p CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
supernatant. Although the concentration of intracellular nonprotein
thiols are much higher than that of protein thiols, the decrease of
peak current of piazselenole induced by the latter is more distinct
than that by the former. Taken together, piazselenole is more
sensitive to protein thiols than nonprotein thiols. This conclusion
is in good agreement with the result reported by Tang et al.14 The
different pKa, and implicitly then the nucleophilicity of the
sulfhydryl group surrounding the proteins, might account for the
difference in sensitivity for protein thiols.
In summary, we provide for the first time with an extremely
sensitive and specific electrochemical probe of piazselenole a
method to detect GSH and related cellular thiols. The relatively
low detection limit of 83 pM and a broad dynamic range of 5.0 ×
10-10∼2.2 × 10-8 M were achieved. The novel developed
electrochemical assay based on the strong nucleophilicity of
sulfhydry to cleave Se-N bond for electrochemical response is
considerably attractive for rapid, sensitive, and selective detection
of cellular thiols.
Figure 2. Electrochemical responses of piazselenole (0.25 µM) to diverse
bioanalytes in 0.2 M BR buffer (pH 2.0). Light gray bars represent the
addition of analytes: Fe3+, Al3+, Mg2+, Na+, Zn2+, Cu2+, K+, dopamine,
histamine, L-adrenaline (1000, 5.0 µM); Ca2+, H2O2 (500, 2.5 µM); Vc
(300, 1.5 µM); uric acid (200, 1.0 µM); GSH (5.0 nM). Black bars represent
the subsequent addition of 5.0 nM GSH to the mixture. All data were
obtained after incubation at 25 °C for 20 min.
Acknowledgment. This work was supported by the National
Natural Science Foundation of China (No.20775038), the Scientific
and Technical Development Project of Qingdao (06-3-1-4-yx), and
theNationalHigh-techR&DProgram(863Program,No.2007AA09Z113).
We thank Fred R Miller for providing the murine breast tumor cell
lines.
Note Added after ASAP Publication. After this paper was
published ASAP July 25, 2008, the y-axis labels were corrected in
Figure 1 and Figures S1-S3, S5, and S6 in the Supporting Information.
The corrected versions were published July 29, 2008.
Figure 3. Electrochemical responses of piazselenole (0.25 µM) toward
the supernatant of 4T-1 cells (A) and responses toward the protein extracts
of 4T-1 cells (B). The gray bars represent no addition of NEM and the
black bars represent the addition of 1.0 mM NEM.
µM); Ca2+, H2O2 (500, 2.5 µM); ascorbic acid (Vc) (300, 1.5 µM);
uric acid (200, 1.0 µM). The results indicated that various
bioanalytes yielded no or low enough for ignorance interference
with GSH detection. That is also to say, piazselenole could
selectively respond to GSH compared with other analytes (Figure
2).
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://pubs.acs.org.
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