A SERS study of oxidation of glutathione under plasma irradiation

Article abstract of DOI:10.1039/c5ra07440a

This paper reports a new application of surface enhanced Raman scattering (SERS) in analysis of oxidation of glutathione (GSH) to oxidized glutathione (GSSG), an important biochemical redox reaction in biological systems, under oxidative stress imposed by dielectric barrier discharge (DBD). Using the silver nanoparticles (NPs) prepared through the reduction of AgNO₃ by beta-cyclodextrin (β-CD), the transformation of GSH to GSSG under DBD irradiation can be probed with only a small quantity of sample at low concentration. Based on the intensity ratio of two characteristic Raman bands, i.e., the band at 1051 cm^-1 (C-N stretching) and the band at 509 cm^-1 (S-S stretching), which stem respectively from GSH and GSSG, the conversion between the reduced and oxidized glutathione can be determined quantitatively. This work demonstrates another useful extension of the SERS technique applied to bioscience research, i.e., rapid probing and quantitative assessing of chemical reactions of biomolecules under oxidative stress conditions.

Full text of DOI:10.1039/c5ra07440a

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A SERS study of oxidation of glutathione under
plasma irradiation†
Cite this: RSC Adv., 2015, 5, 57847
a
ab
*
Shanshan Ma and Qing Huang
This paper reports a new application of surface enhanced Raman scattering (SERS) in analysis of oxidation of
glutathione (GSH) to oxidized glutathione (GSSG), an important biochemical redox reaction in biological
systems, under oxidative stress imposed by dielectric barrier discharge (DBD). Using the silver
nanoparticles (NPs) prepared through the reduction of AgNO3 by beta-cyclodextrin (b-CD), the
transformation of GSH to GSSG under DBD irradiation can be probed with only a small quantity of
sample at low concentration. Based on the intensity ratio of two characteristic Raman bands, i.e., the
band at 1051 cmꢀ1 (C–N stretching) and the band at 509 cmꢀ1 (S–S stretching), which stem respectively
from GSH and GSSG, the conversion between the reduced and oxidized glutathione can be determined
quantitatively. This work demonstrates another useful extension of the SERS technique applied to
bioscience research, i.e., rapid probing and quantitative assessing of chemical reactions of biomolecules
under oxidative stress conditions.
Received 24th April 2015
Accepted 24th June 2015
DOI: 10.1039/c5ra07440a
www.rsc.org/advances
measurement of the involved chemical processes, not to
mention the real-time measurement in living cells.
Introduction
In this sense, SERS just provides the capability of quick and
trace chemical analysis, and therefore, it is intriguing for us to
introduce SERS and test its applicability in the radiation-
biological studies. Previously, Ou et al. have applied SERS for
DNA analysis in the nasopharyngeal carcinoma cells under X-
ray radiation.18 Also, we have employed SERS to study the
chemical reactions of tyrosine, a small biomolecule, under
particle irradiation (including plasma and electron-beam irra-
diations).19 In this work, we intended to make use of SERS to
probe the ROS induced reaction of glutathione (GSH), a peptide
that is composed of three amino acid residue, namely, gluta-
mine, cysteine and glycine. This biomolecule is an important
anti-oxidant in living organisms, and it is the most prevalent
cellular thiol involved in major biologic processes.20,21 The
depletion of GSH in cell would result in the mitochondria
impairment22 or apoptosis.23 The thiol of GSH is reactive, and
can easily be converted into oxidized glutathione (GSSG), the
major product of GSH oxidation.24 On the other hand, GSSG can
also be reduced to GSH by reductase. In cellular redox reactions,
keeping an optimal GSH and GSSG ratio is crucial to cell
survival.21 Breakdown of the balance between GSH and GSSG
upon exposure of ROS can result in protein damage and
apoptosis.21
Because of the advantages of high sensitivity and ngerprint
character, surface-enhanced Raman spectroscopy (SERS) has
become a very powerful analytical tool which has been
successfully applied in many diversied elds.1–5 Especially,
there is an increasing interest in application of this technique in
bioscience research. For example, SERS has been applied in the
rapid trace-detection of biomolecules, such as DNA6,7 and
proteins,8–11 and also sensitive identication of intrinsic cancer
biomarkers in cells.12
However, up to now there are very few reports in the litera-
ture concerning the application of SERS in the research of
radiation-biological eld, although different kinds of radiations
exist ubiquitously and can induce abundant biological effects.13
For ionizing radiation, it possesses the energy that can break up
water molecules and give rise to reactive oxygen species (ROS),
which may subsequently induce complex reactions of biomol-
ecules14–16 and cause cell injury or cell death.17 Although
conventional analytical tools such as chromatography and mass
spectrometry can be utilized to investigate the involved reac-
tions, they are normally time-consuming and expensive in
operation, and moreover, they cannot render direct and in situ
ROS
ƒƒƒƒƒ!
aKey Laboratory of Ion Beam Bio-engineering, Institute of Technical Biology and
Agriculture Engineering, Hefei Institutes of Physical Science, Chinese Academy of
Sciences, Hefei, 230031, Anhui Province, PR China. E-mail: huangq@ipp.ac.cn
bUniversity of Science & Technology of China, Hefei, Anhui 230026, People's Republic
of China
GSc
GSH
GSH
ƒƒƒƒƒ!
GSc
GS ꢀ SG
or GSc
To mimic low-energy particle irradiation induced oxidative
stress, we employed dielectric barrier discharge (DBD), an
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra07440a
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emerging non-thermal plasma technique which is regarded as solution was removed from the reactor, and treated with cata-
one of advanced oxidation processes (AOPs).25,26 During plasma lase to deplete the surplus hydrogen peroxide in the solution.
discharge, energetic particles including electrons and ions are
produced, together with the plasma induced ROS such as
hydrogen peroxide and hydroxyl radicals.27 As for the SERS
measurements, we employed Ag nanoparticles (Ag NPs) which
were synthesized through reduction of AgNO3 by beta-
cyclodextrin (b-CD) under alkaline condition. We then probed
the damage of GSH and production of GSSG based on SERS, and
due to careful spectral analysis we could make quantitative
assessment of conversion between GSH and GSSG under
oxidative stress.
Preparation of silver colloids
The Ag nanoparticles were synthesized through the reduction of
AgNO3 (0.6 mM) by b-CD under alkaline condition. Considering
the low solubility of b-CD in water (7 mM), the reaction solution
ꢁ
was heated at 80 C. The overall reaction took for 10 min, and
the nal silver solution showed a yellow color. All the glass or
quartz container were cleaned by aqua regia (HNO3 : HCl ¼
1 : 3) and piranha solution (H2O2 : H2SO4 ¼ 1 : 3), followed by
washing with deionized water.
UV-vis absorption and SEM measurements
Experiments
In order to characterize the silver colloids prepared under
different conditions, UV-vis spectroscopy and scanning electron
microscope (SEM) were employed. The UV-visible absorption
property was examined using UV-vis spectrometer (SHIMADZH
UV-2550). The images of the silver NPs were provided by scan-
ning electron microscope (SEM) with a Hitachi S-4800 machine
(SEM, SIRION 200, Toshiba S4800).
Materials and reagents
Silver nitrate (AgNO3), catalase (CAT) were obtained from
Sigma-Aldrich Co, Ltd. Glutathione (GSH), oxidized glutathione
(GSSG), beta-cyclodextrin (b-CD), methyl alcohol were
purchased from Sangon Biotech (Shanghai) Co. Potassium
phosphate monobasic (KH2PO4) and phosphoric acid were
purchased from Sinopharm Chemical Reagent Co., Ltd. All the
chemicals were of analytical grade and used as received without
any further purication.
SERS measurements and spectral analysis
For SERS detection, the silver colloid solution was added to 20
mL GSH/GSSG samples. Then 5 mL of the mixture was dropped
onto the quartz plate, and dried under room temperature. The
SERS spectra were recorded using HORIBA JOBIN YVON XploRA
Raman Spectrometer equipped and a 50ꢂ objective. All the
Raman spectra were excited with 785 nm laser of power about
0.2 mW focused on the samples. The laser focus spot size was
about 2 mm in diameter, and the acquisition time for an indi-
vidual Raman spectrum was typically 10 s.
Non-thermal plasma irradiation
To generate reactive oxygen species (ROS), non-thermal plasma
irradiation was employed. Fig. 1 shows the schematic plot of our
set-up of dielectric barrier discharge (DBD). Briey, two stain-
less steel plates were used as the electrodes, in which the upper
one was connected to the power supply and the other one was
connected to the ground. A circular quartz container covered by
a quartz plate was placed between the electrodes. The discharge
argon gas was introduced into the reactor and the plasma
discharge was formed in the gas–solution interface. The applied
voltage was about 16 kV, and when the discharge was steady, the
current was about 1 mA. For the DBD irradiation experiment, 4
mL 5 mM GSH solution was injected into the reactor, followed
by plasma irradiation. Aer the discharge treatment, the
High performance liquid chromatography (HPLC)
measurement
The HPLC was carried using the instrument Waters-600E
equipped with the chromatographic column RP-C18 column.
Mobile phase was the mixture of 0.05 M KH2PO4 (phosphoric
acid was added to keep the pH 3) and methyl alcohol, with the
volume ratio as 97 : 3. The ow rate was 1 mL minꢀ1 and the
column temperature was 30 ꢁC, while the detection wavelength
was 210 nm.
Results and discussion
Characterization of Ag NPs
Ag NPs were prepared by reduction of AgNO3 with b-CD, a
substance which has medium reduction ability under the
alkaline condition. b-CD is cyclic oligosaccharides and the
interior cavity of CD is relatively hydrophobic while the external
cavity is hydrophobic, it has been used for synthesis of Au
nanoparticles28 and Ag shell on the Au surface29 previously.
Fig. 2 shows the SEM images of the as-prepared Ag nano-
particles (NPs), showing that the morphology of Ag NPs is
spherical and the quite uniform with the indicated diameter ca.
Fig. 1 The schematic plot of the DBD set-up. For the atmospheric
non-thermal discharge, argon gas was filled in the container, and the
discharge voltage was about 16 kV with the current about 1 mA. The
volume of the solution sample was 4 mL.
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40 nm. The surface plasmon resonance (SPR) peak of the Ag NPs conducted normal Raman measurements for comparison
is about at 406 nm, and the zeta potential measurement (Fig. S4†) and noticed that Raman spectra could be inuenced
demonstrates that the Ag NPs are homogeneously negatively- by many other factors under different under different condi-
charged and so the colloids were dispersed well without tions.31,32 Also, we were ensured that the SERS measurements
aggregation for the concentrations used in this work (Fig. S1†). were reproducible (Fig. S3b†). For the spectra of GSH and GSSG,
The SERS activity of the Ag NPs was checked by using the the most prominent band occurs at 657 cmꢀ1 and 659 cmꢀ1
,
standard R6G. The lower detect limitation is 10ꢀ11 M, and respectively, which is attributed to the C–S stretching vibra-
accordingly, the enhancement factors (EFs) is about 5 ꢂ 106 tion.33 Fortunately, this C–S vibration has almost the identical
(Fig. S2, ESI Part 1†).
Raman intensity for both GSH and GSSG per unit molar
In this work we employed the Ag NPs for SERS analysis of (Fig. S8†), so we can use this band as an internal reference for
GSH/GSSG because we found that they were very suitable for our normalization of the spectra, and as will be explained in the
biomolecular study. Compared with Ag NPs obtained by others following section, this property can also facilitate us to give the
methods, these Ag NPs had very low background signals, quantitative evaluation of the GSH/GSSG mixture samples. For
resulting in little interference to the detection of the identication of GSSG, the characteristic band appears at 509
biomolecules.
cmꢀ1, which stems from the –S–S– stretching vibration,34,35 and
this band can be utilized as the unique signature for analysis of
GSSG. To discriminate GSH, the strong signal at 1051 cmꢀ1 can
be identied, which is ascribed to the C–N stretching vibration.
To be noted, the same vibration band for GSSG appears at 1048
cmꢀ1, which is however much weaker in intensity. The Raman
intensity at 1051 cmꢀ1 for GSH is about 6–7 folds stronger than
that of the 1048 cmꢀ1 band for GSSG (Fig. S7†), suggesting that
we can utilize this 1051 cmꢀ1 band as the characteristic band
for quantitative evaluation of GSH. Also, in the evaluation of
GSH/GSSG mixture samples, the signal at 1048 cmꢀ1 can be
neglected for the estimation of the intensity at 1051 cmꢀ1 for
GSH.
Based on the above-approved characteristic bands for GSH
and GSSG, the quantities of GSH/GSSG in the mixture samples
can be evaluated. For this purpose, it also requires to measure
the standard curves for the SERS intensity versus concentration
of GSH (Fig. 4(a)), and also, in order to avoid the uncertainty in
determining the absolute SERS intensity, it is favorable to use
the relative parameters such as the intensity ratio of the 509
cmꢀ1 band to the 1051 cmꢀ1 band for estimating the GSH/GSSG
contents in the DBD-treated samples. The standard curves for
determining the concentration of CGSH and the concentration
SERS identication of GSH and GSSG
The conventional analysis of GSH and GSSG can be achieved by
colorimetric tests using GSH/GSSG assay kits or by HPLC based
on the derivation of thiol group, which however requires bulk
volume samples. Also, for conventional Raman spectroscopy,
although the GSH concentration in realistic bio-samples may
reach millimolar (0.5–10 mM),30 it also requires bulk volume of
samples and the normal Raman signal of GSH is still too weak
to be clearly determined. Therefore, it is critical to improve the
Raman detection sensitivity, and SERS can greatly amplify the
Raman signal and thus provide the solution (Fig. S3a†). Based
on SERS technique, only a tiny volume of sample (5 mL) was
enough for our purpose of examination.
To achieve both qualitative and quantitative analysis, at rst
the SERS spectra of pure GSH and GSSG were recorded, with
which we can therefore readily identify the mixture of GSH/
GSSG in the DBD treated samples. The typical SERS spectra
are shown in Fig. 3 and the assignments are shown in Table 1.
For the assignment of Raman bands in SERS spectra, we also
Fig. 2 The absorption spectrum of the silver colloids, showing the
absorption maximum at 406 nm. Inset: the SEM picture (above) and
the size analysis of the Ag NPs (below), showing the average diameter Fig. 3 The SERS spectra of GSH, GSSG and DBD-treated samples. The
about 40 nm.
assignments of the bands are given in Table 1.
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Table
observed SERS bands for GSH and GSSG
1
The wavenumbers (cmꢀ1) and the assignments of the
GSH
GSSG
Assignment
509
659
796
S–S35
657
791
C–S stretching36
Amide V36
909
909
C–C stretching36
C–N stretching36
–COO-symmetric stretching36
Amide I36
1051
1416
1660
1048
1412
1668
ratio of CGSSG/CGSH are shown in Fig. 4(b). Here we prepared the
GSH/GSSG mixture calibration samples with known contention
ratios, with CGSH in the range of 10ꢀ5 to 10ꢀ4 M, and CGSSG in
the range of 10ꢀ5 to 4.5 ꢂ 10ꢀ5 M. The intensity ratio between
the band at 509 cmꢀ1 and 1051 cmꢀ1 increases with the
concentration ratio of CGSSG/CGSH. Based on the standard curves
provided by Fig. 4, we can estimate the loss of GSH and the
production of GSSG under the oxidative stress imposed by DBD
irradiation.
Then, we measured the intensity ratios for the DBD treated
samples, and the result is shown in Fig. 5. The inset presents
the SERS spectra which show clearly the intensity decrease for
the 1051 cmꢀ1 band and the increase for the 509 cmꢀ1 band,
conrming the loss of GSH and the formation of GSSG under
the DBD irradiation.
Fig. 5 The SERS intensity ratio of I509/I1051 versus the DBD irradiation
time. Inset: the SERS spectra of the DBD treated samples. All the data
points are statistical averages of at least three measurements.
stability of Ag NPs.37,38 To circumvent this problem we added
catalase to remove the residual H2O2. Second, the SERS
measurement could also be affected by the pH change caused
by DBD treatment. So we checked that the pH value of the DBD
treated GSH solution, and found that the pH value was
changed from 4.8 (initial pH value without DBD treatment) to
2.7 (10 min of DBD treatment). Fortunately, within this range,
the SERS intensity was only slightly varied (Fig. S5†), therefore
the pH effect could be ignored in our case. Third, another
difficulty for quantitative evaluation was that the SERS inten-
sity was also dependent on the quantity of Ag NPs applied. To
overcome the uncertainty and ensure the reproducibility of the
measurement, we checked the SERS signals using different
concentration of Ag NPs. It was found that when sufficient
amount of Ag NPs relative to the low concentration of GSH (in
the range of 10ꢀ4) was applied, the SERS signal from GSH was
relatively unchanged (Fig. S6a†); as for assessment of GSSG,
when its concentration was low or comparable with that of
GSH, which was true for our case, the small discrepancy
among the different calibration curves obtained for different
silver colloid concentration could be neglected (Fig. 4(b)).
Therefore, in our SERS experiment we applied an optimal
concentration of 2 nM of silver colloids, and ensured that this
same condition was applied for all the SERS measurements. As
a result, our measurements were reproducible and reliable for
the quantitative assessment.
For the quantitative evaluation of the SERS spectra, some
notes should be taken. First, DBD produces hydrogen peroxide
which displays an adverse effect on the SERS detection
because it can interact with silver colloids and affect the
Determination of GSH/GSSG conversion
As described previously, the discharge plasma can produce
ROS which leads to the transformation from GSH to oxidized
form GSSG. With the aid of the standard curves in Fig. 4, and
based on the SERS intensity ratios given by Fig. 5, we can
estimate contents of GSH and GSSG. The result is shown in
Fig. 6, where CGSH was obtained using the standard curve in
Fig. 4(a), while CGSSG was obtained from CGSH and according to
Fig. 4 (a) The standard curve of SERS intensity of the 1051 cmꢀ1 band
(I1051) versus the concentration of GSH (CGSH). (b) The standard curve
for evaluation of the GSH/GSSG mixture based on the intensity ratio of
the 509 cmꢀ1 band of GSSG to the 1051 cmꢀ1 band of GSH.
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the standard curve regarding the CGSSG/CGSH in Fig. 4(b).
Moreover, in order to conrm the accuracy of SERS, other
methods could be employed. In our previous work, the quan-
titative analysis of GSH and GSSG was achieved by using GSH
and GSSG assay kits.39 Herein HPLC was employed to measure
GSH and GSSG (Fig. S9†). As seen in Fig. 6, the result of SERS
agrees well with the HPLC result, conrming the applicability
of SERS for the reliable quantitative evaluation of GSH/GSSG
transformation.
Furthermore, the conversion percentage can be obtained
according to the following formula:
2 ꢂ CGSSG
X ¼
ꢂ100%
(1)
ðC0 ꢀ CGSH
Þ
where C0 refers to the initial concentration of GSH. Since one
GSSG molecule comes from combination of two GSH mole-
cules, when GSH is completely converted to GSSG, X is 100%. To
estimate the conversion efficiency in the real case, we measured
the ratios of the SERS intensity of S–S and C–S bands (Fig. S8†),
and calculated X according to the following equation:
Fig.
7
The transformation efficiencies for the GSH/GSSG redox
reaction, assessed from both the SERS measurement and the HPLC
measurement.
of hydroxyl radical to hydrogen peroxide,41 which brings more
severe damage to GSH, so that less GSH can be converted to
GSSG. In addition, for the detection method, compared to our
previous work,40 here we employed SERS rather than normal
Raman for the measurement, the sensitivity has been improved
more than 103 times. In that way, we could achieve detection of
trace amount of GSH and GSSG at low concentration. This is an
important advance because it gives us the possibility to monitor
the redox reaction rapidly in cells.
2P ꢂ CGSH
X ¼
ꢂ100%
(2)
ðPo ꢀ PÞꢂðC0 ꢀ CGSH
Þ
where P is dened as I509/I659 for the SERS measurements, and
Po is a constant ratio per unit molar (ESI Part 5†). The derivation
of eqn (2) is given in the ESI-Part S4.† Fig. 7 shows the result
evaluated from the SERS data according to eqn (2). As illustrated
by Fig. 7, about 70%–90% of GSH can be converted to GSSG
under the DBD irradiation. This result is in good agreement of
the evaluation obtained from the HPLC measurement.
Formerly, our group have applied DC glow discharge plasma,
another form of plasma to mimic the oxidative stress on GSH,
and applied conventional Raman spectroscopy to detect the
conversion between GSH and GSSG.40 Compared to the result
obtained from the DC glow discharge, the DBD-induced
damage of GSH is smaller and the conversion efficiency is
higher. This is mainly because that the DC glow discharge gives
rise to higher amount of hydroxyl radical as well as higher ratio
Conclusions
Presently, SERS has become an intensively explored and
powerful tool in analytical chemistry, and there is an
increasing interest for employing SERS to probe or monitor
biochemical reactions in biological systems.42,43 However, one
of big challenges is to identify different mixtures quantita-
tively in the reaction system. Our work is certainly an effort in
this regard. In this work, we have synthesized silver nano-
particles through the reduction of AgNO3 by b-CD under the
alkaline condition, and employed these Ag NPs to achieve
sensitive and quantitative SERS analysis of the redox reaction
of GSH/GSSG transformation under oxidative stress imposed
by non-thermal plasma DBD irradiation. Based on the char-
acteristic bands of 509 and 1051 cmꢀ1 for GSSG and GSH
respectively, we could analyze the mixture of GSH and GSSG
simultaneously and make the quantitative distinction and
evaluation of conversion between GSH and GSSG conveniently
and quickly. As such, we have not only successfully employed
the method SERS for evaluating the mixture of GSH/GSSG
quantitatively, but also introduced it in the study of radia-
tion chemistry involving redox reaction of biomolecules.
Because GSH/GSSG redox reaction is a most important process
in biological systems, and employment of SERS improves the
detection sensitivity tremendously, this work therefore also
suggests the potential applicability of SERS in the radio-
biological study of cells against oxidative stress.
Fig. 6 The comparison in evaluation of the GSH and GSSG contents
between the SERS result and the HPLC result.
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