Electrochemical investigations of 8-hydroxydeoxyguanosine and its determination at an edge plane pyrolytic graphite electrode

Article abstract of DOI:10.1039/c5ra22682a

The electrochemical oxidation of 8-hydroxydeoxyguanosine (8-OHdG) has been studied at the edge plane surface of a pyrolytic graphite electrode (EPPGE) by cyclic sweep, square wave voltammetry, spectral studies, controlled potential electrolysis and related techniques. The oxidation of 8-OHdG occurred in a single well-defined peak over the entire pH range. The effect of pH revealed that the oxidation of 8-OHdG involved an equal number of electrons and protons. The 8-OHdG exhibited a linear calibration curve over a concentration range of 0.5-100 μM and the detection limit and sensitivity are found to be 28 × 10^-9 M and 1.068 μA μM^-1 respectively. The kinetics of the decay of the UV-absorbing intermediate generated during oxidation has been studied and the decay has been found to follow first order kinetics having a rate constant (k) in the range ～2.5 × 10^-3 s^-1. The products of electrochemical oxidation have been characterized by using GC-MS studies. A tentative mechanism for the oxidation of 8-OHdG has also been suggested.

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Electrochemical investigations of 8ꢀhydroxydeoxyguanosine and its
determination at edge plane pyrolytic graphite electrode
Pankaj Guptaa, Munetaka Oyamab, Rajendra N Goyala*
aDepartment of Chemistry, Indian Institute of Technology Roorkee, Roorkee ꢀ247667 (India)
bDepartment of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto ꢀ
615ꢀ 8520, Japan
*E.Mail : rngcyfcy@iitr.ac.in, Tel: +91ꢀ1332ꢀ285794 (O) Fax: +91ꢀ1332ꢀ273560
Abstract
The electrochemical oxidation of 8ꢀhydroxydeoxyguanosine (8ꢀOHdG) has been studied
at edge plane surface of pyrolytic graphite electrode (EPPGE) by cyclic sweep, square wave
voltammetry, spectral studies, controlled potential electrolysis and related techniques. The
oxidation of 8ꢀOHdG occurred in a single wellꢀdefined peak over the entire pH range. The effect
of pH revealed that the oxidation of 8ꢀOHdG involved equal number of electrons and protons.
The 8ꢀOHdG exhibited linear calibration curve over a concentration range of 0.5ꢀ100 µM and the
detection limit and sensitivity are found to be 28× 10ꢀ9 M and 1.068 µAµMꢀ1 respectively. The
kinetics of the decay of the UVꢀabsorbing intermediate generated during oxidation has been
studied and the decay has been found to follow first order kinetics having rate constant (k) in the
range ∼ 2.5 × 10−3 sꢀ1. The products of electrochemical oxidations have been characterized by
using GCꢀMS studies. A tentative mechanism for the oxidation of 8ꢀOHdG has also been
suggested.
Keyword: 8ꢀHydroxydeoxyguanosine, Electrochemical oxidation, Pyrolytic graphite electrode,
Cyclic voltammetry.
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Introduction
8ꢀHydroxydeoxyguanosine (8ꢀOHdG or 8ꢀoxoꢀ7,8ꢀdihydroꢀ2’ꢀdeoxyguanosine, I) is one
of the oxidized nucleosides produced, when DNA is attacked by the reactive oxygen species,
ultraviolet light, or genotoxic agents [1, 2]. The increased level of 8ꢀOHdG has been found in the
body fluids and tissues during an inflammatory conditions, in response to the production of
reactive oxygen species (ROS). The production of 8ꢀOHdG in the tissue DNA has been found to
be a sensitive, stable and integral biomarker of oxidative stress and cancer [3, 4]. It has been
reported that the elevated level of 8ꢀOHdG accumulates in the breast, gastric tumors and renal
cell in comparison to the surrounding tissues, which directly indicates the enhanced oxidative
stress in the tumor tissues and can be related to the initiation of carcinogenesis. In agreement
with this fact, cancer cell lines have been found to contain elevated level of oxidized damaged
DNA, when compared with surrounding cell lines. The oxidized deoxyguanosine may lead to
detrimental consequences like genetic mutations, cancer, thus unrepaired DNA lesions excised
and replaced by DNA repair mechanism and excreted in urine. The urinary excretion of DNAꢀ
repair product 8ꢀOHdG can be used for the quantitative assessment of oxidative DNA damage in
the human system [1, 5ꢀ8]. Oxidative stress and ROSꢀinduced elevations of 8ꢀOHdG have also
been claimed associated with the neurological diseases such as Parkinson's, Alzheimer's,
Huntington's and numerous pathological processes including cardiovascular disease, rheumatoid
arthritis, pancreatitis, chronic hepatitis and inflammatory bowel disease [9ꢀ11]. Several analytical
methods such as coupledꢀcolumn HPLC with electrochemical detection, capillary
32
electrophoresis, gas chromatographyꢀmass spectrometry (GC/MS), Pꢀpostlabeling and ELISA
have been used for the measurement of the oxidative DNA damage [12ꢀ17]. However, most of
these methods are complicated, tedious and require time consuming derivatization, sample
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preparation and extraction steps. An electroanalytical method bypass such complicated
procedures and proved to be a simple, highlyꢀsensitivity, cost effective, rapid, convenient and
effective tool for the analysis of biomolecules in pharmaceutical formulations and human body
fluids [18].
Structure of 8ꢀOHdG (I)
In view of the importance of 8ꢀOHdG as a specific marker of oxidative DNA damage,
attempts have been made to determine this compound at variety of unmodified and modified
electrodes. The determination of 8ꢀOHdG have been extensively carried out and low detection
limit for 8ꢀOHdG is reported [19, 20]. Glassy carbon electrode (GCE) modified with nanocarbon
film coupled with HPLC [21], poly (3ꢀmethylthiophene) [22, 23] and polyethylenimine [24] have
been used for the determination of 8ꢀOHdG. A review on the electrochemical devices for
detection of DNA damage has also appeared in the literature [25]. A comparison of various
methods reported in the literature is compiled in Table 1. The extensive literature survey
indicates that several attempts have been focused to determine 8ꢀOHdG, whereas, practically no
attempt has been made to study the mechanism of electroꢀoxidation of 8ꢀOHdG.
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The main objective of the present work is to study the electrochemical oxidation of 8ꢀ
OHdG at edge surface exposed pyrolytic graphite electrode. The kinetics of the UVꢀabsorbing
intermediate generated during electrochemical oxidation has been studied and the major
oxidation products formed at physiological pH have been identified. A tentative mechanism for
the oxidation of 8ꢀOHdG has also been suggested.
2. Experimental
2.1. Instrumentation
Electrochemical experiments were performed using voltammetric analyzer (BAS, West
Lafayette, USA) Epsilon ECꢀUSB. All potentials are referred to Ag/AgCl (1M KCl) electrode at
an ambient temperature, 25± 2 °C. Controlled potential electrolysis (CPE) for monitoring
spectral changes was carried out by electrolyzing 0.1 mM 8ꢀOHdG, in a single compartment cell
equipped with three electrodes system using pyrolytic graphite plate (1 cm × 8 cm) as working,
cylindrical platinum gauze as auxiliary and Ag/AgCl as reference electrode. The spectral
changes during the electroꢀoxidation and the kinetic studies of the decomposition of the UVꢀ
absorbing intermediates generated during oxidation of 8ꢀOHdG were performed using Shimadzu
spectrophotometer (UVꢀvis) model UVꢀ2450. Gas chromatography mass spectrometry (GCꢀMS)
of the silylated samples were carried out by using PerkinꢀElmer (model Clarusꢀ500) mass
spectrometer using helium as carrier gas at 30 ml/min. The glass column (1.8 m x 2.0 mm id.)
containing 3% SE 30 on Chromosorb ꢀ W was used. Electron impact (EI) spectra were recorded
at an electron beam voltage of 70 eV, whereas chemical ionization (CI) spectra were recorded
using methane as the reagent gas at a pressure of 2x10ꢀ4 Torr. After injection the column was
maintained at 100oC for 10 min followed by an increase at a rate of 5oC per minute. The pH
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determination of buffer solutions was performed using Thermo Fisher Scientific, Singapore
Digital pH meter (Eutech Instruments, model pH 700).
2.2. Chemicals
8ꢀOHdG was purchased from SigmaꢀAldrich (USA). Phosphate buffers of ionic strength
(1.0 M) and appropriate pH were prepared from analytical grade chemicals (NaH2PO4 and
Na2HPO4 from Merck) in double distilled water according to the method of Christian and Purdy
[26]. Silylation grade acetonitrile (CH3CN) and N,Oꢀbis[trimethylsilyl]trifluotoacetamide
(BSTFA) were incurred from Supelco, USA. The 2 mm × 2 mm × 10 mm pieces of pyrolytic
graphite (PGE) obtained from Pfizer, USA as a gift were used for the fabrication of the electrode.
2.3. Preparation of sensor
The sensor was prepared according to the reported literature [27]. Briefly, a Pyrex glass
tube of 80 mm length and 6 mm diameter was cleaned thoroughly and dried. An epoxy resin
(Araldite, Ciba) was applied to the one end of the tube to fill in such a way so that tube is filled
inside with epoxy to about 100 mm and then pyrolytic graphite piece (10 × 2 × 2 mm3) was then
slided from the open end of the tube with the help of a glass tube and pushed till it got covered
with epoxy resin. Air pocketing was avoided in between the resin and the graphite piece inside
the tube. After the resin had solidified, the glass tube end was rubbed on an emery paper gently
(Pꢀ400) to expose the layered edge of the graphite piece. The electrode surface was then washed
with distilled water several times in order to remove adhered fine carbon particles. A sufficient
amount of mercury was then placed into the glass tube and a copper wire of appropriate length
was inserted to make proper contact of the sensor to the outer circuit. To calculate the surface
area of the exposed edge plane surface of the sensor, cyclic voltammograms (CVs) were
recorded in the solution of 5 mM K3[Fe(CN)6] at different scan rates using 0.1 mM KCl as the
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supporting electrolyte. A redox couple was observed due to the Fe+2/Fe+3. The effective surface
area was calculated from the slope of ip vs. ν1/2 plot using RandlesꢀSevcik equation and found as
0.085 cm2.
2.4 Procedure
The stock solution of 8ꢀOHdG (1 mM) was prepared by dissolving the required amount
in double distilled water. Working solutions were prepared by adding required volumes of the
respective stock solution to 2.0 ml of phosphate buffer and the total volume was made to 4.0 ml
with double distilled water. The voltammograms were then recorded after bubbling nitrogen for
~ 10 min. The surface of the electrode was cleaned after each run by rubbing it on an emery
paper followed by washing with water. This procedure caused deviation in current, hence, the
current reported are an average of at least three runs. The optimized parameters used for the
square wave voltammetry were initial potential (E): 0 mV, final potential (E): 800 mV, square
wave amplitude: 25 mV, potential step: 4 mV and square wave frequency (f): 15 Hz.
UVꢀvis spectral changes during the electroꢀoxidation of 8ꢀOHdG were monitored in 200ꢀ
400 nm region at a potential ∼100 mV more positive than the peak potential. Throughout the
electrolysis the solution in the working compartment was bubbled slowly with N2 gas and stirred
with a teflonꢀcoated magnetic stirring bead. Nearly 3 mL of the electrolyzed solution was taken
from the working compartment of the electrolysis cell at 2 min. time intervals and transferred to
1 cm3 quartz cuvette and the spectrum was recorded. When the absorbance at λmax reduced to
about 50%, electrolysis was turned off and spectral changes were monitored at different times to
detect the wavelength of the UVꢀvis absorbing intermediate generated during the oxidation.
For the product identification, about 8ꢀ10 mg of the compound was electrooxidized at a potential
∼100 mV more positive than the oxidation peak potential in a buffer of pH 7.2. The ionic
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strength of the buffer was kept at (0.05M) as phosphate interferes in the silylation procedure. The
progress of the electrolysis was monitored by recording UVꢀspectrum at different time intervals.
When the absorbance at λmax reached to minimal (usually 24 h), the electrolysis was stopped and
the electrolyzed solution was removed from the cell and filtered using Whatman filter paper 42,
lyophilized and the resulting solid was analyzed by GCꢀMS.
2.5. Characterization of the oxidation product
In order to identify the products formed upon electrochemical oxidation of 8ꢀOHdG, few
µg of the lyophilized product was put in a silylation vial and 300 µL of acetonitrile and 300 µL
of BSTFA were added. The vial was heated at 110 ºC in an oil bath for 30 min and then cooled.
After cooling 5 µL of the solution was injected in GCꢀMS.
3. Results and discussions
3.1. Voltammetric studies
Cyclic voltammetry provides the information about the redox process of reaction. Thus,
the cyclic voltammograms for 100 ꢁM of 8ꢀOHdG were recorded using edge plain exposed
pyrolytic graphite electrode (EPPGE) at a sweep rate of 100 mV sꢀ1. At pH 7.2, a wellꢀdefined
oxidation peak at Ep (390 mV) was observed, when the sweep was initiated in the positive
direction. In the reverse sweep no reduction peak was observed which confirmed the irreversible
oxidation of 8ꢀOHdG. A typical cyclic voltammogram observed for the oxidation of 8ꢀOHdG is
presented in Fig. 1. To ascertain the nature of the electrode reaction, the scan rate effect on the
electrochemical response was performed in the range 5ꢀ200 mV sꢀ1. The oxidation peak current
of 8ꢀOHdG was found to increase linearly with the increase in the scan rate over the range used
and the dependence of the peak current on scan rate can be expressed by the following linear
relationship:
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ip = 0.0908 [v] ꢀ 0.337
having a correlation coefficient of 0.998, where v is the scan rate in mV sꢀ1 and ip is the peak
current in ꢁA. The linear relation between ip versus scan rate (ν) (inset of Fig. 1) indicated that
oxidation of 8ꢀOHdG at the EPPGs was adsorption controlled, which was further confirmed by
the linearity of ip/v1/2 vs. log v and log ip vs. log v plots. The following relation was observed for
log ip vs. log v plot:
log ip = 1.0273 log v − 0.1179
having a correlation coefficient 0.999. The slope value (> 0.5) of log ip vs. log v plot further
suggested that 8ꢀOHdG underwent an oxidation involving adsorptionꢀcontrolled process at the
surface of the EPPGE [28, 29].
As square wave voltammetry (SWV) is considered to be more sensitive, further studies
on the oxidation peak of 8ꢀOHdG, are carried out using SWV. The effect of the pH on the anodic
peak potential of 8ꢀOHdG was investigated using EPPGE. The peak potential of 8ꢀOHdG was
considerably influenced by the pH value of the supporting electrolyte. It was found that with the
increasing pH in the range of 2.2–10.5, the oxidation peak potential of 8ꢀOHdG shifted towards
less positive potentials. The nature of Ep versus pH plot was linear and observed the linear
regression equationꢀ
Ep/mV (pH 2.2 – 10.5) = ꢀ60.30 pH + 791.06
with a correlation coefficient of 0.9975. The slope of 60.30 mV per pH of the plot indicated that
the number of electrons and protons involved in the oxidation of 8ꢀOHdG are equal [22, 30].
The dependence of the oxidation peak current (ip) of 8ꢀOHdG on square wave frequency (f) was
also studied in the range of 5ꢀ40 Hz at pH 7.2. The oxidation peak current (ip) of 8ꢀOHdG was
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found to increase linearly with increase in the square wave frequency and the linear relation
between ip and f can be expressed by the relation:
ip (µA) = 0.9401[f] ꢀ 2.80
having a correlation coefficient of 0.999. The slop of the plot is in agreement with an adsorption
controlled electrochemical process, which supports the inferences obtained from cyclic
voltammetric studies [31].
The effect of the concentration of 8ꢀOHdG on the oxidation peak current was examined
at optimized parameters of SWV in the phosphate buffer of pH 7.2. Fig. 2 shows the square wave
voltammograms observed for various concentration of 8ꢀOHdG at the EPPGE. The peak current
(ip) was found to increase linearly with an increase in 8ꢀOHdG concentration. The inset of Fig. 2
shows the corresponding calibration curve, demonstrating that in the range 1ꢀ100 µM, the anodic
peak current has a good linear relationship with 8ꢀOHdG concentration. The linear relationship
between the oxidation peak current and concentration of 8ꢀOHdG can be expressed by the
relation:
ip (µA) = 1.0688C (µM) + 4.1842
with a correlation coefficient of 0.994. The detection limit (LOD) and the limit of quantification
(LOQ) were calculated by using the relation 3σ/b and 10σ/b and found to be 28 nM and 93 nM,
respectively, where σ is the standard deviation of the blank solution and b is the slope of the
calibration plot.
3.2. Spectrophotometric study
The UVꢀvis spectral changes during electrochemical oxidation of 8ꢀOHdG were
monitored at pH 7.2. A typical UVꢀspectrum of 8ꢀOHdG exhibited two well defined absorption
bands at λmax=293 and 245 nm, (Fig. 3, curve 1). Upon application of a potential 100 mV more
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positive than the peak potential of 8ꢀOHdG, absorbance in the region 240ꢀ320 nm decreases
systematically, whereas, the absorbance in the region 225ꢀ235 nm increase systematically (Fig.
3, curve 2ꢀ20). Curve 20 (in Fig. 3) was recorded after 40 min. of electrolysis and both the
absorption peaks (293 nm and 245 nm) completely disappeared and a new band at 224 nm
observed as shown in the inset of Fig 3. Thus, it is clear that the products of oxidation absorb at
shorter wavelength in comparison to the starting material. A clear isosbestic band at 236 nm was
observed during spectral changes.
The kinetics of the decomposition reaction of the UVꢀabsorbing intermediate generated
during electroꢀoxidation was monitored by recording absorbance at selected wavelengths. For
this purpose, electroꢀoxidation was carried out and when the absorbance reached to about 50%,
(corresponding to curve 5 in Fig. 3) the electrolysis was terminated and the decay of the
absorbance at 245 nm and 293 nm were monitored as a function of time. The absorbance vs. time
profile showed an exponential decay and the plots of log (AꢀA∞) vs. time were linear, indicating
thereby that the decomposition reaction of the UVꢀabsorbing intermediate followed firstꢀorder
kinetics (Fig. 4). The values of the rate constants were calculated from the slope value of log (Aꢀ
A∞) versus time plots and are presented in Table 2. The cyclic voltammograms was also recorded
to monitor the progress of electrolysis of 8ꢀOHdG at different times. At pH 7.2, when a potential
∼100 mV more positive than anodic peak potential of 8ꢀOHdG was applied, the peak current of
8ꢀOHdG systematically decreased as shown in Fig. 5.
3.3. Product Characterization
The products of oxidation of 8ꢀOHdG were characterized by recording GCꢀMS of the
silylated lyophilized product. The total ion current chromatogram exhibited a major peak at
32.57 min together with small peak at 22.51, 23.90, 27.10, 28.52 and 28.60 min as shown in Fig.
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6. The peak corresponding to 32.57 min exhibited a molar mass peak at 589 (M+, 10.5%) and
574 (M+ꢀ15, 2.0%). The molar mass of 589 corresponded to hexasilylated 5ꢀ
guanidinoimidazolidineꢀ2,4ꢀdione as shown in Scheme 1.
Scheme 1
The peak at Rt ~ 28.60 min exhibited molar mass of 517 and corresponded to penta
silylated molecule of 5ꢀguanidinoimidazolidineꢀ2,4ꢀdione. The formation of such imidazole
derivative has also been observed during the electroꢀoxidation of guanosine [32ꢀ34]. Thus, it
appears that during electroꢀoxidation the deoxyribose unit is hydrolyzed and removed from 8ꢀ
OHdG.
The chemical ionization mass spectrum was then recorded to confirm the molar mass of
589 for the hexasilylated species using methane. In this case a peak at Rt ~ 33.67 min was
observed in the chromatogram, which showed the molar mass at 590 (M+ +1; 48.4%). Other
major high mass peaks in the spectrum were observed at 574, (M+ ꢀCH3; 100%); 618 (M+ +C2H5;
4%); and 630 (M+ +C3H7; 1%). Thus, it was confirmed that the major product of electroꢀ
oxidation had a molar mass of 157, which on silylation gave peak corresponding to 589 (M+).
The formation of deoxyribose was also confirmed in the electrolyzed product of 8ꢀ
OHdG. The small peak at 22.55 min in the GCꢀMS indicated a molar mass of 350 (M+, 7.4%) as
shown in Scheme 2.
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Scheme 2
The fragmentation pattern gave a clear M+ꢀ15 peak at 335 (2.1%). Hence, it is concluded that
deoxyribose unit is detached from 8ꢀOHdG during electrooxidation. The hydrolysis of sugar
moiety in purine nucleoside and nucleotides is common during oxidation and has been reported
earlier [34, 35].
3.4 Coulometric Experiment
The experiment value of ‘n’, number of electrons involved in the oxidation of 8ꢀOHdG
was determined by plotting change in current with time. The resulting plot was exponential for
the first 15ꢀ20 min of electrolysis, whereas, a significant deviation was observed after that.
Hence, it was concluded that followup chemical reactions play a significant role after 15ꢀ20 min
[36]. The value of ‘n’ calculated was found to be 1.8 ± 0.2 at pH 7.2. Thus, it seems reasonable
to conclude that number of electrons involved in oxidation is ~ 2.0.
3.5 Reaction Mechanism
The observed results indicate that electrooxidation of 8ꢀOHdG occurs in 2eꢀ, 2H+
reaction, to give a UVꢀabsorbing intermediate, which decompose in follow up chemical reactions
to give 5ꢀguanidinoimidazolidineꢀ2,4ꢀdione (IV) as a major product. Following tentative
mechanism can be suggested for the electrooxidation of 8ꢀOHdG. The first step of oxidation
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involves 2eꢀ, 2H+ oxidation of 8ꢀOHdG to give diimine species (II). The diimine species (II) is
readily attacked by water to give imine alcohol (III). The hydration of species II was first order
reaction and the k values observed were 0.0025 sꢀ1 and 0.0024 sꢀ1 at pH 7.2. In a series of
followup reactions imine alcohol (III) gives the major product 5ꢀguanidinoimidazolidineꢀ2,4ꢀ
dione. The major steps involved in the oxidation are shown in Scheme 3. Similar steps for the
oxidation of purines and their nucleosides are already reported in the literature [32, 37ꢀ39]. Thus,
in contrast to adenosine and guanosine, where dimers or oligomers have been observed as
products, the oxidation of 8ꢀOHdG follows a simple path to give (IV) as the major product. One
of the possible reasons for this difference in behavior may be the higher positive potential
required for the oxidation of adenosine and guanosine in contrast to the less potential for 8ꢀ
OHdG.
Conclusion
In this study, electrochemical oxidation of 8ꢀOHdG in phosphate buffers has been studied
by cyclic voltammetry and controlled potential electrolysis. The UVꢀvis spectral behaviour
during oxidation has been studied using EPPGE. Electrochemical studies of 8ꢀOHdG have
provided significant information about its basic oxidation chemistry. The investigations clearly
demonstrate that the electrochemical oxidation of 8ꢀOHdG proceeds via a 2eꢀ, 2H+ mechanism
and it was found that the generated UVꢀabsorbing intermediate (Schemeꢀ1 (I)) decayed to follow
first order kinetics. The major product of oxidation has been characterized as 5ꢀ
guanidinoimidazolidineꢀ2,4ꢀdione (IV) by GCꢀMS. The suggested tentative mechanism was
explained by all the observed voltammetric, coulometric and spectral behaviour. The detection
limit for the 8ꢀOHdG is found to be 28 nM. A comparison of the detection limit at other
electrodes reported in recent literature is presented in Table 1 and indicates that the electrode is
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satisfactory for the determination of 8ꢀOHdG and no complicated modification of the electrode
surface are needed to achieve similar order of detection limit. Thus, it is expected that the
oxidation of 8ꢀOHdG could serve as an important tool for the biochemists to deeply understand
the medicinal applications of the products.
Acknowledgements
Thanks are due to the Japan Society for the Promotion of Science (JSPS) for awarding
Invitation Fellowship for Research in Japan to RNG. One of the authors (PG) is thankful to the
Ministry of Human Resource Development, New Delhi, for the award of Senior Research
Fellowship.
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Figure Caption
Fig. 1: Typical cyclic voltammograms Observed for 100 µM 8ꢀOHdG at scan rate 100 mV sꢀ1 in
phosphate buffer of pH 7.2 at pyrolytic graphite electrode. Inset is showing the graph between ip
and v. The background is shown by the dotted line.
Fig. 2: Square wave voltammograms observed for increasing concentration of 8ꢀOHdG. Curves
were recorded at (a) 0.5; (b) 2; (c) 5; (d) 10; (e) 20; (f) 30; (g) 50; (h) 80ꢁM; (i) 100 ꢁM using
EPPGE in phosphate buffer of pH 7.2. The background curve is shown by dotted line. The
observed calibration plot for 8ꢀOHdG is shown in the inset.
Fig. 3: Spectral changes observed at pH 7.2 for electrooxidation of 0.2 mM 8ꢀOHdG at an
interval of 2 min each after application of 0.5 V vs. SCE. Inset is showing a comparison of 1st
and 20th curves observed during spectral changes.
Fig. 4: Plot of the absorbance vs. time and log (A− A∞) vs. time (inset) plot observed at pH 7.2
at 245 nm for 8ꢀOHdG.
Fig. 5: Cyclic voltammograms recorded for 100 µM 8ꢀOHdG at pH 7.2. Curves were recorded at
(1) 0; (2) 15; (3) 25; (4) 35; (5) 50; (6) 65; (7) 80; (8) 35; (9) 100; (10) 110; (11) 120 and (12)
130 min of electrolysis.
Fig. 6: Total ion current chromatogram obtained for the silylated oxidation products of 8ꢀOHdG.
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Scheme 3: Tentative reaction mechanism proposed for the electrooxidation of 8ꢀOHdG.
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Table 1: A comparison of the EPPGE with recently reported methods for the determination of 8ꢀ
OHdG.
Sr.
No.
Electrode Type
Linear range
Detection Limit Reference
MWNT/GCE
GCE/CNTꢀPnaf
0.08ꢀ5 µM
0.5ꢀ20 µM
9 nM
13 nM
3 nM
19
1
20
2
3
4
5
6
7
Nanoꢀcarbon/GCE
poly(3ꢀmethylthiophene)/GCE
DNA/P3MT/GCE.
GCE/CNTꢀPEI
0.02ꢀ0.5 µM
0.7ꢀ70 ꢁM
21
22
100 nM
56 nM
100 nM
28 nM
0.28ꢀ19.6 µM
0.5ꢀ30 µM
23
24
EPPGE
0.5ꢀ100 µM
This work
*GCEꢀ Glassy carbon electrode, MWNTꢀ multiꢀwall carbon nanotube, Pnafꢀ polyꢀnafion, P3MTꢀ poly(3ꢀ
methylthiophene), PEIꢀ polyethylenimine.
Table 2: Rate constant (k) values observed for the decomposition of UV absorbing generated
intermediate during electrochemical oxidation of 8ꢀOHdG at pH 7.2.
Rate (k×10−3 s−1)a
pH
λ
max (nM)
Electrochemical
7.2
293
2.4
245
2.5
a
Average of at least three replicate determinations.
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Fig. 1
Fig. 2
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Fig. 3
Fig. 4
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Fig. 5
Fig. 6
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209x129mm (96 x 96 DPI)