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Bioelectrochemistry 99 (2014) 24–29
Contents lists available at ScienceDirect
Bioelectrochemistry
journal homepage: www.elsevier.com/locate/bioelechem
A novel nanogold–single wall carbon nanotube modified sensor for the
electrochemical determination of 8-hydroxyguanine, a diabetes
risk biomarker
Sunita Bishnoi a, Rajendra N. Goyal b,⁎, Yoon-Bo Shim c,⁎
a
b
c
Department of Chemistry, Vivekananda Institute of Technology (East), Jaipur 303 012, India
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247 667, India
Department of Chemistry and Institute of Bio-Physico Sensor Technology, Pusan National University, Busan 609-735, South Korea
a r t i c l e
i n f o
Article history:
Received 30 January 2014
Received in revised form 21 May 2014
Accepted 4 June 2014
Available online 15 June 2014
Keywords:
SWCNT
Square wave voltammetry
8-Hydroxyguanine
Diabetes
a b s t r a c t
An electrochemical study of the oxidation of 8-hydroxyguanine (8-OH-Gua) at gold nanoparticles attached to
single walled carbon nanotube modified edge plane pyrolytic graphite electrode (AuNP-SWCNT/EPPGE) has
been carried out to develop a method for the self diagnosis of diabetes. The level of 8-OH-Gua, an important
biomarker of oxidative DNA damage, is higher in urine of diabetic patients than control subjects. A detailed
comparison has been made between the square wave voltammetric (SWV) response of SWCNT/EPPGE and
AuNP-SWCNT/EPPGE towards the oxidation of 8-OH-Gua in respect of several essential analytical parameters
viz. sensitivity, detection limit, peak current and peak potential. The AuNP-SWCNT/EPPGE exhibited a well
defined anodic peak at potential of ~ 221 mV for the oxidation of 8-OH-Gua as compared to ~ 312 mV using
SWCNT/EPPGE at pH = 7.2. Under optimized conditions linear calibration curve for 8-OH-Gua is obtained over
a concentration range of 0.01–10.0 nM in phosphate buffer solution (PBS) of pH = 7.2 with detection limit
and sensitivity of 5.0 (±0.1) pM and 4.9 (±0.1) μA nM−1, respectively. The oxidation of 8-OH-Gua occurred
in a pH dependent process and the electrode reaction followed adsorption controlled pathway. The electrode
exhibited an efficient catalytic response with good reproducibility and stability. The method has been found
selective and successfully implemented for the determination of 8-OH-Gua in urine samples of diabetic patients.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Diabetes, termed ‘diabetes mellitus’ in clinical terminology, is a
serious and growing health care problem worldwide and is associated
with acute and chronic complications [1]. The excessive production of
reactive oxygen species (ROS) due to aerobic cellular metabolism can
accelerate oxidative damage to macromolecules including lipids,
proteins as well as nucleic acids [2]. Reactive oxygen species are
known to cause cleavage of strands and base modifications in nuclear
and mitochondrial DNA which in turn results in the cellular dysfunction
and apoptosis that are critical to the pathogenesis of many diseases
including diabetes and its complications [3]. Substantial evidences
have been demonstrated to suggest the increased production of reactive
oxygen species in diabetic patients [4,5]. Electrochemical oxidation of
DNA can occur at any of the four bases out of which guanine can undergo the easiest oxidative damage [6]. The major product of guanine
oxidation is 8-hydroxyguanine (8-OH-Gua) which is widely accepted
as a biomarker of oxidative DNA damage [7]. 8-OH-Gua is excreted
⁎ Corresponding authors. Tel.: +91 1332 285794.
E-mail addresses: [email protected] (R.N. Goyal), [email protected] (Y.-B. Shim).
http://dx.doi.org/10.1016/j.bioelechem.2014.06.003
1567-5394/© 2014 Elsevier B.V. All rights reserved.
into urine from cellular DNA without further metabolism [8]. Thus, its
urinary excretion reflects oxidative DNA damage and the “whole
body” repair of DNA. Therefore, an assay that is able to determine the
level of 8-OH-Gua in biological fluids can better reflect the extent of
oxidative damage of cellular DNA. Since urine concentration of metabolites symbolizing DNA damage is very low, and urine as such is a complex matrix, measurement of these lesions in urine is an analytical
challenge [9]. Hence, the quantification of 8-OH-Gua in biological fluids
requires sensitive approach. Moreover, method should be selective, as
guanine and other major urinary metabolites viz. uric acid, and ascorbic
acid may also interfere the 8-OH-Gua determination. In addition
common-diabetic patients are subjected to glucose test in blood
serum and are required to undergo painful blood suctions.
Literature survey reveals that various analytical methods including
capillary electrophoresis with precolumn derivatization [10], high performance liquid chromatography (HPLC) equipped with an electrochemical
detector, [11] flow injection biosensor with an online microdialysis
sampling method [12] and gas chromatography–mass spectrometry
(GC/MS) [13] have been discovered for 8-OH-Gua determination with
low detection limit. These methods are complicated, time consuming,
need expensive instrumentation and cannot be used for self-diagnosis.
Therefore, these methods are not widely used, diabetic patients want to
S. Bishnoi et al. / Bioelectrochemistry 99 (2014) 24–29
utilize methods that offer ease of detection, do not require blood suctions,
have numerically sensitive quantification and allow self-diagnosis anywhere. Hence, still there is a vital need to develop a specific method
that is fast, sensitive, easy to use, and cost effective having lower detection limit for the determination of oxidative DNA damage and its effects.
In the present method a new approach is proposed using edge plane
pyrolytic graphite electrode (EPPGE) after surface modification with
gold nanoparticles attached to single walled carbon nanotubes. The
modified electrode was used for the typical analysis of 8-OH-Gua in biological fluids for detecting any oxidative DNA damage which further
clues to the diabetic diagnosis. Square wave voltammetry is a versatile
technique for electroanalytical determination as it has higher sensitivity
and effectively suppresses background current. A detailed comparison
has been made to examine electrochemical response of single walled
carbon nanotube modified edge plane pyrolytic graphite electrode
(SWCNT/EPPGE) and gold nanoparticles attached to single walled
carbon nanotube modified edge plane pyrolytic graphite electrode
(AuNP-SWCNT/EPPGE) towards 8-OH-Gua oxidation. The comparative
study shows that the catalytic effect of nanotubes gets enhanced significantly on attachment with gold nanoparticles. Improved sensitivity,
selectivity, reproducibility and stability of AuNP-SWCNT/EPPGE are
observed in comparison to SWCNT/EPPGE, which make it attractive
for further developments in the field of electrochemical sensors. In
this paper, the concentration of 8-OH-Gua in urine samples of diabetic
patients is determined and compared with non-diabetic (control) samples using the proposed sensor.
2. Experimental
2.1. Materials
Pyrolytic graphite pieces (2 × 2 × 10 mm3) were obtained from
Pfizer Inc. New York, U.S.A. Guanine and 8-OH-Gua were obtained
from Fluka and Adams Chem. Co. Illinois, USA respectively and used
without further purification. Ascorbic acid and uric acid were purchased
from Wako Pure Chemicals Industries Ltd., Japan and Sigma-Aldrich, respectively. All solutions were prepared in double distilled water. The
first urine samples of the day were collected from three departmental
personnel and used as control (male: 53 years, 69 kg, male: 50 years,
76 kg, male: 55 years, 75 kg) whereas, three urine samples of diabetic
patients (male: 52 years, 61 kg, male: 57 years, 72 kg, male: 54 years,
84 kg) were obtained from the hospital of Indian Institute of Technology, Roorkee after clearance from Ethics Committee of I.I.T. Roorkee. The
urine samples collected were stored in a refrigerator until used. Prior to
recording voltammograms, urine samples were diluted 60 times with
phosphate buffer solution of pH = 7.2 to minimize matrix complexity.
Single walled carbon nanotubes (SWCNT) of purity N98% were purchased from Bucky, USA. HAuCl4 was purchased from Aldrich (USA).
The stock solution of 8-OH-Gua was prepared in 2 M Na2CO3 and the
stock solutions of guanine, uric acid and ascorbic acid were prepared
25
in double distilled water and then diluted with phosphate buffer
solution (PBS) of pH = 7.2 to achieve the desired concentration. Other
solvents and chemicals used were of analytical grade obtained
from Merck. Phosphate buffer solutions of pH range of 2.4–10.0 and
ionic strength μ = 1.0 M were prepared according to the method of
Christian and Purdy by mixing standard solutions of Na2HPO4 and
NaH2PO4 [14].
2.2. Apparatus and experimental procedure
All voltammetric measurements were carried out using BAS
(Bioanalytical Systems, West Lafayette, USA) CV-50W voltammetric
analyzer. The voltammetric experiments were performed using three
electrode single compartment cells equipped with a SWCNT/EPPGE
or AuNP-SWCNT/EPPGE as working, platinum wire as counter and
Ag/AgCl (3 M NaCl) as reference electrode (Model MF-2052 RB-5B).
The surface morphology of the modified electrodes was characterized
using Quanta 200-F (FEI Company) FE-SEM instrument. All potentials
are referred to the Ag/AgCl reference electrode at an ambient temperature of 27 ± 2 ° C. The pH of the buffer solutions was measured
using Eutech Instruments pH 510, pH meter after standardization
with 0.05 M potassium hydrogen phthalate (pH 4.0 at 25 °C) and 0.01
M borax (pH 9.2 at 25 °C). The voltammetric experiments were performed in 1.0 M phosphate buffer solution of different pH containing
8-OH-Gua at AuNP-SWCNT/EPPGE in a suitable potential range. The
modified electrodes gave reproducible results for three consecutive
runs in the same solution, however, before the next sample a potential
of −100 mV for 60 s was applied to overcome the problem of adsorption of analyte at the electrode surface. The optimized square wave
voltammetric parameters used were: square wave amplitude (Esw):
25 mV; potential step (E): 4 mV; and square wave frequency (f): 15 Hz.
2.3. Preparation of electrode
A Pyrex glass tube of appropriate length and diameter was cleaned
thoroughly and dried. One end of the glass tube is filled with epoxy
resin (Araldite, Ciba Geigy) up to a height of about 2 cm, with the help
of a thin glass rod. Pyrolytic graphite piece was then slided in the glass
tube carefully from the other open end of the tube so that its edge
plane side reaches at the bottom of the tube. The graphite piece was
pushed with a thin glass rod till 3/4th portion of it got covered with
epoxy resin to avoid any air pocketing between the tube and the graphite piece. The electrode was then allowed to stand for 24 h until resin
gets solidified. The glass tube was rubbed on an emery paper till the
edge plane side of graphite appeared at the resin end. Finally, the electrode was washed several times with double distilled water in order
to remove the fine powder adhered to the electrode surface of pyrolytic
graphite. Mercury was filled into the glass tube and a copper wire was
inserted to make proper contact of electrode to the outer circuit.
Fig. 1. Comparison of typical FE-SEM images of (A) EPPGE, (B) SWCNT/EPPGE and (C) AuNP-SWCNT/EPPGE.
26
S. Bishnoi et al. / Bioelectrochemistry 99 (2014) 24–29
2.4. Modification of electrode surface
The gold nanoparticle (AuNP) solution was prepared by reducing
Au3+ ions to Auo with ascorbic acid [15,16]. For this purpose 50 mL of
2.2 mM aqueous ascorbic acid was added to 50 mL of 1.34 mM aqueous
HAuCl4 under stirring. The change in the color of the solution from
yellow to deep red indicated the formation of gold nanoparticles.
In order to carry out functionalization of SWCNT, carboxylation was
carried out according to the reported method [17].
In order to modify the surface of EPPGE, initial optimization studies
were carried out. In order to find optimum amount for surface modification, different volumes of SWCNT were casted on the surface of EPPGE
and the current response for 8-OH-Gua was determined. It was observed that initially the current increased with increase in volume and
then became constant at μL. Thus, μL was considered as optimum for
SWCNT. In the next step, the amount of AuNP was varied from 5 to
50 μL. The current was found to increase with increase in amount of
AuNP and current became practically constant at 20 μL. Hence, 20 μL
aliquot of SWCNT and AuNP was selected as the optimum amount for
modification for further studies. Thus, initially 20 μL of 1 mg/mL carboxylated SWCNT was drop casted on clean surface of EPPGE and dried at
60 °C for 1 h. In the second step, 20 μL solution of the AuNP prepared
was dropped on the SWCNT layered EPPGE, followed by drying at
60 °C for 2 h. The electrodes were then ready to use for voltammetric
experiments.
3. Results and discussions
3.1. Characterization of modified surface
A comparison of typical FE-SEM images of bare EPPGE, SWCNT/
EPPGE and AuNP-SWCNT/EPPGE is given in Fig. 1. It can be seen that
the surface of bare EPPGE is rough (Fig. 1A) and after deposition of
SWCNT, a layer of SWCNT is clearly observed (Fig. 1B). The shining
white crumb parts observed in Fig. 1(C) are gold nanoparticles and, on
the backdrop, consistently formed SWCNT layer are clearly observed.
The surface properties of SWCNT/EPPGE and AuNP-SWCNT/EPPGE
were also characterized by electrochemical impedance spectroscopy.
The Randles equivalent circuit was used to obtain data, where parallel
combination of resistance to charge transfer (Rct) and interfacial capacity (Cdl) gave rise to a semicircle whose diameter is equal to the Rct. The
experiment was carried out in a 1:1 solution of 5 mM K3[Fe(CN)6]
and 0.1 M KCl solution in the frequency range of 0.001–100 KHz.
Fig. 2. Square-wave voltammograms of 10.0 nM 8-OH-Gua at (a) AuNP-SWCNT/EPPGE
(—) and (b) SWCNT/EPPGE (- - - -), dotted line represents blank phosphate buffer solution
using AuNP-SWCNT/EPPGE at pH = 7.2.
Fig. 3. Square wave voltammograms observed for (i) phosphate buffer solution
(background) (…..), (ii) increasing concentration of 8-OH-Gua; a = 0.25, b = 1.0, c =
2.5, d = 3.75, e = 5.0, f = 6.25, g = 7.5, h = 10.0 nM; and calibration curve for
8-hydroxyguanine [inset] at pH = 7.2 using AuNP-SWCNT/EPPGE.
At AuNP-SWCNT/EPPGE, a charge transfer resistance for [Fe(CN)6]3-/4redox process was observed as 32 Ω, while for SWCNT/EPPGE, it was
about 40 Ω implying that the AuNP increases the rate of electron transfer between sensor and electrolyte. At bare EPPGE, charge transfer resistance was about 100 times larger than SWCNT/EPPGE. These data
showed that the SWCNT and AuNP have been successfully attached to
the electrode surface.
3.2. Voltammetric response of 8-hydroxyguanine
The electrochemical behavior of 8-OH-Gua was studied by square
wave voltammetry using SWCNT/EPPGE and AuNP-SWCNT/EPPGE as
working electrode in order to elucidate the catalytic effect of nanofilm having both single walled carbon nanotubes and gold nanoparticles. Fig. 2 depicts the electrochemical response of 10 nM 8-OH-Gua
under optimal parameters in phosphate buffer of pH = 7.2 using the
abovementioned electrodes. 8-OH-Gua was oxidized at Ep ~ 312 mV
using SWCNT/EPPGE (curve a), whereas, at AuNP-SWCNT/EPPGE, Ep
shifted to ~ 221 mV (curve b) with remarkable enhancement in the
peak current. A comparative study clearly indicates that a substantial
decrease (~ 91 mV) in the peak potential along with significant enhancement in peak current is observed using AuNP-SWCNT/EPPGE as
compared to SWCNT/EPPGE for 8-OH-Gua oxidation. The shift in peak
potential to less positive potentials and enhancement in peak current
indicate that the film containing the combination of carbon nanotubes
with gold nanoparticles exhibits efficient electrocatalysis towards 8OH-Gua oxidation. Hence, AuNP-SWCNT/EPPGE sensor has been used
Fig. 4. Calibration plot observed for the 8-OH-Gua at pH = 7.2.
S. Bishnoi et al. / Bioelectrochemistry 99 (2014) 24–29
Scheme 1. Structural formula of 8-hydroxyguanine.
for further detailed studies of 8-OH-Gua determination in human body
fluids.
3.3. Electrochemical characteristics of 8-hydroxyguanine
Square wave voltammograms corresponding to various concentrations of 8-OH-Gua in phosphate buffer of pH = 7.2 were recorded
using AuNP-SWCNT/EPPGE. A well-defined oxidation peak at 0.22 V is
clearly observed at all concentrations. Peak current was found to
increase with the increase in concentration of 8-OH-Gua as shown in
the inset of Fig. 3. At each concentration three runs were taken and
the current values observed were marked with error bars as shown in
Fig. 4. The calibration curve yielded a linear range from 0.01 × 10−9 to
10.0 × 10−9 mol L−1 with a regression equation of ip (μA) = 4.917 C
(nM) + 0.198 having a correlation coefficient of 0.997. The detection
limit of 8-OH-Gua was calculated to be 0.005 × 10−9 mol L−1 and the
limit of quantification was found as 0.015 × 10−9 mol L−1.
As pH is a very significant parameter for any determination especially for those which are concerned with biological fluids, hence, the effect
of pH on the electro-oxidation of 8-OH-Gua was explored by square
wave voltammetry in a solution containing 5.0 nM 8-OH-Gua. The Ep
versus pH plot showed that the oxidation peak potential of 8-OH-Gua
shifted to negative potentials with the increase of the solution pH,
which indicated that protons were involved in the oxidation of 8-OHGua. The slope dEp/dpH of the plot is ~50 mV pH−1, which reveals the
involvement of equal proportion of the electron and proton in the reaction. A linear relationship was observed between the oxidation peak
potential and the solution pH having a linear regression equation as:
27
Ep (pH 2.4–10.4) = (584.9–50.15 pH) versus Ag/AgCl, with a correlation coefficient of 0.997.
The square wave voltammograms of 8-OH-Gua were recorded at
different square wave frequencies using SWCNT/EPPGE in phosphate
buffer solution of pH = 7.2 in order to determine the effect of square
wave frequency on the current response of 8-hydroxyguanine. The
peak current showed a linear increase with the increase of square
wave frequency in the range of 5 to 180 Hz. The linear relationship
between the oxidation peak current and square wave frequency can
be represented by the equation: ip (μA) = 0.440 f (Hz) + 12.86, having
a correlation coefficient of 0.992 (See Scheme 1).
The increase in peak current was also accompanied by the shift in
peak potential with square wave frequency in the range of 5 to
180 Hz. The oxidation peak potential increased gradually with increase
in frequency and the plot of Ep vs log f was linear. The dependence of
Ep with log f can be expressed by the relation: Ep (mV) = 143.9 log
f + 56.56, with a correlation coefficient of 0.991. These results indicate
that 8-hydroxyguanine strongly adsorbs at the surface of AuNPSWCNT/EPPGE and the redox process involves adsorption complications
[18]. The adsorption of 8-OH-Gua at the surface of electrode was further
confirmed by dipping AuNP-SWCNT/EPPGE in the solution of 8-OH-Gua
for different times followed by recording voltammogram in the buffer
solution. It was observed that peak current for 8-OH-Gua oxidation increased with increase in dipping time up to 2 min after which the current
became practically constant. This behavior further confirmed the adsorption of 8-OH-Gua at the surface of AuNP-SWCNT/EPPGE. A 2e, 2H+
oxidation of 8-OH-Gua has been well documented in the literature
[19,20] to give 2,5-diamino-4-imidazolone and 5-guanidohydantoin as
shown in Scheme 2.
3.4. Analysis of urine samples of diabetic patients
In diabetic patients, 8-OH-Gua is excreted from cellular DNA, into
urine without further metabolism [8]. Therefore, in order to better reflect
the oxidative DNA damage efforts have been made to determine the
level of 8-OH-Gua in urine samples of diabetic patients using AuNPSWCNT/EPPGE. The urine samples (control) were diluted 60 times
with phosphate buffer solution of pH = 7.2 to minimize matrix complexity and transferred into the voltammetric cell. The voltammogram of
Scheme 2. Structure of oxidation products formed on the oxidation of 8-hydroxyguanine.
28
S. Bishnoi et al. / Bioelectrochemistry 99 (2014) 24–29
Table 1
Square wave voltammetric determination of 8-hydroxyguanine in control urine samples
using AuNP-SWCNT/EPPGE.
Fig. 5. Standard addition plot of ip versus spiked concentration of 8-OH-Gua in control
urine samples using AuNP-SWCNT/EPPGE.
control urine samples exhibited up to five peaks in the range of −0.2 to
+0.4 V as shown in Fig. 6. The peak corresponding to oxidation of 8-OHGua was observed at 0.22 V (Peak 4). The prominent peaks are due to
major urinary metabolites, such as ascorbic acid (Peak 1), dopamine
(Peak 2) and uric acid (Peak 3), and remaining small peaks may be due
to the trace amount of other products formed in DNA damage. Hence,
the concentration of 8-OH-Gua in control urine samples was determined
by using standard addition method. The recovery studies were
performed by adding the known concentrations of standard 8-OH-Gua
in urine samples of control. The standard addition plot of control urine
sample 1 is shown in Fig. 5 and the linear relation can be expressed as
ip (μA) = 4.092 [8-OH-Gua] + 6.828 with a correlation coefficient of
0.993. The concentration of 8-OH-Gua after considering dilution factor
(60 times) in control urine sample 1 is found as ~ 0.10 μM. For control
urine samples 2 and 3, the concentration of 8-OH-Gua was found as
0.11 and 0.10 μM. The results observed are listed in Table 1 and show
that recoveries varied in the range from 100.0 to 103.3%. The recovery
studies show that the accuracy of the proposed voltammetric sensor is
good and thus the method can be recommended for monitoring DNA
damage and consecutively diabetes risk biomarkers.
Then square wave voltammograms of urine samples of three diabetic patients were recorded under the same conditions and parameters,
which were used during calibration curve studies. Fig. 6 shows a typical
square wave voltammogram of urine sample of diabetic patient 1 and
consists of oxidation peak at ~ 0.221 V in addition to other peaks. The
Fig. 6. A comparison of square wave voltammograms of (i) phosphate buffer solution
(…..), and (ii) urine sample of diabetic patient 1 (—) at pH = 7.2 using AuNP-SWCNT/
EPPGE.
Spiked amount
(nM)
Detected amount⁎
(nM)
Actual amount
(nM)
Recovery (%)
Sample 1
0.00
0.50
1.00
1.50
1.66
2.16
3.18
4.64
1.66
1.66
1.68
1.64
–
103.32
101.12
100.55
Sample 2
0.00
0.50
1.00
1.50
1.60
2.12
3. 16
4.68
1.60
1.62
1.66
1.68
–
103.24
101.11
100.56
Sample 3
0.00
0.50
1.00
1.50
1.58
2.10
3.22
4.58
1.58
1.60
1.72
1.58
–
103.20
101.15
100.00
⁎ The detected amount represents the total amount of 8-OH-Gua present in the solution.
Actual amount = detected amount − spiked amount.
peak at Ep ~0.221 V is assigned to the oxidation of 8-OH-Gua as the Ep
is similar to the one observed in the standard solution of 8-OH-Gua as
shown in Fig. 3 under identical conditions. This confirmed that the peak
at +0.221 V is due to the oxidation of 8-OH-Gua. The other peaks at
−0.08 (ascorbic acid), +0.07 (dopamine), +0.11 (uric acid), +0.18
and +0.31 were also observed and were essentially similar to control
urine samples. No attempt was made to identify peaks at +0.18 and
+0.31 V as they were not interfering in the determination. The peak at
~0.221 V was further confirmed by spiking of known amount of standard
solution of 8-OH-Gua. Moreover, other urinary metabolites viz. uric acid,
ascorbic acid and dopamine etc. are oxidized at potentials significantly
apart from the oxidation potential of 8-OH-Gua. The actual concentrations
of 8-OH-Gua in urine samples of diabetic patients were then determined
using calibration curve and dilution factor. The concentrations of 8-OHGua in urine samples of diabetic patients 1, 2 and 3 were found as 0.30,
0.28 and 0.32 μM, respectively.
Fig. 7. A comparison of observed concentration of 8-OH-Gua in urine samples of controls
(A) and diabetic patients (B) at pH = 7.2 using AuNP-SWCNT/EPPGE.
S. Bishnoi et al. / Bioelectrochemistry 99 (2014) 24–29
The comparison of concentrations of 8-OH-Gua in urine samples of
normal and diabetic patients determined by using proposed sensor is
presented in column graph in Fig. 7. The column graph clearly indicates
that the concentration of 8-OH-Gua in diabetic patients is almost 3-fold
higher as compared to the control subjects. The results obtained are also
in agreement with earlier report for 8-OH-Gua using HPLC [21].
4. Conclusion
Diabetes is a serious and growing health care problem worldwide
and is likely to be associated with oxidative DNA damage resulting in
increased level of 8-OH-Gua in biological fluids [3]. In the present studies efforts have been made for the development of a simple, fast and
reliable method for the analysis of oxidative DNA damage products in
body fluids. A AuNP-SWCNT/EPPGE sensor has been utilized for the determination of 8-OH-Gua in urine samples of diabetic patients based on
its oxidation. The proposed sensor offers several benefits over conventional diagnostic analysis including simplicity of use, specificity for the
target analyte, rapidity, low-cost and sensitivity. The proposed sensor
showed significant electrocatalytic activity in voltammetric response
of 8-OH-Gua in terms of large peak currents and lower peak potential.
The advantage of the proposed sensor is that there is no need of complex pretreatment or pre-purification steps as required in the methods
reported in earlier literature [22–24] for urine samples.
Literature survey reveals that there is no previous report on using
gold nanoparticles–nanotubes film for the diagnosis of diabetes. As, in
our previous work [25] it has been observed that SWCNT/EPPGE is
more sensitive as compared to bare EPPGE, hence, in the present work
an additional catalytic effect of gold nanoparticles along with nanotubes
is determined. Moreover, 8-OH-Gua existing with uric acid and ascorbic
acid in a homogeneous solution can also be determined simultaneously
using proposed sensor. It indicates specificity of the sensor for target analyte; as our aim is to utilize the sensor in biological fluids having matrix
complexity. Our findings suggest that measuring urinary 8-OH-Gua is a
novel convenient method for evaluating oxidative DNA damage and in
turn diagnosis of diabetes. Good sensitivity, reproducibility and stability
of the proposed sensor together with synergetic effect of nanogold and
SWCNT made itself promising for further development in the field of
electrochemical sensors for trends and challenges envisaged for the
near future. There is also a need of prospective studies to establish
whether increased concentration of 8-OH-Gua in urine is related to
‘life style diseases’ viz. lack of exercise, morning walk and physical
workout. The studies on this aspect are in progress and will be reported
later.
Acknowledgments
One of the authors, (SB) is thankful to Dr. S.S. Dhindsa, Dean R&D
Cell, VIT Campus, Jaipur for helpful discussions. Thanks are also due to
the Korean Federation of Science and Technology, South Korea for
awarding Brain Pool Fellowship to RNG. Financial assistance for this
work was provided by the Department of Biotechnology, New Delhi
vide grant no. BT/PR13954/MED/32/143/2010. The research is also partially supported by Basic Science Research Program through National
Research Foundation of Korea (Grant number: 20100029128). Thanks
are also due to Mr. Pankaj Gupta and Ms. Rosy, JRF, Chemistry Department, IIT Roorkee for their help in biological samples analysis.
29
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