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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 References [1] M. Grotzke, R.E. Jones, Acute and chronic complications of diabetes endocrine secrets, in: Michael T. McDermott (Ed.), Endocrine Secrets, 6th edition, Elsevier Health Science, 2013, pp. 16–27. 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