Branched Platinum Nanostructures on Reduced Graphene: An excellent Transducer for Nonenzymatic Sensing of Hydrogen Peroxide and Biosensing of Xanthine

Accepted Manuscript Title: Branched Platinum Nanostructures on Reduced Graphene: An excellent Transducer for Nonenzymatic Sensing of Hydrogen Peroxide and Biosensing of Xanthine Author: Tapan Kumar Behera Subash Chandra Sahu Biswarup Satpati Bamaprasad Bag Kali Sanjay Bikash Kumar Jena PII: DOI: Reference:

S0013-4686(16)30584-9 http://dx.doi.org/doi:10.1016/j.electacta.2016.03.046 EA 26876

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Electrochimica Acta

Received date: Revised date: Accepted date:

18-1-2016 5-3-2016 8-3-2016

Please cite this article as: Tapan Kumar Behera, Subash Chandra Sahu, Biswarup Satpati, Bamaprasad Bag, Kali Sanjay, Bikash Kumar Jena, Branched Platinum Nanostructures on Reduced Graphene: An excellent Transducer for Nonenzymatic Sensing of Hydrogen Peroxide and Biosensing of Xanthine, Electrochimica Acta http://dx.doi.org/10.1016/j.electacta.2016.03.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Branched Platinum Nanostructures on Reduced Graphene: An excellent Transducer for Nonenzymatic Sensing of Hydrogen Peroxide and Biosensing of Xanthine.

Tapan Kumar Behera,a Subash Chandra Sahu,a Biswarup Satpati,b Bamaprasad Bag,a Kali Sanjaya and Bikash Kumar Jenaa*1

a

CSIR-Institute of Minerals and Materials Technology, Bhubaneswar-751013, India.

Email:[email protected] b

Saha Institute of Nuclear Physics, Kolkata-700 064, India.

1

ISE member

*

Corresponding author

1

Abstract Here, a facile and highly sensitive platform has been developed for synthesizing branched Pt nanostructures on graphene. Graphene support has been employed to enhance the performance of platinum nanostructures (PtNs) in electrochemical sensing/biosensing. The graphene supported PtNs (GPtNs) modified electrode efficiently oxidises the H2O2 at a lower potential in neutral phosphate buffer solution without employing the enzymes or redox mediators. The GPtNs electrode shows high sensitivity and can detect very low nanomolar concentrations (1 nM) of H2O2. It can linearly sense H2O2 from 0-0.32 mM, and the sensitivity of the sensor was estimated to be 811.26 μAmM-1cm-2. The sensor shows excellent reproducibility, long time storage and operational stability. The sensor successfully estimates the H2O2 concentration in the real sample (rainwater) for practical analysis. The interesting response of the sensor towards H2O2 employed for fabrication of amperometric oxidase based biosensor for sensing of xanthine. The biosensor is highly stable and showed fast response in biocatalytic sensing of xanthine.

Key words: Graphene, Pt nanostructures, Hydrogen peroxide, Sensor, Xanthine biosensor

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1. Introduction Hydrogen peroxide is a vital oxidising agent and found to be the enzymatic product of various oxidase based enzymes [1]. It is used as an essential chemical in various potential fields such as the clinic, pharmaceutics, food processing, paper bleaching Industry and so forth [2,3]. It has a great impact on the biological processes of living organisms. H2O2 solution up to permissible percentage is biologically safe but at higher concentration, it is harmful to the primary constituents of food such as vitamins and fatty acid [4,5]. The excess of H2O2 secretion is a threat to the life of living organisms and associated with diseases like cancer, Parkinson’s disease etc [1]. Therefore, quantitative detection of H2O2 with high accuracy is of great importance. Several methods such as fluorometry[6], chemiluminescence[7], spectrophotometer[8] and titrimetry[9] have been applied for detection of H2O2. However, these methods involve the use of expensive instruments, time-consuming processes and complicated strategies. Therefore, the development of efficient, cost effective and reliable method that can provide high sensitivity, facile operation and quick response to the H2O2 molecule is highly desirable. Among the various methods, the electrochemical method has been attracting owing to simplicity, rapid sensitivity and cost effectiveness [10-14]. Nanoparticles of noble metals have been attracting for electrochemical detection of H2O2 due to their high surface to volume ratio and high electrocatalytic activity [15-19]. Especially, Pt nanoparticle possesses excellent catalytic and electrocatalytic activity due to its unique affinity to the hydrogen, oxygen and various other molecules [20-22]. Interestingly, the electrocatalytic and catalytic properties can be tuned by varying size, shape and morphology of Pt nanoparticles. Among the various shape, nanostructures with branched morphology such as nanoflower, nanodendrites, tri- and tetra-pods have gained

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special interest because of large surface to volume ratio, high degree of anisotropy, very reactive edges and sharp tips [23]. For example, Mehdizadeh et al. reported the enhanced activity of hierarchical Pt nanoflower for non-enzymatic detection of H2O2 [24]. Although, great efforts have been dedicated to the development of advanced Pt and Ptbased nanostructures to improve the electrochemical performance as well as to minimise the usage of Pt, the metal nanostructures have relatively low electrocatalytic activity and short range durability. The nanoparticles get agglomerate to microstructures during the electrochemical operation that results in the decrease in their catalytic performance. Therefore, great efforts have been made to enhance the catalytic performance of metal nanostructures by loading on the surface of conducting supports such as carbon black, carbon nanotube, activated carbon fibre and so forth [25]. Recently, the newly discovered graphene has been attracting enormous interests in the field of electrochemistry. Because, it has extraordinary properties like large surface to volume ratio, super electrical conductivity and can be applied in a wide range of the potential window [26-28]. The graphene support provides the large surface area for accommodation and well dispersion of nanoparticles, avoids their aggregation and fastens the electron transfer process [29]. Therefore, significant attempts have been made to the development of graphene-metal nanoparticle as catalysts for electrochemical sensing applications [30]. The noble properties of graphene and unique electrocatalytic activity of platinum have made the graphene-Pt nanostructures as a suitable material for the development of enzymatic biosensor. Xanthine (XA), a precursor of uric acid (UA) is known to be involved in many clinically significant diseases which are genetically related, such as xanthinuria and Lesch-Nyhan syndrome [31-33]. Xanthine is also used for estimating freshness of fish and meat [34-36]. During the conversion of XA to UA by

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Xanthine Oxidase (XOD), superoxide anions are produced, which are reactive species in the body [37,38]. Hence, sensitive and selective detection of XA is necessary. In this article, graphene supported unique branched Pt nanostructures based biosensor has been developed for non-enzymatic detection of H2O2 and fabrication of amperometric oxidase based biosensor for biosensing of xanthine. 2. Experimental Section 2. 1. Materials required Graphite powder (8-10 µM), Potassium permanganate (KMnO4, ≥ 99%), Hydrogen peroxide (H2O2, 30%), Borohydride (NaBH4, 98%) and Chloro platinic hydrate (H2PtCl6. xH2O, ≥99.9%) were obtained from Sigma-Aldrich and used without further purification. Uric acid, Ascorbic acid, (+) β-D glucose, Dopamine, Potassium Ferrocyanide (K4[Fe (CN6)]), Potassium ferricyanide (K3[Fe (CN6)]), Hydrochloric Acid, Barium Chloride, Sodium hydroxide and Xanthine Oxidase were purchased from Himedia, India. All the solutions were prepared using de-ionised water (18 Ωm). 2.2. Instrumentation The morphology of GPtNs was studied with transmission electron microscopy (TEM) using FEI Tecnai G2 20 and F30 ST microscope operated at a voltage of 200 kV and 300 kV respectively. 2 μl of as-prepared nanocomposite was drop casted on carbon coated copper grid for TEM analysis. The structural aspects of the as-synthesized nanostructures were studied by X-ray Diffraction using X’pert PRO pan analytica with Ni filtered Cu Kα ( λ = 1.54 Å ) radiation, Fourier transmission infra-red analysis (Perkin Elmer SPECTRUM GX), Raman Spectra (Reni Shaw in Via Raman microscope) and UVvisible spectroscopy (Schimadzu UV-2600). All the electrochemical measurements were recorded by CHI-760D electrochemical workstation by using two-compartment threeelectrode cell, glassy carbon electrode as working electrode, Pt wire electrode as counter

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electrode and Ag/AgCl (3M KCl) as the reference electrode. Electrochemical impedance spectroscopy (EIS) measurement was carried out in the frequency range from 10 MHz to 100 kHz. 2.3. Synthesis of graphene oxide (GO) and Graphene–Pt nanocomposites (GPtNs) Modified Hummer’s method was employed for the synthesis of GO as described in our earlier report [25]. In brief, 1g of graphite powder was slowly mixed with 25 ml of conc. H2SO4 in a conical flask. 3.5 g of KMnO4 was slowly added to the mixture solution under a stirring condition in an ice-bath. The mixture was left for stirring for 2 h in a water-bath at room temperature. After that, the solution was diluted with 50 ml of water by reducing the temperature with ice-bath. Then, the sufficient amount of 30% hydrogen peroxide was added until the evolution of gas ceases. The solution was centrifuged and washed with copious amount of 0.1 M HCl followed by de-ionised water until the complete removal of sulphate ion occurs. The GO solution was dried and stored in a desiccator for further use. The synthesis of GPtNs was carried out according to our earlier report [29]. In brief, 1 mg of GO was well dispersed in 10 ml water and 0.054 g of β-Dglucose was added followed by 80 μl of 25 mM Chloroplatinic acid and pH of the solution was adjusted to 7.7. To this mixture, 186 μl of 50 mM NaBH4 was added for insitu reduction of Pt (IV) and graphene oxide in a stirring condition for 30 minutes. The colour of the solution turns from yellowish brown to deep black within the reaction period of 10 minutes. For comparison study, the reduced graphene oxides (RGO) were synthesized by similar reduction condition without using the platinum precursor. 2.4. Fabrication of GPtNs modified electrode and Oxidase enzyme biosensor The glassy carbon (GC) electrodes were polished with the aqueous slurry of 0.05 µm of α-Al2O3 powder and sonicated with de-ionised water. The process was repeated to get the mirror finish surface. The well cleaned GC electrodes were dried in an argon

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atmosphere for modification with the sample. A mixture of GPtNs with 5% nafion was prepared and drop casted onto the GC electrode followed by vacuum drying for prior use. Hereafter, the GPtNs modified GC electrode is referred as GPtNs. A well cleaned GC electrode was used for fabrication of amperometric Xanthine oxidase (XOD) enzyme biosensor. A composite solution of 10 mg XOD and 1 mg GPtNs hybrid material was prepared by mixing with 5 µL of nafion solution in 0.1M of PBS (pH 7.2). The GCE was modified with the enzyme mixture solution and dried at 4° C for 12 h. Hereafter, the GPtNs and XOD modified biosensor is referred GPtNs/XOD. 3. Results and Discussion 3. 1. Characterization of graphene supported PtNs The GO was successful synthesized and characterized with different techniques [29]. The graphene supported PtNs were carried out by one step simultaneous reduction of GO and Pt (IV). The morphology of graphene and structure of PtNs on graphene of graphene supported PtNs (GPtNs) were analysed by high-resolution TEM measurement (Fig.1). From Fig.1A it can be seen that the graphene sheets are highly transparent and the PtNs are well dispersed on the transparent surface of graphene. The PtNs are grown in irregularly branched structures and well dispersed on graphene surface [29]. The branching structure of PtNs is irregular in shape and differs from particle to particle (Fig.1B). The HRTEM image of one PtNs structure is represented in the inset of Fig.1B. The scanning transmission electron microscopy high angle annular dark-field (STEMHAADF) observation also confirms that the branched PtNs are on the surface of thin layer graphene support (Fig. S1A, Supporting Information). The composition of GPtNs was examined with energy-dispersive X-ray (EDX) spectroscopy analysis which supports the existence of C, Pt (Fig. S1B). The Cu signal in EDX spectrum is due to copper grid used for TEM study.

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The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) technique were applied to measure the kinetics of electron transfer in bare glassy carbon electrode(GCE), RGO and GPtNs modified electrodes using Fe(CN)63-/4- redox couple. Fig. 2A represents their CV response in 0.1M PBS at a sweep rate of 10 mVs-1. A pair of well-defined redox peaks for GPtNs with ΔEp of 110 mV was observed, whereas the RGO and GCE showed sluggish electron transfer behaviour with a ΔEp of 162 mV and 200 mV respectively [39,40]. The enhanced electron transfer capacity of GPtNs can be ascribed to the presence of PtNs on the graphene surface. The EIS measurement was used to study the electron-transfer kinetics at the GPtNs and RGO modified electrodes and GCE (Fig. 2B). The GPtNs modified electrode showed a very lower Rct compared to RGO modified electrode and GCE. The result of EIS measurement is in good agreement with the CV response. The result confirmed that the synergetic effect of graphene and PtNs enhanced the electron transfer rate of GPtNs significantly and can be utilised for the electrocatalytic application. The CV technique was employed to measure the electrochemical active surface area of the PtNs of GPtNs modified electrode (Fig. S2). A characteristic CV response of a zero valent state of Pt nanoparticles was observed [29]. The electrochemical accessible surface area (ECSA) of GPtNs electrode was calculated by integrating the charge consumed during hydrogen adsorption (QH) on the surface of nanoparticles. The ECSA value of GPtNs was estimated to be 1.42 cm2 with reference to the standard value 210 μC/cm2 [29]. 3.2. Electrochemical study of Hydrogen peroxide oxidation Attracted by the excellent electron tunnelling properties of GPtNs, it was utilised for the electrocatalytic oxidation of a vital analyte of interest, H2O2. Fig. 3 represents CV towards the oxidation of H2O2 on GPtNs. A well-defined oxidation peak was observed at

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a lower potential of +0.37 V (vs. Ag/AgCl) for GPtNs electrode. For comparison, the CV response on RGO modified electrode was recorded. The oxidation of H2O2 on RGO modified electrode occurred at a higher potential of +0.82 V and the CV response is illdefined as compared to GPtNs (Fig. S3). This result suggests that the GPtNs efficiently catalyses the electrooxidation of H2O2. Further, the electrocatalytic performance of GPtNs has been compared with that of PtNs by cyclic voltammetric technique. Figure S4 shows the CV profiles of GPtNs and PtNs electrodes in 0.1 M PBS solution containing 1 mM H2O2. It can be observed that GPtNs shows a sharp oxidation peak and higher current towards H2O2 as compared to PtNs. The PtNs electrode catalyses the oxidation of H2O2 at lower potential but the oxidation current is significantly small. This indicates that GPtNs effectively catalyses the oxidation of H2O2.The CV response on GPtNs was recorded at different sweep rates (Fig. S5). A slight shift of peak potential (Ipa) to the positive side was observed on increasing the sweep rates (). The plot of Ipa vs. 1/2 is linear showing the oxidation of H2O2 on GPtNs is a diffusion controlled process. The CV response of GPtNs towards the successive addition of H2O2 at a fixed scan rate was recorded. A gradual increase in the current on increasing the concentration of H2O2 was observed (Fig. 4). The sensitivity of the electrode was estimated from the calibration plot of the CV response. It shows good sensitivity and stable response. The CV response at higher concentrate level was monitored to explore the performance of GPtNs in a wide range of concentration. It shows a linear increasing response up to 5 mM and get saturated further addition of H2O2 (Fig. S6). For practical application, the constant potential amperometry technique is used to evaluate the real performance of the sensor. Here, the amperometric response of GPtNs electrode was evaluated on increasing the concentration of H2O2. Fig. 5 manifests the amperometric signal obtained towards the detection of H2O2. The electrode was polarised

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at its oxidation potential and successive concentration of H2O2 was injected into the electrolyte at a particular interval of time. Interestingly, the GPtNs electrode shows fast response and stable current profile upon every injection of the analyte. The current response was linearly increased on increasing the concentration of H2O2 and the sensitivity of the electrode was calculated to be 811.26 μAmM-1cm-2. The limit of detection (LOD) of the present electrode was also evaluated from the amperometric response at very low concentration. It could detect very low concentration of H2O2 as low as 1 nM concentrations (S/N>3). Further, the i-t response of GPtNs electrode has been carried out to compare with the PtNs modified electrode towards the oxidation of H2O2 (5nM) in 0.1 M PBS (Fig. S7).The amperometric current for GPtNs electrode was found to be higher for oxidation of H2O2 compared to that of PtNs modified electrode under similar experimental condition. Thus, it indicates that the graphene support plays vital role in the performance of GPtNs electrode towards the oxidation of H2O2. Interestingly, the graphene supported branched Pt nanostructure shows very sensitive towards H2O2 and can monitor the concentration at nanomolar level without using any enzymes and redox mediators. This may be attributed to the synergistic effect of both graphene and the branched Pt nanostructures in excellent electrocatalytic activity and high electron tunnelling capacity towards the electrooxidation of H2O2. It is worth to compare the present observation with the recent graphene based literature on H2O2 sensing is shown in Table 1(Fig. S8) [41-52]. It reveals the excellent sensitivity and low detection capability of the present sensor. This can be attributed to the unique structure of PtNs and the synergistic role with graphene for excellent activity towards H2O2. Further, the i-t response of GPtNs electrode has been studied at the higher concentration of the analyte to optimize the concentration of H2O2 (Fig. S9A). It can be observed that the sensor shows linear response towards H2O2 in the range 0-0.32 mM (Fig.S9B) and further increase in

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concentration of the analytes decreases the performance. The interference test was also performed to check the selectivity of GPtNs electrode towards H2O2. A certain concentration of ascorbic acid, uric acid, dopamine and glucose were injected after a stable amperometric response obtained from H2O2 oxidation. The result (Fig.S10) convincingly indicates that there was no significant current change occurs for the interfering analytes. 3.3. Stability study of the electrode The relative longevity and durability of the sensors are essential for continuous monitoring of the analytes. Therefore, the operational stability of the present GPtNs electrode was evaluated from the amperometric response of the polarised electrode containing 10 nM of H2O2. The amperometric response was quite stable over a long duration of time (Fig. S11) and supports its practical applications for monitoring the concentration of analytes. Then, the storage stability was employed to know the reproducibility and validation for the long-term use of the electrode. The amperometric response of the GPtNs modified electrode was recorded towards a particular concentration of H2O2 in every day after 24 h interval and stored in PBS. The initial current response of the electrode on first day of the experiment was compared with the subsequent days. The electrode shows a stable response up to 14 days of the continuous experiments. The operational and storage stability of the present electrode towards H2O2 demonstrates its worth for practical application. 3.4. Real Sample analysis (H2O2 in Rainwater) The practical utility of the present sensor was validated by measuring the concentration of H2O2 in a real sample (rainwater). The rainwater samples were collected from the rooftop of our laboratory during the month of April before monsoon and filtered by 0.45 µM filter paper to remove small amount of insoluble dry deposition. Two

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samples were taken and three repeated experiments on each sample were recorded. The rainwater sample was injected into the PBS solution and analysed by constant potential amperometric technique. The current response of the rainwater samples showed a linear response along with the standard H2O2 concentrations injected into the solution during the measurement (Fig. S12). The recovery percentage of the spiked H2O2 sample was estimated to more than 100%. The concentration of the H2O2 present in the real sample (rainwater) was estimated to the range of 103-108 nm (Table 2, Fig. S13). It is important to note that the concentration of H2O2 varies from dawn to dusk, weather condition, air pollution and also the place of collection [53-57]. The GPtNs electrode proved its successful utilization for sensing of H2O2 in rainwater and can be applied for practical application. 3.5. Biosensing of Xanthine Attracted by the fast response, good selectivity and excellent stability of GPtNs electrode towards the oxidation of H2O2, it was utilised for the development of oxidase enzyme based amperometric biosensor. Generally, the oxidase enzyme converts the analytes in the presence of molecular O2 and the H2O2 is formed as the product of the enzymatic reaction. So the concentration of enzymatically generated H2O2 is directly proportional to the amount of analyte in solution. Hence, a sensor showing high sensitivity towards H2O2 can effectively monitor the concentration of the analyte of interest. In the present work, GPtNs electrode was effectively applied for biosensing of xanthine using xanthine oxidase (XOD). The biosensor was fabricated by integrating the enzyme and GPtNs on the electrode platform and schematically presented in Fig. 6. The enzyme xanthine oxidase converts xanthine to uric acid and generates hydrogen peroxide enzymatically [31-33]. The GPtNs-enzyme modified electrode was first characterised by cyclic voltammetric techniques in 0.1M PBS (Fig. S14). It can be seen from the cyclic voltammograms of the enzyme modified electrode that there is an increase of oxidation 12

current observed in the presence of xanthine. However, it is not showing sharp oxidation peak the cyclic voltammograms. Therefore, biosensing detection of xanthine by the enzyme electrode was monitored with the amperometric i-t technique. The amperometric potential hold at +0.45 V could oxidise to the bi-product H2O2 produced from Xanthine (Fig. 7A).The amperometric response is stable. A linear increase in current response was observed at the lower concentration of xanthine and got saturated at the higher concentration as anticipated for an enzymatic Michaelis–Menten kinetics (Fig. 7B) [58]. Interestingly, the as-designed xanthine biosensor could detect very low concentration of 0.1 μM xanthine (S/N>3) which is worth comparison to the reported literatures (Fig. S15). The amperometric response was diagnosed from the Lineweaver-Burk plot of 1/icat vs. 1/Cs to deduce the apparent Michaelis-Menten constant (Kmapp). The Kmapp was estimated to be 1.21 mM. Further, the interference effect of substances like ascorbic acid, glucose, uric acid and dopamine on the i-t response of the xanthine biosensor was performed in 0.1 M PBS(Fig. S16). It can be observed that no such appreciable interfering effect on the amperometric response of the xanthine biosensor was observed. From the above data, it can be concluded that, the analytical performance of the GPtNs/XOD electrode provides an ideal environment for the xanthine biosensor and can be employed for practical applications. 3.6 Effect of Concentration of Enzyme The amperometric response of the xanthine biosensor depends on the concentration of XOD enzyme. Fig. S17 shows the analytical performance of the GPtNs/XOD at different concentration of enzyme loading. It can be seen that the amperometric current increases linearly with the higher loading of XOD. The maximum amperometric current was obtained at an enzyme loading of 5 mg. However, the further increase in the loading of enzyme decreases the analytical performance of the biosensor. We assume that the higher

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concentration of enzymes on the electrode surface may create the diffusion barrier for the enzymatically generated product to the surface of the electrode. 3.7 Effect of concentration of GPtNs The amount of catalyst loading on the electrode surface plays a vital role for the efficiency of electrochemical devices. Fig. S18 shows the amperometric response of the GPtNs/XOD at different loading amount of GPtNs. It can be seen that the amperometric current increases with the loading of higher amount of GPtNs catalyst in the range of 0.52 mg. However, the amperometric current of GPtNs electrode towards H2O2 oxidation gets saturated on further increase in the amount of catalyst loading. 3.8 Effect of concentration of Nafion The concentration of nafion is also playing a vital role on the electrochemical performance of the biosensor. The interaction of the electrocatalysts on the biosensor with the electrolyte solution depends upon the amount of nafion mixed with it. Fig. S19 shows the amperometric response of the biosensor at different loading of nafion. It can be observed that the amperometric current gave the good response with increase of nafion content in the range of 1-5 μl. But the catalytic current of the amperometric response decreases significantly at higher concentration of nafion. Therefore, all the experiments were carried out at the optimum nafion content of 5 μl. 4. Conclusion In summary, graphene supported branched Pt nanostructure (GPtNs) showed a very good response for the detection of hydrogen peroxide at the nanomolar range. At a favourable lower potential, hydrogen peroxide was oxidised in comparison to other interference analytes. The successful detection of hydrogen peroxide in rainwater validated its practical application. The interesting sensing performance of GPtNs electrode towards H2O2 was effectively applied for biosensing of xanthine using xanthine oxidase (XOD). The combining activity of the PtNs and the XOD was utilised to 14

fabricate the integrated biosensor for biosensing xanthine. The integrated biosensor successfully detects the xanthine and can be employed for practical applications.

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Graphene-induced

Pd

nanodendrites:

A

high

performance

hybrid

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Figure caption:

Figure 1: (A) TEM image of GPtNs showing the graphene and PtNs [29]. Inset of (B) shows the HRTEM image of one single branched PtNs

Figure 2: (A) Cyclic voltammograms of GPtNs (a) and RGO (b) modified electrode and GCE (c) towards the Fe2+/Fe3+ redox couple in 0.1 M PBS. Scan rate: 10 mV/s and (B) Corresponding EIS data.

Figure 3:

Cyclic voltammograms of GPtNs modified electrode in absence (a) and

presence (b) of 1 mM H2O2 in 0.1 M PBS. Scan rate: 10 mV/s.

Figure 4: (A) Cyclic voltammetic response of GPtNs electrode for oxidation of H2O2 in 0.1 M PBS. Scan rate: 10 mVs-1. Each addition increased the concentration by 5 µM. (B) Corresponding calibration plot.

Figure 5: (A) Constant potential amperometry (i-t) responses of GPtNs electrode on successive injection of 5 nM of H2O2 into 0.1 M PBS. (B) Corresponding calibration plot. Inset of A shows the i-t response for 1 nM H2O2.

Figure 6: Scheme illustrating the biosensing of Xanthine on XOD-GPtNs integrated platform. Figure 7: (A) Amperometric i-t response for biosensing of xanthine on XOD-GPtNs integrated biosensor. (B) Corresponding calibration plot.

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