An efficient electrode for simultaneous determination of guanine and adenine using nano-sized lead telluride with graphene

Susmita Pradhan a, Sudip Biswas b, Dipak K. Das c, Radhaballabh Bhar a, Rajib Bandyopadhyay b and Panchanan Pramanik *c
aDepartment of Instrumentation Science, Jadavpur University, Kolkata-700032, India
bDepartment of Instrumentation and Electronics Engineering, Jadavpur University, Salt Lake Campus, Sector-III, Kolkata-700098, India
cDepartment of Chemistry and Nanoscience, GLA University, Mathura-281406, India. E-mail: pramanik1946@gmail.com

Received 8th September 2017 , Accepted 9th November 2017

First published on 22nd November 2017


Herein, lead telluride (PbTe) nanocrystals were chemically synthesized at room temperature via reduction of homogeneous mixtures of tartrate complexes of Pb2+ and Te4+ with sodium borohydride. Graphene (GR) was synthesized through thermal reduction of graphene oxide (GO) in the presence of Zn dust. The structure, phase, and morphology of the synthesized PbTe and GR were characterized by XRD, FESEM, and EDX. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to study the electro-oxidation behaviors of guanine and adenine molecules on the surface of the composite of lead telluride and graphene-modified graphite paste electrode (PbTe/GRGP). The voltammetric responses revealed that the electrocatalytic properties of these biomolecules (GU and AD) were significantly improved after incorporation of both PbTe nanoparticles and graphene into graphite powder in comparison with those of bare graphite (bare GP) and the graphene graphite paste electrode (GR/GP). CV studies indicate well defined and distinct irreversible oxidation peaks for both biomolecules upon treatment of the PbTe/GRGP electrode with a solution of biomolecules in phosphate buffer (pH 5). DPV measurements illustrated the appearance of two well defined oxidation peaks of GU and AD with a peak potential separation of 289 mV. Under optimized experimental conditions, the modified electrode exhibited wider linear ranges of 4–140 μM and 3–50 μM with the detection limits of 80 nM (S/N = 3) and 70 nM for GU and AD, respectively. Furthermore, the modified electrode was successfully used in spiked human urine and serum samples to determine the GU and AD contents for real time applications.


1. Introduction

Guanine (GU) and adenine (AD) are two important purine bases of deoxyribonucleic acid, which play crucial roles in the biological synthesis of proteins, cerebral circulation, and cell signaling and even as storage units for biological heredity information.1 Abnormal changes in GU and AD in nucleic acids create deficiencies and mutations in the immune system, which may cause various diseases including carcinoma, epilepsy, Parkinson's disease, AIDS, and liver diseases.2 Thus, simultaneous determination of these purine bases has become extremely important for clinical diagnosis. Various techniques such as fluorescence,3 high performance liquid chromatography,4 electrophoresis,5 and surface enhanced Raman scattering6 have been developed for the detection and measurement of these two purine bases. However, these methods are expensive and time consuming. Moreover, skilled operators are required to handle these large and complicated instruments. Hence, electrochemical techniques7–9 have been preferred as a perfect alternative to the above mentioned techniques due to their advantages such as high sensitivity, low time consumption, excellent selectivity, fast response, and simple operation.10–13 However, electrochemical methods have some shortcomings such as overlapping of oxidation peaks and a slow rate of electron transfer. These problems are mitigated by modifying the surface of the electrode with suitable materials. For this reason, the surface of the electrode has been modified chemically to investigate the electrochemical oxidation of these biomolecules. To date, literature reports indicate that many materials such as polyaniline/MnO2,14 graphene–Nafion composite films,15 MWCNT decorated with NiFe2O4 nanoparticles,16 3D porous nitrogen-doped graphene,17 Ag nanoparticle–polydopamine@graphene composites,18 β-cyclodextrin-based composites,19 MWCNT–Fe3O4@PDA–Ag nanocomposites,20 porous silicon-supported Pt–Pd alloys,21 graphene–COOH,22 graphene/platinum nanoparticles,23 carbon-supported NiCoO2 nanoparticles,24 and TiO2–graphene nanocomposites25 have been developed for this purpose. However, these modified electrodes have certain limitations such as low stability, poor reproducibility, narrow linear range, high background current, and complicated material preparation; therefore, the development of a novel, cheap, and stable electrode modifier material with high selectivity and sensitivity is very challenging. Recently, graphene-based electrochemical sensors have attracted significant attention in the field of electrochemistry for the detection of biomolecules due to some unique properties, such as excellent electrochemical transport, high conductivity, and strong adsorption of organic molecules, of graphene.26,27 Moreover, the two-dimensional sp2 hybridized carbon sheet of graphene provides large surface area, which makes graphene attractive as a supporting material for different nanocatalyst materials.28 Among these materials, nanoscale materials are being extensively studied because of their unique properties such as high surface area, high conductivity and electrocatalytic activity, sufficient sensitivity, and simple synthetic procedure.29 Recently, semiconductor nanocrystals have attracted significant attention from the scientific community due to the quantum confinement effect and their various applications in different fields such as in electronic and optical devices, electrochemical sensors, etc.30–33

Basically, metal chalcogenides are synthesized by the direct combination of precursor materials in an evacuated silica tube for several hours at high temperatures.34 This method is not appropriate for the synthesis of metal chalcogenide nanoparticles. To overcome this problem, various alternative approaches such as hydrothermal, solvothermal, hot injection method, etc. have been explored to synthesize chalcogenide nanoparticles.35 Gholamrezaei et al. have synthesized PbTe nanostructures through the hydrothermal method and used them as a photocatalyst in the degradation of organic pollutants.36 Ganguly and Brock have explored the sol–gel method of nanoparticle assembly for the synthesis of lead telluride aerogel and xerogels.37 James and coworkers have also synthesized lead telluride via the hot injection method in common organic solvents.38 However, these techniques require high temperature, high pressure, and large and sophisticated instruments and are costly as well. Moreover, due to the reaction conditions, contamination with impurities during the course of the reaction is possible. In this regard, the synthesis of lead telluride nanoparticles from a homogeneous solution of precursor materials via a clean efficient chemical route is always beneficial.

In this study, we have prepared nanoparticles of lead telluride through the chemical reduction of a homogeneous solution of tartrate complexes of lead salt and tellurium salt with sodium borohydride. Graphene was synthesized via thermal reduction of graphene oxide by Zn dust. The electrocatalytic properties of the two purine bases were investigated using an electrode of the composite of lead telluride nanoparticles with graphene. Although several studies have been reported on the determination of small molecules using graphene-based materials as electrode modifier agents, to the best of our knowledge, no studies have been reported on the use of a lead telluride and graphene-modified graphite paste electrode for the simultaneous determination of the above mentioned two purine bases. In addition, the analytical performance of the electrode was compared with previous reports, and it was found that our prepared electrode showed highest sensitivity.

2. Experimental

2.1 Materials

Lead acetate dihydrate [Pb(OAc)2·2H2O], tartaric acid (C4H4O6), 30% ammonia solution (NH3), sodium hydroxide (NaOH), sodium borohydride (NaBH4), and zinc dust were purchased from E. Merck, India. Graphite powder, tellurium dioxide (TeO2), guanine (C5H5N5O), and adenine (C5H5N5) were obtained from Sigma-Aldrich. All reagents were of analytical grade and used without further purification. All experimental studies were carried out using Millipore water (Resistance 18 MΩ).

2.2 Synthesis of PbTe nanoparticles

PbTe nanoparticles were synthesized according to our previously reported study.39 Briefly, 1 mmol lead acetate dihydrate was dissolved in 2 mmol of tartaric acid. The pH of the solution was maintained at 10 (solution 1). Similarly, 1 mmol TeO2 powder was dissolved in 2 mmol ammoniacal tartaric acid at pH = 10 (solution 2). Then, the solution 2 was poured into solution 1 with simultaneous addition of NaBH4. After the addition of 10 mmol NaBH4 solution to the mixture, black coloured PbTe nanoparticles were obtained, which were suspended in the solution. Finally, the products were centrifuged and washed with distilled water, followed by ethanol. Then, the as-prepared PbTe nanoparticles were dried in an oven at 70 °C under an inert atmosphere.

2.3 Synthesis of graphene

Graphene oxide (GO) was synthesized according to the modified Hummers method.40 Then, 500 mg of GO was mixed with 1 gram of Zn dust, and the mixture was ground well. The resulting mixture was carbonized at 950 °C for 1 h at the heating rate of 5 °C min−1 under the flow of N2 gas. Finally, the black powder of graphene (GR) was prepared, which was used for the modification of electrodes.

2.4 Preparation of electrodes

Graphite powder, synthesized GR, and PbTe were placed in a morter in a 2[thin space (1/6-em)]:[thin space (1/6-em)]3[thin space (1/6-em)]:[thin space (1/6-em)]5 (w/w) ratio. After this, 2–3 drops of paraffin oil were added to the mixture, and the mixture was then ground well until a uniform paste was obtained. The paste was put in a 2.5 mm inner diameter capillary glass tube, and it was then tightly packed by pressing with a metal rod. For electrical contact, a Pt wire was inserted from the backside of the capillary tube. Bare GP, GR/GP, and PbTe/GP electrodes were fabricated using the same procedure. The surfaces of the prepared electrodes were cleaned with 0.3 μM and 0.05 μM Al2O3 slurries, followed by rinsing with ethanol. Then, the electrodes were dried under a N2 atmosphere before conducting the experiments.

2.5 Characterization techniques

A Philips PW 1710 X-ray diffractometer (Eindhoven, the Netherlands) operated at 40 kV and 40 mA with CuKα radiation (λ = 1.5406 Å) was used to obtain X-ray diffraction (XRD) patterns of the as-prepared materials. A JEM 2100 transmission electron microscope and a JEOL JEM6700F field emission scanning electron microscope along with an Oxford energy-dispersive X-ray spectroscopy detector (EDX) were used to study the morphology, structure, and chemical composition of the synthesized materials. The TEM sample was prepared by dispersing the prepared PbTe in acetone, and then, a drop of the dispersed solution was put on the carbon-coated copper TEM grid. The selected-area electron diffraction (SAED) pattern was obtained at an accelerating voltage of 200 kV. The optical property of the as-prepared material was investigated using a Shimadzu UV-3600 UV-visible spectrometer. A Beckman Coulter SA3100 at 77 K was used for Brunauer–Emmett–Teller (BET) surface area analysis and nitrogen adsorption–desorption isotherms measurements. An Autolab Potentiostat/Galvanostat Model-101 (the Netherlands) was employed for all electrochemical studies using a Pt wire as the counter electrode, Ag/AgCl as the reference electrode, and the prepared PbTe/GRGP as the working electrode in a 0.1 M phosphate buffer solution (PBS) of pH 5. Electrochemical behaviors of the two biomolecules (GU and AD) were investigated by CV and DPV techniques at a scan rate of 100 mV s−1 and 50 mV s−1, respectively. Cyclic voltammograms of individual GU and AD were obtained from 0.6 V to 1.2 V and 0.6 V to 1.4 V, respectively. However, for the binary mixture of GU and AD, CV and DPV measurements were conducted in the voltage ranges from 0.6 V to 1.4 V and 0.7 V to 1.3 V, respectively. Human urine and serum samples were obtained from volunteers in our laboratory. The clinical studies of human urine and serum samples complied with relevant institutional and national guidelines (GCLP guidelines, ICMR 2008); the experiment was approved by the Department of Instrumentation Science, Jadavpur University, Kolkata, India, and all participants provided written informed consent.

3. Results and discussions

3.1 XRD study

Fig. 1a and b represent the X ray diffraction patterns of the synthesized PbTe and GR, respectively. For PbTe, the diffraction patterns are well matched with the standard JCPDS card no. 78-1905; this affirms the formation of the cubic phase of PbTe nanoparticles. The Debye–Scherrer method41 was used to calculate the crystallite size of the nanomaterial, and it was found to be ∼19.89 nm. The presence of two graphitic peaks at 2θ = 26.48° for the (002) plane and at 2θ = 43.33° for the (101) plane in Fig. 1b confirms the successful thermal reduction of GO to GR. No peak for the Zn species is found in the XRD pattern; this confirms its complete removal; this may be due to evaporation of metallic zinc at higher temperatures (>900 °C).42
image file: c7nj03308g-f1.tif
Fig. 1 X-ray diffraction patterns of the synthesized (a) PbTe nanopowder and (b) graphene.

3.2 FESEM & TEM measurements

To study the surface morphologies of the synthesized PbTe nanoparticles and graphene, FESEM studies were carried out, and the images are presented in Fig. 2a and b, respectively. In Fig. 2a, it can be clearly observed that the prepared PbTe nanoparticles are composed of fine nanocrystallites, and in Fig. 2b, it is also seen that the synthesized GR has a wrinkled thin nanoflake-like morphology with a well-defined porous structure. Furthermore, TEM and HRTEM measurements were carried out to obtain detailed information about the surface morphology and structural characteristics of the synthesized PbTe nanoparticles. Fig. 2c represents the TEM image of the as-prepared PbTe nanomaterial, indicating the formation of smaller fine crystallites with a spherical shape. The inset of Fig. 2c illustrates that the average particle size of the PbTe nanoparticles is 21.98 ± 3.29 nm, and this result is consistent with the particle size obtained from the XRD measurements. From the HRTEM image (Fig. S1, ESI), the calculated d spacing of the lattice fringes was found to be 0.32 nm and 0.22 nm, which corresponded to the (2 0 0) and (2 2 0) planes of the PbTe nanoparticles, respectively. This result is also in good agreement with the XRD results. The inset of Fig. S1 (ESI) displays the SAED pattern of the PbTe nanoparticles, which confirms that the calculated diffraction patterns are again well matched with the d values of the XRD patterns. The presence of Pb and Te peaks in the EDX spectrum (Fig. 2d) confirms the expected composition of the synthesized PbTe nanomaterial. In the EDX spectrum, the Pt peaks are observed due to the coating for FESEM analysis.
image file: c7nj03308g-f2.tif
Fig. 2 FESEM images of the synthesized (a) PbTe nanoparticles and (b) graphene; (c) TEM image of the synthesized PbTe nanopowder; the inset shows the particle size distribution graph; and (d) EDX spectrum of the as-prepared PbTe nanoparticles.

3.3 UV-vis spectroscopy

The optical properties of the as-prepared PbTe nanomaterial were studied using UV visible spectroscopy. The diffuse reflectance measurement was transformed into equivalent absorption coefficients using the Kubelka–Munk function.43 From the Tauc plot (Fig. S2, ESI), the band gap was calculated by extrapolating a straight line to the energy axis for zero absorption coefficient. The band gap value is found to be 1.58 eV, which is larger than that of the bulk PbTe material. A wider band gap value implies that the material is in the nano phase, and this is due to a stronger quantum confinement effect in the nanomaterials. This result is in good agreement with previous studies reported by Gholamrezaei et al.36 and Kungumadevi et al.,44 in which the obtained band gap values have been reported to be 3 eV and 1.64 eV, respectively.

3.4 BET analysis of GR

Fig. S3a and b (ESI) represent the BET N2 adsorption–desorption isotherm at 77 K and the corresponding BJH (Barrett–Joyner–Halenda) pore size distribution plot of the synthesized GR. The isotherms (Fig. S3a, ESI) illustrate reversible adsorption character, which is similar to the type-II isotherm curve, and the corresponding BET surface area is found to be 252 m2 g−1. This value is lower than the theoretical surface area (2630 m2 g−1) of the individual graphene sheet; this may be due to the multilayered structure of graphene, indicating the formation of micropores. The BJH pore diameter distribution curve (Fig. S3b, ESI) illustrates that the synthesized GR is mesoporous cum microporous in nature with a pore size distribution in the range from 1.8 nm to 7.4 nm. It has been reported that a pore diameter larger than 2 nm improves the diffusion of ions in an aqueous electrolyte and facilitates a faster mass transfer of biomolecules.45 Based on these results, the GR-modified electrode is expected to have excellent electrocatalytic properties due to its high surface area with a narrow pore diameter.

3.5 Electrochemical properties of the prepared bare GP, GR/GP, PbTe/GP, and PbTe/GRGP electrodes

To study the electrochemical properties of the prepared bare GP, GR/GP, PbTe/GP, and PbTe/GRGP electrodes, a standard [Fe(CN)6]4−/[Fe(CN)6]3− redox system was used as a reference. Fig. 3 shows a comparison between the cyclic voltammogramms of a 2 mM K4[Fe(CN)6] solution at bare GP, GR/GP, and PbTe/GRGP electrodes. From Fig. 3, it is observed that the peak separation (ΔEp) value at bare GP is 402 mV, whereas at GR/GP, PbTe/GP, and PbTe/GRGP electrodes, the ΔEp values are 191 mV, 182 mV, and 155 mV, respectively.
image file: c7nj03308g-f3.tif
Fig. 3 CV plots for a 2 mM [Fe(CN)6]4−/[Fe(CN)6]3− redox system at bare GP (black), GR/GP (blue), PbTe/GP (pink), and PbTe/GRGP (green) electrodes.

This result is in well accordance with the Velasco equation, which states that the rate of electron transfer is inversely proportional to the peak separation value. Thus, we can conclude that the electron transfer rate is in the order PbTe/GRGP > PbTe/GP > GR/GP > bare GP electrodes. Moreover, at the PbTe/GRGP electrode, the oxidation peak current was found to be 3.35-, 2.31-, and 1.54-times higher as compared to that at the bare GP, GR/GP, and PbTe/GP electrodes, respectively. This may be due to smaller particle size, large electroactive surface area, and faster electron transfer on the surface of the PbTe/GRGP electrode. The Randles–Sevcik equation46 was used for a reversible process to calculate the effective surface area of the bare GP, GR/GP, and PbTe/GRGP electrodes. According to the Randles–Sevcik equation,

Ip = (2.69 × 105)n3/2D1/2ν1/2AC
where Ip, n, ν, D, and C represent the anodic peak current, total no. of electrons transferred (1), scan rate (100 mV s−1), diffusion coefficient (6.5 × 10−6 cm2 s−1), and concentration of K4[Fe(CN)6], respectively. The calculated effective surface areas of the prepared bare GP, GR/GP, PbTe/GP, and PbTe/GRGP electrodes are 0.017, 0.024, 0.038, and 0.059 cm2, respectively.

3.6 Electrocatalytic behavior of the GU and AD molecules

Fig. 4 shows the comparative electrochemical study of individual GU and AD molecules as well as the mixture of GU and AD molecules at the bare GP, GR/GP, PbTe/GP, and PbTe/GRGP electrodes by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). In Fig. 4a and b, for all the electrodes, only the oxidation peaks of GU and AD are observed, indicating that both oxidation processes are irreversible in nature. It is observed from Fig. 4a that the oxidation peak potential of the GU molecule on the PbTe/GRGP electrode surface is shifted negatively to 17 mV, 15 mV, and 12 mV as compared to that on the bare GP, GR/GP, and PbTe/GP electrode surfaces, respectively. Moreover, the oxidation peak current at PbTe/GRGP is 41.61 μA, which is 2.9- and about 1.5-times higher than that of the bare GP and GR/GP electrodes, respectively. The voltammetric responses of the GU molecule at the GR/GP and PbTe/GP electrodes appear very similar; the only exception is the oxidation peak current of the GU molecule. At PbTe/GP, the oxidation peak current is 1.02-times higher than that at the GR/GP electrodes, and it is about 1.46-times higher at PbTe/GRGP as compared to that at the PbTe/GP electrode. For AD, at the PbTe/GRGP electrode surface, a sharp oxidation peak appears as compared to the cases of GR/GP and bare GP, which display broad peaks. However, at the PbTe/GP electrode, a sharp oxidation signal for the individual AD molecule is observed at 1233 mV, which is shifted positively to 27 mV as compared to the case of the PbTe/GRGP electrode. In addition, for PbTe/GRGP, the anodic peak current increases up to 55.34 μA, which is 1.37-, 1.5-, and 4.8-times higher than those of PbTe/GP, GR/GP, and bare GP electrodes.
image file: c7nj03308g-f4.tif
Fig. 4 CV plots of (a) 0.1 mM GU and (b) 0.1 mM AD at bare GP (orange), GRGP (blue), PbTe/GP (red), and PbTe/GRGP (green) electrodes in a 0.1 M phosphate buffer (pH 5); CVs of the mixtures of (c) 0.1 mM GU + 0.1 mM AD at bare GP (orange), GRGP (blue), PbTe/GP (red), and PbTe/GRGP (green) electrodes; DPV plot of a mixture of (d) blank buffer solution (brown line) and a mixture of 0.1 mM GU + 0.1 mM AD solutions (green line) in a 0.1 M phosphate buffer (pH 5) at the PbTe/GRGP electrode. Scan rates for CV and DPV are 100 mV s−1 and 50 mV s−1, respectively.

Fig. 4c shows the cyclic voltammogramms of a mixture of GU and AD molecules at different electrodes. It can be seen that at bare GP, GR/GP, and PbTe/GP electrodes, the anodic peaks of the GU and AD molecules are broad. However, for the PbTe/GRGP electrode, two well separated sharp oxidation peaks of GU and AD are observed at 937 mV and 1230 mV, respectively. Furthermore, at PbTe/GRGP, the peak current values of GU and AD are significantly enhanced as compared to the electrochemical responses of the bare GP and GR/GP electrodes. Based on these results, it is documented that PbTe/GRGP has better electrocatalytic activities towards the oxidation of GU and AD. A tentative mechanism for this excellent electrocatalytic activity at PbTe/GRGP may be due to the combination of both synthesized PbTe and graphene materials. Graphene has a high surface area of 252 m2 g−1 with a narrow pore diameter distribution between 1.8 nm and 7.4 nm and the average particle size of PbTe is 22 nm, which is essential for the mass transfer of biomolecules in an aqueous electrolyte.47 Both GU and AD have an aromatic ring, and graphene has a layered structure; thus, there is a possibility of π–π stacking between graphene and biomolecules resulting in an increase of interaction.48 Moreover, the smaller particle size of PbTe nanoparticles helps to increase the number of electroactive interaction sites more suitably to facilitate faster electron transfer kinetics between the surface of the electrode and the biomolecules. Thus, the combination of these two materials in PbTe/GRGP enhances the electronic conductivity as well as increases the adsorption property with biomolecule accessibility. All these parameters provide a suitable microenvironment for the electrode reactions to occur for these biomolecules.

To check the specificity of the peak of the biomolecules, we have investigated the electrochemical performance of the PbTe/GRGP electrode in the absence of GU and AD in 0.1 M PBS (pH = 5) in the potential range from 0.7 V to 1.3 V at a scan rate of 50 mV s−1 by DPV (Fig. 4d). It can be seen that there are no redox peaks in the absence of biomolecules; this indicates that PbTe/GRGP is non-electroactive in the selected potential window. In contrast, after the addition of GU and AD to the PBS (pH = 5) solution, PbTe/GRGP provides two well-separated redox peaks for GU and AD, respectively, with a sufficient peak potential separation of 289 mV, indicating that the two oxidation processes are independent and not interfering with each other when analyzed using a modified electrode.

3.7 Effect of pH and scan rate

The pH of the supporting electrolyte plays an important role in the electrochemical reaction; therefore, the effect of pH on the electrochemical behaviors of GU and AD was investigated in the pH range of 3–7 using PBS (pH = 5) as the supporting electrolyte. In Fig. 5a, it can be seen that for both biomolecules (GU and AD), the oxidation peak potentials are shifted negatively with an increase in the solution pH; this suggests that protons have participated in the electrode reaction processes.
image file: c7nj03308g-f5.tif
Fig. 5 Variation of (a) pH vs. peak potential and (b) pH vs. current plot in 0.1 mM GU and 0.1 mM AD at the PbTe/GRGP electrode, respectively.

The linear regression equations between the oxidation peak potentials of the biomolecules (GU and AD) and the pH of the solution are as follows:

 
EpGU (V) = −0.054 pH + 1.166 (R2 = 0.981)(1)
 
EpAD (V) = −0.055 pH + 1.458 (R2 = 0.970)(2)

From the abovementioned equations, it has been found that the calculated slope values for GU and AD are −0.054 and −0.055, respectively, which are very close to the theoretical value (−0.059) provided by Nernst.49 These results indicate that an equal number of electrons and protons participate in the electrode reactions. Thus, the electrocatalytic oxidation of GU and AD is a two-electron and a two-proton transfer process.50 From Fig. 5b, it is found that for both molecules, the highest peak current occurs at pH 5, and it decreases when the pH of the solution increases further. In addition to this, the peak current of AD was decreased at pH 4, and the peak current of GU was increased when the pH was over 6. These results are consistent with those reported in previous studies on the determination of these molecules.51,52 The exact reason for this phenomenon is unclear. This may be due to several reasons such as formation of various kinds of anionic, cationic, and dimeric species of analyte molecules, which may originate from the difference in the pKa values of GU (pKa = 3.46) and AD (pKa = 4.24), thereby resulting in mutual influence between them when they coexist in a solution as well as different adsorption capacity of analyte molecules on the electrode surface. However, a detailed study of this phenomenon is yet to be explored, and this will be the subject of our future research. Moreover, the maximum peak potential separation for GU and AD molecules was observed at pH 5. Therefore, considering the maximum separation of peak potential for GU–AD as well as the effect of pH for both peak currents and to obtain the highest detection sensitivity, pH 5 of the PBS buffer solution was chosen as the optimum pH value for further measurements.

The effect of the scan rate on the electrocatalytic oxidation of GU and AD was investigated by CV in the range of 50–500 mV s−1. As depicted in Fig. S4 (ESI), an enhancement of the anodic peak current with the increasing scan rates by following the regression equations Ipa (μA) = 0.138ν (mV s−1) + 31.17 (R2 = 0.992) and Ipa (μA) = 0.36ν (mV s−1) + 53.97 (R2 = 0.995) for GU and AD, respectively, can be observed. This indicates that the electrochemical oxidation of GU and AD on the PbTe/GRGP surface is an adsorption-controlled process.51 Therefore, the adsorbed amounts of GU and AD on the surface of PbTe/GRGP were calculated using the following equation, ip = nFQν/4RT = n2F2AνΓc/4RT.53 Herein, n is the number of electrons transferred, A is the surface area of the electrode, ν is the scan rate, F is Faraday's constant, Γc is the surface concentration of GU and AD, and Q is the quantity of charge consumed during the electrochemical oxidation processes. Thus, the number of electrons transferred (n) during the redox processes of GU and AD are 2.4 and 1.8, respectively. In addition, the surface concentrations (Γc) of GU and AD on the modified electrode surface are estimated to be 2.58 × 10−9 mol cm−2 and 9.15 × 10−9 mol cm−2, respectively, which are larger than the earlier reported values on the surfaces of the graphene–COOH-modified GCE electrode (2.86 × 10−10 mol cm−2 and 2.84 × 10−10 mol cm−2 for GU and AD, respectively)22 and for the NiAl-layered doubled hydroxide/GO–MWCNT-modified glassy carbon electrode (Γc of GU and AD are 3.19 × 10−10 mol cm−2 and 3.41 × 10−10 mol cm−2, respectively),52 indicating that the modified PbTe/GRGP electrode possesses an excellent adsorption property. Furthermore, the Laviron equation53 was used to calculate the electron transfer rate constant (ks) of the abovementioned biomolecules on the modified electrode surface. Fig. S5 (ESI) shows the linear relationship between peak potential (Epa) vs. logarithm of scan rates (log[thin space (1/6-em)]ν) for both GU and AD with the slope of 2.3RT/(1 − α)nF. The linear regression equation can be expressed as Epa (V) = 0.133[thin space (1/6-em)]log[thin space (1/6-em)]ν (V s−1) + 1.069 (R2 = 0.999) and Epa (V) = 0.224[thin space (1/6-em)]log[thin space (1/6-em)]ν (V s−1) + 1.490 (R2 = 0.999) for GU and AD, respectively. From the values of the slopes, the charge transfer coefficient (α) for GU and AD was found to be 0.81 and 0.85, respectively. According to the Laviron equation,54 log[thin space (1/6-em)]ks = α[thin space (1/6-em)]log(1 − α) + (1 − α)log[thin space (1/6-em)]α − log(RT/nFν) − α(1 − α)nFΔEp/2.3RT. Thus, the calculated value of ks is 8.81 × 10−3 s−1 and 1.32 × 10−2 s−1 for GU and AD, respectively. For both biomolecules, our calculated value of ks is comparable to or even better than previously reported values, which are summarized in Table 1.

Table 1 Comparison of the electron transfer kinetics at different modified electrodes
Electrodes Charge transfer coefficient (α) Standard rate constant (ks/s−1) Ref.
GU AD GU AD
Graphene–COOH/GCE 0.54 0.60 9.82 × 10−4 1.02 × 10−3 22
Mesoporous carbon-modified carbon ionic electrode 0.75 0.85 3.13 × 10−3 3.17 × 10−3 55
Carbon ionic liquid electrode 0.65 0.58 2.39 × 10−3 7.42 × 10−4 56
PbTe/GRGP electrode 0.81 0.85 8.81 × 10 −3 1.32 × 10 −2 Our work


3.8 Simultaneous determination of GU and AD

Simultaneous determination of GU and AD in their binary mixture has been investigated by DPV using PbTe/GRGP by varying the concentration of one constituent while maintaining the other constituent at a constant concentration. Fig. 6 depicts the DPVs of GU at different concentrations while keeping AD at a fixed concentration.
image file: c7nj03308g-f6.tif
Fig. 6 DPV plots obtained on the surface of the PbTe/GRGP electrode (a) containing 0.05 mM AD and different concentrations of GU from 4 to 140 μM; the corresponding concentration vs. peak current curve is shown in the inset and (b) containing different concentrations of AD from 3 to 50 μM and 0.05 mM GU in 0.1 M phosphate buffer, pH 5.0; the inset shows the corresponding concentration vs. peak current plot.

From the oxidation peak current vs. concentration plot (inset of Fig. 6a), it can be seen that with the increasing concentration of GU, there is a linear enhancement of the peak current. As documented from the calibration curve of GU, there are two linear segments: one linear segment is from 4 μM to 10 μM, which follows the regression equation of IGU = 0.261CGU + 4.648 (R2 = 0.998). The second linear segment is within the range of 10 μM and 140 μM, and the corresponding regression equation is IGU = 0.096CGU + 6.096 (R2 = 0.993). It can also be seen from Fig. 6b that with the increasing concentration of AD, the anodic peak current of AD increases gradually, whereas the peak current of GU remains almost constant. As shown in the inset of Fig. 6b, there are two linear regions in the calibration curve of AD. The first linear region is in the concentration rage from 3 μM to 15 μM, which follows the regression equation IAD = 0.349CAD + 2.699 (R2 = 0.996), and the second linear region is obtained from 15 μM to 50 μM with a regression equation of IAD = 0.311CAD + 3.485 (R2 = 0.996). From these results, it can be concluded that PbTe/GRGP can be used for the simultaneous determination of GU and AD. We have compared our results with those obtained for previously reported relevant electrochemical sensors, as listed in Table 2.

Table 2 Comparison of different electrochemical sensors for simultaneous determination of GU and AD
Electrode materials Linear range (μM) Limit of detection (μM) Ref.
GU AD GU AD
Polyaniline/MnO2 10–100 10–100 4.8 2.9 14
Graphene–Nafion/GCE 2–120 5–170 0.58 0.75 15
MWCNT–Fe3O4@PDA–Ag 8–130 10–120 1.47 5.66 20
NiCoO2/C 390.63–943.4 3.9 24
TiO2–graphene nanocomposite 0.5–200 0.5–200 0.15 0.10 25
Polyimidazole/graphene oxide 3.3–103.3 9.6–215 0.48 1.28 57
Graphene-new fuchsin 10–180 2–9 19 0.9 58
Graphitised mesoporous carbon/GCE 25–200 25–150 0.76 0.63 59
Graphene–ionic liquid–chitosan 2.5–150 1.5–350 0.75 0.45 60
Nano ZSM-5/MIM 10–500 5–500 4.2 7 61
PbTe/GRGP 4–10–140 3–15–50 0.08 0.07 Our work


From these results, it was observed that our sensor offered a shorter linear range but lower detection limit as compared to other reported sensors for the simultaneous determination of these two biomolecules. The probable explanation of this phenomenon may be due to the electron transfer kinetics between the analytes and the electrode surface. It is worth nothing that the enhanced surface concentration of GU and AD on the PbTe/GRGP electrode surface as compared to other reported studies indicates a strong adsorption property, which is attributed to the enhanced electron transfer rate constant in the PbTe/GRGP material. Due to this strong adsorption, the outer more accessible active surface area was fully covered by the first layer of molecules even at relatively lower concentrations of analytes. Thus, in the higher concentration range, the interactions between second-layer molecules were screened by first-layer molecules; this ensured that the interaction strength of the second-layer molecules differed from that of the first-layer molecules; this led to higher sensitivity and excellent adsorption capacity of the proposed electrode at lower concentrations. Hence, in the calibration curve of GU and AD, two different linear regions appear, and this can be explained by the Langmuir adsorption isotherm behavior,62 which demonstrates the monolayered adsorption of biomolecules followed by multilayered adsorption. Similar results were also found previously for simultaneous determination of these molecules (GU and AD).63,64

3.9 Interference study

The effect of some interfering agents (Na+, K+, Mg2+, Cu2+, sucrose, glucose, citric acid, ascorbic acid, uric acid, and dopamine) on the voltammetric responses of GU and AD have also been studied. However, it has been observed from Fig. S6 (ESI) that 1000 μM of these foreign compounds does not have any significant influence on the peak current of these two biomolecules; thus, this confirms the selectivity of the modified PbTe/GRGP electrode.

3.10 Repeatability, reproducibility, and stability of the PbTe/GRGP electrode

To check the repeatability of the modified electrode, five successive measurements have been carried out by DPV in the binary mixture of 0.1 mM GU and 0.05 mM AD (Fig S7a, ESI). The values of relative standard deviation (RSD) of these five repetitive measurements for GU and AD are 1.76% and 3.45%, respectively. The reproducibility of the as-prepared PbTe/GRGP was determined by preparing three electrodes independently using the same procedure, and the fabricated electrodes were tested in the binary mixture (0.1 mM GU and 0.1 mM AD) of biomolecules. From the DPV plots (Fig. S7b, ESI) of these measurements, it was noticed that the RSD value of GU and AD was 1.67% and 2.78%, respectively, thereby suggesting good reproducibility of the modified electrode. To evaluate the long-term stability of the modified electrode, it was stored at room temperature in air for 21 days. Then, the modified electrode was tested again in the same binary mixture of GU and AD solutions at different time intervals between 21 days. The results indicated that (Fig. S7c, ESI) there was no change in the peak positions for both molecules, and the corresponding current signals showed no significant change in the peak currents (RSD values for GU and AD were 3.86% and 4.25%, respectively). All these results indicate that PbTe/GRGP has good repeatability, reproducibility, and stability, which make it attractive for the fabrication of a new electrochemical sensor.

3.11 Clinical sample analysis

The practical performance of the modified PbTe/GRGP electrode was investigated in clinical samples by measuring the GU and AD content in spiked human urine and serum samples. Human urine samples were obtained from healthy volunteers. Serum samples were prepared by centrifuging the blood samples at 3000 rpm for 10 minutes followed by collection with a syringe. Then, 100 μL of human urine and serum samples were diluted 100 times with a PBS (pH = 5) solution. The DPV technique was applied on a diluted 10 mL solution where the known concentrations of GU and AD were added for analysis. These results are summarized in Table S1 (ESI). From this table, it is observed that the recovery values of GU and AD in the human urine and serum samples are 101.6% and 100.5% and 99% and 100.6%, respectively. All these results indicate that the proposed sensor has good accuracy and reliability.

4. Conclusion

In conclusion, we have successfully synthesized PbTe nanoparticles with an average particle size of 22 nm through a simple chemical method, and graphene oxide has been reduced thermally in the presence of Zn dust to synthesize graphene. Herein, PbTe/GRGP has been manifested as a new electrode modifier agent for the construction of a new electrochemical sensor for simultaneous determination of GU and AD. The modified PbTe/GRGP electrode showed excellent electrocatalytic activity towards the oxidation of GU and AD. Furthermore, excellent sensitivity, comparable linear range, low detection limits, long-term stability, and reproducibility have been achieved on the surface of the modified electrode in comparison with other previously reported studies. Furthermore, the fabrication methodology for the preparation of the modified electrode is easy, simple, and cost effective in comparison with that stated in earlier reports. In addition, the modified electrode was used successfully for the assessments of GU and AD content in biological samples with satisfactory results. This superiority of PbTe/GRGP in analytical applications may be due to the combination of the smaller particle size of the PbTe nanomaterial and the high surface area and narrow pore size distribution of graphene. These results indicate that PbTe/GRGP is a promising material for the development of a new electrochemical sensor for future uses in clinical diagnosis and genetic research.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

The authors are thankful to UGC-DAE consortium scientific research, the Kolkata centre, for providing the XRD facility and the Centre for Research in Nanoscience and Nanotechnology (CRNN), Calcutta University, for the FESEM, TEM, and HRTEM imaging facilities. The authors are also grateful to Dr P. Sujatha Devi, CGCRI Kolkata, for providing the UV-visible spectroscopy measurements.

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Footnote

Electronic supplementary information (ESI) available: HRTEM image of the PbTe nanoparticles, Tauc plot of the synthesized PbTe nanoparticles, BET analysis of the synthesized graphene, scan rate variation of both the biomolecules at the PbTe/GRGP electrode, effect of the interfering agent, repeatability, reproducibility, and stability of the modified PbTe/GRGP electrode, real sample analysis. See DOI: 10.1039/c7nj03308g

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