Tuning the direct growth of Ag_seeds into bimetallic Ag@Cu nanorods on surface functionalized electrochemically reduced graphene oxide: enhanced nitrite detection

Abstract

We describe a facile and an efficient method for the direct growth of bimetallic Ag@Cu nanorods (Ag@Cu NRDs) on electrochemically reduced graphene oxide (ERGO) nanosheets by a seed mediated growth approach. Surface functionalization of ERGO is a key step carried out using L-tryptophan that directs the growth of one dimensional Ag@Cu NRDs on its surface. The generated bimetallic Ag@Cu NRDs on L-tryptophan functionalized ERGO (Ag@Cu NRDs/L-ERGO) modified electrodes were characterised by SEM, XRD, EDAX, FT-IR and Raman spectroscopy. Anodic stripping voltammetry analysis and electrochemical impedance spectra were used to understand the electrochemical properties of the synthesized nanostructured material. The modified electrode enabled the electrooxidation of nitrite ions (NO₂⁻) with high sensitivity and a good detection limit of 1 nM. Importantly, the modified electrodes were successfully tested for the analyses of generated NO₂⁻ in urinary tract infection (UTI) affected patient's urine samples. In addition, the modified electrode was satisfactorily used in the electrooxidation of NO₂⁻ generated from in vitro biochemical reduction of nitrate ions using microbial species such as Escherichia coli (E. coli).

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1. Introduction

A network of one dimensional (1D) metallic nanomaterials such as nanorods or nanowires on graphene surface has been considered as a promising material for new generation supercapacitors, fuel cells, sensors and optical devices.^1–3 Growing of bimetallic nanoparticles on a graphene surface greatly alters its physical and chemical properties.^4,5 Compared to other nanostructures, 1D nanostructure such as nanorods and nanowires possess unique characteristics which result in intriguing material characteristics due to synergistic effects such as high optical transparency, specific surface area, good mechanical strength, electrical and thermal conductivity.⁶ Lee et al. constructed transparent and stretchable electrodes using graphene–metal nanowire hybrid structures.⁷ Liu et al. developed conductive films using graphene–silver nanowire sandwich like structures.⁸

Although various metallic nanostructures have been generated on the graphene surface through different techniques like hybridisation, in situ reduction, microwave assisted synthesis and hydrothermal process,^7–11 very few reports are available on the direct growth of 1D nanostructures on graphene surface. Kim et al. studied the direct growth of gold nanorods on graphene surface by seedless and seed mediated method.¹² Surface functionalization and uniform dispersion in aqueous medium are significant challenges in the growth of 1D nanostructure on graphene surface. Kim et al. have used long chain organic molecules like pyrine ethylene glycol amine and decylpyrine for surface functionalization of reduced graphene oxide (RGO) nanosheets which in turn grow as gold nanorods.¹³ In seed-mediated growth method, high immobilisation of seed density on RGO surface leads to the growth of spherical nanostructures.¹³ On the other hand, tuning the surface functionalization on graphene could result in low immobilisation of seed density which in turn paves a way to uniformly generate 1D nanostructure such as nanorods or nanowires on its surface. In the present investigation, two key aspects were taken into consideration for simple and effective growth of bimetallic nanorods on ERGO surface. (1) The poor dispersion quality of RGO in wet chemical method was overcomed by electrochemical reduction of graphene oxide as ERGO on the surface of electrode and (2) in the next step, a simple amino acid L-tryptophan would be introduced to carryout stringent functionalization on ERGO surface. It enabled the immobilization of low density Ag_seeds on ERGO surface. Currently, various techniques were used to synthesize graphene nanosheets including epitaxial growth,¹⁴ chemical vapour deposition,¹⁵ mechanical exfoliation¹⁶ and chemical reduction of graphene oxide.¹⁷ Importantly, in order to scale up the RGO on electrode surface, ERGO was considered as an efficient tool for the synthesis of better quality graphene sheets.^18,19 Accordingly, ERGO nanosheets possess low oxygen moieties and better conductivity compared to chemically reduced GO sheets.²⁰

Transition metals like Ag, Au, Pt, Pd, and Cu were commonly used in the formation of heterogeneous catalysts due to its high catalytic efficiency.^21,22 Yin et al. developed graphene and Pd-based bimetallic nanocomposites for electrocatalytic oxygen reduction reaction.²³ Feng et al. synthesised PtAg alloy–graphene hybrid composites for studying the catalytic activity in methanol electrooxidation.²⁴ Even though various metal nanostructures loaded graphene were extensively studied, the direct growth of bimetallic Ag@Cu NRDs on ERGO nanosheets is not yet explored. In the present work, a simple and an efficient seed mediated growth method was developed for the direct growth of bimetallic Ag@Cu NRDs on L-tryptophan functionalized ERGO.

Nitrite ion (NO₂⁻) is commonly present in food, soil and physiological systems.^25,26 It is used as a preservative in food industries and an overdose is reported to seriously affect human health. It is formed as carcinogenic nitrosamines in the presence of amine which causes blue body and gastric cancer.^27,28 Excess amount of NO₂⁻ secreted in the urine samples is known to result in urinary tract infection (UTI). Thus, a positive NO₂⁻ test in the urine sample is considered as a crucial indication of UTI.²⁹ Different techniques were developed for the detection of NO₂⁻ including spectrophotometry,³⁰ polarography,³¹ chromatography³² and electrochemical methods.^33,34 Electrochemical detection of NO₂⁻ has been considered as the most efficient tool with high sensitivity, reproducibility and detection limit. Liu et al. studied Au–Fe(III) nanoparticle modified glassy carbon electrode for electrochemical sensing of NO₂⁻ with a detection limit of 2.0 × 10⁻⁷ M.³³ Lin et al. used poly(3,4-ethylenedioxythiophene) modified SPCEs for NO₂⁻ with the detection limit of 0.96 μM.³⁴ Liu et al. used gold nanoparticles for the electrochemical detection of NO₂⁻.³⁵ Kalimuthu et al. studied electropolymerized film of functionalized thiadiazole modified glassy carbon electrode for the detection of NO₂⁻.³⁶ Pal et al. used Ag nanoparticles modified electrode for the oxidation of NO₂⁻.³⁷ Ning et al. used PAMAM dendrimer-stabilized Ag nanoparticles for the electrochemical sensing of NO₂⁻ with a detection limit of 0.4 μM.³⁸ Gao et al. used Ag nanorods for the electrooxdiation of NO₂⁻.³⁹ Jayabal et al. developed Au/Ag bimetallic nanorods for the electrochemical sensing of NO₂⁻.⁴⁰ Teymourian et al. used Fe₃O₄ magnetic nanoparticles/reduced graphene oxide nanosheets as a biosensor for NO₂⁻ detection.⁴¹ Nevertheless, bimetallic nanorods or nanowires decorated reduced graphene oxide nanosheets were used in the development of NO₂⁻ sensor. In the present investigation, bimetallic Ag@Cu NRDs decorated L-tryptophan functionalized ERGO electrode exhibits an efficient electrochemical sensing of NO₂⁻ with high sensitivity and good detection limit in wide linear range. The interference of various cations and anions were studied in the detection of NO₂⁻. In addition, hospital urine samples of UTI affected patients were analysed for the effective practice of NO₂⁻ assay at bimetallic Ag@Cu NRDs decorated L-tryptophan functionalized ERGO electrode. Finally, in vitro biochemical conversion of NO₃⁻ to NO₂⁻ was carried out using E. coli to study NO₂⁻ generation at the modified electrode.

2. Experimental section

2.1. Chemicals

Graphite powder (300 mesh) was purchased form Alfa Aesar, UK. Sodium nitrate, sulphuric acid, hydrogen peroxide, sodium borohydride, copper nitrate, L-tryptophan, sodium hydroxide, sodium nitrite, disodium hydrogen phosphate and monosodium dihydrogen phosphate were purchased form Merck, India. Cetyl trimethyl ammonium bromide (CTAB) was obtained from Himedia, India, and trisodium citrate, ascorbic acid (AA) and silver nitrate were obtained from Sigma-Aldrich, USA. All reagents were of analytical grade and used without further purification. All solutions were prepared using deionized (DI) water.

2.2. Characterizations

In order to exhibit the structural and morphological properties, bimetallic Ag@Cu NRDs decorated ERGO nanosheets were grown on Indium tin oxide (ITO) coated electrode surface. Prior to the growth process, ITO plates were cleaned and pretreated in H₂O, NH₄OH and H₂O₂ with the ratio of 5 : 1 : 1. Surface morphology of bimetallic Ag@Cu NRDs on ERGO nanosheet was confirmed by SEM (VEGA 3 TESCAN, USA) analysis. X-ray diffraction (XRD) patterns were studied using Shimadzu XRD 6000 (Japan) with Cu-Kα radiation (λ = 1.5418 Å). EDAX mapping analyses were carried out using energy dispersive X-ray analyser (Bruker, Germany). FT-IR (Jasco 4000 series, Japan) analyses were carried out to study the surface functionalization of graphene nanosheets using L-tryptophan. Raman spectra were recorded using Horiba-Jobin, LabRAM HR equipped with Argon (514 nm) ion laser excitation source.

2.3. Electrochemical measurements

All electrochemical measurements were carried out using CHI 660D electrochemical workstation (CH Instruments, USA). The electrochemical cell consist of a three electrode system, Ag@Cu/L-ERGO modified glassy carbon electrode (0.07 cm²) serves as a working electrode, platinum wire as a counter electrode and Ag/AgCl filled with saturated KCl solution was used as a reference electrode.

2.4. Synthesis of L-tryptophan functionalized ERGO electrode

Graphite oxide was prepared by modified hummers method.^42,43 Briefly, 2 g of natural graphite powder was mixed with 1 g of NaNO₃ and 25 mL of conc. H₂SO₄ and stirred for 30 min at 5 °C, followed by stepwise addition of 7 g KMnO₄ over a period 1 h. The reaction mixture was stirred further for another 2 h at 35 °C, followed by the addition of 92 mL of DI water slowly into the reaction mixture that eventually turned into brown colour. Finally, 10 mL H₂O₂ (30%) and 140 mL of warm DI water were added. The filtrate was collected by washing with 5% HCl and DI water and dried overnight at 50 °C. The synthesised graphite oxide should be dispersed in a fine concentration of 0.2 mg mL⁻¹ in DI water under ultrasonication for 3 h and thereby resulted in exfoliation of graphene oxide (GO). The well dispersed GO thus synthesized is stable in DI water for more than 6 months.

Glassy carbon electrodes (GCEs) were polished well with alumina (0.3 micron) using buehler cloth and sonicated in ethanol–water mixture for 5 min. Then, 10 μL of exfoliated GO was drop casted on GCE surface and dried at room temperature for 1 h. GO was electrochemically reduced in 0.05 M phosphate buffer (pH 5).^18,44 Using three electrode cell set up, 30 successive cycles were performed in the potential range between 0.0 and −1.5 V at a scan rate of 50 mV s⁻¹. Further, the ERGO modified electrode was dipped in L-tryptophan (1 mg mL⁻¹) for 24 h at room temperature for surface functionalization.

2.5. Synthesis of bimetallic Ag@Cu NRDs on L-tryptophan functionalized ERGO electrode

Bimetallic Ag@Cu NRDs were grown on functionalized ERGO surface by seed mediated growth method. First, Ag seeds (Ag_seeds) solution was prepared by adding 0.6 mL of 10 mM NaBH₄ to an aqueous solution containing 0.25 mM AgNO₃ and 0.25 mM trisodium citrate. L-tryptophan functionalized ERGO/GCE was dipped in Ag_seeds solution for 30 min and dried for 1 h. In the next step, Ag_seeds/ERGO/GCEs were dipped into growth solution containing 80 mM CTAB for 3 min. Subsequently, 1 mL of 10 mM Cu(NO₃)₂, 2 mL of 100 mM AA, 0.4 mL of 1 M NaOH were added to the growth solution for the anisotropic growth of bimetallic Ag@Cu NRDs on the electrode surface. A gradual change in colour of the growth solution from colourless to bright yellow over a period of 15 min indicated the generation of bimetallic Ag@Cu NRDs on functionalized ERGO surface. Importantly, it is possible to control the aspect ratio of bimetallic NRDs generated by varying the concentration of Ag_seeds on ERGO surface.^45,46 Thus, low loading Ag_seeds on functionalized ERGO surface would tend to grow long uniform bimetallic NRDs. Accordingly, the growth process was repeated to increase the loading density of bimetallic Ag@Cu NRDs on functionalized ERGO surface.

3. Results and discussion

The direct growth of bimetallic Ag@Cu NRDs on ERGO surface by seed mediated growth method is outlined in Scheme 1. It explained the 1D growth of Ag_seeds into bimetallic Ag@Cu NRDs. It was carried out by the interaction of crystal faces of Ag_seeds with the soft template CTAB and the anisotropic reduction of Cu²⁺ ions using mild reducing agent such as AA. The direct growth of 1D NRDs or nanowires on RGO surfaces is a challenge due to poor surface functionalization methodologies. Seed immobilisation density on graphene surface is altered by effective surface functionalization. In the present work, the direct growth of Ag_seeds in the presence and absence of functionalizing agent were compared. L-tryptophan was used as the surface functionalizing agent paving a way for the direct growth of bimetallic Ag@Cu NRDs at ERGO surface. L-Tryptophan plays a vital role in the coordination of metal ions by the electrostatic interaction, reduction of metal ions and in the direct growth of bimetallic NRDs at ERGO surface. Fig. 1 shows the SEM images of ERGO (A), Ag@Cu nanoparticles on ERGO (B) and bimetallic Ag@Cu NRDs generation on L-tryptophan functionalized ERGO (C) nanosheets decorated electrodes. Fig. 1A represents the sheet like morphology of ERGO nanosheets. Fig. 1B clearly shows the formation of spherical nanostructures on graphene surface. In the absence of L-tryptophan, Ag_seeds grew densely as Ag@Cu spherical nanoparticles on bare ERGO surface. On the other hand, the presence of L-tryptophan on ERGO surface facilitated the successful growth of Ag_seeds into Ag@Cu bimetallic NRDs. Fig. 1C confirmed the formation of 1D Ag@Cu NRDs on L-tryptophan functionalized ERGO surface with an average nanorods diameter as 180 ± 2 nm and length as ∼1 μm.

Scheme 1 Schematic representation of 1D growth of bimetallic Ag@Cu NRDs on L-tryptophan functionalized ERGO nanosheets at GCE surface.

Fig. 1 SEM images of ERGO (A), Ag@Cu nanoparticles decorated at ERGO in the absence of L-tryptophan (B) and Ag@Cu NRDs decorated at L-ERGO (C).

Functionalization of ERGO surface with L-tryptophan was confirmed by FT-IR spectroscopy. Fig. 2 shows the FT-IR spectra of GO (a), ERGO (b), L-ERGO (c) and L-tryptophan (d). The broad band observed for GO at 3461 cm⁻¹ corresponds to O–H stretching vibration. Sharp bands at 1730 cm⁻¹, 1620 cm⁻¹ 1224 cm⁻¹ and 1048 cm⁻¹ were due to stretching vibrations of C=O, C=C, epoxy groups and C–O group, respectively. Subsequently, the corresponding bands for oxygen functionalities and epoxy groups were absent which confirmed the electrochemical reduction of GO (a) into ERGO (b). Importantly, FT-IR spectral tool inferred the pure form of electrochemically reduced graphene nanosheets compared to chemically reduced graphene oxide nanosheets.²⁰ The C=C stretching occur at 1650 cm⁻¹ and the broad bands at 1085 cm⁻¹and 950 cm⁻¹ were responsible for C–H vibrations. L-Tryptophan functionalized ERGO (c) shows intense bands at 3604 cm⁻¹, 3401 cm⁻¹, and 1700 cm⁻¹ corresponding to O–H stretching, N–H stretching, and C=O stretching, respectively. The C=C stretching and C–H bending vibrations occur at 1514 cm⁻¹ and 680 cm⁻¹. A weak band at 2920 cm⁻¹ is responsible for the stretching vibrations of CH₂ group. All these bands were correlated with the FT-IR spectra of L-tryptophan (d). Thus, FT-IR spectral results confirmed the N–H stretching which validates the effective functionalization of L-tryptophan on ERGO surface (c). The ERGO surface intrinsically does not contain heteroatom like nitrogen, and it must therefore solely be derived from N–H functionalization of L-tryptophan where Ag_seeds immobilize and direct the seed mediated growth of bimetallic Ag@Cu NRDs.

Fig. 2 FT-IR transmittance spectra of GO (a), ERGO (b), L-ERGO (c) and L-tryptophan (d).

EDAX spectra and elemental mapping analyses were used to confirm the coexistence of bimetallic Ag and Cu on the surface of ERGO. Fig. 3 shows the EDAX pattern and elemental mapping analyses of bimetallic Ag and Cu on L-ERGO surface. Briefly, in the first step, Ag_seeds were immobilised on L-tryptophan functionalized ERGO surface which corresponds to the identification of metallic Ag (Fig. 3A). In the next step, using mild reducing agent, the existing Ag_seeds tend to mediate the growth of Cu on its surface. The existence of Cu on Ag surface was confirmed with the identification of Cu in mapping analysis (Fig. 3B). Fig. 3C clearly confirmed the coexistence of Ag and Cu on bimetallic Ag@Cu NRDs on ERGO surface. Finally, EDAX spectra (Fig. 3D) authenticated the presence of bimetallic Ag and Cu at NRDs on functionalized ERGO surface with a weight percentage ratio of ∼10 : 1. Further, XRD pattern (Fig. 4) justified the presence of bimetallic Ag and Cu at L-tryptophan functionalized ERGO surface. Graphene oxide shows an intense diffraction at 10.1° whereas it is minimised and a new diffraction is observed at 23.9° for ERGO surface or at Ag@Cu NRDs decorated L-ERGO surface. The change in the diffraction pattern is due to the complete reduction of GO with an interlayer spacing of 3.7 Å.⁴⁷ In addition, the XRD pattern at 17.1°, 20.6°, 27.5° corresponds to Ag with (1 1 1), (2 0 0) and (2 2 0) facets and at 20.6° (1 1 1), 23.5° (2 0 0) and 38.2° (3 1 1) corresponds to the metallic Cu. All diffraction patterns exhibited good correlation with JCPDS no. 011167 and 011242, respectively.

Fig. 3 EDAX elemental mapping analyses of Ag_seeds (A) or Cu at Ag@Cu (B) or bimetallic Ag@Cu at L-ERGO (C), and EDAX spectra of Ag@Cu NRDs on L-ERGO nanosheets coated electrode (D).

Fig. 4 XRD patterns of ERGO (a), Ag@Cu NRDs/L-ERGO (b) nanosheets coated electrodes. Inset: XRD patterns of GO.

Raman spectroscopy is an effective and non-destructive method to characterize the crystal structure and defects in graphene based materials. D and G band in the Raman spectra are responsible for the κ-point phonons of the A_1g symmetry and E_2g vibration mode of sp² carbon atoms. The intensity ratios of D and G bands (I_D/I_G) are the measure of defects on graphene layers. Fig. 5 shows the Raman spectra of ERGO and bimetallic Ag@Cu NRDs decorated L-ERGO. GO shows sharp bands at 1350 cm⁻¹ and 1594 cm⁻¹ corresponding to D and G with an I_D/I_G ratio of 0.90 (Fig. 5 (inset)). The ratio increased to 1.42 due to RGO formation, reflecting the rise in defect concentration as well as disorderliness at RGO surface.^48,49 On the other hand, the I_D/I_G ratio decreased to 1.03 at bimetallic Ag@Cu NRDs decorated L-ERGO with sharp bands at 1345 cm⁻¹ and 1586 cm⁻¹. This is due to the growth of bimetallic Ag@Cu NRDs on ERGO producing a healing effect which reflects in the increase of sp² domain character. 2D and S3 bands at 2681 cm⁻¹ and 2943 cm⁻¹ confirmed better graphitization process. In addition, the intensity of 2D band was attributed to the layer stacking in graphene.⁵⁰

Fig. 5 Raman spectra of ERGO (a) and Ag@Cu NRDs/L-ERGO (b) modified electrodes. Inset: Raman spectra of GO.

Anodic stripping voltammetry (ASV) is the most effective, reproducible and sensitive method for the detection of trace amounts of metal ions at the electrode surface. The existence of metallic Ag and Cu at L-ERGO modified electrode was analyzed by ASV. Fig. 6 shows the cyclic voltammograms (CVs) of Ag_seeds/L-ERGO/GCE (a) and bimetallic Ag@Cu NRDs/L-ERGO/GCE (b) in 0.5 M H₂SO₄. The anodic peak appeared at 0.23 V corresponding to Ag oxidation at Ag_seeds/L-ERGO/GCE (a) and at 0.12 V corresponding to bimetallic Cu and Ag oxidation at Ag@Cu NRDs/L-ERGO/GCE (b), which coincides with the stripping potential of Cu which is in the range of 0.1 V.⁵¹ Hence, the decrease in the anodic stripping potential at Fig. 6b was attributed to the presence of Cu at Ag surface.⁵² Thus, the presence of Cu at the bimetallic NRDs surface tends to decrease the over potential of Ag metal dissolution and a concurrent oxidation of bimetallic Cu and Ag with decreased oxidation potential. This result confirms the presence of bimetallic Ag and Cu at L-ERGO surface.

Fig. 6 CVs of Ag_seeds/L-ERGO (a) and Ag@Cu NRDs/L-ERGO/GCE (b) in 0.5 M H₂SO₄ at a scan rate of 50 mV s⁻¹.

Electrochemical impedance spectroscopy is an effective tool for studying the interfacial properties of modified electrodes. Fig. 7 shows the Nyquist plot for ERGO/GCE (a), Ag_seeds/L-ERGO/GCE (b) and Ag@Cu NRDs/L-ERGO/GCE (c) in the electrolyte containing 0.5 mM K₄Fe(CN)₆ and 0.5 M KCl. All the impedance data are fitted with Randle's equivalent circuit. Ag@Cu bimetallic NRDs decorated at L-ERGO (c) shows a very low charge transfer resistance (R_ct) compared to ERGO (a) and Ag_seeds decorated L-ERGO electrode (b). This indicates the importance of bimetallic NRD formation at graphene surface resulting in enhancement of electron transfer kinetics between the electrolyte and electrode interface with increase in conductivity.

Fig. 7 Electrochemical impedance spectra of ERGO (a), Ag_seeds/L-ERGO (b) and Ag@Cu NRDs/L-ERGO (c) in 0.5 mM K₄Fe(CN)₆. The data's are fitted with Randle's equivalent circuit.

3.1. Electrooxidation of NO₂⁻ at bimetallic Ag@Cu NRDs/L-ERGO/GCE

The bimetallic modified electrodes were tested for the electrochemical sensing of NO₂⁻ by investigating the electrooxidation of NO₂⁻ in phosphate buffer (PB), pH 6. Fig. 8 shows the CVs at Ag@Cu NRDs/L-ERGO/GCE in the presence (a) and absence (b) of 1 mM NO₂⁻ in PB at a scan rate of 50 mV s⁻¹. In the presence of NO₂⁻, Ag@Cu NRDs/L-ERGO/GCE (a) shows an intense electrooxidation peak at 0.78 V with an onset potential at 0.46 V due to the oxidation of NO₂⁻ to NO₃⁻ through a two electron process.⁵³ On the other hand, in the absence of NO₂⁻, no such oxidative behaviour was observed (Fig. 8b). Further, for comparison, bare GCE (a), L-ERGO/GCE (b) and Ag_seeds/L-ERGO/GCE (c) were used to investigate the electrooxidation of NO₂⁻ under identical conditions as displayed in Fig. 8 (inset). Bare GCE shows a broad oxidation peak at 0.92 V with an increase in the overpotential towards NO₂⁻ oxidation, Fig. 8 (inset a). Interestingly, after L-ERGO deposition GCE shows an increased peak current towards NO₂⁻ electrooxidation with a broad peak at 1.05 V (Fig. 8 (inset b)). On the other hand, no noticeable difference in the peak current towards NO₂⁻ electrooxidation was observed after Ag_seeds was introduced at L-ERGO/GCE surface (Ag_seeds/L-ERGO/GCE), Fig. 8 (inset c). A new oxidative peak at 0.26 V which corresponds to the dissolution of Ag_seeds in PB buffer was observed in addition to NO₂⁻ oxidation. Scheme 2 illustrates the unfavorable and favorable conditions for electrooxidation of NO₂⁻ at Ag_seeds/L-ERGO/GCE (A) and Ag@Cu NRDs/L-ERGO/GCE (B), respectively. Scheme 2(A) shows the poor electrooxidation of NO₂⁻ at Ag_seeds modified electrode which is due to electrostatic repulsive behaviour between the surface plasmon of Ag_seeds and NO₂⁻ ions. On the other hand, in the case of bimetallic Ag@Cu NRDs, the heterometallic bonding interactions (ligand effect) between the metals resulted in the modification of surface plasmon of Ag_seeds.⁵⁴ It greatly enhanced the catalytic effect of Ag@Cu NRDs on ERGO for the electrooxidation of NO₂⁻ which is depicted in Scheme 2(B). Thus, seed mediated growth approach of Cu coated Ag as bimetallic Ag@Cu NRDs generation is absolutely essential at ERGO surface to minimize the electrostatic repulsion and to subsequently enhance the electrooxidative behaviour towards NO₂⁻ oxidation. Accordingly, Ag@Cu bimetallic NRDs decorated ERGO electrode shows an increased electrochemical activity for the electrooxidation of NO₂⁻ with decrease in overpotential of 0.26 V compared to L-ERGO/GCE or Ag_seeds/L-ERGO/GCE.

Fig. 8 Cyclic voltammetric response of Ag@Cu NRDs/L-ERGO/GCE in the presence (a) and absence of 1 mM NO₂⁻ in PB, pH 6. Inset: CVs of bare GCE (a), L-ERGO/GCE (b) and Ag_seeds/L-ERGO/GCE (c) at 1 mM NO₂⁻ in PB.

Scheme 2 Schematic representation of electrooxidation of NO₂⁻ at Ag_seeds/L-ERGO/GCE (A) and Ag@Cu/L-ERGO/GCE (B).

The potential scan rate study is an important factor that might affect the kinetics of the electrode process. Therefore, the effect of scan rate on the electrooxidation of NO₂⁻ at Ag@Cu NRDs/L-ERGO/GCE was studied. Fig. 9 shows the CVs of Ag@Cu NRDs/L-ERGO/GCE in PB containing 1 mM NO₂⁻ under different scan rate from 0.01 to 0.1 mV s⁻¹. It shows a positive shift in the oxidation potential with respect to increase in scan rate. Fig. 9 (inset) shows a linear relationship between the anodic peak current and the square root of scan rate with the linear regression equation I_pa = 85.72 (V s⁻¹) + 13.09. The linear increase in the anodic peak current with respect to the square root of scan rate indicates that the electrooxidation of NO₂⁻ at Ag@Cu NRDs/L-ERGO/GCE is a diffusion controlled process. It is used to calculate the number of electrons transferred in the electrooxidation process with Randles–Sevcik equation.

I_p = 0.4463(F³/RT)^1/2An_α^3/2D_o^1/2C_oν^1/2 (1)

Fig. 9 CVs of Ag@Cu NRDs/L-ERGO/GCE in 1 mM NO₂⁻ in PB, pH 6 at 0.01 (a), 0.03 (b), 0.05 (c), 0.07 (d) and 0.09 (e) V s⁻¹. Inset: plot of the peak currents vs. square root of scan rates.

Here n_α the no. of electrons transferred, D_o is the diffusion coefficient of the electroactive species, C_o is the concentration of NO₂⁻ and ν^1/2 is the square root of scan rate. Hence, the number of electrons transferred is estimated as 2.1 using diffusion coefficient as 2.1 × 10⁻⁵ cm² s⁻¹ for NO₂⁻ electrooxidation in 0.1 M PB.^55,56 The oxidation of NO₂⁻ to NO₃⁻ involves two step reactions as eqn (2) and (3).^34,36

2NO₂⁻ ⇌ 2NO₂ + 2e⁻ (2)

2NO₂ + H₂O → NO₃⁻ + NO₂⁻ + 2H⁺ (3)

The oxidation of NO₂⁻ to NO₂ is the rate determining step. The electron transfer coefficient (α) of the reaction can be calculated from eqn (4)

where n_α is the number of electrons transferred in the rate determining step. Since it is a two electron process the estimated value of α is 0.674.

The effect of pH on the electrooxidation of NO₂⁻ with respect to anodic peak current and anodic peak potential were investigated between pH 4 and 8 at Ag@Cu NRDs/L-ERGO/GCE. Fig. 10 shows the variations in the anodic peak current with respect to pH from 4 to 8. Fig. 10 (inset) shows the plot of current vs. pH where a slight decrease and linear increase in anodic peak current was observed from pH 4 to 5 and up to pH 6. Further increase in pH resulted in a drastic decrease in the anodic peak current. On the other hand, there was no change in the anodic peak potential between pH 4 and 5, whereas the oxidation potential shifted to a less positive range at pH 6. From pH 6 to 8, a linear increase in the oxidation potential was observed with a linear regression equation y = 0.0155x + 0.6978 (R² = 0.9914). The number of exchanged protons were calculated from the equation dE/dpH = 0.059X/αn_α. Here, α is the electron transfer coefficient and n_α is the no. of electrons. From the slope, the no. of protons is estimated as 0.3. It indicates that in acidic media, there is no change in peak potential and in basic media, the estimated proton range is very low. Thus, pH 6 offers an appropriate proton environment for the electrocatalytic oxidation of NO₂⁻ due to the electrocatalytic effect of Ag@Cu NRDs decorated L-ERGO nanosheets. The number of protons calculated from the slope of peak potential vs. pH is inconsistent with theoretical two proton oxidation reaction of NO₂⁻. Therefore, the electrooxidation of NO₂⁻ is speculated as a kinetically controlled process as discussed elsewhere.^57,58 Thus, pH 6 was found to be optimal for enhanced electrooxidation of NO₂⁻ at Ag@Cu NRDs/L-ERGO/GCE. Subsequent NO₂⁻ electrooxidation studies were carried out in PB at pH 6.

Fig. 10 CVs of Ag@Cu NRDs/L-ERGO/GCE at different pH for 1 mM NO₂⁻ [pH: 4 (a), 5 (b), 6 (c), 7 (d) and 8 (e)]. Inset: plot of pH vs. anodic peak current.

Ag@Cu NRDs/L-ERGO/GCE was used to study the linear range of NO₂⁻ assay and detection limit. Fig. 11A shows the chronoamperometric current response for the successive addition of an aliquot of NO₂⁻ after every 40 s in PB. It indicates the steady increase in the oxidation current for [NO₂⁻] from 0.1 mM to 0.9 mM. Fig. 11A (inset) shows the linear relationship between the oxidative current vs. [NO₂⁻] with a correlation coefficient of 0.998. The detection limit of NO₂⁻ was estimated as 1 × 10⁻⁹ M with a signal to noise ratio 3 (S/N = 3) at Ag@Cu NRDs/L-ERGO modified electrode (Fig. 11B). The interference study of a 100 fold higher concentration of different cations and anions such as Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, PO₄³⁻, SO₄²⁻, NO³⁻, Cl⁻ and CO₃⁻ were carried out in the presence of 0.1 mM NO₂⁻ using chronoamperometry. None of the aforementioned ions interfered in the electrochemical sensing of NO₂⁻ at Ag@Cu NRDs/L-ERGO/GCE. The reproducibility and stability of the Ag@Cu NRDs/L-ERGO/GCE were studied by cyclic voltammetry. After 20 successive cycles, Ag@Cu NRDs/L-ERGO/GCE shows a very good response toward NO₂⁻ electrooxidation with a relative standard deviation of 3.3%. Further, the modified GCE was stored and studied after 3 weeks at room temperature and exhibit a similar current response toward NO₂⁻ electrooxidation. This indicates that the modified electrode possess good reproducibility and stability.

Fig. 11 (A) Chronoamperometric response of Ag@Cu NRDs/L-ERGO/GCE for 0.1–0.9 mM [NO₂⁻] in PB, pH 6. The electrode potential is fixed at −0.85 V. Inset plot shows the [NO₂⁻] vs. anodic peak current. (B) CVs of modified electrode in the presence (a) and absence (b) of 1 nM NO₂⁻ in PB.

3.2. Real sample analysis

UTI is one of the most common bacterial infection affecting both men and women. E. coli (80–85%) is the prominent bacteria for the cause of UTI and it converts the urinary nitrate into NO₂⁻. A positive NO₂⁻ test is therefore an indication of UTI. Thus the modified electrode should enable the analysis of hospital urine samples of UTI affected patients for detecting the presence of NO₂⁻ at Ag@Cu NRDs/L-ERGO/GCE. Prior to the test, the hospital urine samples were centrifuged and supernatant solutions were used for analyses. Fig. 12 shows the electrooxidation signal of NO₂⁻ in the urine samples of UTI affected patient (a) compared with non infected urine sample (b) at Ag@Cu NRDs/L-ERGO/GCE. UTI patient sample shows a broad oxidation in the range of 0.6–0.9 V in Fig. 12a whereas no such oxidation was observed in the control experiment (Fig. 12b). In order to validate the result, differential pulse voltammetry was used to confirm the electrooxidation corresponding to NO₂⁻. Fig. 12 (inset a) shows a noticeable oxidation peak at 0.75 V due to NO₂⁻ oxidation in the urine sample of UTI patient whereas no such oxidation peak was observed in the case (Fig. 12 (inset b)) of non infected person. The concentration of nitrite at UTI infected samples was calculated as 0.1 mM through chronoamperometry and 90 ± 4% recovery of nitrite was obtained with urine samples. The decrease in the recovery is due to the matrix effect that exist in urine samples. The modified electrode was successfully used in the rapid screening of UTI affected urinary samples, proving it to be a highly sensitive electrochemical sensor for NO₂⁻ detection.

Fig. 12 Cyclic voltammetric responses of Ag@Cu NRDs/L-ERGO/GCE for hospital urine sample of UTI patient (a) and a urine sample of non-infected person (b) in PB, pH 6. Inset: differential pulse voltammograms of same urine samples (a) and (b).

In addition, E. coli bacteria were used for in vitro biochemical reduction of sodium nitrate to sodium nitrite and the generated NO₂⁻ was analyzed at Ag@Cu NRDs/L-ERGO/GCE. 5 × 10⁷ cfu mL⁻¹ of E. coli was added into 10 mL of 5 mM NO₃⁻ in PB and incubated for 20 h at room temperature.²⁹ The control experiment did not contain E. coli. The solutions were centrifuged and the supernatants were subjected for electrochemical analyses. Fig. 13 shows the cyclic voltammetric response of supernatant solutions obtained in the presence (a) and absence (b) of E. coli. An oxidative peak was observed at 0.72 V in the case of supernatant containing E. coli (a) which is due to the electrooxidation of NO₂⁻. Thus, the biochemical reduction of nitrate into nitrite ions in the presence of E. coli was confirmed. On the other hand, there was no such oxidation peak was observed in the absence of E. coli for 5 mM NO₃⁻. In addition, in the absence of 5 mM NO₃⁻ under identical conditions of PB and E. coli was incubated for 20 h. Then the resultant supernatant was subjected for electrochemical analysis, Fig. 13c. The control experiments didn't show any characteristic cyclic voltammetric responses. Fig. 13 (inset) shows a characteristic DPV peak corresponding to electrooxidation of NO₂⁻ generated in the presence of E. coli. These investigations permit us to validate the successful utilization of bimetallic Ag@Cu NRDs decorated L-ERGO electrodes in the sensing of NO₂⁻ at hospital urine samples.

Fig. 13 Cyclic voltammetric responses of biochemically generated NO₂⁻ due to the presence (a) and absence (b) of E. coli containing 5 mM NO₃⁻ and in the absence of 5 mM NO₃⁻ (c) under identical conditions of PB and E. coli at Ag@Cu NRDs/L-ERGO/GCEs. Inset: differential pulse voltammogram of biochemically generated NO₂⁻ due to the presence of E. coli.

4. Conclusions

Ag@Cu bimetallic NRDs were successfully grown on ERGO surface by simple seed mediated method. Importantly, a dual role for L-tryptophan was identified; (1) as a functionalizing agent for controlling the Ag_seeds density on ERGO surface and (2) in the direct growth of bimetallic Ag@Cu NRDs on the surface of ERGO. The modified electrode was characterized with ease using SEM, XRD, EDAX, FT-IR and Raman spectroscopy. Ag@Cu NRDs/L-ERGO modified electrodes were used for the electrooxidation of NO₂⁻ with amplified current response and good detection limit was achieved as low as 1 nM. In addition, the overpotential for NO₂⁻ oxidation was decreased at the modified electrode compared to bare GCE. It indicates the synergistic effect produced at the coexistence of bimetallic Ag@Cu NRDs on L-ERGO surface which drastically enhanced the electrochemical and catalytic effect of graphene nanosheets towards NO₂⁻ detection with a fast electron transfer rate. The real time application of the modified electrode was satisfactorily proved in the analyses of NO₂⁻ containing hospital urine samples of UTI patients with ease. Further, the modified electrode could be commercialized by generating the patterns of low cost bimetallic Ag@Cu NRDs at L-ERGO surface based screen printed electrode for analyzing the hospital urine sample of UTI in short duration of time with very high accuracy.

Acknowledgements

Financial support by DST-SERB, New Delhi (File no. SR/FT/CS-44/2011 dated 04.05.2012) is gratefully acknowledged. We thank Karunya University, Coimbatore for providing instrumental facilities. This work is dedicated to Professor R. Ramaraj's 60th birthday and his pioneering contribution in the fields of Photochemistry & Electrochemistry.

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