Polyelectrolyte stabilized bi-metallic Au/Ag nanoclusters modified electrode for nitric oxide detection

Abstract

In situ synthesis of bi-metallic Au/Ag nanoclusters (NCs) embedded in poly(acrylamide-co-diallyldimethylammonium chloride) (PADA) matrix and its application towards the detection of nitric oxide (NO) in nanomolar concentration range are reported. The cationic PADA matrix was used as stabilizer and support material for the preparation of bi-metallic Au/Ag NCs. The electrocatalytic oxidation and electrochemical sensing of NO were studied using the bi-metallic Au/Ag NCs modified electrodes. The modified electrode exhibited synergistic electrocatalytic activity, sensitivity and selectivity towards NO detection when compared to the mono-metallic Au and Ag nanostructures (NSs) modified electrodes. The present bi-metallic Au/Ag NCs modified electrode showed a sensitivity of 3.77 nA nM⁻¹, a lowest experimental detection limit of 10 nM with S/N = ∼4.9, a fast response time (1 s) and a wide linear range of 10 nM to 0.9 μM towards the detection of NO. Selective sensing of NO was studied in the presence of NO₃⁻, CO₂, NH₃ and other common physiological interferences.

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1 Introduction

Nitric oxide (NO), an endogenous free radical produced by a collection of enzymes known as nitric oxide synthases (NOS), is a physiological mediator of the cardiovascular, immune and nervous systems.¹ In the brain, NO is produced by neuronal NOS functions as a neurotransmitter and is involved in memory formation.² Conversely, the overproduction of NO is related to neurotoxicity in the brain tissue, such as tissue injury, cellular apoptosis, and ischemia.³ Furthermore, gas-phase NO levels in exhaled oral and nasal breath may be diagnostically useful for detecting asthma⁴ and sinus infections,⁵ respectively. In cancer biology, NO acts as a tumor progressor or suppressor, depending on its concentration and lifetime.⁶ Due to the short half-life time and low concentration, it is not easy to detect NO in biological samples. Moreover, NO produced by biological means reacts rapidly with oxygen and superoxides present in the bio fluids, which makes the detection of NO even more difficult. The location and concentration of NO governs its biological activity and hence establishing a valid analytical method for measuring NO at physiological pH is important due to its physiological significance. Among techniques available for NO sensing, such as Griess assay, chemiluminescence, electron spin and paramagnetic resonance spectroscopy, UV-vis spectroscopy, fluorescence and electrochemical methods,⁷ electrochemical techniques have an advantage over other techniques due to the small electrode size, real-time and direct analysis with high sensitivity.⁸ An ideal electrochemical sensor should be cheap, selective and highly sensitive towards the detection of NO. Efforts have been made to improve the sensor performance by way of decreasing the over potential of NO oxidation.

In this connection, nanoscale materials have received significant attention as electrochemical sensors.^9,10 Bi-metallic nanoparticles (NPs) comprising two mono-metallic counterparts in a single particle exhibit interesting electronic, optical and catalytic properties due to the bi-functional or synergistic effect induced by charge transfer from one metal to the other.^11,12 Among the different bi-metals, Au and Ag have gained increasing interest due to their unique optical and electronic properties.¹³ In particular, cluster-like structures are interesting because the properties of nanomaterials are influenced by their inter-particle distance and aggregation state and thus the collective properties are different from the properties of a single nanoparticle.

Our group has previously explored the possible analytical applications of bimetallic Au/Ag nanostructures embedded in polymer matrices.^12,14,15 Herein, we report the preparation of bi-metallic Au/Ag NCs embedded in PADA polymer matrix and the development of a modified electrode for selective and sensitive detection of NO. The mono-metallic Au and Pt NPs have been used as an electrochemical sensor material for NO detection.⁹ To the best of our knowledge, this is the first report on NO sensing using bimetallic Au/Ag NCs modified electrode. The bi-metallic Au/Ag NCs were synthesized in aqueous solution at room temperature without using seeds or surfactants. The present electrochemical sensor showed synergistic activity and selectivity towards the detection of NO with fast response time.

2 Experimental section

2.1 Materials and methods

Chloroauric acid (HAuCl₄), silver nitrate (AgNO₃), poly(acrylamide-co-diallyldimethylammonium chloride) (PADA), uric acid and D-glucose were received from Sigma-Aldrich. Pure CO₂ gas saturated 0.1 M KHCO₃ solution was used as CO₂ source. All other chemicals were analytical grade and received from Merck. All glassware was thoroughly cleaned with aqua regia (1 : 3 HNO₃/HCl v/v) (caution: aqua regia is a powerful oxidizing agent and it should be handled with extreme care) and rinsed extensively with distilled water before use. UV-visible absorption spectra were recorded using an Agilent Technologies 8453 spectrophotometer with a 1 cm quartz cell. High resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED) analyses were conducted in a JEOL JEM 2100 instrument operating at 200 kV. The specimen for the HRTEM analysis was prepared by dropping the colloidal solution onto carbon coated copper grid and dried at room temperature. Energy dispersive X-ray (EDX) analysis was carried out using a JEOL Model JSM-6390LV. FT-IR spectra were recorded using Thermo Scientific Nicolet 6700 FT-IR Spectrometer. Electrochemical characterization of mono- and bi-metallic Au/Ag NCs were performed by using a CH Instruments electrochemical workstation (Model-760D).

2.2 Synthesis of mono-metallic Au and Ag NPs

50 μL of 0.1 M aqueous solution of HAuCl₄ or AgNO₃ was added to 4.95 mL of 1% PADA solution under vigorous stirring. Then, 200 μL of 0.05 M freshly prepared ice-cold NaBH₄ was added slowly to the above solution. The color of the solution immediately turned into wine red for Au NPs and dark yellow for Ag NPs. The solution was further stirred for 3 h. A homogenous solution was obtained after 3 h without any aggregation which confirmed the successful formation of PADA-Au and PADA-Ag NPs.

2.3 One-step synthesis of bi-metallic Au/Ag NCs

PADA-Au₅₀Ag₅₀ NCs was prepared by using the following procedure. In a typical experiment, each 25 μL of 0.1 M solutions of HAuCl₄ and AgNO₃ was added to 4.95 mL of 1% PADA solution under vigorous stirring. Then, 200 μL of 0.05 M freshly prepared ice-cold NaBH₄ was slowly added to the above solution. The color of the solution immediately turned brown and the solution was further stirred for 3 h. The PADA-Au₂₅Ag₇₅ and PADA-Au₇₅Ag₂₅ NCs were prepared using corresponding molar ratios of 0.1 M solutions of HAuCl₄ and AgNO₃ by following the same procedure. The subscripts shown in Au₂₅Ag₇₅, Au₅₀Ag₅₀ and Au₇₅Ag₂₅ NCs refer to the percentage molar composition of the corresponding metal precursors.

2.4 Electrochemistry

All the electrochemical experiments were conducted in a single compartment three electrode cell using a CHI760D Electrochemical Workstation, CH Instruments, USA. A GC electrode (3 mm dia) and a platinum wire were used as working and counter electrodes, respectively. A calomel electrode was used as reference electrode. A 5 μL of prepared NCs solution was drop cast onto the cleaned GC surface and allowed to dry for 2 h at room temperature. The modified electrode was soaked in double distilled water for 5 min to allow swelling and used for electrochemical experiments. 0.1 M phosphate buffer solution (PBS) (pH 7.2) was used as electrolyte for electrocatalysis and sensor studies. Nitrogen gas was bubbled into the cell solution for 25 min prior to each experiment unless otherwise stated.

2.5 Preparation of NO saturated PBS

All glassware and PBS solution were bubbled with nitrogen gas prior to NO preparation. The NO saturated PBS was prepared using a reported procedure.¹⁶ Briefly, 2 M of H₂SO₄ was added dropwise to a saturated NaNO₂ solution leading to the production of NO gas through disproportionation reaction of NO₂⁻ ions in acidic solution. The produced NO gas was bubbled through a 5% (w/v) pyrogallol solution saturated with potassium hydroxide and then 10% (w/v) potassium hydroxide in order to remove other forms of nitrogen oxides. Finally, NO gas was bubbled in PBS and stored in nitrogen-protected environment. The NO standard solutions were prepared by making successive dilutions of the NO saturated PBS. Freshly prepared NO standard solutions were used for all experiments and the solutions were kept in a tightly closed glass flask and light-free septum wrapped in black foil. The concentration of saturated NO solution at 20 °C was reported as 1.8 mM.¹⁶

3 Results and discussion

3.1 Absorption spectral studies

The formation of mono-metallic Au, Ag NPs and bi-metallic Au/Ag NCs was primarily confirmed by recording their absorption spectra. Fig. 1 shows the absorption spectra obtained for mono- and bi-metallic Au/Ag NCs embedded in PADA matrix. The surface plasmon resonance (SPR) bands of mono- and bi-metallic Au/Ag NCs are strongly dependent on their size, shape, composition of Au or Ag NPs and their local environment.^17–20 Mono-metallic Ag and Au NPs embedded in PADA matrix showed their characteristic SPR absorption maximum (λ_max) at 406 (Fig. 1(a)) and 521 nm (Fig. 1(e)), respectively. Bi-metallic Au/Ag NCs prepared using different molar ratios of Au and Ag precursors showed their typical SPR absorption bands in between the mono-metallic NPs absorption. Au₇₅Ag₂₅ (Fig. 1(d)) and Au₅₀Ag₅₀ NCs (Fig. 1(c)) embedded in PADA matrix showed absorption bands at 515 and 496 nm, respectively. Au₂₅Ag₇₅ NCs embedded in PADA matrix showed a broad absorption band at 417 nm (Fig. 1(b)). The SPR band of the bimetallic Au/Ag NCs was blue-shifted with increasing concentration of Ag (shift is not linear) (Fig. S1†) and this observation suggests that the Au/Ag NCs may be an alloy²¹ or hybrid nanostructures. The absorption bands of mono- and bi-metal NCs were plotted against the gold mole fraction (x_Au) (inset figure in Fig. S1†). The observed broadness and blue shift in the SPR band of Au₂₅/Ag₇₅ NCs is attributed to the surface to surface interparticle plasmon coupling of NCs. The interparticle distance in Au₂₅/Ag₇₅ NCs is very low compared to that of other two bi-metal ratios, which is evidenced from the HRTEM images (Fig. 2(E)).^15,22 The electromagnetic field enhancement may be very large when two resonant particles are brought close to each other and most of the energy is located between the particles. As the surface electron density depends on the shape or arrangement of the metal atoms in nanoparticle, the electric field on the particle surface varies and this causes variations in the oscillation frequency of the electrons. This indicates that the dimensionality plays a crucial role in determining the position and shape of the SPR band. The observation of SPR band and the noticeable color change of the solutions suggest that the formed bi-metallic Au/Ag NCs are neither a core–shell nor a physical mixture of the mono-metallic Au and Ag NPs. It is evident from the literature that two distinguishable SPR bands were observed for physical mixture of mono-metallic Ag and Au NPs.²¹ As the absorption spectrum of PADA did not show any absorption band in the wavelength range of 300–1100 nm, the spectral bands obtained for the NCs are essentially due to SPR of Au and Ag NPs.

Fig. 1 Absorption spectra obtained for PADA-Ag NPs (a), PADA-Au₂₅Ag₇₅ NCs (b), PADA-Au₅₀Ag₅₀ NCs (c), PADA-Au₇₅Ag₂₅ NCs (d) and PADA-Au NPs (e) solutions.

Fig. 2 HRTEM images of PADA-Ag NPs (A), PADA-Au NPs (C), PADA-Au₂₅Ag₇₅ NCs (E), PADA-Au₅₀Ag₅₀ NCs (G) and PADA-Au₇₅Ag₂₅ NCs (H). SEAD patterns of PADA-Ag NPs (B), PADA-Au NPs (D) and PADA-Au₂₅Ag₇₅ NCs (F).

3.2 HRTEM and FT-IR studies

The morphology of the PADA stabilized mono-metallic Au, Ag NPs and bi-metallic Au/Ag NCs were studied by HRTEM analysis. It was observed that the well dispersed spherical Ag NPs (Fig. 2(A)) and Au NPs (Fig. 2(C)) with average particle sizes of 13.5 and 7 nm, respectively, were formed in the presence PADA matrix. Both Ag and Au NPs showed their characteristic SAED patterns and the d spacing values obtained from the SAED patterns correspond to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) crystal planes of Ag (Fig. 2(B)) and (1 1 1), (2 0 0) and (2 2 0) crystal planes of Au (Fig. 2(D)). When compared to the HRTEM images of the mono-metallic Ag and Au NPs, the bi-metallic Au/Ag NCs are smaller in size and have relatively narrow size distribution. The HRTEM images of PADA-Au₂₅Ag₇₅ NCs (Fig. 2(E)) reveal that closely associated spherical NCs are formed in the presence of PADA matrix with average particle size of 4.3 nm. The d spacing values obtained from the SAED pattern of PADA-Au₂₅Ag₇₅ NCs (Fig. 2(F)) correspond to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) crystal planes of Ag and Au. This observation suggests that the Au₂₅Ag₇₅ NCs were enriched with both Ag and Au contents. Furthermore, the presence of Au and Ag in the bi-metallic PADA-Au₂₅Ag₇₅ NCs were confirmed by EDX spectrum (Fig. S2†). The atomic percentage obtained from the EDX analysis was merely equal to that of the experimentally calculated atomic percentage of PADA-Au₂₅Ag₇₅ NCs. The HRTEM images recorded for PADA-Au₅₀Ag₅₀ (Fig. 2(G)) and PADA-Au₇₅Ag₂₅ (Fig. 2(H)) NCs also show the spherical shape particles with average particle size of 5.3 and 3.7 nm, respectively. From the HRTEM analysis of mono- and bi-metallic Au/Ag NCs embedded in PADA matrix, it is confirmed that bi-metallic NCs have smaller size and relatively narrow size distribution than the mono-metallic NPs. The Au/Ag NCs embedded in PADA matrix was further confirmed from the FT-IR spectra (Fig. S3†). The bands observed for PADA at 1665 and 3180 cm⁻¹ are due to the stretching vibrations of C=O and N–H bonds, respectively, and the bands at 1607 and 1458 cm⁻¹ are due to the bending vibration of N–H and C–H bonds,^23,24 respectively. In comparison to the PADA, mono- and bi-metallic Au/Ag nanostructures showed the same FT-IR spectra observed for PADA, which confirms that the prepared mono- and bi-metallic nanostructures are embedded in PADA matrix.

3.3 Electrochemical characterization

In order to understand the electrochemical characteristics of mono- and bi-metallic Au/Ag NCs embedded in PADA matrix, cyclic voltammograms were recorded for mono- and bi-metallic Au/Ag NCs modified GC electrodes in 0.1 M PBS. The characteristic oxidation and reduction peaks due to Au NPs present at the modified electrode were clearly observed at 0.950 and 0.414 V, respectively (Fig. 3(b)). Similarly, the characteristic oxidation peak was observed for Ag NPs at 0.252 V (Fig. 3(a)). In the case of GC/PADA-Au₂₅Ag₇₅ NCs modified electrode, the characteristic oxidation peak and the corresponding reduction peak were observed at 0.256 V and 0.077 V, respectively, for Ag and also the oxidation and the corresponding reduction peaks for Au were observed at 0.873 V and 0.392 V, respectively, along with the increment in the anodic and cathodic peak currents (Fig. 3(c)). The cyclic voltammograms recorded for the three different bi-metallic Au/Ag NCs modified electrodes are shown in Fig. S4.† The characteristic oxidation and reduction peaks obtained for Au and Ag at the GC/PADA-Au₂₅Ag₇₅, GC/PADA-Au₅₀Ag₅₀ and GC/PADA-Au₇₅Ag₂₅ modified electrodes confirmed the presence of Au and Ag in the bi-metallic NCs. The two distinguished redox peaks and the enhanced peak currents observed for bimetallic Au/Ag NCs suggest that the NCs are in good electrical contact with the electrode.²⁵ Furthermore, the two distinguished redox peaks characteristics of metals were usually absent for the alloy nanostructures^26,27 and the HRTEM studies confirmed that the bi-metallic Au/Ag NCs are not the core–shell structure. Therefore, it is understood that the synthesized bi-metallic Au/Ag NCs may be hybrid nanostructures consisting of both Au and Ag clusters fused together in a single particle (for a perfect alloy, it is supposed to be atom level fusion). The higher peak currents were attributed to the catalytically active hetero-junctions between Au and Ag nanoclusters and not as mono-metal entity. Moreover, the Au/Ag bi-metallic nanostructures modified electrode may enhance the redox peak currents due to the synergistic effect of Au/Ag.

Fig. 3 Cyclic voltammograms recorded for GC/PADA-Ag NPs (a), GC/PADA-Au NPs (b) and GC/PADA-Au₂₅Ag₇₅ NCs (c) modified electrodes in 0.1 M PBS at a scan rate of 50 mV s⁻¹.

3.4 Electrocatalytic oxidation of NO

The electrocatalytic oxidation of NO was carried out at the mono- and bi-metallic Au/Ag NCs modified electrodes in 0.1 M PBS (pH 7.2). Fig. 4 shows the comparison of cyclic voltammograms recorded for 100 μM NO at the GC/PADA-Ag NPs (a), GC/PADA-Au₂₅Ag₇₅ NCs (b), GC/PADA-Au₅₀Ag₅₀ NCs (c), GC/PADA-Au₇₅Ag₂₅ NCs (d), GC/PADA-Au NPs (e), GC/PADA (f) and bare GC (g) electrodes. As shown in Fig. 4, electrocatalytic oxidation of NO at all the modified electrodes via the following reactions were observed: NO–e⁻ → NO⁺, NO⁺ + OH⁻ → HNO₂ and HNO₂ → H⁺ + NO₂⁻.¹⁶ In the absence of NO, no oxidation peak was observed at the GC/PADA-Au₂₅Ag₇₅ NCs (Fig. 4(h)) modified electrode and in the presence of NO, an observable oxidation peak was observed at 0.90 V. When compared to the GC/PADA-Au₂₅Ag₇₅ NCs (Fig. 4(b)) electrode, the GC/PADA-Au NPs (Fig. 4(e)) electrode showed less intense oxidation peak current for NO at 0.95 V, whereas the GC/PADA-Ag NPs (Fig. 4(a)) electrode showed almost equal current intensity but the oxidation peak potential for NO was noticed at 1.01 V. The bare GC (Fig. 4(g)) electrode and GC/PADA (Fig. 4(f)) electrode showed NO oxidation at 1.01 and 0.95 V, respectively. The observed trend from Fig. 4 clearly explains the synergistic electrocatalytic effect of the bi-metallic Au₂₅Ag₇₅ NCs towards NO oxidation, when compared to the mono-metallic Au and Ag NPs. Hence, the observed synergistic catalytic effect of the GC/PADA-Au₂₅Ag₇₅ NCs modified electrode can be attributed to the suitable composition of Au and Ag, which is responsible for the observed higher catalytic current and reduced over potential towards the NO oxidation. Among the three different Au/Ag compositions, the Au₂₅Ag₇₅ NCs modified electrode exhibited better catalytic response towards NO. Furthermore, the GC/PADA electrode showed better catalytic response towards NO oxidation than the bare GC electrode, which reveals the electrocatalytic and/or pre-concentrating ability of the PADA matrix. The synergistic catalytic effect of Au₂₅Ag₇₅ NCs was further supported by the PADA matrix at the GC/PADA-Au₂₅Ag₇₅ NCs electrode towards NO oxidation.

Fig. 4 Cyclic voltammograms obtained for 100 μM NO at GC/PADA-Ag NPs (a), GC/PADA-Au₂₅Ag₇₅ NCs (b), GC/PADA-Au₅₀Ag₅₀ NCs (c), GC/PADA-Au₇₅Ag₂₅ NCs (d), GC/PADA-Au NPs (e), GC/PADA (f) and bare GC (g) electrodes in 0.1 M PBS at a scan rate of 50 mV s⁻¹. CV obtained in the absence of NO at GC/PADA-Au₂₅/Ag₇₅ NCs (h) electrode in PBS.

3.5 Amperometric sensing of NO and interference studies

The GC/PADA-Au₂₅Ag₇₅ NCs modified electrode was used to prepare the amperometric sensor for the detection of NO. The amperometric i–t curve was recorded at the GC/PADA-Au₂₅Ag₇₅ NCs modified electrode at an applied potential of 0.9 V upon each addition of 10 nM NO to the stirred solution of 0.1 M PBS (pH 7.2) (Fig. 5(A)). The amperometric responses observed at the GC/PADA-Ag and GC/PADA-Au NPs modified electrodes are shown in Fig. S5.† Comparison of the corresponding calibration plots (concentration of NO versus current) obtained for the GC/PADA-Ag NPs, GC/PADA-Au NPs and GC/PADA-Au₂₅Ag₇₅ NCs electrodes upon each addition of 10 nM NO are shown in Fig. 5(B). The GC/PADA-Au₂₅Ag₇₅ NCs electrode showed synergistic sensing response when compared to the mono-metallic Ag and Au NPs modified electrodes. The sensitivity of GC/PADA-Au₂₅Ag₇₅ NCs electrode was found to be 3.77 nA nM⁻¹ (Fig. 6(B)). The amperometric i–t curve response time for 10 nM NO addition at the GC/PADA-Au₂₅Ag₇₅ NCs electrode is represented in the inset of Fig. 5(A) and the response time was found to be 1 s, which is much lower than the physiological lifetime (∼5 s) of NO and also much lower than the reported NO detection systems (Table 1). This indicates the fast electron transfer process at the GC/PADA-Au₂₅Ag₇₅ NCs modified electrode.

Fig. 5 (A) Amperometric i–t curve obtained for NO at GC/PADA-Au₂₅Ag₇₅ NCs electrode during the successive addition of 10 nM NO to a solution of 0.1 M PBS (pH 7.2) at an applied potential of 0.9 V. Inset: amperometric i–t curve response time observed for 10 nM NO addition at the GC/PADA-Au₂₅Ag₇₅ NCs electrode. (B) Comparison of calibration plots of current versus NO concentration obtained for GC/PADA-Ag NPs (a), GC/PADA-Au NPs (b) and GC/PADA-Au₂₅Ag₇₅ NCs (c) electrodes.

Fig. 6 (A) Amperometric i–t curve obtained for NO at GC/PADA-Au₂₅Ag₇₅ NCs electrode during successive addition of various concentrations of NO to the solution of 0.1 M PBS (pH 7.2) at an applied potential of 0.9 V. (B) Corresponding calibration plot.

Table 1 Performance comparison of NO amperometric sensors based on GC electrode modified with metal NPs

Modified electrode	Linear range	Sensitivity	Response time (s)	S/N	Ref.
a RGO: reduced graphene oxide and TPDT: tri-amine functionalized silicate sol–gel matrix. b Nf: Nafion membrane. c MWNTs: multiwalled carbon nanotubes. d MP: microperoxidase, PTTCA: poly-5,2′,5′,2′′-terthiophene-3′-carboxylic acid, SOD: superoxide dismutase. e Gold nanoparticles.
GC/PADA-Au25/Ag75 NCs	10 nM to 0.9 μM	3.77 nA nM^−1	1	4.9	Present work
GC/RGO/Au–TPDT NRs^a	10 nM to 0.14 μM	0.598 nA nM^−1	2	—	29
GC/Nf–Au^b	1 nM to 500 μM	—	4	3	30
GCE/MWNTs-chitosan–Au NPs/Nf^c	19 nM to 54 mM	1.73 μA μM^−1	4	3	31
GCE/AuNPs/MWCNT-PTTCA/MP/SOD/catalase^d	1.0 mM to 40 mM	1.20 μA μM^−1	3.6	—	32
GCE/nano-Au	50 nM to 10 mM	∼0.5 μA μM^−1	0.5 s	3	33
GCE/chitosan–GNPs^e	36.0 nM to 43.2 mM	0.04 μA μM^−1	—	—	34

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Fig. 6(A) represents the amperometric i–t curve obtained for NO at the GC/PADA-Au₂₅Ag₇₅ NCs electrode during the successive addition of NO at different concentrations and the corresponding calibration plot (Fig. 6(B)) showed the linear response from 10 nM to 0.9 μM. In general, the bare electrode is often modified to eliminate interferences and to facilitate the selective detection of NO. The amperometric determination of NO in the presence of common physiological interferences such as glucose, urea, oxalate, sodium chloride, nitrate, CO₂ and NH₃ was studied at the GC/PADA-Au₂₅Ag₇₅ NCs electrode and the results are shown in Fig. 7. The interference study was carried out without purging with N₂ gas in PBS in order to check the interfering effect of oxygen. The addition of each 10 nM NO to the cell solution showed amperometric current response and further addition of each 100 μM of glucose (b), urea (c), oxalate (d) sodium chloride (e) and nitrate (f) followed by 1.5 μM of CO₂ (g) and 150 μM of NH₃ (h) separately at an interval of 40 s to the cell solution did not show any electrochemical response. The addition of 10 nM NO after the addition of all interferences clearly showed the amperometric current response. These observations reveal that the GC/PADA-Au₂₅Ag₇₅ NCs modified electrode is selective towards the determination of NO in the presence of excess physiological interferences as well as oxygen.

Fig. 7 Amperometric i–t curve obtained at GC/PADA-Au₂₅Ag₇₅ NCs electrode in aerated solution of 0.1 M PBS (pH 7.2) at each addition of 10 nM NO (a) and each 100 μM addition of common physiological interferences such as glucose (b), urea (c), oxalate (d), sodium chloride (e) and nitrate (f) followed by 1.5 μM of CO₂ (g) and 150 μM of NH₃ (h). The applied potential was 0.9 V.

The GC electrode is the most widely used electrode for NO detection due to its inertness, good conductivity, high hardness, high hydrogen over potential and stability. In addition, it can also be easily modified.²⁸Table 1 shows the comparison of previously reported NO sensors based on metal NPs modified GCE with the present modified electrode.

4 Conclusions

The polyelectrolyte PADA matrix stabilized bi-metal Au/Ag NCs was prepared and the development of a selective and sensitive sensing platform for NO using PADA-Au/Ag NCs was demonstrated. An amperometric technique was used to sense NO and the sensitivity was found to be 3.77 nA nM⁻¹. The sensing platform was also selective and high concentration of common physiological interfering agents did not interfere with the amperometric detection of NO. The presence of a polyelectrolyte PADA matrix favored the electrocatalytic oxidation of NO at the PADA-Au/Ag NCs modified electrodes. The performance of the sensing platform was accounted by the enhancement in the S/N ratio (∼4.9), fast response time (1 s) and the linear range from 10 to 900 nM. The electrocatalytic oxidation and electrochemical sensing of NO at the bi-metallic PADA-Au/Ag NCs modified electrode showed synergistic electrocatalytic activity, sensitivity and selectivity towards NO when compared to the corresponding mono-metallic Au and Ag NPs modified electrodes.

Acknowledgements

RR acknowledges the financial support received from the Science and Engineering Research Board (SERB), New Delhi. PV is the recipient of a Junior Research Fellowship under UGC-BSR scheme.

References

1. F. H. Epstein, S. Moncada and A. Higgs, N. Engl. J. Med., 1993, 329, 2002–2012.
2. S. H. Snyder, Curr. Opin. Neurobiol., 1992, 2, 323–327.
3. V. Calabrese, C. Mancuso, M. Calvani, E. Rizzarelli, D. A. Butterfield and A. M. G. Stella, Nat. Rev. Neurosci., 2007, 8, 766–775.
4. K. Alving, E. Weitzberg and J. M. Lundberg, Eur. Respir. J., 1993, 6, 1368–1370.
5. M. Jorissen, L. Lefevere and T. Willems, Allergy, 2001, 56, 1026–1033.
6. A. W. Carpenter and M. H. Schoenfisch, Chem. Soc. Rev., 2012, 41, 3742–3752.
7. E. M. Hetrick and M. H. Schoenfisch, Annu. Rev. Anal. Chem., 2009, 2, 409–433.
8. Y. Lee and J. Kim, Anal. Chem., 2007, 79, 7669–7675.
9. X. Dang, H. Hu, S. Wang and S. Hu, Microchim. Acta, 2015, 182, 455–467.
10. P. K. Rastogi, V. Ganesan and S. Krishnamoorthi, J. Mater. Chem. A, 2014, 2, 933–943.
11. T. Shibata, B. A. Bunker, Z. Zhang, D. Meisel, C. F. Vardeman and J. D. Gezelter, J. Am. Chem. Soc., 2002, 124, 11989–11996.
12. S. Manivannan and R. Ramaraj, J. Chem. Sci., 2009, 121, 735–743.
13. L. Wang, F. Wang, L. Shang, C. Zhu, W. Ren and S. Dong, Talanta, 2010, 82, 113–117.
14. S. Jayabal and R. Ramaraj, Appl. Catal., A, 2014, 470, 369–375.
15. S. Jayabal and R. Ramaraj, Electrochim. Acta, 2013, 88, 51–58.
16. S. L. Tinga, C. X. Guob, K. C. Leongc, D.-H. Kima, C. M. Lia and P. Chena, Electrochim. Acta, 2013, 111, 441–446.
17. L. M. Liz-Marzan, Mater. Today, 2004, 7, 26–31.
18. E. Hao, G. C. Schatz and J. T. Hupp, J. Fluoresc., 2004, 14, 331–341.
19. X. Liu, R. Huang and J. Zhu, Chem. Mater., 2008, 20, 192–197.
20. K. Jia, M. Y. Khaywah, Y. Li, J. L. Bijeon, P. M. Adam, R. Déturche, B. Guelorget, M. François, G. Louarn and R. E. Ionescu, ACS Appl. Mater. Interfaces, 2014, 6, 219–227.
21. S. Link, Z. L. Wang and M. A. El-Sayed, J. Phys. Chem. B, 1999, 103, 3529–3533.
22. J. J. Mock, D. R. Smith and S. Schultz, Nano Lett., 2003, 3, 1087–1090.
23. A. Akelah, P. Kelly, S. Qutubuddin and A. Moet, Clay Miner., 1994, 29, 169–178.
24. W. P. McKenna and G. Apai, J. Phys. Chem., 1992, 96, 5902–5907.
25. G. Maduraiveeran and R. Ramaraj, J. Electroanal. Chem., 2007, 608, 52–58.
26. L. Ur-Rahman, A. Shah, S. Bahadar Khan, A. M. Asiri, H. Hussain, C. Han, R. Qureshi, M. N. Ashiq, M. A. Zia, M. Ishaq and H.-B. Kraatz, J. Appl. Electrochem., 2015, 5, 463–472.
27. S. N. Mailu, T. Waryo, P. M. Ndangili, F. R. Ngece, A. A. Baleg, P. G. Baker and E. I. Iwuoha, Sensors, 2010, 10, 9449–9465.
28. T. Xu, N. Scafa, L.-P. Xu, L. Su, C. Li, S. Zhou, Y. Liu and X. Zhan, Electroanalysis, 2014, 26, 449–468.
29. S. Jayabal, P. Viswanathan and R. Ramaraj, RSC Adv., 2014, 4, 33541–33548.
30. S. Thangavel and R. Ramaraj, J. Phys. Chem. C, 2008, 112, 19825–19830.
31. X. Deng, F. Wang and Z. Chen, Talanta, 2010, 82, 1218–1224.
32. A. A. Abdelwahab, W. C. Koh, H. B. Noh and Y. B. Shim, Biosens. Bioelectron., 2010, 26, 1080–1086.
33. Y.-J. Li, C. Liu, M.-H. Yang, Y. He and E. S. Yeung, J. Electroanal. Chem., 2008, 622, 103–108.
34. W. Fang, D. Xiaocui, W. Wei and C. Zilin, J. Solid State Electrochem., 2011, 15, 829–836.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra06098b

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