Monitoring of Anthracene Using Nanoscale Au-Cu Bimetallic Alloy Nanoparticles Synthesized with Various Compositions.

Bimetallic alloy Au-Cu nanoparticles (Au-Cu alloy NPs) were synthesized using a chemical reduction method for sensing applications. Electronic absorption spectroscopy (UV-visible spectroscopy), X-ray diffraction (XRD), and scanning electron microscopy (SEM) were used for the confirmation and morphological studies of the synthesized nanoparticles. The composition of Au-Cu alloy NPs was studied by energy-dispersive spectroscopy (EDS). The high crystallinity of Au-Cu alloy NPs was demonstrated by XRD analysis. Both XRD and SEM analyses revealed that the nanoparticles' size ranges from 15 to 25 nm. Pyrrole was polymerized into polypyrrole (PPy) over a neat and clean glassy carbon electrode (GCE) by potentiodynamic polymerization. The sensitivity of GCE was improved by modifying it into a composite electrode. The composite electrode was developed by coating GCE with an overoxidized PPy polymer followed by Au-Cu alloy NPs. The ratio of Au and Cu was carefully controlled. The composite electrode (PPyox/Au-Cu/GCE) successfully detected an environmental toxin anthracene with a detection limit of 0.15 μM, as evidenced by cyclic voltammetry (CV), square-wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS).

Introduction

For the synthesis of bimetallic alloy nanoparticles (BMANPs), a number of protocols are available. Among the protocols, chemical reduction is the most convenient and simple but more accurate method to follow.[1] Ag–Cu alloy nanoparticles were synthesized by Rahman et al. for environmental applications.[2] However, particles with a narrow distribution and small size could not be synthesized via the protocols. To overcome these drawbacks, the chemical reduction method has become a useful choice in the synthesis of finely tuned bimetallic alloy nanoparticles. One of the chemical reduction methods that is more convenient, economically favorable, and pollution-free is the polyol process.[2] Cu has a high electrical conductivity and acts to quench the emission of light, additionally being cost-effective. Au is inert and nontoxic, having a higher affinity for Cu to make a bimetallic alloy of Au–Cu, thus preventing corrosion of Cu.[3] These Au–Cu bimetallic alloy nanoparticles have outperformed monometallic nanocrystals in their properties owing to their improved electronic, optical, and catalytic performances.[3] Thus, Au–Cu alloy nanoparticles were prepared by a method in which ethylene glycol performs dual function, i.e., acts as a solvent to dissolve the precursor salts of Au and Cu as well as a reducing agent to chemically reduce Au3+ and Cu2+ to their respective metals.[3]

Polycyclic aromatic hydrocarbons (PAHs) are a class of homocyclic organic compounds, which do not contain elements other than carbon in their ring skeleton.[3] Trace amounts of these compounds are naturally available (in some plant extracts), but they mostly enter the atmosphere due to anthropogenic activities. Moreover, these compounds are usually found in coal reservoirs and also dumped into the environment as byproducts during partial burning of coal. They are widely present in areas where chemical industries and vehicular emissions are abundant.[3] Also, sewage waste water from petroleum, coal tars, and perfume industries contains PAHs with high concentrations.[4] Combustion of plants as fuel in desert areas is another source of PAHs. Existence of PAHs in the environment is a serious problem because they are hazardous organic compounds, with teratogenic, carcinogenic, and mutagenic effects. They cause skin and lungs cancers and retard activities of growth and sex hormones.[5] In addition, they adversely influence the reproduction processes and cause developmental toxicity in some animals.[5] They disturb the digestive and respiratory systems of human beings. Moreover, PAHs contribute majorly to environmental contamination as reported by the Environmental Protection Agency (EPA).[6] They affect the growth and thickness of hairs as well as alimentary canal. They also cause heartburn diseases in human beings.[7] EPA has declared PAH contamination in the environment as a global problem. Emergency steps should be taken to monitor PAHs for keeping a clean and safe environment.[7] Therefore, there is an urgent need to develop a reliable and fast technique to monitor and control PAHs in the environment, especially the atmosphere.

PAHs can be determined in the atmosphere with different methods, including UV–visible spectroscopy, fluorescence spectroscopy, capillary electrophoresis, gas chromatography, and high-performance liquid chromatography.[8,9] Even though these methods are commonly used, they require high cost, well-trained persons, and a long processing time for operation. More importantly, since these methods need large quantity of samples, they may not be used for the detection of PAHs at trace levels of concentration. Researchers have focused on the use of electrochemical sensing devices as a further advanced tool in the detection of trace-level PAHs due to their easy sensing setup, fast response, and smooth operation even by a semiskilled operator.[10] There is also a need to emphasize the use of small samples and high sensitivity and selectivity of the electrochemical sensors. However, electrode fouling is the main problem of electrochemical sensors during PAH detection. To overcome this, polypyrrole (PPy) is overoxidized to PPyox and then PPyox is coated on the surface of the glassy carbon electrode (GCE).[11] Thus, the PPyox-modified electrode can successfully detect PAHs in trace levels.[12] In addition, the detection power of the polymer-coated electrode can be improved with a modification using bimetallic alloy nanoparticles.[13] The alloy nanoparticles and polymer-coated electrode will not only lower the detection limits but also minimize the electrode fouling to a greater extent.[14]

PPy is a conducting polymer that shows high sensitivity toward the aromatic ring. It also provides nanoporous structures for immobilization of alloy nanoparticles.[15] Currently, the PPy-based BMANPs have been receiving special attention due to its numerous applications in various fields. It is also observed that researchers are interested in employing PPy as a capping agent for BMANPs because of its high conductivity and environmental stability.[16] Therefore, the improved properties of synthesized PPy/Au–Cu nanocomposite electrode can be useful for developing functional electrochemical sensors to monitor and detect carcinogenic, polycyclic aromatic hydrocarbons (PAHs).[17] Also, recent studies have shown that the PPy and BMANP composite electrode (electrochemical sensor) has high conductivity based on its excellent sponge-like morphology for the detection of volatile organic compounds and environmental toxins.[18] Electrodeposition is an effective way to make PPy and Au–Cu composite films with a large variety of tunable parameters.[19] Additionally, PPys have been prepared to enhance its electrochemical capacitance performance.[20] PPy, polythiophene, and poly ortho aminophenol represent a group of conjugate-electron materials that can detect a combination of various carcinogenics.[21] A new type of crystalline porous materials, metal–organic frameworks (MOFs), have attracted significant attention because of their unique properties such as high surface area and high pore volume with a uniformly distributed size.[22] Electrochemical synthesis of PPy and Au–Cu nanoparticle composites in which nanomaterials are dispersed in PPy is the best way to detect environmental toxin anthracene.[23] To the best of our knowledge, no previous report is available for the detection of a representative PAHs, i.e., anthracene in trace-level concentration, using composite electrode of Au–Cu. This article seeks the synthetic protocol of Au–Cu bimetallic nanoparticles with various compositions of Au and Cu. This article also demonstrates detection of anthracene using Au-, Cu-, PPyox-, and Au–Cu-modified GCE. The GCE coated with PPyox and modified with various compositions of Au–Cu alloy nanoparticles is also investigated. The PPyox-coated GCE modified with Au–Cu alloy nanoparticles having a Au-to-Cu ratio of 1:3 is found to be most effective for PAH sensing. The composite electrode (PPyox/Au–Cu 1:3 NPs/GCE) demonstrates excellent performance for the detection of the worst toxin anthracene with a lower limit of detection (Table ). The composite electrode may also be used to monitor penthracene and pyrene in trace levels.[24]

Table 1

Experimental Conditions Required for the Preparation of Various Compositions of Au–Cu Alloy Nanoparticles

s. no.	ratio of Au–Cu	volume of AuCl3 (mL)	volume of CuCl2 (mL)	PVP (mL)	purging (min)	heating at 175 °C (min)
1	3:1	6	2	4	35	15
2	2:1	4	2	3	30	20
3	1:1	3	3	3	30	25
4	1:2	2	4	3	40	30
5	1:3	2	6	4	45	60

Results and Discussion

Electronic Absorption Studies of Au, Cu, and Au–Cu Nanoparticles

Electronic absorption spectra of the synthesized NPs were recorded. Monometallic NPs of each Au and Cu demonstrated the maximum absorption at wavelengths 455 and 558 nm, respectively. Their λmax is closer to the reported values (450 and 560 nm for Au and Cu, respectively).[25] The signals of the Au–Cu alloy nanoparticle spectrum were observed between the spectra of pure Au and Cu nanoparticles (i.e., λmax at 485 nm), indicating the formation of Au–Cu bimetallic alloy NPs (Figure A) by the synthesis method. Figure B demonstrates the spectral response of various compositions of Au–Cu alloy NPs. A bathochromic shift is observed with an increase in the composition of Cu in the Au–Cu alloy (Figure B). It also confirmed the formation of bimetallic Au–Cu alloy NPs by the synthesis method.

Figure 1

UV–visible spectra: (A): Au (a), Au–Cu (b), and Cu (c); (B): Au–Cu 3:1 (a), Au–Cu 1:1 (b), and Au–Cu 1:3 (c) alloy nanoparticles.

Composition and Morphology of Au–Cu Nanoparticles

Composition and morphology of the alloy nanoparticles were examined with X-ray diffraction (XRD). The obtained XRD spectrum was used to calculate the size of the Au–Cu alloy NPs using the Debye–Scherrer formula. The XRD patterns of all of the samples showed that the position of Au signal shifted when the Cu ratio increased during the synthesis.[26] It indicates that more Cu was incorporated into the Au–Cu alloy NPs, with a high loading of Cu precursor, as shown in the Supporting Information. Three intense peaks were observed for Au–Cu 1:3 at 36, 42, and 65° corresponding to 111, 200, and 210 for Au–Cu bimetallic alloy NPs (Figure ). These three peaks were used to calculate the size of alloy NPs based on the Debye–Scherer formula (eq ).The XRD signal also confirmed that the sample contains two metals, i.e., Au and Cu only. The size changed due to different Cu loading in the samples.[27] The sizes of bimetallic alloy nanoparticles calculated are tabulated in Table . Energy-dispersive spectroscopy (EDS) highlights various compositions of Au and Cu in Au–Cu alloy NPs (Figure A–C).

Figure 2

XRD pattern of Au–Cu (1:3) alloy nanoparticles synthesized by the polyol process from AuCl3 and CuCl2 in the presence of PVP, which is used as a capping agent.

Table 2

Size of Nanoparticles Calculated from SEM and XRD

s. no.	Au–Cu	average size (nm)
1	3:1	25
2	2:1	23
3	1:1	22
4	1:2	19
5	1:3	15

Figure 3

EDS images of Au–Cu 3:1 (A), Au–Cu 1:1 (B), and Au–Cu 1:3 (C) alloy nanoparticles.

Morphological studies of Au–Cu alloy nanoparticles were also carried out by SEM analysis. The sizes of the samples are not completely uniform, as evidenced by the SEM images of the samples (Figure A–E). However, the sample Au–Cu 1:3 showed higher uniformity in shape compared to other samples (Figure E). The mismatch in the spacing shows that Au and Cu have very small diameters and closed lattice parameters.[28] Hence, the sample Au–Cu 1:3 is more suitable to be used for the development of electrochemical sensor for detection of anthracene. The sizes of bimetallic alloy NPs determined from XRD and SEM are shown in Table . Interestingly, the sizes obtained from both techniques are quite similar. Table shows that with an increase in composition of Cu in the Au–Cu alloy, the size of bimetallic alloy NPs decreases and the smallest size for Au–Cu 1:3 (15 nm) is observed. This decrease in size will cause an increase in their surface area. Thus, the sample Au–Cu 1:3 with the smallest size having more surface area has been chosen as the best to detect anthracene.

Figure 4

SEM images of Au–Cu 3:1 (A), Au–Cu 2:1 (B), Au–Cu 1:1 (C), Au–Cu 1:2 (D), and Au–Cu 1:3 (E) alloy nanoparticles coated at accelerating voltages between 10 and 20 kV.

Electrochemical Behaviors of Au–Cu Nanoparticles

Electrochemical characterization of alloy nanoparticles was carried out using cyclic voltammetry (CV), square-wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). CVs of Au NPs/GCE, Cu NPs/GCE, and Au–Cu 1:3 NPs/GCE were obtained at a potential scan rate of 50 mV/s in a deoxygenated medium of pH 7 using a 0.1 M phosphate buffer. The comparison of CVs reveals that Au registers no anodic peak while Cu/GCE and Au–Cu NPs/GCE show pronounced anodic signals (Figure A). The current intensities of the oxidation signals of Cu and Au–Cu 1:3 NPs are summarized in Table .

Figure 5

CVs of Au–Cu ANPs showing an increase in the anodic peak with increasing scan rate (A); plot of current versus scan rate showing a linear relationship between current and scan rate (B).

Table 3

Peak Potential and Anodic Current Response Data of the Designed Sensor

electrode	Epa (mV)	Ipa (μA)
Cu	8	35
AuCu13	13	62
PPyox/GCE	10	40
Au–Cu 1:1 NPs/GCE	11	55
Au–Cu 1:2/Ppyox/GCE	12	65
Au–Cu 1:3/Ppyox/GCE	15	80

It is revealed that the anodic peak current increases when scan rate is accelerated, and the value reaches to optimum 100 mV/s scan rate (Figure B). Direct proportion between the current intensity of signals and potential scan rate offers evidence of the stitching of nanoparticles and conducting polymer at the surface of the GCE and the lack of their leaching to the electrolytic solution (Figure B). This demonstrates that the electron transfer process is adsorption-controlled, having no contribution of diffusion-controlled processes.[29]

Detection of Anthracene Using a Au–Cu NP-Based Electrochemical Sensor

Figure A shows no peak for bare GCE and a clear oxidation (anodic) peak for GCE coated with Cu NPs (a) and GCE coated with Au–Cu 1:3 (b). Also, an irreversible oxidation peak for Cu nanoparticles at 7.0 mV appeared, as seen in Figure A. No pronounced oxidation peaks can be observed by Au nanoparticles. Au–Cu 1:3 NPs/GCE gives an oxidation peak at 12 mV. A large oxidation peak is evidenced by Au–Cu 1:3 alloy nanoparticles, at the potential ranging from 0 to 20 mV. The appearance of this peak negates the presence of metal domain exclusively for Au and Cu but gives a clue to the atomic mixture (alloy) of Au and Cu.

Figure 6

(A) CV curves obtained with different working electrodes, i.e., bare GCE, GCE modified with Cu (a), and Au–Cu alloy nanoparticles (b) stabilized with PVP. (B) Amperometric response of GCE and GCE modified with various ratios of Au–Cu alloy nanoparticles in the highest anodic current response shown by Au–C 1:3 for the sensing of 1 μM anthracene.

Figure B shows the amperometric response of GCE and GCE modified with various ratios of Au–Cu alloy NPs. The highest anodic current response was shown by Au–Cu 1:3 when dipped in 1 μM anthracene. The anodic peak current was enhanced to 62 μA when GCE was modified with Au–Cu 1:3 alloy nanoparticles compared to Cu NP-coated GCE anodic peak current (35 μA). Polypyrrole was overoxidized on the surface of GCE to avoid its susceptibility to a nucleophilic attack.[30]Figure B also indicates that the polymer-coated GCE showed lower anodic peak currents (40 μA) than bimetallic nanoparticle-coated glassy carbon electrodes in anthracene solution. The negative charge on the overoxidized polypyrrole (PPyox) film hindered the electron transfer process, causing a decrease in the anodic peak current. The negatively charged layer of the PPyox film was not suitable for the approach of ferricyanide anions. However, when Au–Cu 1:3 NPs were coated on already overoxidized polymer-coated GCE, the anodic peak current was increased to 80 μA in the presence of 1 μM anthracene. It may have resulted from larger surface area and good conductivity of Au–Cu 1:3 alloy NPs, which provides more active centers for electron transfer processes to happen. As a result, much more of the K3Fe(CN)6 is accommodated at the composite electrode. The overoxidation may bring the conducting polymer (having pores) at the surface that facilitates the attachment of nanoparticles.[31,32]

Interestingly from Figure B, it is observed that the composite electrode (Au–Cu 1:3 NPs/PPyox/GCE) showed about 100% enhancement in the anodic peak current (80 μA) compared to the anodic peak current shown by PPyox-coated electrode (40 μA) during the detection of anthracene (Table ). The PPyox provides better sites for the attachments of Au–Cu 1:3 alloy nanoparticles that yielded a fruitful increase in the surface area, accelerating the kinetics involved in the detection mechanism.[33] This resulted in a rapid increase in the anodic peak that is an important finding of our research work. It is claimed that no such huge enhancement in the anodic peak current has been observed before this study.

The lowest concentration of anthracene was sensed using an electrochemical sensor employing the square-wave voltammetry (SWV) technique. Applying this electrochemical technique, the lowest concentration of anthracene (0.15 μM) was detected (Figure A). Figure B shows a plot of oxidation peak current versus concentration of anthracene. It shows a direct proportion with a positive potential shift.[34]

Figure 7

Square-wave voltammetry of Au–Cu 1:3 ANPs showing increase in the anodic peak with increasing concentration of anthracene (A); plot of current versus anthracene concentration showing the lowest possible concentration of anthracene, i.e., 0.15 μM at 7 mV (B).

Electrochemical Impedance Studies of Au–Cu NP-Based Sensor

More informative results were achieved, and the roles of Au–Cu 1:3 alloy NPs for the detection of anthracene were studied by applying an electrochemical impedance spectroscopy (EIS) technique. Using this technique, the detection limit of nanoparticle-modified GCE, PPyox-modified GCE, and a composite electrode (NP- and polymer-coated GCE) for anthracene sensing was studied. EIS data demonstrate that the nanoparticles cause prominent changes in values of charge transfer resistance (Rct), which enhances the anodic peak current (Figure ). This figure clarifies that Rct for bare GCE is high (7.91 × 103 Ω), showing the lowest electron transfer process. PPyox-coated GCE showed a slight decrease in the Rct valve (7.10 × 103 Ω) due to the negatively charged PPyox offering resistance to the layer of ferrocyanide anions.[34,35] A decrease in the Rct value and accelerating behavior of PPyox-coated GCE toward the electron transfer process was observed (Figure b). A further decrease in the Rct value (4.66 × 103 Ω) was observed for Au–Cu 1:3 alloy nanoparticle-coated GCE (Figure c). It shows that alloy nanoparticles were further accelerating the electron transfer process. The composite electrode shows the largest decrease in the Rct value (1.95 × 103 Ω) (Figure d). Au–Cu 1:3 alloy nanoparticles along with the PPyox further accelerate the electron transfer process and improve the sensing nature of the composite electrode (electrochemical sensor) for the detection of anthracene. The EIS data are in good agreement with CV and SWV.

Figure 8

Nyquist plots of the EIS recorded in the presence of [Fe(CN)6]3 redox system in aq. KCl (0.1 M) for bare GCE (a), PPyox/GCE (b), Au–Cu 1:3 NPs/GCE (c), and PPyox/Au–Cu 1:3 NPs/GCE (d).

Stability and Reproducibility of Au–Cu NP-Based Electrochemical Sensor

The stability and reproducibility of the composite electrode were investigated. The reproducibility of the composite electrode was tested in the presence of 1.46 × 10–4 M anthracene in acetonitrile and 0.1 M LiClO4. The relative standard deviation of 1.2% (n = 6) shows that the response of the composite electrode for the 1.46 × 10–4 M concentration of anthracene was checked after every 10 days by SWV. It gives approximately the same concentration, confirming the electrode’s stability.

Conclusions

Bimetallic alloy nanoparticles of Au–Cu were synthesized by a convenient, environmentally friendly, and low-cost method. Various compositions of Au–Cu alloy nanoparticles were manufactured and used for the development of composite electrodes to detect the environmental toxin anthracene. CVs, SWV, and EIS were used for testing the electroanalytical characteristics of the developed sensors. The best electrochemical response of the analyte was observed by coating Au–Cu NPs with the ratio of 1:3 on the surface of GCE along with PPyox, where pyrrole was potentiodynamically polymerized to polypyrrole. The excellent electrocatalytic role of Au–Cu NPs (1:3) is related to their high surface area. The electrocatalytic properties of the designed sensor using bimetallic alloy nanoparticles were found to be more effective compared to monometallic nanoparticles. The developed sensor exhibited a favorable sensing response for anthracene in the form of robust voltammetric signal with a detection limit of 0.15 μM. Using Au–Cu (1:3) nanoparticle-based electrochemical sensor, it is suggested that PAHs other than anthracene may also be sensed, which needs further intensive work.

Methods

Gold chloride (AuCl3), copper chloride (CuCl2), polypyrrole (PPy), poly(vinyl pyrrolidone) (PVP), and ethylene glycol (C2H6O2) were obtained from Thermo Fisher Scientific, Inc. and used as received. Analytical-grade lithium chlorate (LiClO4), potassium chloride (KCl), anthracene (C14H10), acetonitrile (C2H3N), and potassium ferrocayanide K4[Fe(CN)6] were purchased from Acros Organics. A microwave oven (model: MG 605 AP) was used for the preparation of Au, Cu, and Au–Cu alloy NPs.[36]Table summarizes the experimental conditions required for the synthesis Au–Cu alloy NPs (with various compositions).

Monometallic (Au or Cu) NPs were synthesized by a chemical reduction method, known as the polyol process. In the polyol process, ethylene glycol was used as a solvent and a reducing agent. Copper NPs were prepared by mixing 10 mL of CuCl2 (1 mM) and PVP (1 mM) followed by purging with argon gas for 30 min. The purged sample was heated at 175 °C for 20 min. The formation of Cu NPs was ensured when a blackish-brown color appeared. UV–visible spectrophotometry was used for further confirmation of Cu NP production. The reduction mechanism of Cu2+ to Cu by ethylene glycol is shown in Scheme .

Scheme 1

Reduction of Cu2+ by Ethylene Glycol

Similarly, Au NPs were synthesized by mixing 10 mL of AuCl3 (1 mM) and 10 mL of PVP (1 mM). The sample was kept in an oven at 125 °C for 20 min. The appearance of reddish-brown color indicated completion of the process. Electronic absorption spectroscopy was used for the confirmation of the formation of Au nanoparticles. The overall reduction reaction showing the conversion of Au3+ to Au is presented in Scheme .[37]

Scheme 2

Mechanism Showing the Reduction of Au3+ by Ethylene Glycol

Bimetallic (Au–Cu) alloy nanoparticles (BMANPs) were prepared by adding 5 mL of CuCl2 (1 mM) to 5 mL of PVP (1 mM). The medium was deoxygenated by blowing argon gas for 30 min. The mixture was heated at 100 °C for 20 min. HAuCl4 (5 mL, 1 mM) was added to the purged sample at 100 °C. Immediately, the color of the mixture became black, and after 10 min, it turned yellowish-brown. This color change indicated the formation of Au–Cu alloy nanoparticles with a ratio of 1:1. This is how BMANPs of various Au and Cu ratios were manufactured from their respective precursor salts. For all compositions, the same protocol was applied, but the ratio was varied accordingly. The amount of PVP was also changed, nevertheless keeping it half of the total mixture volume (one sample). The time required for heating and purging in the synthesis was found to be directly proportional to the amount of Cu in samples, as shown in Table .

Sample Preparation for Characterization Techniques

1 mM solutions of all of the samples were prepared. These specific concentrations were chosen because the UV–vis spectra gave distinct peaks with no noise. The cells were cleaned thoroughly with chromic acid and then with distilled water. The baseline was set for ethylene glycol used as the solvent and then for each concentration of the samples. The recorded spectra were collected, and after each operation, the cells were washed regularly so as to minimize the possibility of impurity.

All of the samples prepared were centrifuged for 30 min by centrifugation instrument at 6000 rpm, which were kept overnight at 25 °C at a constant temperature in an oven for drying. Powdered alloy nanoparticles were prepared and washed many times with acetone for the complete removal of PVP. They were then scratched from the tubes for XRD and high-vacuum SEM studies. For XRD, a normal scan rate of 1 deg 2θ per min was applied. For SEM analysis, PPy fabricated alloy nanoparticles were coated at acceleration voltages between 10 and 20 kV for coating analysis.

The glassy carbon electrode (GCE) was cleaned with distilled water. Then, it was placed in a colloidal solution of Au–Cu alloy nanoparticles for 10 min. Extreme care was taken during the synthesis and deposition processes because even a minute quantity of contamination or any other disturbances could cause nonuniformity of the deposition, resulting in a drastic change in the data.[38]

Pyrrole was polymerized and overoxidized on well-polished GCE. The polymer-coated electrode was kept in the colloidal solution of Au–Cu alloy nanoparticles until the formation of a uniform layer over the surface of the electrode is completed. The alloy NPs were uniformly coated upon polymer-fabricated GCE, and this electrode was called composite electrode (PPyox/Au–Cu NPs/GCE). The GCE was also modified with monometallic NPs of Au and Cu denoted as Au NPs/GCE and Cu NPs/GCE, respectively. For reference, Au–Cu (1:3) NP-coated GCE (Au–Cu NPs/GCE) was also prepared. This modified electrode was developed just for comparing the sensing power of the PPyox-modified electrode and BMANP-modified electrode. Electrochemical impedance spectroscopy was carried out for Au–Cu (1:3) NPs with the same concentration.