The voltammetric measurements were recorded by employing the electrochemical work station CHI-6038E (CH. Instruments, USA) with the three electrode compartment, and the analyser was connected to a personal computer for the control and storage of the data. BNPCPE, P(DLPA)MNPMCPE served as the working electrode, the platinum electrode was kept as an auxiliary electrode, and an aqueous saturated calomel electrode was employed as a reference electrode. The different pH solutions were prepared using the EQ-160 digital pH device. The double-distilled water was acquired by VITSIL-VBSD/VBDD water purifier instrument. FE-SEM (operating in keV) data for the surface study of P(DLPA)MNPMCPE and BNPCPE were received from DST-PURSE Laboratory, Mangalagangotri, Mangalore University, India. XRD data (Co Kα radiation) of the nanoparticle was collected from JSSSTU Mysore, India.

AN was acquired from Tokyo Chemical Industry Company Ltd., Japan. Graphite powder and Silicone oil were procured from Nice Company, India. Monosodium hydrogen phosphate (NaH₂PO₄), Disodium hydrogen phosphate (Na₂HPO₄) and DL-Phenylalanine were obtained from Molychem, India. The chemicals that were utilized in this experiment were of analytical grade and used without any purification. The AN stock solution (25×10^-4 M) and DL-Phenylalanine (25×10^-4 M) were prepared in distilled water. Appropriate amounts of 0.2 M NaH₂PO₄.H₂O and 0.2 M Na₂HPO₄.H₂O were mixed for the experiments at proper pH, and used as the supporting electrolytes. All the experiments were conducted at the lab temperature (25 ± 1°C).

DyCuO nanoparticle was synthesized by the simple co-precipitation method. Briefly, 50ml of 0.05 M (DyNO₃)₂ and 50 ml of 0.1 M Cu(NO₃)₂ were prepared using triple distilled water and mixed in a 1:1 ratio to yield a homogeneous mixture. Subsequently, 0.1 M NaOH solution was added dropwise to attain an approximate pH of 10 until the precipitate has been formed. The precipitate was kept for magnetic stirring for about 3 to 4hrs at 80°C. The resultant precipitate was washed with distilled water and ethanol several times and dried in a hot air oven. Finally, the solid product was heated in a muffle furnace at 650°C for 6 hrs and was used for further experimental procedures.

A homogeneous paste was prepared by blending the graphite powder, DyCuO NP and silicone oil in the proportion of 60:10:30 (w/w) by grinding it in an agate mortar. A Teflon tube of 3 mm diameter was taken, which consisted of a copper wire for the electrical bonding, and the paste thus obtained was tightly packed into the crater of the Teflon tube; smoothening was done by rubbing the surface of the tube on the filter paper, and thus, an unmodified electrode has been obtained as a result. The modified electrode was constructed by the method of electropolymerization of DL-Phenylalanine in supporting electrolyte of optimum pH, which was swept continuously for 10 cycles on the surface of the unmodified electrode. Finally, the electrode was rinsed with distilled water to remove the physically unadsorbed DL-Phenylalanine. The electrode was coined as P(DLPA)MNPCPE, and the modified electrodes were utilized for the electrochemical detection of AN throughout the experiment.

Among various modification methodologies employed, electropolymerization is a simple method for altering the CPE’s electrocatalytic activity. Hence, electropolymerization of DL-Phenylalanine was performed in 0.2 M supporting electrolyte of pH 7.0 at a constant sweep rate of 0.1 Vs^-1, keeping the potential window at 0 V to 1.6 V by running 20 continuous segments of CV (Fig. 1a). As can be inferred from the Cyclic Voltammograms (CVs), the layer of the electropolymerisation intensifies by escalating the CV cycles signifying that the poly DLPA covers the BNPCPE film. Furthermore, in order to gain the highest response of the analyte, the electropolymerisation was conducted by differing polymerization cycles from 5 to 20. The bar graph of peak current with respect to electropolymerization cycles (Fig. 1b) confirms the enhancement of peak current till the 10th cycle. As we moved beyond the 10th cycle, the peak current diminished gradually due to the electron transfer of AN at P(DLPA)MNPMCPE in an inappropriate proportion. Hence, the number of sweep cycles for the electropolymerization was kept constant for 10 cycles.

To analyse the surface morphology of the designed electrode, FE-SEM technique was employed for the prepared electrodes. The captured FE-SEM images for the different electrodes are displayed in Fig. (2). The flakes of the graphite and nanoparticles have been observed from the FE-SEM images of the BCPE (Fig. 2a) and DyCuO nanoparticles (Fig. 2b and c) portrays the FE-SEM of BNPMCPE containing both flakes and particles. The FE-SEM photos of P(DLPA)MNPMCPE reveal that poly DLPA covers all the surface of BNPMCPE. Thus, the FE-SEM analysis indicates that the bare electrode was successfully modified by electropolymerisation. The crystalline nature of the material was analysed using XRD. The XRD (Fig. 3) of the DyCuO nanoparticles was performed which depicted the polycrystalline pattern of the CuO phase. The distinctive diffraction peaks at 2θ 29.32⁰, 35.77⁰, 39.01⁰ and 48.69⁰ were found to correspond to the planes of DyCuO, respectively.

Fig. (1). (a) CVs documented for 1×10-4 M DLPA in 0.2 M supporting electrolyte (pH 7.0) at a scan rate of 0.1 Vs -1 for 20 segments, (b) The graph of peak current against the number of polymerization cycles.

Fig. (2). FESEM images of the BCPE (a), DyCuO Nanoparticles (b), BNPCPE (c) and P(DLPA) MNPMCPE (d).

Fig. (3). XRD patterns for DyCuO Nanoparticles.

CV is an influential tool that indicates the material catalytic behaviour towards the redox reaction of the molecule. Fig. (4) shows the CVs of 0.1 mM K₄Fe(CN)₆ in 0.1 M KCl as a buffer solution on the surface of BNPMCPE (curve a) and P(DLPA)MNPMCPE (curve b) at a sweep rate of 0.1 Vs^-1. Here, we observed that the bare electrode provided a lower oxidation peak current compared to the modified electrode due to the effect of the active surface area. The calculation of active surface area was based on the following Randles–Sevcik relation (Eq. 1),

Where, A is the active surface area of the electrode, n is the number of electrons, C is the concentration of the analyte, D is the diffusion coefficient, and the physical meaning of other terms is already known [27]. The calculated active surface area for BNPMCPE and P(DLPA)MNPMCPE is 0.05 cm² and 0.02 cm² for modified and bare electrodes, respectively.

The property of the surface of the electrode after modification was studied by using EIS. The impedance measurement was carried out at a frequency of 1mM of K₄Fe(CN)₆ with 0.1 M KCl as the supporting electrolyte. In the Nyquist plot, the semicircle found at a higher frequency referred to the restriction of electron transfer phenomenon and the linear section at a lower frequency range represented the diffusion electron transfer process. As can be noted from the Nyquist diagram shown in Fig. (5), the largest semicircle at the high frequency was observed for bare electrode with an R_ct value of 9145 Ω. After modifying with polymer, the R_ct value was found to minimise to a value of 206 Ω. This confirms that the P(DLPA)MNPMCPE film is capable of accelerating the electron transfer signifying good conductance of the electrode. In turn, the P(DLPA)MNPMCPE proved to have a catalytic effect on the voltammetric behaviour of the redox probe. The heterogeneous electron transfer rate constant (k⁰) can be determined by considering the obtained R_ct values from the below Eq. (2),

Fig. (4). CVs documented for the voltammetric response of 1×10-3 M l,K 4 [Fe (CN6)] in 0.2 M KCl at a scan rate of 0.1 Vs-1 on the surface of the modified (curve b) and unmodified electrode (curve a).

Where, n represents the number of electrons transferred, A depicts the area of the electrode, C is the concentration of the solution, and other symbols are already known [28]. The calculated k⁰ value for the modified electrode (2.5×10^-5 cms^-1) is higher than the bare electrode (1.4 × 10^-6 cms^-1), clearly signifying that the modified electrode promotes redox reaction better than the bare electrode.

The voltammetric behaviour of 0.1 mM AN in 0.2 M buffer solution (pH 7.0) on BCPE (curve a), BNPMCPE (curve b) and P(DLPA)MNPMCPE (curve c) has been examined by the CV method at a sweep rate of 0.1 Vs^-1 within the potential of -0.10 to 0.70 V. It can be seen from Fig. (6) that the P(DLPA)MNPMCPE shows excellent current response, in that the peak current was located at the peak potential of 0.340 V compared to BNPMCPE (0.420 V). This result signifies that the modified electrode reduces the overpotential with enhanced catalytic nature due to the influence of the high active surface area and anti-fouling property than the unmodified electrode. Additionally, the modified electrode did not give any of the redox peaks in the absence of AN in the supporting electrolyte, indicating that the response of the electrode is completely dependent on the mutual interaction of the AN and buffer solution.

The pH has a distinct effect on peak current, peak potential, stability of analyte, and the shape of voltammograms in the voltammetric detection of bioactive molecules. So in order to optimize pH, the determination of AN was performed at P(DLPA)MNPMCPE in pH range 5.5-8.0 with a potential scan rate of 0.1 Vs^-1, as depicted in Fig. (7a). The anodic current of AN was found optimum at pH 7.0, as represented in Fig. (7b); this pH was considered as the calibrated pH for the complete analysis of AN. It can be observed from the plot of E_pa and E_pcvs. pH (Fig. 7c) that as the solution pH increases, the basicity increases and the peak of AN shifts to a higher negative scale. The linear fit of E_pa and E_pcvs. pH is described by linear regression as

The slope 0.056 (experimental) is close to 0.059 (theoretical), so as per the Nernst equation, the electrochemical interface of AN at P(DLPA)MNPMCPE involves the same quantity of electrons and protons [29]. With the help of the obtained slope from E_pc and E_pa, the ratio of number of electrons was calculated by using the following formula,

where, m is the number of protons and n is the number of electrons [30]. The estimated m/n ratio was found to be 1.040 and 1.376; these values convey that the same quantity of protons and electrons was present in the redox reaction of AN at the prepared electrode.

The impact of sweep rate from 0.1 to 0.30 Vs^-1on the redox reaction of AN at the PDLPAMNPMCPE was evaluated in 0.2 M buffer solution at pH 7.0 having 0.1 mM AN, as documented in Fig. (8a). This, in turn, results in an increase of peak current against the change of sweep rate. A graph of the square root of sweep rate v/s I_pa is plotted, and it is depicted in Fig. (8b); the linearity is expressed using the following Eq. (6).

Fig. (5). Nyquist plots for modified (curve a) and unmodified electrode (curve b).

Fig. (6). CVs recorded for the voltammetric response of 1×10-4 M AN and without AN (curve d) in 0.2 M supporting electrolyte (pH 7.0) at a scan rate of 0.1 Vs -1 on the surface of BCPE (curve a), BNPCPE (curve b), and P (DLPA) MNPMCPE (curve c).

Fig. (7). (a) CVs recorded for the electrochemical response of 1×10-4 M AN in 0.2 M supporting electrolyte with varying pH of 5.5- 8.0 at a scan rate of 0.1 Vs-1; (b) the plot of I pavs. pH; (c) the graph of potential against pH.

Fig. (8). (a) CVs attained for the voltammetric behaviour of 1×10-4 M AN in 0.2 M supporting electrolyte (pH 7.0) changing the scan rate range from 0.1- 0.3 Vs-1 ; (b) The graph of the square root of scan rate with respect to peak current, (c) The plot of the logarithm of scan rate vs. logarithm of peak current, (d) The graph of potential against the logarithm of scan rate.

The electrode-analyte interface process (adsorption/diffusion) of AN has been investigated, and slope 0.61 obtained from the plot of log I_pa v/s. log v (Fig. 8c); thus, we can conclude that the process was diffusion-controlled [31].

The graph of E_pa and E_pc against log v (Fig. 8d) has been constructed; the fitted linear equation is represented as follows

By using the above-obtained regression equations, it can be assumed that the electrons participated in the redox reaction of the AN at the prepared sensor. The slopes of the graph of E_pa and E_pc against log v have been found equal to the subsequent formulas,

Here, n indicates transferred electrons, α is a charge transfer coefficient, and other symbols are commonly known [32]. The electrons transferred during the redox reaction of the AN are estimated as two by Eqs (10 and 11).

DPV was accomplished for the oxidation of AN by differing its concentration at P(DLPA)MNPMCPE between 2 µM to 50 µM under optimum conditions. The graph of the concentration of AN with respect to peak current (Fig. 9) has been constructed. As can be inferred from Fig. (9), as the concentration of AN increased, the current response also intensified with the linearity remaining up to 10 µM; further, the linearity deviated from 10 µM to 50 µM. The regression equation measured for the first linearity from the concentration ranging between 2µM to 10µM is as follows,

The limit of detection and limit of quantification of the P (DLPA) MNPMCPE have been assessed by Eq. (11),

Here, S_D is defined as the standard deviation obtained for 5 blank measurements, and M is the obtained slope of the calibration curve [33]. The measured LOD and LOQ values were 1.19 × 10^-7 M and 3.9 × 10^-7 M, respectively. The LOD values obtained for various classical techniques and electrodes are shown in Table 1 [34-40]. This devised electrode was found to be efficient in contrast to the other reported electrodes.

The reproducibility, repeatability and stability of the electrodes were examined using CV by using 1 × 10^-4 M AN in 0.2 M supporting electrolyte at optimum pH 7.0 with a scan rate of 0.1 Vs^-1. Reproducibility of the proposed electrode was attained and examined by five repeated measurements with respect to five different P(DLPA)MNPMCPE, and the RSD value was observed to be 3.8%. Also, the repeatability of the proposed P(DLPA)MNPMCPE was carried out by varying the standard solution by keeping the electrode constant for 5 different measurements, and thus, we observed the RSD value to be 3.2%. The analyte solution was taken for 30 successful runs of CV to check the stability of the proposed sensor. Even after the 30 cycles, the prepared electrode did not lose its stability in sensing the analyte, and hence, its voltammetric activity remained unaltered. Thus, the entire outcome certifies that the prepared electrode has excellent repeatability, sufficient reproducibility and agreeable antifouling feature.

Fig. (9). The graph of peak current vs. concentration of AN.

Scheme 1. Redox reaction of AN.

In biological samples, many interfering elements exist; thus, simultaneous separation of AN and DA at BNPMCPE and P(DLPA)MNPMCPE was conducted by DPV method with a scan rate 0.05 V/s, and the documented differential pulse voltammograms (DPVs) have been illustrated in Fig. (10). In the figure, the BNPMCPE identifies the AN and DA with low voltammetric responses, while the modified electrode gives two clear and characteristic peaks for AN and DA with a high current response. This shows that the equipped P(DLPA)MNPMCPE can be efficiently utilized for the instantaneous determination of AN and DA in a mixture. Hence, the suggested electrode possesses worthy selectivity for the detection of AN in the presence of DA Table 2.

To confirm the working capability and applicability of the projected sensor, the analysis of AN was accomplished in the tablet sample. The tablet containing AN content was procured from the drug store. The tablet was weighed, and using a mortar, the tablet sample was grinded and processed into a fine powder. The fine powder of the tablet was dissolved using distilled water and directly taken for CV scanning in 0.2 M supporting electrolyte. A good recovery range was observed from the current method. Tablet sample analysis and recoveries are displayed in Table 3.

Fig. (10). DPVs profile for the simultaneous electroanalysis of 1×10-4 M AN and DA in 0.2 M supporting electrolyte at the scan rate of 0.1 Vs-1 on the surface of P(DLPA)MNPMCPE and BNPMCPE.