From the IR assessment withinside the activated SPCE, the absorption band has become observed at 3500 cm-1, at 1600 cm-1, and at 1200 cm-1 this is as a result of the formation of hydroxyl and carbonyl containing groups in the route of activation of SPCE Fig. S1 [28, 29]. The SEM imaging for the activated SPCE exhibits a greater cracked surface together with massive defects as compared to the bare SPCE. It is seen that the small ball-like structures have been allocated over the activated SPCE surface with an average length of 56.4 to 86 nm Fig. S2. This confirms that the electrochemical activation significantly affects the surface morphology of SPCE. The Nyquist plot diameter of the semicircle is quite large on the bare SPCE in contrast to the activated one suggesting that the surface of the bare SPCE acts has an insulating property. And the smaller diameter length of the activated SPCE is a piece of evidence for superior electrical conductivity of the activated SPCE Fig. S3.
Electrochemical Property of the Electrodes
Upon scanning from positive to negative potentials, a well-defined irreversible cathodic peak (F1) was observed at -0.59 V. Also, one additional anodic peak (F2) is obtained at -0.069 V during the second scanning a new cathodic peak (S1) at -0.036 V was observed only after the second run. The irreversible cathodic peak at −0.59 V (F1) as a result of the nitro functional group reduction in the CAP to phenylhydroxylamine, which is due to four-electrons and four-protons transfer mechanisms. Whereas the anodic peak (F2) is due to the oxidation of hydroxylamine to the nitroso group derivative and the cathodic peak (S1) is the reverse of the nitroso group derivative to hydroxylamine. This redox process follows a two-electron and two-proton transfer mechanism. The displayed result in the Figure 1 show, no obvious electrochemical response for CAP was observed at the bare SPCE. On the contrary, a well-deﬁned peak was observed for the activated SPCE indicating its high electrocatalytic activity Fig. S4.
The Effect of pH
The result of varying pH for CAP oxidation was investigated in the pH range 4.0 to 8.0, using cyclic voltammetry. It was noted that the current response and the peak potential of CAP changed with increasing pH from 4.0 to 6.5 which implies the oxidation is pH-dependent. As displayed in Figure 2, the oxidation peak current after increasing up to pH 6.5, then gradual decrease was observed till it reaches 8.0 therefore pH 6.5 was selected for further analysis. A decrease in the current peak of CAP with increase in pH after 6.5 revealed that the detection of CAP at the activated SPCE is feasible only in a neutral medium.
A potential peak shift of the CAP to the negative way was observed when the electrolyte pH solution increases suggesting that proton is involved in the redox reaction process. The linear regression equation obtained from the cathodic peak potential vs. pH plot is Epc(V)= −0.05153pH − 0.25276; R2= 0.99534. The slope value from the linear graph 51.53 mV.pH indicates that complete reversible redox reaction of CAP the surface of activated SPCE involving the transfer of equal number of protons and electrons [30, 31].
The Effect of Scan Rate
The impact of changing the scan speed of on the electrochemical property of CAP was investigated using cyclic voltammetry (Figure 3) in PBS pH 6.5 with 50 µM CAP the reduction due to nitro functional group on CAP was small and not investigated. The plot of the peak current vs. scan rate study in the range 50–275 mV s-1 for the oxidation and reduction of CAP and is linear with equations of: Ipa (µA) = 0.00285v (mV s-1) +1.175; with R2 = 0.99342 and Ipa (µA) = -0.004887ν (mV s-1) + 1.175; with R2 = 0.99343. The peak potential shows little shift positively while the scan rate was increased. Moreover, the plot of log Ipc vs log ν is linear with equation: log Ipc (µA) = 0.83171log ν ‒ 0. 84933, R2 = 0.9919 and gave slope value of 0.83171which is near to 1. The obtained results suggest that the oxidation-reduction of CAP at the surface of Activated-SPCE is principally an adsorption controlled process .
Optimization of SWV Parameters
The three parameters of square wave were evaluated with the objective of obtaining highest signal for CAP determination. The peak current rely on the values of the parameters step potential, amplitude and frequency and this was studied in by varying 1–15 mV, 10–120 mV and 10–100 Hz range respectively. The study was done varying one parameter by maintaining the other two at constant value. By considering the optimum signal and good square wave voltammetric peak shape for CAP, the optimized parameters were 8 mV step potential; 90 mV amplitude and 30 Hz frequency.
The Effect of Accumulation Potential and Time
The effect of varying the accumulation potential on the determination current of CAP was studied in the range 0.0 to -1.0 V with a difference of 0.1 V at an accumulation time of 15 s. The current response increased when the potential is reduced up to -0.8 V, then after the peak current starts to decrease (Figure 4 (A)). Therefore, the accumulation potential of -0.8 V was chosen as an optimum potential for further measurements. The effect of accumulation time on the reduction peak current of CAP was also investigated by varying from 15–90 s with a difference of 15 s. As illustrated in Figure 4 (B), the reduction peak current increased with increasing accumulation time till it reached 60 s, and then a plateau was observed as a result of surface saturation. Therefore, 60 s was selected as the optimal time for the accumulation of CAP in the activated SPCE.
The inﬂuence of various potentially interfering substances in the determination of CAP was studied under the optimum conditions. Oxidation peak currents of CAP were compared with and without the potential interferents: ascorbic acid, lactose, urea, D-glucose, glycine, p-nitrophenol, Zn2+, Ca2+, Mg2+, K+, Na+, Cl- and NO3- and the result shows that the percent changes in the peak currents were less than 5% for the studied substances except for, p-nitrophenol. This point out that the activated SPCE exhibited no response to potentially interfering excipients and the p-nitrophenol must be separated before analysis.
Determination of NA by Square Wave Voltammetric Techniques
The determination of CAP was performed using the optimized square-wave parameter. Figure 5 shows the SW voltammograms of varying concentration of CAP in 0.1 M PBS of pH 6.5 at the Activated-SPCE. The peak current versus concentration plot for CAP is linear within 0.05 to 100 µM range, Figure 5 (B) with linear equation: Ip(µA) = -1.68[CAP](µM) – 3.6 and R² = 0.99517. The calculated value for quantification and detection limit for the blank measurements (n = 6) were discovered as 0.067 µA and 0.02 µA, respectively.
As shown in the Table 1 the developed electrode has wider linear range and better sensitivity to all except to graphene oxide hierarchical zinc oxide nanocomposite modified glassy carbon electrode.
The practical applicability of the developed sensor was demonstrated by analysing CAP-eye drop from commercial pharmaceutical products (Table 2) and pasteurized milk sample (Table 3). The experimentally detected values were compared with the labelled one. And it was found that the results obtained using the proposed sensor is in good agreement with the labelled values. Moreover, the accuracy and reliability of the proposed method was checked by adding CAP standard to the pharmaceutical product and pasteurized milk sample and calculating the percent recovery values. As can be seen from Table 2 and 3, the recovery results lie between 95% and 108%, indicating that the effect of the sample matrix is not significant and hence the activated SPCE has excellent potential to be used in real sample analysis.