Differential Pulse Voltammetric Electrochemical Sensor for the Detection of Etidronic Acid in Pharmaceutical Samples by Using rGO-Ag@SiO2/Au PCB

An rGO-Ag@SiO2 nanocomposite-based electrochemical sensor was developed to detect etidronic acid (EA) using the differential pulse voltammetric (DPV) technique. Rapid self-assembly of the rGO-Ag@SiO2 nanocomposite was accomplished through probe sonication. The developed rGO-Ag@SiO2 nanocomposite was used as an electrochemical sensing platform by drop-casting on a gold (Au) printed circuit board (PCB). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) confirmed the enhanced electrochemical active surface area (ECASA) and low charge transfer resistance (Rct) of the rGO-Ag@SiO2/Au PCB. The accelerated electron transfer and the high number of active sites on the rGO-Ag@SiO2/Au PCB resulted in the electrochemical detection of EA through the DPV technique with a limit of detection (LOD) of 0.68 μM and a linear range of 2.0–200.0 μM. The constructed DPV sensor exhibited high selectivity toward EA, high reproducibility in terms of different Au PCBs, excellent repeatability, and long-term stability in storage at room temperature (25 °C). The real-time application of the rGO-Ag@SiO2/Au PCB for EA detection was investigated using EA-based pharmaceutical samples. Recovery percentages between 96.2% and 102.9% were obtained. The developed DPV sensor based on an rGO-Ag@SiO2/Au PCB could be used to detect other electrochemically active species following optimization under certain conditions.


Introduction
Bisphosphonate compounds are a category of drugs in the contemporary pharmacological arsenal that avert bone density damage. Etidronic acid (EA), which is also called hydroxyethylidene diphosphonic acid (HEDP), is a member of the bisphosphonate group. EA is used as an active ingredient for cosmetic agents, medication, water treatment, and chelating agents [1]. Etidronate has the lowest potency of all bisphosphonates, which causes high diffusional distribution through the bone surface. In medication, EA is mainly used to reduce osteoclastic bone resorption (Paget's disease and osteoporosis), and only very low concentrations (200-400 mg) of etidronate are used to treat bone damage [2,3]. Etidronate has been approved in Canada and many European countries to treat osteoporosis. To treat symptomatic Paget's disease, the US FDA approved etidronate, and thus it is widely used in the United States [4]. However, excess etidronate could lead to severe renal failure, joint pain, and low levels of calcium in the blood. The estimated overdose concentration of To recognize the Ag-embedded SiO2 nanoparticles, TEM analysis was carried out, and Figure 2a shows the Ag@SiO2 nanostructure. The image confirms the presence of spherical SiO2 nanoparticles, with Ag nanoparticles embedded on the surface of the SiO2. Figure 2b shows the high-angle annular dark-field (HAADF) image of Ag@SiO2. The elements in Ag@SiO2 were classified by energydispersive X-ray spectroscopy (EDX) mapping, which confirmed the presence of a spatial distribution of elements such as Ag (Figure 2c  To recognize the Ag-embedded SiO 2 nanoparticles, TEM analysis was carried out, and Figure 2a shows the Ag@SiO 2 nanostructure. The image confirms the presence of spherical SiO 2 nanoparticles, with Ag nanoparticles embedded on the surface of the SiO 2 . Figure 2b shows the high-angle annular dark-field (HAADF) image of Ag@SiO 2 . The elements in Ag@SiO 2 were classified by energy-dispersive X-ray spectroscopy (EDX) mapping, which confirmed the presence of a spatial distribution of elements such as Ag (Figure 2c The elemental compositions of the Au PCB, rGO/Au PCB, and rGO-Ag@SiO 2 /Au PCB were analyzed through EDX spectral analysis. The predominant elements observed at the Au PCB were Ni, Au, C, and O, with corresponding weight percentages of 63.4 wt.%, 30.7 wt.%, 5.2 wt.%, and 0.7 wt.% ( Figure S2a). The rGO-modified Au PCB contained 57.9 wt.% C and 4.9 wt.% O ( Figure S2b). EDX analysis of the rGO-Ag@SiO 2 /Au PCB confirmed the presence of C, O, Ag, and Si, with corresponding weight percentages of 16.0 wt.%, 8.8 wt.%, 48.6 wt.%, and 1.4 wt.% ( Figure S2c).

Electrochemical Characterization of rGO-Ag@SiO2/Au PCB
Two methods were used to estimate the electrochemical active surface area (ECASA). The first method involved the estimation of the roughness factor, and the second method involved the evaluation of ECASA in the presence of a redox probe [(K3Fe(CN)6]. The roughness factor of the electrode (Rf) was calculated from the double-layer capacitance (Cdl). The non-faradaic current was

Electrochemical Characterization of rGO-Ag@SiO 2 /Au PCB
Two methods were used to estimate the electrochemical active surface area (ECASA). The first method involved the estimation of the roughness factor, and the second method involved the evaluation of ECASA in the presence of a redox probe [(K 3 Fe(CN) 6 ]. The roughness factor of the electrode (R f ) was calculated from the double-layer capacitance (C dl ). The non-faradaic current was captured at different scan rates (25 to 200 mV/s) with a potential window from −0.7 to −0.2 V. The current density at −0.45 V was considered to construct the C dl [32]. Figure S3a Figure S3d,e show the CV responses of the bare Au PCB and the rGO-Ag@SiO 2 -modified Au PCB. The Randles-Sevcik equation was applied to estimate the ECASA values [8]. The calculated ECASA values of the Au PCB and rGO-Ag@SiO 2 /Au PCB were found to be 2.210 and 13.812 mm 2 , respectively ( Table S2). The percent real value (%) in terms of surface area is the ratio between ECASA and the geometric surface area of the Au PCB, which was found to be 122.3% [33,34].
In the presence of 5 mM K 3 Fe(CN) 6 , as a redox probe, electrochemical impedance spectroscopy (EIS) was used to characterize the rGO-Ag@SiO 2 -modified Au PCB electrodes in the frequency range between 0.1 mHz and 1 MHz. Higher frequencies could generate resistance, whereas diffusion was facilitated at lower frequencies in the electrodes. The bare Au PCB showed a higher charge transfer resistance (R ct ) of 8.62 kΩ ( Figure 5).  (Table S2). The percent real value (%) in terms of surface area is the ratio between ECASA and the geometric surface area of the Au PCB, which was found to be 122.3% [33,34].
In the presence of 5 mM K3Fe(CN)6, as a redox probe, electrochemical impedance spectroscopy (EIS) was used to characterize the rGO-Ag@SiO2-modified Au PCB electrodes in the frequency range between 0.1 mHz and 1 MHz. Higher frequencies could generate resistance, whereas diffusion was facilitated at lower frequencies in the electrodes. The bare Au PCB showed a higher charge transfer resistance (Rct) of 8.62 kΩ ( Figure 5). The rGO-modified Au PCB exhibited an excellent Rct (5.53 KΩ) compared to the other modified electrodes in this study. The rapid electron transport of rGO caused fast charge transfer at the rGO/Au PCB electrode interface, which might be attributed to the low Rct of the rGO/Au PCB. The Ag@SiO2/Au PCB demonstrated a high Rct (7.73 KΩ) as a result of the generation of an electron blocking layer at the Ag@SiO2-modified Au PCB electrode. The formation of electronegative charges on the Ag@SiO2/Au PCB led to electrostatic repulsion between the Ag@SiO2/Au PCB and ferricyanide ions, which also generated the electron blocking layer. The rGO-Ag@SiO2-modified Au PCB generated a low Rct (6.60 KΩ). The presence of rGO in rGO-Ag@SiO2 could be responsible for the The rGO-modified Au PCB exhibited an excellent R ct (5.53 KΩ) compared to the other modified electrodes in this study. The rapid electron transport of rGO caused fast charge transfer at the rGO/Au PCB electrode interface, which might be attributed to the low R ct of the rGO/Au PCB. The Ag@SiO 2 /Au PCB demonstrated a high R ct (7.73 KΩ) as a result of the generation of an electron blocking layer at the Ag@SiO 2 -modified Au PCB electrode. The formation of electronegative charges on the Ag@SiO 2 /Au PCB led to electrostatic repulsion between the Ag@SiO 2 /Au PCB and ferricyanide ions, which also generated the electron blocking layer. The rGO-Ag@SiO 2 -modified Au PCB generated a low R ct (6.60 KΩ). The presence of rGO in rGO-Ag@SiO 2 could be responsible for the enhanced charge transfer of the rGO-Ag@SiO 2 /Au PCB. This EIS study confirmed the importance of rGO in the rGO-Ag@SiO 2 nanocomposite-modified Au PCB and that diffusion occurred at the rGO-Ag@SiO 2 /Au PCB.

Electrochemical Oxidation of EA in the rGO-Ag@SiO 2 /Au PCB
To study the effect of EA on the rGO-Ag@SiO 2 /Au PCB, 1 mM EA in 0.1 M NaOH was used. To investigate the oxidation potential of EA and the electrocatalytic activity of the rGO-Ag@SiO 2 /Au PCB toward EA, CV responses in the presence and absence of EA were obtained ( Figure 6a). After adding 1 mM EA, the current response at 0.67 V was increased to 19.4 µA. This shows the enhanced electrocatalytic ability and improved ECASA of the rGO-Ag@SiO 2 /Au PCB. The strengthening of the hydrogen bond between the SiO 2 and the -OH group of EA might be the reason for the high selectivity toward EA as EA contains numerous hydroxyl groups in its structure.

Electrochemical Oxidation of EA in the rGO-Ag@SiO2/Au PCB
To study the effect of EA on the rGO-Ag@SiO2/Au PCB, 1 mM EA in 0.1 M NaOH was used. To investigate the oxidation potential of EA and the electrocatalytic activity of the rGO-Ag@SiO2/Au PCB toward EA, CV responses in the presence and absence of EA were obtained ( Figure 6a). After adding 1 mM EA, the current response at 0.67 V was increased to 19.4 μA. This shows the enhanced electrocatalytic ability and improved ECASA of the rGO-Ag@SiO2/Au PCB. The strengthening of the hydrogen bond between the SiO2 and the -OH group of EA might be the reason for the high selectivity toward EA as EA contains numerous hydroxyl groups in its structure. The electrochemical oxidation of EA at the individual layers of the rGO-Ag@SiO2/Au PCB electrode was investigated. The bare Au PCB, rGO/Au PCB, Ag@SiO2/Au PCB, and rGO-Ag@SiO2/Au PCB electrodes were studied in the presence of 1 mM EA in 0.1 M NaOH electrolyte (pH = 13). Figure  6b shows the CV responses of the individual layers of the rGO-Ag@SiO2-modified Au PCB electrodes. The bare Au PCB failed to show a significant current response to EA. At 0.61 V, a very negligible current (0.25 μA) was obtained for EA. A significant increase in the current response (3.45 μA) was observed at 0.67 V for the rGO-modified Au PCB. The Ag@SiO2-modified Au PCB produced an enhanced current response (5.99 μA) at 0.68 V. Although the individual layer-modified Au PCB failed to show an elevated current response, the rGO-Ag@SiO2-modified Au PCB generated a current response of 19.4 μA, which was 77.6 times higher than that of the bare Au PCB. The higher current response for EA at the rGO-Ag@SiO2/Au PCB confirmed the improved electrocatalytic activity and enhanced electronic transport.

Effect of Scan Rate and pH
To evaluate the effect of the scan rate on the electrochemical oxidation of 1 mM EA in 0.1 M NaOH, the CV responses were recorded at scan rates from 25 to 200 mV/s and the results are shown in Figure 7a. At 0.67 V, the anodic peak current corresponding to the oxidized EA increases linearly as the scan rate increases. The electrochemical oxidation of EA at the individual layers of the rGO-Ag@SiO 2 /Au PCB electrode was investigated. The bare Au PCB, rGO/Au PCB, Ag@SiO 2 /Au PCB, and rGO-Ag@SiO 2 /Au PCB electrodes were studied in the presence of 1 mM EA in 0.1 M NaOH electrolyte (pH = 13). Figure 6b shows the CV responses of the individual layers of the rGO-Ag@SiO 2 -modified Au PCB electrodes. The bare Au PCB failed to show a significant current response to EA. At 0.61 V, a very negligible current (0.25 µA) was obtained for EA. A significant increase in the current response (3.45 µA) was observed at 0.67 V for the rGO-modified Au PCB. The Ag@SiO 2 -modified Au PCB produced an enhanced current response (5.99 µA) at 0.68 V. Although the individual layer-modified Au PCB failed to show an elevated current response, the rGO-Ag@SiO 2 -modified Au PCB generated a current response of 19.4 µA, which was 77.6 times higher than that of the bare Au PCB. The higher current response for EA at the rGO-Ag@SiO 2 /Au PCB confirmed the improved electrocatalytic activity and enhanced electronic transport.

Effect of Scan Rate and pH
To evaluate the effect of the scan rate on the electrochemical oxidation of 1 mM EA in 0.1 M NaOH, the CV responses were recorded at scan rates from 25 to 200 mV/s and the results are shown in The Randles-Sevcik equation was applied to study the electrochemical oxidation process of EA at the rGO-Ag@SiO2-modified Au PCB: Ip = 2.69 × 10 5 n 3/2 AD 1/2 Cʋ 1/2 , where Ip is the peak current of EA, n is the number of electrons participating in the reaction, A is the ECASA, D is the diffusion coefficient, C is the concentration of EA (mol/cm 3 ), and ʋ is the scan rate (mV/s) [35]. The linear graph (Figure 7b) between the square root of the scan rate (ʋ 1/2 ) and the corresponding anodic peak current of EA provides concrete confirmation that the electrochemical oxidation of EA is a diffusional process.
To enhance the stability and performance of the electrochemical sensor, a pH study was conducted in three different pH media containing 1 mM EA: 0.1 M acetate buffer (pH = 4.4), 10 mM PBS (pH = 7.4), and 0.1 M NaOH (pH = 13) [36]. It was observed (Figure 7c) that the oxidation potential (Ep) shifted negatively with increasing pH of the electrolyte. A low current response (3.29 μA) was observed in the acidic medium. At neutral pH, an enhanced peak current (10.3 μA) was observed compared to the acidic medium. The highest peak current of EA (18.4 μA) was observed at 0.68 V in 0.1 M NaOH, and pH = 13. The Randles-Sevcik equation was applied to study the electrochemical oxidation process of EA at the rGO-Ag@SiO2-modified Au PCB: Ip = 2.69 × 10 5 n 3/2 AD 1/2 Cʋ 1/2 , where Ip is the peak current of EA, n is the number of electrons participating in the reaction, A is the ECASA, D is the diffusion coefficient, C is the concentration of EA (mol/cm 3 ), and ʋ is the scan rate (mV/s) [35]. The linear graph (Figure 7b) between the square root of the scan rate (ʋ 1/2 ) and the corresponding anodic peak current of EA provides concrete confirmation that the electrochemical oxidation of EA is a diffusional process.
To enhance the stability and performance of the electrochemical sensor, a pH study was conducted in three different pH media containing 1 mM EA: 0. The Randles-Sevcik equation was applied to study the electrochemical oxida at the rGO-Ag@SiO2-modified Au PCB: Ip = 2.69 × 10 5 n 3/2 AD 1/2 Cʋ 1/2 , where Ip is th EA, n is the number of electrons participating in the reaction, A is the ECASA, coefficient, C is the concentration of EA (mol/cm 3 ), and ʋ is the scan rate (mV/s) [35 (Figure 7b) between the square root of the scan rate (ʋ 1/2 ) and the corresponding an of EA provides concrete confirmation that the electrochemical oxidation of EA process.
To enhance the stability and performance of the electrochemical sensor, conducted in three different pH media containing 1 mM EA: 0.1 M acetate buffer PBS (pH = 7.4), and 0.1 M NaOH (pH = 13) [36]. It was observed (Figure 7c  The Randles-Sevcik equation was applied to study the electrochemical oxidation process at the rGO-Ag@SiO2-modified Au PCB: Ip = 2.69 × 10 5 n 3/2 AD 1/2 Cʋ 1/2 , where Ip is the peak curre EA, n is the number of electrons participating in the reaction, A is the ECASA, D is the diff coefficient, C is the concentration of EA (mol/cm 3 ), and ʋ is the scan rate (mV/s) [35]. The linear g (Figure 7b) between the square root of the scan rate (ʋ 1/2 ) and the corresponding anodic peak cu of EA provides concrete confirmation that the electrochemical oxidation of EA is a diffus process.
To enhance the stability and performance of the electrochemical sensor, a pH study conducted in three different pH media containing 1 mM EA: 0. The Randles-Sevcik equation was applied to study the electrochemical oxidation process of E at the rGO-Ag@SiO2-modified Au PCB: Ip = 2.69 × 10 5 n 3/2 AD 1/2 Cʋ 1/2 , where Ip is the peak current EA, n is the number of electrons participating in the reaction, A is the ECASA, D is the diffusio coefficient, C is the concentration of EA (mol/cm 3 ), and ʋ is the scan rate (mV/s) [35]. The linear grap (Figure 7b) between the square root of the scan rate (ʋ 1/2 ) and the corresponding anodic peak curren of EA provides concrete confirmation that the electrochemical oxidation of EA is a diffusion process.
To enhance the stability and performance of the electrochemical sensor, a pH study wa conducted in three different pH media containing 1 mM EA: 0.1 M acetate buffer (pH = 4.4), 10 mM PBS (pH = 7.4), and 0.1 M NaOH (pH = 13) [36]. It was observed (Figure 7c) that the oxidation potenti (Ep) shifted negatively with increasing pH of the electrolyte. A low current response (3.29 μA) wa a b c 1/2 ) and the corresponding anodic peak current of EA provides concrete confirmation that the electrochemical oxidation of EA is a diffusional process.
To enhance the stability and performance of the electrochemical sensor, a pH study was conducted in three different pH media containing 1 mM EA: 0.1 M acetate buffer (pH = 4.4), 10 mM PBS (pH = 7.4), and 0.1 M NaOH (pH = 13) [36]. It was observed (Figure 7c) that the oxidation potential (E p ) shifted negatively with increasing pH of the electrolyte. A low current response (3.29 µA) was observed in the acidic medium. At neutral pH, an enhanced peak current (10.3 µA) was observed compared to the acidic medium. The highest peak current of EA (18.4 µA) was observed at 0.68 V in 0.1 M NaOH, and pH = 13.

Mechanism of the Detection of EA
It is well known that primary and secondary alcohols may be oxidized directly to produce their corresponding aldehydes or ketones. However, tertiary alcohols can never be oxidized directly, thus, the electrochemically active functional groups for tertiary alcohols are oxidized. Hence, multiple steps are involved in the oxidation of electrochemically active functional groups in tertiary alcohols. EA followed a similar oxidation procedure ( Figure S5). Initially, C-P bond breaking occurs via oxidation, which causes the formation of phosphate ions [37,38]. The oxidation process is driven by the participation of 1.5 times more protons than the number of electrons ( It is well known that primary and secondary alcohols may be oxidized directly to produce their corresponding aldehydes or ketones. However, tertiary alcohols can never be oxidized directly, thus, the electrochemically active functional groups for tertiary alcohols are oxidized. Hence, multiple steps are involved in the oxidation of electrochemically active functional groups in tertiary alcohols. EA followed a similar oxidation procedure ( Figure S5). Initially, C-P bond breaking occurs via oxidation, which causes the formation of phosphate ions [37,38]. The oxidation process is driven by the participation of 1.5 times more protons than the number of electrons (

DPV Study
Validation of the rGO-Ag@SiO2-modified electrodes at various concentrations of EA was performed using the DPV technique. As the concentration of EA increased, the oxidation peak current of EA increased. Figure 8a shows the DPV curves obtained with different concentrations of EA. The validated Ep of EA was obtained at 0.68 V. The Ep of EA remained constant and showed the greater stability of the rGO-Ag@SiO2-modified Au PCB electrodes. The EA concentration vs. oxidation peak current plot (Figure 8b) demonstrates that the oxidation peak current of EA increases linearly with increasing EA concentration. The linearity expression of EA concentration vs. EA peak current can be written as Y = 0.0497X + 1.0293 (R 2 = 0.9890). The error bar signifies the standard deviation of the five determinants. To calculate the limit of detection (LOD) in the electrochemical detection of EA, the IUPAC method was used. As per this method, 10 blank injections of 0.1 M NaOH were added and the DPV responses were recorded. LOD = 3SB/S (SB: standard deviation of 10 blank samples, S: slope of the calibration curve) [39,40]. The LOD of the electrochemical detection of EA in the rGO-Ag@SiO2-modified Au PCB was calculated to be 0.68 μM. The constructed linear equation could be used to determine the unknown concentration of EA in the real sample analysis. The effectiveness of

DPV Study
Validation of the rGO-Ag@SiO 2 -modified electrodes at various concentrations of EA was performed using the DPV technique. As the concentration of EA increased, the oxidation peak current of EA increased. Figure 8a shows the DPV curves obtained with different concentrations of EA. The validated E p of EA was obtained at 0.68 V. The E p of EA remained constant and showed the greater stability of the rGO-Ag@SiO 2 -modified Au PCB electrodes. The EA concentration vs. oxidation peak current plot (Figure 8b) demonstrates that the oxidation peak current of EA increases linearly with increasing EA concentration. The linearity expression of EA concentration vs. EA peak current can be written as Y = 0.0497X + 1.0293 (R 2 = 0.9890). The error bar signifies the standard deviation of the five determinants. To calculate the limit of detection (LOD) in the electrochemical detection of EA, the IUPAC method was used. As per this method, 10 blank injections of 0.1 M NaOH were added and the DPV responses were recorded. LOD = 3S B /S (S B : standard deviation of 10 blank samples, S: slope of the calibration curve) [39,40]. The LOD of the electrochemical detection of EA in the rGO-Ag@SiO 2 -modified Au PCB was calculated to be 0.68 µM. The constructed linear equation could be used to determine the unknown concentration of EA in the real sample analysis. The effectiveness of the rGO-Ag@SiO 2 /Au PCB-based EA sensor was compared with previously evaluated sensors, and the results are presented in Table S1. Nanomaterials 2020, 10, x FOR PEER REVIEW 13 of 18 the rGO-Ag@SiO2/Au PCB-based EA sensor was compared with previously evaluated sensors, and the results are presented in Table S1.

Stability, Reproducibility, and Reusability
The operational stability of the rGO-Ag@SiO2/Au PCB electrochemical sensor was evaluated through CV in the presence of 1 mM EA in 0.1 M NaOH, pH = 13. Twenty CV cycles were recorded, and at the end of the 20th cycle, 92.7% of the initial peak current was retained. To assess the longterm stability of the rGO-Ag@SiO2/Au PCB in the detection of EA, the rGO-Ag@SiO2/Au PCB was stored at room temperature (25 °C), and a storage stability study was conducted at intervals of seven days for four weeks. At the end of the fourth week, 93.1% of the DPV peak current response of the initial peak current was retained. The evaluation of the reusability of the rGO-Ag@SiO2/Au PCB in the electrochemical detection of 100.0 μM EA was carried out using the DPV technique. A single rGO-Ag@SiO2/Au PCB was used continuously for up to eight runs. At the end of each run, the rGO-Ag@SiO2/Au PCB was cleaned with a 0.1 M NaOH buffer to eliminate the oxidized form of EA adsorbed on the PCB. The observed DPV current responses were plotted in a bar chart ( Figure S6) to compare the eight runs. The evaluated percent relative standard deviation (%RSD) between the eight runs was calculated to be 3.51%. The reproducibility of the rGO-Ag@SiO2/Au PCB was investigated in the presence of 100.0 μM EA in 0.1 M NaOH using four independent rGO-Ag@SiO2/Au PCBs. The %RSD between the DPV current responses of EA was evaluated to be 2.88%. Table 1 shows the sensor parameters of rGO-Ag@SiO2 in EA detection.

Stability, Reproducibility, and Reusability
The operational stability of the rGO-Ag@SiO 2 /Au PCB electrochemical sensor was evaluated through CV in the presence of 1 mM EA in 0.1 M NaOH, pH = 13. Twenty CV cycles were recorded, and at the end of the 20th cycle, 92.7% of the initial peak current was retained. To assess the long-term stability of the rGO-Ag@SiO 2 /Au PCB in the detection of EA, the rGO-Ag@SiO 2 /Au PCB was stored at room temperature (25 • C), and a storage stability study was conducted at intervals of seven days for four weeks. At the end of the fourth week, 93.1% of the DPV peak current response of the initial peak current was retained. The evaluation of the reusability of the rGO-Ag@SiO 2 /Au PCB in the electrochemical detection of 100.0 µM EA was carried out using the DPV technique. A single rGO-Ag@SiO 2 /Au PCB was used continuously for up to eight runs. At the end of each run, the rGO-Ag@SiO 2 /Au PCB was cleaned with a 0.1 M NaOH buffer to eliminate the oxidized form of EA adsorbed on the PCB. The observed DPV current responses were plotted in a bar chart ( Figure S6) to compare the eight runs. The evaluated percent relative standard deviation (%RSD) between the eight runs was calculated to be 3.51%. The reproducibility of the rGO-Ag@SiO 2 /Au PCB was investigated in the presence of 100.0 µM EA in 0.1 M NaOH using four independent rGO-Ag@SiO 2 /Au PCBs. The %RSD between the DPV current responses of EA was evaluated to be 2.88%. Table 1 shows the sensor parameters of rGO-Ag@SiO 2 in EA detection.

Interference Study
The selectivity of the EA detection at the rGO-Ag@SiO 2 /Au PCB was evaluated in the presence of other excipients, co-existing interferents, and ionic interferents. For the selectivity and to determine the effect of the interferents at the EA peak current and its E p , 50 µM EA was added to 5 mM each of different interferents: glucose (Glu), uric acid (UA), ascorbic acid (AA), potassium hydroxide (KOH), starch, nitric acid (HNO 3 ), sodium chloride (NaCl), magnesium stearate (MS), and cetyl trimethyl ammonium bromide (CTAB) [41]. The recorded DPV responses are shown in Figure 9a. A comparison of the effect of different interferents on the EA peak current is displayed in Figure 9b. The selectivity of the EA detection at the rGO-Ag@SiO2/Au PCB was evaluated in the presence of other excipients, co-existing interferents, and ionic interferents. For the selectivity and to determine the effect of the interferents at the EA peak current and its Ep, 50 μM EA was added to 5 mM each of different interferents: glucose (Glu), uric acid (UA), ascorbic acid (AA), potassium hydroxide (KOH), starch, nitric acid (HNO3), sodium chloride (NaCl), magnesium stearate (MS), and cetyl trimethyl ammonium bromide (CTAB) [41]. The recorded DPV responses are shown in Figure 9a. A comparison of the effect of different interferents on the EA peak current is displayed in Figure 9b.  Table 2 shows the current responses and the Ep values of EA after the addition of the different interferents. No significant change in the Ep of EA was observed. The change in the EA peak current owing to the addition of interferents was less than 5%. This proved that the developed sensor was highly selective toward EA.   Table 2 shows the current responses and the E p values of EA after the addition of the different interferents. No significant change in the E p of EA was observed. The change in the EA peak current owing to the addition of interferents was less than 5%. This proved that the developed sensor was highly selective toward EA.

Real Sample Analysis
To verify the real-time application of the proposed sensor, an rGO-Ag@Sio 2 -modified Au PCB-based electrochemical sensor was used to analyze the concentration of EA in etidronate tablets (25.0, 50.0, and 100.0 µM). The obtained DPV current responses ( Figure 10) were fitted into the calibration curve (Y = 0.0497X + 1.0293) to calculate the actual concentration of the EA in the real sample. The recovery of EA in the pharmaceutical samples was calculated to be between 96.2% and 102.9% (Table S2). 50.0, and 100.0 μM). The obtained DPV current responses ( Figure 10) were fitted into the calibration curve (Y = 0.0497X + 1.0293) to calculate the actual concentration of the EA in the real sample. The recovery of EA in the pharmaceutical samples was calculated to be between 96.2% and 102.9% (Table  S2).

Conclusions
Rapid construction of an rGO-Ag@SiO2/Au PCB-based electrochemical sensing platform was achieved through probe sonication. The self-assembled rGO-Ag@SiO2 nanocomposite was characterized by SEM, UV, FTIR, and XPS Steps involved in the electrochemical oxidation of EA were studied by CV and DPV. Electrochemical detection of EA in the rGO-Ag@SiO2/Au PCB was found to be a diffusion-controlled process with a linear range of 2.0-200.0 μM, and the obtained LOD was 0.68 μM. Using 100-fold higher concentrations of various interferent, detection of EA was carried out, and the effect of the various interferents at the EA peak current was less than 5%. The constructed rGO-Ag@SiO2/Au PCB-based DPV sensor exhibited excellent repeatability (%RSD = 3.51 for eight runs), high reproducibility (%RSD = 2.88 for four independent Au PCB electrodes), and long-term stability over four weeks. The rGO-Ag@SiO2/Au PCB sensor was tested on etidronate tablets, and the obtained recovery values were excellent (96.2-102.9%). Thus, the optimized rGO-Ag@SiO2/Au PCB electrochemical sensor could be used to detect EA in real samples.

Conclusions
Rapid construction of an rGO-Ag@SiO 2 /Au PCB-based electrochemical sensing platform was achieved through probe sonication. The self-assembled rGO-Ag@SiO 2 nanocomposite was characterized by SEM, UV, FTIR, and XPS Steps involved in the electrochemical oxidation of EA were studied by CV and DPV. Electrochemical detection of EA in the rGO-Ag@SiO 2 /Au PCB was found to be a diffusion-controlled process with a linear range of 2.0-200.0 µM, and the obtained LOD was 0.68 µM. Using 100-fold higher concentrations of various interferent, detection of EA was carried out, and the effect of the various interferents at the EA peak current was less than 5%. The constructed rGO-Ag@SiO 2 /Au PCB-based DPV sensor exhibited excellent repeatability (%RSD = 3.51 for eight runs), high reproducibility (%RSD = 2.88 for four independent Au PCB electrodes), and long-term stability over four weeks. The rGO-Ag@SiO 2 /Au PCB sensor was tested on etidronate tablets, and the obtained recovery values were excellent (96.2-102.9%). Thus, the optimized rGO-Ag@SiO 2 /Au PCB electrochemical sensor could be used to detect EA in real samples.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2079-4991/10/7/1368/s1: Figure S1: Photographs of Au PCB; Figure S2: (a), (b), and (c) EDX spectra of bare Au PCB, rGO/Au PCB, and rGO-Ag@SiO 2 /Au PCB, respectively; Figure  The Randles-Sevcik equation was applied to study at the rGO-Ag@SiO2-modified Au PCB: Ip = 2.69 × 10 5 EA, n is the number of electrons participating in the coefficient, C is the concentration of EA (mol/cm 3 ), and (Figure 7b) between the square root of the scan rate (ʋ 1/ of EA provides concrete confirmation that the electr process. To enhance the stability and performance of th conducted in three different pH media containing 1 mM PBS (pH = 7.4), and 0.1 M NaOH (pH = 13) [36]. It was ob (Ep) shifted negatively with increasing pH of the electr observed in the acidic medium. At neutral pH, an enh compared to the acidic medium. The highest peak curr 0.1 M NaOH, and pH = 13.

Mechanism of the Detection of EA
) and current density, (d) and (e) CV response of bare Au PCB and rGO-Ag@SiO 2 /Au PCB in 0.1 M KCl containing 5 mM K3Fe(CN)6; Figure S4: (a) CV responses of rGO-Ag@SiO 2 at various concentrations of rGO (0, 0.5, and 1.0 mg) and at a fixed concentration of Ag@SiO 2 (1 mg) in the presence of EA in 0.1 M NaOH. (b) The plot of CrGO /CrGO + CAg@SiO 2 vs. oxidation peak current (Ipa); Figure S5: Plausible steps involved in the electrochemical oxidation of EA; Figure S6: Eight consecutive DPV responses of 100.0 µM EA in 0.1 M NaOH at rGO-Ag@SiO 2 /Au PCB; Figure S7: Raman spectral analysis of graphite, GO, and rGO. Table S1: Comparison of previously reported materials in the detection of EA to the current study; Table S2: ECASA calculation; Table S3. Real-time analysis of EA in the pharmaceutical samples; Table S4. Raman spectral analysis of graphite, GO, and rGO.