nanomaterials

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

Sathish Panneer Selvam 1 , Somasekhar R. Chinnadayyala 1 , Sungbo Cho 1,2,* and Kyusik Yun 3,*

1 Department of Electronics Engineering, Gachon University, Seongnam-si, Gyeonggi-do 13210, Korea; [email protected] (S.P.S.); [email protected] (S.R.C.) 2 Gachon Advanced Institute for Health Science & Technology, Gachon University, Incheon 21999, Korea 3 Department of Bionanotechnology, Gachon University, Seongnam-si, Gyeonggi-do 13210, Korea * Correspondence: [email protected] (S.C.); [email protected] (K.Y.)

 Received: 22 June 2020; Accepted: 11 July 2020; Published: 14 July 2020 

Abstract: 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 (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.

Keywords: etidronic acid; differential pulse voltammetry; self-assembly; ultrasonic irradiation

1. 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

Nanomaterials 2020, 10, 1368; doi:10.3390/nano10071368 www.mdpi.com/journal/nanomaterials Nanomaterials 2020, 10, 1368 2 of 17 etidronate was found to be above 500 mg. The continuous accumulation of EA in the human body might also cause acute oral and dermal damage in humans [5]. Therefore, it is important to monitor the concentration of EA in real samples and pharmaceutical formulations [6]. Because of the absence of chromophoric and fluorophore agents, direct detection of EA through photometric and fluorometric estimation is impossible. The available conventional methods for determining EA involve direct determination by evaporative light-scattering detection and indirect detection through chemical derivatization. Chemical derivatization is time-consuming, and the resultant derivatives are at times unstable, making it difficult to carry out complete analysis [7]. The recently published United States Pharmacopeia defines the detection method of sodium etidronate as liquid chromatography assisted by a conductivity detector. The efficient design of the sensing platform, low sample volume consumption, and the lack of need of further pretreatment of the samples in the electrochemical sensors are the main factors in contemporary quantitative analysis [8]. Although conventional methods have been developed to detect EA, sensors with higher sensitivities and stabilities have not yet been achieved. Electrochemical sensors possess nanomaterial-modified for enhancing the catalytic activity for analyte detection [9]. Enhancement of the electrocatalytic activity relies on the active surface area of the nanomaterials and the number of active sites in the nanomaterials [10,11]. Nanomaterials based on graphene and its derivatives possess excellent conductivity, high thermal stability, and inexpensive functionalization through chemical processes. For these reasons, a wide variety of electrochemistry-based studies on graphene-based nanomaterials have been carried out [12–14]. Electrochemical characteristic studies have confirmed the enhanced electron transport behavior of reduced graphene oxide (rGO), and thus it has been widely used as an electrochemical sensing platform. The vital role of rGO lies in increasing the number of adsorptive sites, which enhances the electrocatalytic ability of the rGO-modified electrodes [15–17]. Metal nanoparticles have been predominantly utilized in the construction of sensing platforms in electrochemical sensors [11,18,19]. Because metal nanoparticles possess a greater surface area-to-volume ratio, they are adsorbed onto graphene-like sheets to form novel nanocomposites to achieve excellent electrochemical performance. The adsorbed metal nanoparticles could be responsible for the improved electron transfer at the interface, resulting in the excellent conductivities of these electrodes [20,21]. Silver nanoparticles are most frequently used with nanosheet structures to enhance the electrochemical performance of electrochemical sensors [22,23]. In this study, probe sonication-assisted construction was used to develop an rGO-Ag@SiO2 nanocomposite as an electrochemical sensing platform. This is the first differential pulse voltammetric (DPV)-based electrochemical sensor for the electrochemical detection of EA.

2. Experimental

2.1. Chemicals and Apparatus Refer to the supplementary information for details about the chemicals and apparatus.

2.2. Synthesis of Ag@SiO2

An ultrasonication-assisted method was used to prepare Ag@SiO2 nanoparticles. Solution-A containing 7.5 wt.% tetraethyl orthosilicate (TEOS) in ethanol was injected into a solution containing 4.25 wt.% NH4OH in ethanol through a syringe pump at a flow rate of 0.8 mL/min. Then, the solution mixture was ultrasonicated at 30% amplitude for 30 min under a probe sonication temperature of 65 ◦C. To the above solution, an aqueous mixture containing 4.75 wt.% AgNO3 and 4.25 wt.% NaBH4 was injected dropwise, and the solution was ultrasonicated for 30 min. The as-prepared solution was centrifuged, washed twice with ethanol, and dried at 80 ◦C for 3 h [24]. Nanomaterials 2020, 10, 1368 3 of 17

2.3. Preparation of rGO A modified Hummers method was used to prepare rGO. Initially, 1.0 g of graphite powder was dispersed in 40 mL of H2SO4 in a round-bottom flask, and the solution was kept in an ice bath. Next, 3.5 g of KMnO4 was added to the above-prepared solution and the reaction mixture was stirred for 2 h, followed by the addition of 150 mL of Milli-Q water. The dropwise addition of H2O2 was carried out when the reaction mixture no longer formed bubbles. The prepared solution was subsequently filtered to remove undesirable metal ions, and the obtained solution was washed with 10% HCl and completely dried. The prepared graphene oxide (GO) was dispersed in Milli-Q water, and under continuous stirring for 1 h at 80 ◦C, the reduction of GO to rGO was maintained by the introduction of 1 mL of hydrazine hydrate. The color of the obtained solution color changed to black, which confirmed the formation of rGO. The collected precipitate was filtered and subsequently dried in a vacuum oven [8].

2.4. Construction of the rGO-Ag@SiO2 PCB Sensing Platform

2.4.1. Self-Assembly of rGO-Ag@SiO2

One milligram (1 mg) each of rGO and Ag@SiO2 (Figure S4a,b) were dispersed in 1 mL of Milli-Q water and probe sonicated for 5 min at 45% amplitude.

2.4.2. Surface Modification of the Au PCB An integrated circuit-based Au PCB was used as an electrode. The Au PCB comprised a with a diameter of 1.48 mm, a counter electrode 4.3 mm long and 1.6 mm wide, and a reference electrode 4.1 mm long and 0.8 mm wide (Figure S1). The Au PCB was sequentially washed with acetone, ethanol, and Milli-Q water. After the Au PCB was cleaned and dried, plasma treatment was applied to the Au PCB. Approximately 5 µL of the as-prepared rGO-Ag@SiO2 nanocomposite was drop-casted on the cleaned Au PCB [25].

2.5. Real Sample Preparation Etidronate drug tablets (200 mg) were used to prepare three different concentrations (25.0, 50.0, and 100.0 µM) of etidronate real samples in a 0.1 M NaOH diluent. The tablets were finely ground with the help of a mortar and pestle and dispersed in a 0.1 M NaOH electrolyte as a diluent. The samples were then filtered to remove undissolved excipients. The detergent samples were diluted 100 times with 0.1 M NaOH to fit the peak current response of EA into the calibration curve. In the final calculation, the obtained concentration of EA was multiplied by a dilution factor.

3. Results and Discussion

3.1. Characterization

The surface morphologies of the bare Au PCB, rGO-modified Au PCB, Ag@SiO2-modified Au PCB, and rGO-Ag@SiO2–modified Au PCB were examined by scanning electron microscopy (SEM). A smooth surface was observed for the bare Au PCB (Figure1a), which turned into a thin sheet-like monolayer structure at the surface of the Au PCB owing to rGO (Figure1b). The Ag@SiO 2-modified Au PCB showed homogenous, evenly distributed Ag@SiO2 microspheres (Figure1c). Examination of the rGO-Ag@SiO2–modified Au PCB electrode revealed the successful adsorption of Ag@SiO2 microspheres onto the rGO monolayer (Figure1d). Nanomaterials 2020, 10, 1368 4 of 17 Nanomaterials 2020, 10, x FOR PEER REVIEW 4 of 18

Figure 1. ScanningScanning electron electron microscopy microscopy (SEM (SEM)) images images of of (a)( a bare) bare Au Au PCB, PCB, (b) ( rGO/Aub) rGO/ Au PCB, PCB, (c) (c) Ag@SiO /Au PCB, and (d) rGO- Ag@SiO /Au PCB. Ag@SiO2/Au2 PCB, and (d) rGO- Ag@SiO2/Au2 PCB.

To recognize the Ag-embedded SiO2 nanoparticles, TEM analysis was carried out, and Figure2a 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, shows the Ag@SiO2 nanostructure. The image confirms the presence of spherical SiO2 nanoparticles, with Ag nanoparticles embedded on the surface of the SiO2. Figure2b shows the high-angle annular 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 energy-dispersive dark-field (HAADF) image of Ag@SiO2. The elements in Ag@SiO2 were classified by energy- X-ray spectroscopy (EDX) mapping, which confirmed the presence of a spatial distribution of elements dispersive X-ray spectroscopy (EDX) mapping, which confirmed the presence of a spatial distribution such as Ag (Figure2c), Si (Figure2d), and O (Figure2e) in Ag@SiO 2. of elements such as Ag (Figure 2c), Si (Figure 2d), and O (Figure 2e) in Ag@SiO2. The elemental compositions of the Au PCB, rGO/Au PCB, and rGO-Ag@SiO2/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@SiO2/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). Absorption spectral analyses of rGO, Ag@SiO2, and rGO-Ag@SiO2 were carried out in the wavelength range of 200–800 nm (Figure3a). An absorption peak of rGO was observed at 272.7 nm, and a highly intense absorption peak of Ag@SiO2 was observed at 410.2 nm [26,27]. The rGO-Ag@SiO2 nanocomposite showed absorption peaks for rGO and Ag@SiO2 at wavelengths of 276.8 and 408.6 nm, respectively. The presence of the two different absorption peaks for the rGO-Ag@SiO2 nanocomposite proved the presence of rGO and Ag@SiO2.

Nanomaterials 2020, 10, 1368 5 of 17 Nanomaterials 2020, 10, x FOR PEER REVIEW 5 of 18

a) b) SiO2

Ag

c) Ag d) Si

e) O

Figure 2. (a) Transmission electron microscopy (TEM) image of Ag@SiO2,(b) high-angle annular dark-field (HAADF) image of Ag@SiO2, and EDX mapping of (c) Ag, (d) Si, and (e) O. Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 18

Figure 2. (a) Transmission electron microscopy (TEM) image of Ag@SiO2, (b) high-angle annular dark- field (HAADF) image of Ag@SiO2, and EDX mapping of (c) Ag, (d) Si, and (e) O.

The elemental compositions of the Au PCB, rGO/Au PCB, and rGO-Ag@SiO2/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@SiO2/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). Absorption spectral analyses of rGO, Ag@SiO2, and rGO-Ag@SiO2 were carried out in the Nanomaterials 2020, 10, 1368 6 of 17 wavelength range of 200–800 nm (Figure 3a).

a

b

Figure 3. (a) The UV absorption spectra and (b) Fourier transform infrared (FTIR) spectra of rGO, Ag@SiO2, and rGO-Ag@SiO2. Figure 3. (a) The UV absorption spectra and (b) Fourier transform infrared (FTIR) spectra of rGO, The individual functional groups of rGO, Ag@SiO2, and rGO-Ag@SiO2 were studied by Fourier Ag@SiO2, and rGO-Ag@SiO2. transform infrared (FTIR) spectroscopy. The analysis was carried out using wavenumbers from 400 to 1 1 1 1 4000 cm− (Figure3b). C–OH (1190 cm − ), C=C (1556 cm− ), and O–H (3695 cm− ) vibrations were 1 identified in rGO. The bands recorded in Ag@SiO2 at wavenumbers of 831, 978, 2896, and 2981 cm− corresponded to Si–O, Si–OH, –CH2, and –CH2, respectively. After the rGO-Ag@SiO2 nanocomposite was formed, the individual vibrations present in the rGO and Ag@SiO2 were confirmed to be the C–OH 1 1 1 1 (1212 cm− ), C=C (1553 cm− ), and O–H (3675 cm− ) vibration bands of rGO and the Si–O (827 cm− ), 1 1 1 Si–OH (983 cm− ), –CH2 (2900 cm− ), and –CH2 (2985 cm− ) bands of Ag@SiO2. The presence of the individual bands of rGO and Ag@SiO2 at rGO-Ag@SiO2 proved that the nanocomposite was successfully formed owing to a simple probe sonication process [27]. X-ray photoelectron spectroscopy was used to analyze the elements and their electronic states in the rGO-Ag@SiO2 nanocomposite. The overall survey (Figure4e) showed the presence of C, Nanomaterials 2020, 10, 1368 7 of 17

O, Ag, and Si in the rGO-Ag@SiO2 nanocomposite. In the expanded C 1S spectrum (Figure4a), the deconvoluted peaks were at 284.4, 286.8, and 288.9 eV, attributed to the C–C, C–O, and C=O spectra, respectively [28]. The expanded O 1s spectrum (Figure4b) showed deconvoluted peaks corresponding to the C–O, Si–O, and C=O spectra at 534.3, 532.5, and 530.8 eV, respectively [29,30]. The expanded Ag 3dNanomaterials spectrum, 20 shown20, 10, x in FOR Figure PEER4 c,REVIEW contained two orbitals attributed to Ag 3d 5/2 (368.5 eV) and Ag8 3d of3 18/2 (374.4 eV). An expanded Si 2p spectrum (Figure4d) was observed at 103.2 eV [31].

a b

c d

e

Figure 4. X-ray photoelectron spectroscopy (XPS) spectrum of rGO-Ag@SiO2.(a) Deconvoluted C 1S

spectrum,Figure 4 (.b X)- deconvolutedray photoelectron O 1S spectroscopy spectrum, (c) (XPS) expanded spectrum Ag 3d of spectrum, rGO-Ag@SiO (d) expanded2. (a) Deconvoluted Si 2p spectrum C 1S ofspectrum, rGO-Ag@SiO (b) 2 deconvoluted, and (e) full survey. O 1S spectrum, (c) expanded Ag 3d spectrum, (d) expanded Si 2p spectrum of rGO-Ag@SiO2, and (e) full survey.

3.2. 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 probe [(K3Fe(CN)6]. The roughness factor of the electrode (Rf) was calculated from the double-layer capacitance (Cdl). 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 Cdl [32]. Figures S3a and S3b show the cyclic

Nanomaterials 2020, 10, 1368 8 of 17

3.2. 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 calculatedNanomaterials from 2020, the10, x double-layer FOR PEER REVIEW capacitance (Cdl). The non-faradaic current was captured at different9 of 18 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 − − − wasvoltammetry considered (CV) to construct responses the ofC thedl [ 32bare]. Figure Au PCB S3a,b and show the rGO the cyclic-Ag@SiO voltammetry2/Au PCB in (CV) 0.1 responses M KCl. The of thecalculated bare Au C PCBdl values and the of rGO-Ag@SiO the bare Au 2 and/Au PCB rGO- inAg@SiO 0.1 M KCl.2/Au The PCBs calculated were 1.58Cdl andvalues 2.20 of mF/mm the bare2, 2 Aurespectively and rGO-Ag@SiO (Figure S3c).2/Au T PCBshese wereresults 1.58 show and that 2.20 the mF /roughnessmm , respectively factor of (Figure the Au S3c).PCB Theseincreased results 1.4 showtimes, that owing the roughnessto the modification factor of theof the Au PCBAu PCB increased with the 1.4 times,rGO-Ag@SiO owing to2 nanocomposite. the modification Evaluation of the Au PCBof the with ECASA the rGO-Ag@SiO values of the2 barenanocomposite. Au PCB and Evaluation the rGO-Ag@SiO of the ECASA2–modified values Au of PCB the was bare performed Au PCB and in thethe rGO-Ag@SiOpresence of 52 –modifiedmM K3Fe(CN) Au PCB6 in was0.1 M performed KCl. Figures in the S3d presence and S3e of show 5 mM the K3Fe(CN) CV responses6 in 0.1 M of KCl. the Figurebare Au S3d,e PCB showand the the rGO CV- responsesAg@SiO2–modified of the bare Au Au PCB. PCB The and Randles the rGO-Ag@SiO–Sevcik equation2–modified was applied Au PCB. to Theestimate Randles–Sevcik the ECASA equation values [8]. was The applied calculated to estimate ECASA the values ECASA of the values Au PCB [8]. Theand calculated rGO-Ag@SiO ECASA2/Au 2 valuesPCB were of the found Au PCB to be and 2.210 rGO-Ag@SiO and 13.8122/ Aumm PCB2, respectively were found (Table to be 2.210S2). The and percent 13.812 mm real, value respectively (%) in (Tableterms S2).of surface The percent area is real the valueratio between (%) in terms ECASA of surface and the area geometric is the ratio surface between area ECASAof the Au and PCB, the geometricwhich was surface found areato be of122.3% the Au [33,34 PCB,].which was found to be 122.3% [33,34]. InIn thethe presencepresence ofof 55 mMmM KK33Fe(CN)Fe(CN)66, as a redox probe, electrochemicalelectrochemical impedanceimpedance spectroscopyspectroscopy (EIS)(EIS) waswas usedused toto characterizecharacterize thethe rGO-Ag@SiOrGO-Ag@SiO22–modified–modified AuAu PCBPCB electrodeselectrodes inin thethe frequencyfrequency rangerange betweenbetween 0.10.1 mHzmHz andand 11 MHz.MHz. HigherHigher frequenciesfrequencies couldcould generategenerate resistance,resistance, whereaswhereas didiffusionffusion waswas facilitatedfacilitated atat lowerlower frequenciesfrequencies inin thethe electrodes.electrodes. TheThe barebare AuAu PCBPCB showedshowed aa higherhigher chargecharge transfertransfer resistanceresistance ((RRctct) of 8.62 kkΩΩ (Figure5 5).).

FigureFigure 5.5. ElectrochemicalElectrochemical impedanceimpedance spectroscopy (EIS) Nyquist plot of bare Au PCB, rGO//AuAu PCB, Ag@SiO /Au PCB, and rGO-Ag@SiO /Au PCB in 0.1 M KCl containing 1 mM K Fe(CN) . Ag@SiO22/Au PCB, and rGO-Ag@SiO2/Au PCB in 0.1 M KCl containing 1 mM K3Fe(CN)66.

The rGO-modified Au PCB exhibited an excellent Rct (5.53 KΩ) compared to the other modified 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 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 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 enhanced charge transfer of the rGO-Ag@SiO2/Au PCB. This EIS study confirmed the importance of rGO in the rGO-Ag@SiO2 nanocomposite-modified Au PCB and that diffusion occurred at the rGO- Ag@SiO2/Au PCB.

Nanomaterials 2020, 10, 1368 9 of 17

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 enhanced charge transfer of the rGO-Ag@SiO2/Au PCB. This EIS study confirmed the importance of rGO in the rGO-Ag@SiO2 nanocomposite-modifiedNanomaterials 2020, 10, x FOR PEER Au REVIEW PCB and that diffusion occurred at the rGO-Ag@SiO2/Au PCB. 10 of 18

3.3. Electrochemical Oxidation of EA in the rGO rGO-Ag@SiO-Ag@SiO2/Au/Au PCB

To study the effect effect of of EA EA on on the the rGO rGO-Ag@SiO-Ag@SiO2/Au2/Au PCB, PCB, 1 mM 1 mM EA EA in 0.1 in 0.1M NaOH M NaOH was was used. used. To Toinvestigate investigate the the oxidation oxidation potential potential of of EA EA and and the the electrocatalytic electrocatalytic activity activity of of th thee rGO rGO-Ag@SiO-Ag@SiO2/Au/Au PCB toward EA, CV responses in the presence and absence of EA were obtained (Figure6 6a).a). AfterAfter addingadding 1 mM EA, the current response at 0.67 V was increased to 19.4 µ μA.A. This shows the enhanced electrocatalytic ability and improved ECASA of the rGO-Ag@SiOrGO-Ag@SiO2/Au/Au PCB. The strengthening of the hydrogen bond bond between between the the SiO SiO2 and2 and the the–OH – groupOH group of EA of might EA be might the reason be the for reason the high for selectivity the high towardselectivity EA toward as EA containsEA as EA numerous contains hydroxylnumerous groups hydroxyl in its groups structure. in its structure.

a b

Figure 6. 6. (a(a)) Comparison Comparison of of cyclic voltammetry (CV) (CV) response response at rGO-Ag@SiO at rGO-Ag@SiO2/Au PCB2/Au in PCB the absence in the absenceand presence and presence of 1 mM of Etidronic 1 mM Etidronic acid (EA) acid in 0.1(EA) M in NaOH. 0.1 M (NaOH.b) CV response (b) CV response of 1 mM of EA 1 atmM bare EA Au at barePCB, Au rGO PCB,/Au rGO/Au PCB, Ag@SiO PCB, 2Ag@SiO/Au PCB,2/Au and PCB, rGO-Ag@SiO and rGO-2Ag@SiO/Au PCB2/Au in0.1 PCB M in NaOH. 0.1 M NaOH.

The electrochemical oxidation of EA at the individual layers of the rGO-Ag@SiO /Au PCB electrode The electrochemical oxidation of EA at the individual layers of the rGO-2Ag@SiO2/Au PCB was investigated. The bare Au PCB, rGO/Au PCB, Ag@SiO /Au PCB, and rGO-Ag@SiO /Au PCB electrode was investigated. The bare Au PCB, rGO/Au PCB, Ag@SiO2 2/Au PCB, and rGO-Ag@SiO2 2/Au electrodesPCB electrodes were were studied studied in the in presence the presence of 1 mMof 1 mM EA inEA 0.1 in M0.1 NaOH M NaOH electrolyte electrolyte (pH (pH= 13). = 13). Figure Figure6b shows the CV responses of the individual layers of the rGO-Ag@SiO –modified Au PCB electrodes. 6b shows the CV responses of the individual layers of the rGO2-Ag@SiO2–modified Au PCB Theelectrodes. bare Au The PCB bare failed Au to PCB show failed a significant to show current a significant response current to EA. response At 0.61 V, to a veryEA. At negligible 0.61 V, currenta very (0.25negligibleµA) was current obtained (0.25 forμA) EA. was A obtaine significantd for increase EA. A significant in the current increase response in the (3.45 currentµA) response was observed (3.45 at 0.67 V for the rGO-modified Au PCB. The Ag@SiO -modified Au PCB produced an enhanced μA) was observed at 0.67 V for the rGO-modified Au PCB.2 The Ag@SiO2-modified Au PCB produced currentan enhanced response current (5.99 responseµA) at 0.68 (5.99 V. μA) Although at 0.68 the V. individualAlthough the layer-modified individual layer Au PCB-modified failed Au to show PCB an elevated current response, the rGO-Ag@SiO –modified Au PCB generated a current response of failed to show an elevated current response, the rGO2 -Ag@SiO2–modified Au PCB generated a current 19.4responseµA, whichof 19.4 wasμA, 77.6which times was higher77.6 times than higher that of than the that bare of Au the PCB. bare TheAu PCB. higher The current higher response current for EA at the rGO-Ag@SiO /Au PCB confirmed the improved electrocatalytic activity and enhanced response for EA at the rGO2-Ag@SiO2/Au PCB confirmed the improved electrocatalytic activity and electronicenhanced transport.electronic transport. 3.4. Effect of Scan Rate and pH 3.4. 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 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 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.

Nanomaterials 2020, 10, 1368 10 of 17 Nanomaterials 2020, 10, x FOR PEER REVIEW 11 of 18 Nanomaterials 2020, 10, x FOR PEER REVIEW 11 of 18 Figure7a. At 0.67 V, the anodic peak current corresponding to the oxidized EA increases linearly as the scan rate increases. a b a b

c c

FigureFigure 7. 7. (a(a) )EEffecffectt of of the the scan scan rate rate (25 (25–200–200 mV/s) mV/s) on on the the rGO rGO-Ag@SiO-Ag@SiO2/Au2/Au PCB PCB in inthe the presence presence of of1 mM1Figure mM etidronic etidronic 7. (a) Effecacid acid t(EA) of (EA) the in inscan a a0.1 0.1 rate M M NaOH(25 NaOH–200 electrolyte electrolytemV/s) on (pHthe (pH rGO== 13).13).-Ag@SiO (b (b) )Linear Linear2/Au relationship relationshipPCB in the presencebetween between theof the 1 11/2/2 squaresquaremM etidronic root root of of theacid the scan (EA) rate in a( ʋ0.1) ) Mand and NaOH the the oxidation oxidationelectrolyte peak peak (pH current current= 13). ( b( (II)pa paLinear) )for for E EA.relationshipA. ( (cc)) D Diifferentialfferential between pulse pulse the voltammetricvoltammetricsquare root of (DPV) the scan responseresponse rate ( ofofʋ1/21 1) mM mMand EA EAthe in inoxidation di differentfferent pHpeak pH media mediacurrent [0.1 [0.1 M(Ipa acetateM) for acetate E buA.ff (buffererc) (pHDifferential (pH= 4.4), = 4.4), 10 pulse mM 10 mMPBSvoltammetric PBS (pH (pH= 7.4) = (DPV) 7.4) and and 0.1 response M0.1 NaOH M NaOH of (pH1 mM (pH= 13)].EA = 13)].in different pH media [0.1 M acetate buffer (pH = 4.4), 10 mM PBS (pH = 7.4) and 0.1 M NaOH (pH = 13)]. TheThe Randles Randles–Sevcik–Sevcik equation equation was was applied applied to to study study the the electrochemical electrochemical oxidation oxidation process process of of EA EA 5 3/2 1/2 1/2 atat the the rGO rGO-Ag@SiO-Ag@SiO22––modifiedmodified Au Au PCB: PCB: IIpp == 2.692.69 × 10105 n3/2nADAD1/2Cʋ1/2C, where, where Ip isIp theis thepeak peak current current of The Randles–Sevcik equation was applied to study× the electrochemical oxidation process of EA EA,ofat EA,the n isrGOn theis- the Ag@SiOnumber number2 –ofmodified ofelectrons electrons Au participating PCB: participating Ip = 2.69 in in×the 10 the 5reaction, n reaction,3/2AD1/2 CAʋA is1/2is ,the where the ECASA, ECASA, Ip is theDD ispeakis the the currentdiffusion diffusion of 3 coefficient,coeEA,ffi ncient, is the CC isisnumber the the concentration concentration of electrons of of participating EA EA (mol/cm (mol/cm 3),in), and andthe ʋreaction, isis the the scan scan A rateis rate the (mV/s) (mV ECASA,/s) [35] [35 ]..D The The is thelinear linear diffusion graph graph 1/2 (Figu(Figurecoefficient,re 77b)b) betweenbetween C is the theconcentrationthe square square root root of of ofEA the the (mol/cm scan scan rate rate3), ( and (ʋ1/2) ʋ) and andis the the the scan corresponding corresponding rate (mV/s) anodic [35] anodic. The peak peak linear current current graph of ofEA(Figu EA providesre provides 7b) between concrete concrete the confirmation square confirmation root that of the the that scan electrochemical the rate electrochemical (ʋ1/2) and oxidation the corresponding oxidation of EA is of a EAdianodicff usional is a peak diffusional process. current process.of EATo providesenhance the concrete stability confirmation and performance that of the the electrochemical electrochemical oxidation sensor, a pH of study EA is was a diffusional conducted inprocess. threeTo dienhance fferent pH the media stability containing and performance 1 mM EA: 0.1 of M the acetate electro buffchemicaler (pH = 4.4), sensor, 10 mM a pH PBS study (pH = was7.4), conductedand 0.1To M enhance NaOHin three (pH thedifferent = stability13) [pH36 ]. andmedia It was performance containing observed(Figure 1 of mM the 7EA: c) electro that 0.1 M thechemical acetate oxidation sensor,buffer potential (pH a pH = 4.4), ( E studyp) 10 shifted mM was PBSnegativelyconducted (pH = 7.4), within three and increasing 0.1 different M NaOH pH pH of(pH media the = electrolyte.13) containing [36]. It was A 1 low observedmM current EA: 0.1(Figur response M acetatee 7c) that (3.29 buffer theµ A)oxidation (pH was = observed4.4), potential 10 mM in (thePBSEp) acidicshifted (pH = medium.7.4), negatively and 0.1 At withM neutral NaOH increasing pH, (pH an = 13) enhancedpH [36] of the. It was peakelectrolyte. observed current A (Figur (10.3low currentµeA) 7c) was that response observedthe oxidation (3.29 compared μA) potential was to µ observedthe(Ep) acidic shifted in medium. thenegatively acidic The medium.with highest increasing peakAt neutral current pH ofpH, of the EAan electrolyte. enhanced (18.4 A) wasApeak low observed current current at(10.3 response 0.68 μA) V in was(3.29 0.1 observed M μA) NaOH, was comparedandobserved pH = into13. the acidicacidic medimedium.um. TheAt neutralhighest pH,peak an current enhanced of EA peak (18.4 current μA) was (10.3 observed μA) was at 0.68observed V in 0.1compared M NaOH, to theand acidic pH = 13.medi um. The highest peak current of EA (18.4 μA) was observed at 0.68 V in 0.1 M NaOH, and pH = 13. 3.5. Mechanism of the Detection of EA 3.5. Mechanism of the Detection of EA

Nanomaterials 2020, 10, 1368 11 of 17

3.5. Mechanism of the Detection of EA Nanomaterials 2020, 10, x FOR PEER REVIEW 12 of 18 It is well known that primary and secondary alcohols may be oxidized directly to produce their correspondingIt is well known aldehydes that primary or ketones. and secondary However, alcohols tertiary may alcoholsbe oxidized can directly never beto produce oxidized their directly, thus,corresponding the electrochemically aldehydes or active ketones. functional However, groups tertiary for alcohols tertiary can alcohols never arebe oxidized oxidized. directl Hence,y, thus, multiple stepsthe are electrochemically involved in the active oxidation functional of electrochemically groups for tertiary active alcohols functional are oxidized. groups Hence, in tertiary multiple alcohols. EAstep followeds are involved a similar in the oxidation oxidation procedureof electrochemically (Figure S5).active Initially, functional C–P groups bond in breakingtertiary alcohols occurs. via oxidation,EA followed which a similar causes oxidation the formation procedure of phosphate (Figure S5). ions Initially, [37,38 ]. C– TheP bond oxidation breaking process occurs is via driven byoxidation, the participation which causes of 1.5 the times formation more of protons phosphate than ions the [37,38 number]. The of oxidation electrons process (3:2). Theis driven second by step involvesthe participation the removal of of 1.5 H times2O from more the pro intermediatetons than the product. number The of final electrons step (3:2).deals The with second the cleavage step of C–Pinvolves bonding the andremoval generates of H2O a from phosphate the intermediate ion by the product. participation The final of step three deals protons with the and cleavage two electrons. of C–P bonding and generates a phosphate ion by the participation of three protons and two electrons. Scheme1 demonstrates how the rGO-Ag@SiO 2–modified Au PCB facilitates improved electron transfer Scheme 1 demonstrates how the rGO-Ag@SiO2–modified Au PCB facilitates improved electron in the electrochemical detection of EA. The enhanced ECASA of the rGO-Ag@SiO2–modified Au PCB, transfer in the electrochemical detection of EA. The enhanced ECASA of the rGO-Ag@SiO2–modified in addition to the high number of active sites possessed by the rGO-Ag@SiO2 composite, could be Au PCB, in addition to the high number of active sites possessed by the rGO-Ag@SiO2 composite, attributed to the improved electrochemical detection of EA. could be attributed to the improved electrochemical detection of EA.

Scheme 1. Mechanism in the electrochemical oxidation of etidronic acid (EA) at rGO-Ag@SiO2/Au PCB.

Scheme 1 Mechanism in the electrochemical oxidation of etidronic acid (EA) at rGO-Ag@SiO2/Au 3.6. DPV Study PCB. Validation of the rGO-Ag@SiO2–modified electrodes at various concentrations of EA was performed3.6. DPV Study using the DPV technique. As the concentration of EA increased, the oxidation peak currentValidation of EA increased. of the rGO Figure-Ag@SiO8a shows2–modified the DPV electrodes curves obtained at various with concentrations di fferent concentrations of EA was of EA.performed The validated using theEp DPVof EA technique. was obtained As the concentration at 0.68 V. The of EEAp ofincreased, EA remained the oxidation constant peak and current showed theof greater EA increased. stability Figure of the 8a rGO-Ag@SiOshows the DPV2 –modifiedcurves obtained Au PCBwith electrodes.different concentrations The EA concentration of EA. The vs. oxidationvalidated peak Ep of current EA was plot obtained (Figure at8 0.68b) demonstrates V. The Ep of EA that remained the oxidation constant peak and current showed of the EA greater increases linearlystability with of the increasing rGO-Ag@SiO EA concentration.2–modified Au PCB The electrodes. linearity expression The EA concentration of EA concentration vs. oxidation vs. peak EA peak currentcurrent can plot be (Figure written 8b) as demonstratesY = 0.0497X that+ 1.0293 the oxidation (R2 = 0.9890). peak current The of error EA barincreases signifies linearly the standardwith deviationincreasing of EA the concentration. five determinants. The linearity To calculate expression the limitof EA of concentration detection (LOD) vs. EA in peak the electrochemicalcurrent can be written as Y = 0.0497X + 1.0293 (R2 = 0.9890). The error bar signifies the standard deviation of the detection of EA, the IUPAC method was used. As per this method, 10 blank injections of 0.1 M NaOH five determinants. To calculate the limit of detection (LOD) in the electrochemical detection of EA, were added and the DPV responses were recorded. LOD = 3S /S (S : standard deviation of 10 blank the IUPAC method was used. As per this method, 10 blank injectionsB B of 0.1 M NaOH were added samples, S: slope of the calibration curve) [39,40]. The LOD of the electrochemical detection of EA in the and the DPV responses were recorded. LOD = 3SB/S (SB: standard deviation of 10 blank samples, S: µ rGO-Ag@SiOslope of the2 calibration–modified curve) Au PCB [39,40 was].calculated The LOD of to the be 0.68electrochemicalM. The constructed detection of linear EA in equation the rGO could- beAg@SiO used to2 determine–modified Au the PCB unknown was calculated concentration to be of0.68 EA μM. in theThe real constructed sample analysis.linear equation The eff couldectiveness be of used to determine the unknown concentration of EA in the real sample analysis. The effectiveness of

Nanomaterials 2020, 10, 1368 12 of 17 Nanomaterials 2020, 10, x FOR PEER REVIEW 13 of 18 thethe rGO-Ag@SiO rGO-Ag@SiO2/2Au/Au PCB-based PCB-based EA EA sensor sensor was was compared compared with with previously previously evaluated evaluated sensors, sensors, and theand resultsthe results are presented are presented in Table in Table S1. S1.

a b

FigureFigure 8. 8. ((aa)) DiDifferentialfferential pulse pulse voltammetric voltammetric (DPV)(DPV) responseresponse of of etidronicetidronic acid acid (EA) (EA) atat di differentfferent concentrationsconcentrations (2.0, (2.0, 5.0, 5.0, 10.0, 10.0, 20.0, 20.0, 50.0, 50.0, 100.0, 100.0, 150.0, 150.0, andand 200.0 200.0 µ μM)M) in in 0.1 0.1 M M NaOH. NaOH. (b(b)) Linear Linear relationshiprelationship between between the the EA EA concentration concentration and and peak peak current. current. 3.7. Stability, Reproducibility, and Reusability 3.7. Stability, Reproducibility, and Reusability

The operational stability of the rGO-Ag@SiO2/Au PCB electrochemical sensor was evaluated 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, 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 and at the end of the 20th cycle, 92.7% of the initial peak current was retained. To assess the long- stability of the rGO-Ag@SiO2/Au PCB in the detection of EA, the rGO-Ag@SiO2/Au PCB was stored at term stability of the rGO-Ag@SiO2/Au PCB in the detection of EA, the rGO-Ag@SiO2/Au PCB was room temperature (25 C), and a storage stability study was conducted at intervals of seven days for four stored at room temperature◦ (25 °C), and a storage stability study was conducted at intervals of seven weeks. At the end of the fourth week, 93.1% of the DPV peak current response of the initial peak current days for four weeks. At the end of the fourth week, 93.1% of the DPV peak current response of the was retained. The evaluation of the reusability of the rGO-Ag@SiO2/Au PCB in the electrochemical initial peak current was retained. The evaluation of the reusability of the rGO-Ag@SiO2/Au PCB in detection of 100.0 µM EA was carried out using the DPV technique. A single rGO-Ag@SiO /Au the electrochemical detection of 100.0 μM EA was carried out using the DPV technique. A single2 rGO- PCB was used continuously for up to eight runs. At the end of each run, the rGO-Ag@SiO2/Au PCB Ag@SiO2/Au PCB was used continuously for up to eight runs. At the end of each run, the rGO- was cleaned with a 0.1 M NaOH buffer to eliminate the oxidized form of EA adsorbed on the PCB. Ag@SiO2/Au PCB was cleaned with a 0.1 M NaOH buffer to eliminate the oxidized form of EA The observed DPV current responses were plotted in a bar chart (Figure S6) to compare the eight adsorbed on the PCB. The observed DPV current responses were plotted in a bar chart (Figure S6) to runs. The evaluated percent relative standard deviation (%RSD) between the eight runs was calculated compare the eight runs. The evaluated percent relative standard deviation (%RSD) between the eight to be 3.51%. The reproducibility of the rGO-Ag@SiO2/Au PCB was investigated in the presence of runs was calculated to be 3.51%. The reproducibility of the rGO-Ag@SiO2/Au PCB was investigated 100.0 µM EA in 0.1 M NaOH using four independent rGO-Ag@SiO2/Au PCBs. The %RSD between in the presence of 100.0 μM EA in 0.1 M NaOH using four independent rGO-Ag@SiO2/Au PCBs. The the DPV current responses of EA was evaluated to be 2.88%. Table1 shows the sensor parameters of %RSD between the DPV current responses of EA was evaluated to be 2.88%. Table 1 shows the sensor rGO-Ag@SiO2 in EA detection. parameters of rGO-Ag@SiO2 in EA detection. 3.8. Interference Study Table 1. Performance of rGO-Ag@SiO2/Au PCB sensor parameters. The selectivity of the EA detection at the rGO-Ag@SiO2/Au PCB was evaluated in the presence of Sensor Parameters EA other excipients, co-existing interferents, and ionic interferents. For the selectivity and to determine Oxidation potential (V) 0.67 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),Linearity uric acid range (UA), (µM) ascorbic acid (AA), potassium2.0–200.0 hydroxide (KOH), Slope of calibration curve 0.0497 starch, nitric acid (HNO3), sodium chloride (NaCl), magnesium stearate (MS), and cetyl trimethyl −8 ammonium bromide (CTAB) [41Standard]. The recorded deviation DPV of responsesblanks are shown in Figure1.12 ×9 10a. A comparison of the effect of different interferents on theLOD EA (µM) peak current is displayed in Figure0.689b. Operational stability (Retained peak current after 20 cycles) 92.7% Repeatability (%RSD) 3.51% Reproducibility (%RSD) 2.88% Storage stability (Retained peak current after four weeks) 93.1%

3.8. Interference Study

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Table 1. Performance of rGO-Ag@SiO2/Au PCB sensor parameters.

Sensor Parameters EA Oxidation potential (V) 0.67 Linearity range (µM) 2.0–200.0 Nanomaterials 2020, 10, x FOR PEER REVIEW 14 of 18 Slope of calibration curve 0.0497

The selectivity of the EAStandard detection deviation at the of rGO blanks-Ag@SiO2/Au PCB was evaluated1.12 10 8 in the presence × − of other excipients, co-existing interferents,LOD (µ M)and ionic interferents. For the selectivity 0.68 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 Operational stability (Retained peak current after 20 cycles) 92.7% different interferents: glucose (Glu), uric acid (UA), ascorbic acid (AA), potassium hydroxide (KOH), Repeatability (%RSD) 3.51% starch, nitric acid (HNO3), sodium chloride (NaCl), magnesium stearate (MS), and cetyl trimethyl ammonium bromide (CTAB)Reproducibility [41]. The recorded (%RSD) DPV responses are shown 2.88% in Figure 9a. A comparison of Storagethe effect stability of different (Retained interferents peak current on afterthe EA four peak weeks) current is displayed 93.1% in Figure 9b.

a b

FigureFigure 9. 9. (a)( aD)ifferential Differential pulse pulse voltammetric voltammetric (DPV (DPV)) response response of an of interferent an interferent study study of etidronic of etidronic acid (EA)acid (50 (EA) μM) (50 withµM) different with di ffinterferents.erent interferents. (b) Bar chart (b) Bar of peak chart current of peak response current responseof EA with of different EA with interferents.different interferents.

Table2 shows the current responses and the E values of EA after the addition of the different Table 2 shows the current responses and the Epp values of EA after the addition of the different interferents. No significant change in the E of EA was observed. The change in the EA peak current interferents. No significant change in the Epp of EA was observed. The change in the EA peak current owingowing to to the the addition addition of of interferents interferents was was less less than than 5%. 5%. This This proved proved that that the the developed developed sensor sensor was was highlyhighly selective selective toward toward EA. EA. 3.9. Real Sample Analysis Table 2. Interference study results in the electrochemical detection of EA. To verify the real-time application of the proposed sensor, an rGO-Ag@Sio –modified Au Concentration of 2 PCB-basedInterferent electrochemical sensor was used toConcentration analyze the concentration of EA Ep ofof EA inPeak etidronate Current tablets Interferent Added (25.0,Added 50.0, and 100.0 µM). The obtained DPV currentAdded responses(µM) (FigureEA (V) 10 ) wereof EA fitted (µA) into the (mM) calibration curve (Y = 0.0497X + 1.0293) to calculate the actual concentration of the EA in the real - 0 50 0.675 4.43 sample. The recovery of EA in the pharmaceutical samples was calculated to be between 96.2% and Glucose 5 50 0.668 4.52 102.9% (Table S2). UA 5 50 0.679 4.31 AA 5 50 0.679 4.26 KOH 5 50 0.676 4.27 Starch 5 50 0.678 4.43 HNO3 5 50 0.670 4.47 NaCl 5 50 0.673 4.55 MS 5 50 0.673 4.49 CTAB 5 50 0.671 4.58

3.9. Real Sample Analysis

To verify the real-time application of the proposed sensor, an rGO-Ag@Sio2–modified Au PCB- based electrochemical sensor was used to analyze the concentration of EA in etidronate tablets (25.0,

Nanomaterials 2020, 10, 1368 14 of 17

Table 2. Interference study results in the electrochemical detection of EA.

Interferent Concentration of Concentration of Peak Current of E of EA (V) Added Interferent Added (mM) EA Added (µM) p EA (µA) - 0 50 0.675 4.43 Glucose 5 50 0.668 4.52 UA 5 50 0.679 4.31 AA 5 50 0.679 4.26 KOH 5 50 0.676 4.27 NanomaterialsStarch 2020, 10, x FOR PEER REVIEW 5 50 0.678 4.43 15 of 18

HNO3 5 50 0.670 4.47 50.0, and 100.0 μM). The obtained DPV current responses (Figure 10) were fitted into the calibration NaCl 5 50 0.673 4.55 curve (Y = 0.0497X + 1.0293) to calculate the actual concentration of the EA in the real sample. The MS 5 50 0.673 4.49 recovery of EA in the pharmaceutical samples was calculated to be between 96.2% and 102.9% (Table S2). CTAB 5 50 0.671 4.58

Figure 10. 10. DDiifferentialfferential pulse pulse voltammetric voltammetric (DPV (DPV)) response response of of etidronate etidronate tablets tablets at didifferentfferent concentrations (25.0, 50.0, and 100.0 µμM)M) inin 0.1 MM NaOH.NaOH.

4. Conclusions Rapid construction of an rGO-Ag@SiO /Au PCB-based electrochemical sensing platform Rapid construction of an rGO-Ag@SiO2/Au2 PCB-based electrochemical sensing platform was was achieved through probe sonication. The self-assembled rGO-Ag@SiO nanocomposite was achieved through probe sonication. The self-assembled rGO-Ag@SiO2 2 nanocomposite was characterized by SEM, UV, FTIR,FTIR, andand XPS Steps involved in the electrochemical oxidation of EA were studied by CV and DPV. Electrochemical detection of EA in the rGO-Ag@SiO /Au PCB was found studied by CV and DPV. Electrochemical detection of EA in the rGO-Ag@SiO2/Au2 PCB was found to tobe bea diffusion a diffusion-controlled-controlled process process with with a line a linearar range range of 2.0 of– 2.0–200.0200.0 μM,µ andM, andthe obtained the obtained LOD LOD was was0.68 0.68μM. µUsingM. Using 100-fold 100-fold higher higher concentrations concentrations of various of various interferent, interferent, detection detection of EA of was EA carried was carried out, and out, andthe effect the e ffofect the of various the various interferents interferents at the at EA the peak EA peakcurrent current was less was than less 5%. than The 5%. constructed The constructed rGO- rGO-Ag@SiO /Au PCB-based DPV sensor exhibited excellent repeatability (%RSD = 3.51 for eight Ag@SiO2/Au PCB2 -based DPV sensor exhibited excellent repeatability (%RSD = 3.51 for eight runs), runs),high reproducibility high reproducibility (%RSD (%RSD = 2.88 for= 2.88four forindependent four independent Au PCB Auelectrodes), PCB electrodes), and long and-term long-term stability stability over four weeks. The rGO-Ag@SiO /Au PCB sensor was tested on etidronate tablets, and the over four weeks. The rGO-Ag@SiO2/Au PCB2 sensor was tested on etidronate tablets, and the obtained obtained recovery values were excellent (96.2–102.9%). Thus, the optimized rGO-Ag@SiO /Au PCB recovery values were excellent (96.2–102.9%). Thus, the optimized rGO-Ag@SiO2/Au2 PCB electrochemical sensor could be used to detect EAEA inin realreal samples.samples.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/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@SiO2/Au PCB, respectively.; Figure S3: (a), (b) CV responses of non-faradaic current response of bare Au PCB and rGO-Ag@SiO2/Au PCB, respectively, in 0.1 M KCl., (c) relationship between scan rate (ʋ) and current density, (d) and (e) CV response of bare Au PCB and rGO-Ag@SiO2/Au PCB in 0.1 M KCl containing 5 mM K3Fe(CN)6.; Figure S4: (a) CV responses of rGO-Ag@SiO2 at various concentrations of rGO (0, 0.5, and 1.0 mg) and at a fixed concentration of Ag@SiO2 (1 mg) in the presence of EA in 0.1 M NaOH. (b) The plot of CrGO /CrGO

+ CAg@SiO2 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@SiO2/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.

Nanomaterials 2020, 10, x FOR PEER REVIEW 11 of 18

a b

c

Nanomaterials 2020, 10, 1368 15 of 17

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/10/7/1368/s1: Figure 7. (a) Effect of the scan rate (25–200 mV/s) on the rGO-Ag@SiO2/Au PCB in the presence of 1 Figure S1: Photographs of Au PCB; Figure S2: (a), (b), and (c) EDX spectra of bare Au PCB, rGO/Au PCB, mM etidronic acid (EA) in a 0.1 M NaOH electrolyte (pH = 13). (b) Linear relationship between the and rGO-Ag@SiO2/Au PCB, respectively; Figure S3: (a), (b) CV responses of non-faradaic current response of 1/2 bare Au PCB and rGO-Ag@SiO2/Au PCB, respectively, in 0.1 M KCl, (c) relationshipsquare root between of the scan scan rate ( ʋ) and) and the oxidation peak current (Ipa) for EA. (c) Differential pulse current density, (d) and (e) CV response of bare Au PCB and rGO-Ag@SiO2/Auvoltammetric PCB in 0.1 M (DPV) KCl containing response 5of mM 1 mM EA in different pH media [0.1 M acetate buffer (pH = 4.4), 10 K3Fe(CN)6; Figure S4: (a) CV responses of rGO-Ag@SiO at various concentrations of rGO (0, 0.5, and 1.0 mg) 2 mM PBS (pH = 7.4) and 0.1 M NaOH (pH = 13)]. and at a fixed concentration of Ag@SiO2 (1 mg) in the presence of EA in 0.1 M NaOH. (b) The plot of CrGO /CrGO + CAg@SiO2 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 EAThe in 0.1 Randles M NaOH–Sevcik at rGO-Ag@SiO equation2/ Auwas applied to study the electrochemical oxidation process of EA PCB; Figure S7: Raman spectral analysis of graphite, GO, and rGO. Table S1: Comparison of previously reported at the rGO-Ag@SiO2–modified Au PCB: Ip = 2.69 × 105 n3/2AD1/2Cʋ1/2, where Ip is the peak current of 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 ofEA, graphite, n is the GO, number and rGO. of electrons participating in the reaction, A is the ECASA, D is the diffusion coefficient, C is the concentration of EA (mol/cm3), and ʋ is the scan rate (mV/s) [35]. The linear graph Author Contributions: S.P.S. conceptualized the experiment, analysis methodology and carried out all the experiments. S.R.C. is responsible for data curation and validation of the(Figu experimentalre 7b) between results. the S.C. square acquired root the of the scan rate (ʋ1/2) and the corresponding anodic peak current required funding for this study and reviewed the original manuscript.of K.Y. EA is responsible provides concretefor reviewing confirmation the final that the electrochemical oxidation of EA is a diffusional manuscript and data accuracy. All authors have read and agreed to theprocess. published version of the manuscript. Funding: This research was funded by the National Research Foundation ofTo Korea, enhance Republic the of Koreastability (Grant and No. performance of the electrochemical sensor, a pH study was NRF- 2019R1A2C1088680 and 2018M3A9F1023691). conducted in three different pH media containing 1 mM EA: 0.1 M acetate buffer (pH = 4.4), 10 mM Conflicts of Interest: The authors declare no conflict of interest. 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 References observed in the acidic medium. At neutral pH, an enhanced peak current (10.3 μA) was observed 1. Rott, E.; Steinmetz, H.; Metzger, J.W. Organophosphonates:compared A review onto the environmental acidic medi relevance,um. The highest peak current of EA (18.4 μA) was observed at 0.68 V in biodegradability and removal in wastewater treatment plants. 0.1Sci. M Total NaOH, Environ. and 2018pH =, 61513., 1176–1191. [CrossRef][PubMed] 2. Ioachimescu, A.; Licata, A. Etidronate: What is its place in treatment3.5. Mechanism of primary of osteoporosis the Detection and of EA other demineralizing diseases today? Curr. Osteoporos. Rep. 2007, 5, 165–169. [CrossRef][PubMed]

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