# TiO2 Nanoparticles Decorated Graphene Nanoribbons for Voltammetric Determination of an Anti-HIV Drug Nevirapine

Authors: Daniel Apath; Mambo Moyo; Munyaradzi Shumba
Journal: Journal of Chemistry
 (2020)

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## Abstract

In the present study, electrochemical behavior of nevirapine on a glassy carbon electrode (GCE) modified with TiO2 nanoparticles decorated graphene nanoribbons was investigated. Characterization of different components used for modifications was achieved using Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). The electrochemical behavior of nevirapine on the modified electrodes was examined using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), chronoamperometry (CA), and differential pulse voltammetry (DPV). A considerable oxidation potential decrease of +352 mV for nevirapine in 0.1 M phosphate-buffered saline (PBS), pH 11.0, was achieved due to synergy offered by graphene nanoribbons and TiO2 compared to graphene nanoribbons (+252 mV) and TiO2 (−37 mV), all with respect to the glassy carbon electrode. Under optimized conditions, DPV gave linear calibrations over the range of 0.020–0.14 µM. The detection limit was calculated as 0.043 µM. The developed sensor was used for determination of nevirapine in a pharmaceutical formulation successfully.


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## Body

## 1. Introduction

The need for reliable, fast, cheap sensing devices for monitoring biomolecules is important in everyday life. Nevirapine (NVP) (11-cyclopropyl-4-methyl-5, 11-dihydro-6H-dipyrido [3,2-b:2′,3′-e] [1, 2] diazepin-6-one) is a nonnucleoside reverse transcriptase inhibitor. Its uses have been highlighted [1, 3]. The continued use of nevirapine in treatment and management of HIV/AIDS in the health sector thus calls for a great need to improve on its reported analytical work. Several analytical methods, such as reversed phase high-performance liquid chromatography (RP-HPLC) [4] and capillary electrophoresis [2], have been used for quantifying NVP in pharmaceutical combinations and some real samples. The growing demand for NVP biomolecule stimulates a search for new and even more effective monitoring techniques, which give better insight on their reported analytical work. Therefore, it is appropriate to develop new and/or improve on the existing analytical techniques regarding their qualitative and quantitative determination in various matrices, namely, human serum, urine, breast milk, and pharmaceutical formulations. The development of sensors with high sensitivity to promote safety and efficiency during administration to patients who depend on these different biomolecules for life support is important. For example, nevirapine in HIV patients’ treatment can coexist together with other biomolecules; hence, simultaneous determination is critical, helping management of antiretroviral treatment by minimizing drug-food interactions. Furthermore, nevirapine is also easily oxidized; hence development of an electrochemical sensor is significant.Electrochemical techniques based on modification of electrode surfaces can grant some remarkable advantages on applications due to provision of high selectivity and improved sensitivity and stability [5–7]. Among the various modifiers, those based on carbon nanomaterials have been shown to be highly promising in electrochemical sensing [8, 9]. In recent years, graphene nanoribbons (GNRs), a one-dimensional form of graphene strip, have shown promising application in fabrication of composites, batteries, supercapacitors, and fuel cells as shown by a high length-to-width ratio, intrinsic energy band gap, and straight edges, availability of larger surface area, and higher electrical conductivity [10–12]. The electron confinement in GNRs has been reported to offer good electronic properties, hence transforming semimetallic to semiconducting properties [13]. Interestingly, GNRs’ attractiveness in a variety of electrochemical applications is owed to their outstanding electronic, catalytic, charge transport and surface passivation properties in sensing various organic and inorganic compounds [14, 15]. Oxygen functionalities at the edges of nanoribbons after synthesis reportedly offer homogeneous distribution of metal oxide nanoparticles on the surface of carbon [16].Metallic oxide nanoparticles have been reported to show favorable properties in electrocatalysis [17, 18]. In this regard, metal oxide nanoparticles such as TiO2 have been used in sensor fabrication [19–22] due to low cost, nontoxicity, large surface area, biocompatibility, strong adsorptive ability, high uniformity, and excellent catalytic activity [22]. Based on the aforementioned properties, the hybridization of GNRs with metal oxide nanoparticles can provide nanocomposite with synergic properties [23].In the present study, we have fabricated a simple, cheap glassy carbon electrode (GCE) modified with TiO2/GNRs as an electrochemical sensor for nevirapine. In our investigation, the atypical properties of GNRs as a good support for making nanoparticle dispersions caused by large surface area, high electrical conductivity, and electrochemical stability in acidic and alkaline electrolytes were an attractive factor. Based on the attractive properties of GNRs and TiO2, synthesis of TiO2, GNRs, and a TiO2/GNRs nanocomposite through environmental friendly methods was carried out. The results showed that the developed sensor had good performances such good reproducibility and good selectivity, owing to the synergic effects of catalysis characters of TiO2 and GNRs. Besides, to evaluate the applicability of the proposed sensor, it was used to determine nevirapine quantities in a pharmaceutical sample.


## 2. Experimental

### 2.1. Chemicals and Solutions

All chemicals used were of analytical grade. Phosphate-buffered saline (PBS) solutions (as supporting electrolyte) with different pH values (6–12) were prepared by mixing standard stock solutions of 0.10 M Na2HPO4 and 0.10 M NaH2PO4. NaNO3, K3Fe(CN)6, K4Fe (CN)6, N,N-dimethylformamide (DMF), D (+) glucose monohydrate, 5% nitric acid, nevirapine, 98% H2SO4, KMnO4, 30% H2O2, and MWCNT (purity of 95%, diameter∼20–40 nm, and length∼5–15 µm) were obtained from Sigma-Aldrich (South Africa). Titanium acetate dihydrate [Ti(O2CCH3)2(H2O)2] and ethanol (C2H5OH) were obtained from Associated Chemical Enterprises (South Africa). All solutions were prepared using ultra-Millipore water from Milli-Q Water Systems (Millipore Corp., Bedford, MA, USA). The MWCNTs were purified to remove metal oxide catalysts as reported [18]. The stock solution of appropriate solution of nevirapine was prepared by weighing and dissolving the drug in a 1 : 1 mixture of distilled water and ethanol. The working solutions were prepared by dilution of the stock standard solution with PBS (0.1 M, pH 11). A solution of glucose was prepared by dissolving appropriate amount of glucose in ultrapure water and left for 24 h for mutarotation at room temperature.


### 2.2. Equipment

Fourier transform infrared spectroscopy (FT-IR, Nicolet 6700 model) was used in IR characterization. The scanning electron microscopy (SEM) image was obtained using a TESCAN Vega TS 5136LM Electron microscope. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), chronoamperometry, and differential pulse voltammetry (DPV) were performed using an Autolab potentiostat PGSTAT 302N (Eco Chemie, Utrecht, Netherlands) equipped with NOVA 1.10 software. Sonicator model KQ-250B was used for agitation of samples. The pH of the solutions was measured and adjusted by a Thermo Scientific Orion Star A211 pH meter.


### 2.3. Preparation of Graphene Nanoribbons

Graphene nanoribbons (GNRs) were prepared as described in literature with some little modifications [24, 25]. Briefly, a suspension was formed by dissolving 100 mg of MWCNT in 3.4 mL of 98% H2SO4 and then homogenized via ultrasonication for 1 h. Thereafter, the suspension solution was placed in an ice bath accompanied with vigorous stirring and 75 mg of NaNO3 was then added. Next, 450 mg of KMnO4 was added to the suspension. Upon completion of reaction, 20 mL of 5% sulphuric acid solution was added and reaction allowed to cool. As soon as bubble formation started, 2 mL of 30% H2O2 was added dropwise. After approximately 30 min, centrifugation and washing with 5% nitric acid three times and deionized water five times were carried out followed by filtration and drying in an oven at 90°C for 12 h under vacuum. The prepared GNRs were ascertained to contain oxygen-containing functional groups.


### 2.4. Preparation of TiO2 Nanoparticles

The sol-gel process was used for the synthesis of TiO2 nanoparticles at 80–90°C. Briefly, 2.195 g of titanium acetate dihydrate was dissolved in 100 mL ethanol followed by stirring in ambient atmosphere. 1.122 g KOH was dissolved in 10 mL distilled water and then added to the titanium acetate dihydrate-ethanol solution dropwise under continuous stirring. The mixed solution turned into a jelly form and a milky white solution was obtained after a few minutes. Furthermore, the mixture was then heated for 3 h at 80–90°C without stirring. Centrifugation was applied to the resulting suspension to obtain intended product. Finally, the mixture was washed with ultra-Millipore water in an ultrasonic bath and then the powder dried at 70°C overnight.


### 2.5. Preparation of TiO2/GNR and TiO2/GNR/GCE

The suspension of TiO2 (1 mg/mL) was added to GNRs (2 mg/mL) DMF solution (1 : 2) and sonicated for 4 h at room temperature to obtain the TiO2/GNR nanocomposite homogeneous suspension. The GCE was polished to a mirror finish with alumina slurry (0.3 μm). The electrode was sonicated in Millipore water for 5 min during the three successive cleaning stages and finally dried in a stream of nitrogen. The drop dry technique was employed for electrode modification. The optimized volume of the composite, 5 µL of TiO2/GNR suspension, was drop-casted on the surface of GCE and dried in the oven to obtain TiO2/GNRs/GCE. The fabrication procedure of the electrochemical sensor is shown in Scheme 1.Scheme 1
The fabrication procedure of the electrochemical sensor.


### 2.6. Assay of Nevirapine Tablets

Five nevirapine tablets (each containing 200 mg per tablet) were obtained from local commercial sources. The drugs were crushed to obtain a finely homogenized powder using the mortar. A portion of the powder suitable to prepare 1 mM was weighed and transferred into a 10 mL volumetric flask containing water, sonicated to allow dissolution, and then diluted to the mark. Appropriate amounts of solutions were taken and analyzed by DPV method.


## 2.1. Chemicals and Solutions

All chemicals used were of analytical grade. Phosphate-buffered saline (PBS) solutions (as supporting electrolyte) with different pH values (6–12) were prepared by mixing standard stock solutions of 0.10 M Na2HPO4 and 0.10 M NaH2PO4. NaNO3, K3Fe(CN)6, K4Fe (CN)6, N,N-dimethylformamide (DMF), D (+) glucose monohydrate, 5% nitric acid, nevirapine, 98% H2SO4, KMnO4, 30% H2O2, and MWCNT (purity of 95%, diameter∼20–40 nm, and length∼5–15 µm) were obtained from Sigma-Aldrich (South Africa). Titanium acetate dihydrate [Ti(O2CCH3)2(H2O)2] and ethanol (C2H5OH) were obtained from Associated Chemical Enterprises (South Africa). All solutions were prepared using ultra-Millipore water from Milli-Q Water Systems (Millipore Corp., Bedford, MA, USA). The MWCNTs were purified to remove metal oxide catalysts as reported [18]. The stock solution of appropriate solution of nevirapine was prepared by weighing and dissolving the drug in a 1 : 1 mixture of distilled water and ethanol. The working solutions were prepared by dilution of the stock standard solution with PBS (0.1 M, pH 11). A solution of glucose was prepared by dissolving appropriate amount of glucose in ultrapure water and left for 24 h for mutarotation at room temperature.


## 2.2. Equipment

Fourier transform infrared spectroscopy (FT-IR, Nicolet 6700 model) was used in IR characterization. The scanning electron microscopy (SEM) image was obtained using a TESCAN Vega TS 5136LM Electron microscope. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), chronoamperometry, and differential pulse voltammetry (DPV) were performed using an Autolab potentiostat PGSTAT 302N (Eco Chemie, Utrecht, Netherlands) equipped with NOVA 1.10 software. Sonicator model KQ-250B was used for agitation of samples. The pH of the solutions was measured and adjusted by a Thermo Scientific Orion Star A211 pH meter.


## 2.3. Preparation of Graphene Nanoribbons

Graphene nanoribbons (GNRs) were prepared as described in literature with some little modifications [24, 25]. Briefly, a suspension was formed by dissolving 100 mg of MWCNT in 3.4 mL of 98% H2SO4 and then homogenized via ultrasonication for 1 h. Thereafter, the suspension solution was placed in an ice bath accompanied with vigorous stirring and 75 mg of NaNO3 was then added. Next, 450 mg of KMnO4 was added to the suspension. Upon completion of reaction, 20 mL of 5% sulphuric acid solution was added and reaction allowed to cool. As soon as bubble formation started, 2 mL of 30% H2O2 was added dropwise. After approximately 30 min, centrifugation and washing with 5% nitric acid three times and deionized water five times were carried out followed by filtration and drying in an oven at 90°C for 12 h under vacuum. The prepared GNRs were ascertained to contain oxygen-containing functional groups.


## 2.4. Preparation of TiO2 Nanoparticles

The sol-gel process was used for the synthesis of TiO2 nanoparticles at 80–90°C. Briefly, 2.195 g of titanium acetate dihydrate was dissolved in 100 mL ethanol followed by stirring in ambient atmosphere. 1.122 g KOH was dissolved in 10 mL distilled water and then added to the titanium acetate dihydrate-ethanol solution dropwise under continuous stirring. The mixed solution turned into a jelly form and a milky white solution was obtained after a few minutes. Furthermore, the mixture was then heated for 3 h at 80–90°C without stirring. Centrifugation was applied to the resulting suspension to obtain intended product. Finally, the mixture was washed with ultra-Millipore water in an ultrasonic bath and then the powder dried at 70°C overnight.


## 2.5. Preparation of TiO2/GNR and TiO2/GNR/GCE

The suspension of TiO2 (1 mg/mL) was added to GNRs (2 mg/mL) DMF solution (1 : 2) and sonicated for 4 h at room temperature to obtain the TiO2/GNR nanocomposite homogeneous suspension. The GCE was polished to a mirror finish with alumina slurry (0.3 μm). The electrode was sonicated in Millipore water for 5 min during the three successive cleaning stages and finally dried in a stream of nitrogen. The drop dry technique was employed for electrode modification. The optimized volume of the composite, 5 µL of TiO2/GNR suspension, was drop-casted on the surface of GCE and dried in the oven to obtain TiO2/GNRs/GCE. The fabrication procedure of the electrochemical sensor is shown in Scheme 1.Scheme 1
The fabrication procedure of the electrochemical sensor.


## 2.6. Assay of Nevirapine Tablets

Five nevirapine tablets (each containing 200 mg per tablet) were obtained from local commercial sources. The drugs were crushed to obtain a finely homogenized powder using the mortar. A portion of the powder suitable to prepare 1 mM was weighed and transferred into a 10 mL volumetric flask containing water, sonicated to allow dissolution, and then diluted to the mark. Appropriate amounts of solutions were taken and analyzed by DPV method.


## 3. Results and Discussion

### 3.1. Characterization

FT-IR spectra of TiO2, GNR, and TiO2/GNR composite are shown in Figure 1(a). The characteristic peaks in (b) were assigned as follows: 1654 cm−1 attributed to the stretching vibrations of carboxyl (-COOH) and at 1130 cm−1 corresponding to C-O-H bending and C-OH. The presence of such peaks can be used to explain the hybrid structure of graphene nanoribbons. After making the composite, the small peaks observed in (a) disappeared and peak intensity of C = O in GNR/TiO2 shows some changes as a result of incorporation of nanoparticles (Figure 1(c)). The wavenumber region 3400 cm−1 is the stretching vibration of the hydroxyl group in all spectra. The morphology of prepared materials was observed using SEM (Figure 1(b)). As shown in Figure 1(a) TiO2 nanospheres are aggregated in larger agglomerates. Figure 2(b) shows long curved rod-like structures of GNR [26]. The TiO2 structures can be seen to be closely and homogeneously grown on the GNR (Figure 2(c)). For comparison, the GCE was included (Figure 1(d)).Figure 1
(a) FT-IR; (b) SEM images for (a) TiO2 and (b) GNR; (c) TiO2/GNR nanocomposite; (d) GCE.
(a)
(b)Figure 2
(a) EIS behavior of modified GCE in the presence of 1 mM [Fe(CN)6]3−/4− in 0.1 M KCl; (b) CVs of the modified GCE in the presence of 1 mM [Fe(CN)6]3−/4− in 0.1 M KCl at a scan rate of 100 mV s−1. (c) Plot of ipa and Retversus modified GCEs; (a) GCE, (b) TiO2/GCE, (c) GNR/GCE, and (d) TiO2/GNR/GCE.
(a)
(b)
(c)EIS studies were done in the [Fe(CN)6]3−/4− redox system to deduce the resistance to electron transfer. Nyquist plots (Figure 2) are shown by semicircle portions at high frequencies, while diffusion-limiting steps of the electrochemical process are shown by linear parts at lower frequencies. The equivalent circuit model used to fit impedance data into Ret values is shown as inset in Figure 2. From Table 1, it can be seen that modification of the GCE showed lowering of Ret except for TiO2 with a larger diameter (2.2 kΩ) suggesting that TiO2 acted as an insulating layer and barrier [18]. The incorporation of GNRs significantly lowered Ret values of GCE by 33.3% and that of TiO2 in composite by 77.8%. The order as deduced from impedance values is TiO2/GCE > GCE > GNR/GCE > TiO2/GNR/GCE. The changes in Ret suggested proper modification of modified electrodes; hence, the electrode based on TiO2/GNR composite was used throughout the study.Table 1
Parameters for modified electrodes.
Electrodes
R
et/kΩ [Fe(CN)6]3−/4−(0.1 M KCl)
ΔEP [Fe(CN)6]3 −/4−(0.1 M KCl)
Nevirapine detection current (ipa, μA)
Nevirapine detection potential (Epa, mV)
GCE
0.9
100
6.6
742
TiO2
2.2
120
14.0
779
GNR
0.6
85
14.4
490
TiO2/GNR
0.20
83
18.9
390CV involving modified electrodes was performed in 1 mM [Fe(CN)6]3−/4− and 0.1 M KCl electrolyte at a scan rate of 100 mV/s (Figure 2(b)). Redox peaks were observed on all tested electrodes with different peak current (ip) and change in peak potential separation (ΔEP) (Table 1). The effect of different modifiers was shown by ΔEP, with lower ΔEP showing better electron transfer ability. After drop-casting TiO2/GNR on surface of GCE, ipa, increased ipc, and reduced Ep were observed compared to ipa, ipc, and Ep of GCE. The observation is attributed to TiO2/GNR particles offering a large surface and good conductivity. Furthermore, the GNR acted as a suitable pathway to shuttle electrons; hence, improved peak currents were displayed. TiO2/GCE showed a sluggish electron transfer process. The electrodes gave peak potential differences with the following trend: TiO2<GCE < GNR/GCE < TiO2/GNR/GCE. The obtained results are in good agreement with Ret values obtained from EIS results (Figure 2(c)). To see the change in area of modified electrode, electrode surface area of the TiO2/GNR/GCE was determined in 1 mM K3[Fe(CN)6]3−/4− by applying the Randles–Sevcik equation:(1)ip=2.69×105n3/2AeffCv1/2,where ip is the peak current, n is the number of electrons transferred (n = 1) during the redox couple Fe (II)/Fe (III), Dis the diffusion coefficient of the analyte in solution (7.6 × 10−6 cm2 s−1), C is the solution concentration in mol/cm3, Aeff is the effective surface area, and v is the scan rate (V/s). Voltammograms at different scan rates (50–300 mV/s) were run and gave a linear plot for ipversusv1/2. From the slope, TiO2/GNR/GCE had an effective surface area of 0.208 cm2 relative to the GCE area of 0.0712 cm2 [27].


### 3.2. Effect of pH

The electrochemical reaction of a biomolecule is usually affected by pH; hence, influence of pH onipa and Epa was examined by cyclic voltammetry in the pH range of 7–12 in phosphate-buffered solutions at 100 mV/s (Figure 3). From the voltammograms in Figure 3, it can be seen that increasing pH results in biomolecule being easily oxidized, causing peak current and peak sharpness to increase as well. It should be noted that voltammograms in acidic media were not included, since no oxidation could be observed as a result of protonation on biomolecule. The repulsive forces between the biomolecule and the electrode surface of TiO2/GNR/GCE slowed down arrival of biomolecules to the surface. As depicted in the inset, the current increases up to maximum of pH 11 and then decreases. Therefore, pH 11 was chosen as optimum pH for further studies. The peak potential was affected by pH as shown in the inset (Figure 3). The Epa values of nevirapine were shifted to less positive values with increasing pH, showing deprotonation in the oxidation process at higher pHs [18]. A linear relationship was observed between Epa and pH for the biomolecule with the following regression equations:Figure 3
CVs for pH studies using TiO2/GNR/GCE for nevirapine in 0.1 M PBS; 100 mV/s. Insets: plots of Ipa and Epaversus pH.E
pa (mV) = −63.9 pH + 1123; R2 = 0.9508 for nevirapine.The slope for nevirapine (63.9 mV per pH)≈59.6 mV per pH indicates involvement of equal number of protons and electrons during oxidation reactions. This is in agreement with previous reports [5, 6, 28].


### 3.3. Cyclic Voltammetry Behavior of Nevirapine

It is pertinent to study the behavior of modified electrodes in PBS alone before any determinations (Figure4(a)).Figure 4
CVs in (a) 0.1 M PBS alone and (b) 1 mM nevirapine in 0.1 M PBS (pH 11.0): (a) GCE, (b) TiO2/GCE, (c) GNR/GCE, and (d) TiO2/GNR/GCE. (c) Simultaneous analysis of nevirapine and glucose at TiO2/GNR/GCE, at a scan rate of 100 mV/s.
(a)
(b)
(c)There was an increase in the peak background corrected current from GCE to TiO2/GNR/GCE.Graphene nanoribbons assisted in faster electron transport as well as providing a large surface area compared to TiO2 alone. The electrochemical oxidation behavior of nevirapine was investigated by CV in 0.1 M PBS (pH 11.0) at modified electrodes (Figure 4(b)). The obtained voltammograms indicated electroactiveness of the compound and irreversible behavior. As shown in Figure 4(b), ipa increased for nevirapine following modification on GCE; TiO2/GCE < GNR/GCE < TiO2/GNR (Table 1). The GCE exhibits very low ipa at different potentials, whereas with the TiO2 modified glassy carbon electrode under same conditions, ipa increases compared to that of GCE (Figure 4(b)). On the other hand, when GNR was deposited on GCE, ipa of nevirapine was found to be 2.2 times greater compared to GCE (Figure 4(c)). This may be attributed to fast electron transfer as well as high surface area available on the GNR for electrooxidation of nevirapine. The electrocatalytic oxidation of nevirapine with TiO2/GNR composite modified GCE showed improved behaviors. An increase of ipa, 186% for nevirapine, was deduced (Figure 4(d)), accompanied with a decrease in overpotential (352 mV) compared to glassy carbon electrode. The decrease in overpotential is caused by the synergetic effect of GNRs and TiO2. TiO2 nanoparticles help in electrocatalytic oxidation, whereas the GNRs provide larger surface area for TiO2 as well as for nevirapine and then allow faster electrode kinetics. Hence, the TiO2/GNR/GCE was used for the determination of nevirapine in the presence of interferent glucose in 0.1 M PBS (pH 11.0) using CV (Figure 4(c)). CV shows that it is possible for the biomolecules to be determined simultaneously with no overlap as shown by ΔEpa of 410 mV.In order to study nature of the electrode process, the influence of scan rate (v) on ipa and Epa for a mixture of 0.1 mM glucose (interferent) and nevirapine in a 0.1 M PBS (pH 11.0) was examined by CV with scan rate ranging from 50 to 400 mV/s (Figure 5). From Figure 5 inset (a, b), ipa is directly proportional to scan rate (v) with a correlation coefficient of 0.9991 (nevirapine) and 0.9948 (glucose) showing electrochemical behavior of biomolecules on TiO2/GNR modified electrode as adsorption controlled processes [4, 29, 30]. The adsorption mechanism process has also been reported for nevirapine [5, 6]. The presence of functional groups at edges of GNR in the composite might have facilitated adsorption of nevirapine by π–π stacking, hydrogen bonding, and covalent interactions [31]. Furthermore, plot of log ipa against log v (Figure 5(c), inset) gave a linear plot with the following equation:Figure 5
CVs of 0.1 mM nevirapine (1) and 0.1 mM glucose (2) in 0.1 M PBS (pH 11.0) at TiO2/GNR/GCE (d) at various scan rates (50–400 mV/s). Inset: (a) plot ipa (μA) versus v(mV/s); (b) log ipa (μA) versus log v (mV/s) (nevirapine); (c) plot ipa (μA) versusv (mV/s) (glucose).
(a)
(b)
(c)logipa (µA) = 0.804 log v (mV/s) + 0.493 for nevirapine.The slope of nevirapine (0.804) is greater than the theoretical value of 0.5 V/s, which further confirms that electrochemical oxidation exhibits mixed behavior [32]. Additionally, from voltammograms, both biomolecules show ipa increasing and Epa shifting in the less positive value with increase in scan rate. The obtained results depict a totally irreversible electrochemical process.


### 3.4. Catalytic Rate Constants

Catalytic rate constants for nevirapine at TiO2/GNR/GCE (Figure 6) were determined by chronoamperometry based on favorable oxidation results from voltammetry. The rate constants were evaluated using the following equation [33]:(2)IcatIbuf=γ1/2π1/2erfγ1/2+exp−yγ1/2,where Icat and Ibuf are currents on TiO2/GNR/GCE in presence and absence of nevirapine, respectively, γ = kCot (Co is the bulk concentration of nevirapine), and erf is the error function. The error function is almost equal to 1 when γ exceeds 2 and hence equation (2) reduces to the following equation:(3)IcatIbuf=γ1/2π1/2=π1/2kCot1/2,where k is the catalytic rate constant (cm3/mol/s) and t is the time elapsed in (s). The catalytic rate constants for nevirapine were calculated based on information obtained from chronoamperometry (Figure 6). A plot of Icat/Ibuf versus t1/2 for oxidation of nevirapine gave a linear plot. The calculated values of catalytic constants were 7.9 × 105 cm3/mol/s. The calculated values further elucidate sharp features of catalytic ipa of nevirapine at the surface of TiO2/GNR/GCE.Figure 6
Chronoamperograms for different nevirapine concentrations in 0.1 M PBS (pH 11), curves 2–9 : 0.1–0.7µM. Insets: (a) plots of icat/ibuffer versus t1/2; (b) Slope2versus [nevirapine].
(a)
(b)


### 3.5. Nevirapine Determination by Differential Pulse Voltammetry

Determination of nevirapine in the absence of interferents was investigated (Figure7(a)). In all cases, ipa increased with increase in concentration of analyte as shown by voltammograms and the analytical parameters shown in Table 2.Figure 7
DPVs of TiO2/GNR/GCE in different concentrations of nevirapine. Inset plot of ipa (μA) versus [nevirapine]/μM in 0.1 M PBS solution, pH 11.0.Table 2
Analytical parameters for electrochemical determination of nevirapine in 0.1 M PBS at pH 11.0.
Analyte
E(mV)
LDR (μM)
Linear regression equation (y ↔i/µA; x ↔  [analyte], µM)
R2
LOD (μM)
RSD (%)
Nevirapinea
+400
0.020–0.14
y = 25.36 x + 1.73
0.9912
0.041
2.65
Glucoseb
+800
0.020–0.14
y = 321.43x + 7.94
0.9914
0.012
1.42
Nevirapineb
+400
0.020–0.14
y = 269.52x + 1.13
0.9872
0.043
2.60
Glucosec
+800
0.020–0.14
y = 402.91x + 6.12
0.9971
0.012
1.48
Nevirapinec
+400
0.020–0.14
y = 75.00x + 6.07
0.9911
0.043
2.65
Nevirapined
−
Log (0.20–1.2)
R
et (kΩ) = −2.28 log x + 1.06
0.9842
0.070
2. 75
aStatistical data for DPV single analyte determination. bStatistical data from DPV for single analyte when the concentration of the other analyte is kept constant. cStatistical data from DPV for both analytes simultaneously determined. dImpedimetric determination.


### 3.6. Behavior of Nevirapine in the Presence of Interferent

The effect of different concentrations of analyte on electrochemical response of nevirapine and interferent glucose on TiO2/GNR modified electrode was investigated (Figures 8(a)–8(c)). Different cases were studied, where the concentration of only one compound was varied, while the concentration of the other compound was kept constant. In the first case, the concentration of nevirapine was changed, while the concentration of glucose was kept constant (Figure 8(a)). As shown in Figure 8(a), DPP was obtained with different concentrations (0.20–1.4 µM) of nevirapine in presence of 0.20 µM glucose on TiO2/GNR modified electrode. The oxidation peak currents increased linearly when the bulk concentration of nevirapine was also increased. Furthermore, the oxidation peak currents of glucose remained the same as the numbers of cycles were increased. The inset (Figure 8(a)) shows a plot of ipaversus different concentrations of nevirapine.Figure 8
DPVs of TiO2/GNR/GCE for (a) 0.2 μM glucose (kept constant) and different concentrations of nevirapine. Inset plot of ipa (μA) versus [nevirapine]/μM; (b) 0.2 μM nevirapine (kept constant) and different concentrations of glucose. Inset: plot of ipa (μA) versus [glucose]/μM; (c) DPV for different concentrations of nevirapine and glucose, all in 0.1 M PBS solution, pH 11.0. Different concentrations ranged from 0.02 to 0.14 μM.
(a)
(b)
(c)On the other hand, Figure8(b) shows DPVs obtained in different concentrations (0.20–1.4 µM) of glucose in presence of 0.20 µM nevirapine. The same trend was observed with the glucose molecule, where ipa corresponding to oxidation of glucose showed linearity with an increase in the bulk concentration of glucose, and ipa for the oxidation of nevirapine remained the same as the number of cycles increased. A plot of ipaversus different concentrations of glucose is shown as inset (Figure 8(b)). The analytical parameters are shown in Table 2. Figure 8(c) shows DPVs obtained at TiO2/GNR modified electrode when equal concentrations of nevirapine and glucose were simultaneously changed. As shown in Figure 8(c), clearly resolved voltammetric signals were observed for addition of increasing concentrations with calibration curves as insets. The analytical parameters of the calibration plots are listed in Table 2. From the use of TiO2/GNR modified electrode, it is interesting to note the detection of glucose and nevirapine is independent as shown by detection limits and clear peak potential separation. A comparison of the detection limits of the present method with those reported in recent years at other surfaces is tabulated in Table 3. The values of limit of detection (LOD) were calculated using 3s/k (where s is the standard deviation of the blank and k is the slope of the calibration curve) and using Figures 7, 8(a)–8(c), and 9. It can be clearly seen that the LOD observed using TiO2/GNR is much lower. The synergic effects between TiO2 and excellent physicochemical properties of GNR make the composite highly suitable for nevirapine and glucose sensing.Table 3
A comparison of analysis results of nevirapine at TiO2/GNR sensor with recently reported sensors.
Modifier
Analysis technique
Electrolyte type, pH
Type of analysis
LOD (µM)
LR (µM)
Reference
AuNPs/p (MB)/f-MWCNTs/GE)
DPASV
PBS, 11
Single
53
0.1–50
[6]
CuO-CNP-GCE
LSV
BR buffer 7
Single
66
0.1–100
[3]
Ura/CPE
DPV
0.1 M NaOH, 13
Single
0.05
0.1–70
[1]
CP-Bi2O3
DPV
PBS, 8
Single
0.110
0.05–50
[29]
Pd@rGO/MoS2 QDs
DPV
PBS, 10
Single
0.05
0.1–80
34
TiO2/GNR
DPV
PBS, 11
Simultaneous
0.043
0.020–0.14
Present work
Au NPs, gold nanoparticles; DPASV, differential pulse anodic stripping voltammetry, Ura/CPE, uracil-modified carbon paste electrode; CuO-CNP-GCE, copper oxide carbon nanoparticles glassy carbon electrode; LSV, linear sweep voltammetry; CP-Bi2O3, carbon paste electrode modified with Bi2O3.


### 3.7. Impedimetric Determination

EIS was also employed to study the oxidation behavior of nevirapine at TiO2/GNR/GCE (Figure 9(a)). The results showed that, in absence of nevirapine, Nyquist diagram comprises a large semicircle at high frequencies due to large charge transfer resistance (2.5 kΩ). In the presence of interferent glucose and biomolecule nevirapine, diameter of the semicircle decreases, confirming the electrocatalytic capability of composite. However, glucose showed a smaller Ret value of 0.5 kΩ, while nevirapine had a large value of 0.75 kΩ. The lower Ret in glucose showed that TiO2/GNR provided more favorable orientation and conductive pathway for transfer of electrons. Overall, it can be concluded that TiO2/GNR sensor was specific towards oxidation of glucose and nevirapine.Figure 9
Nyquist diagrams of TiO2/GNR/GCE (a) in the absence (a) and in the presence of 0.2 µM nevirapine and (b) in the presence of 0.2 µM glucose (c). Different concentrations of (b) nevirapine in 0.1 M PBS (pH 11.0). Insets: calibration plots.
(a)
(b)The EIS responses of fabricated sensors to different concentrations of nevirapine were carried out further (Figure9(b)). It can be seen in Figure 9(a) that Ret decreased with increasing nevirapine concentration. The observations can be attributed to TiO2/GNR offering a suitable platform for oxidation of the biomolecule. Increasing the concentrations of biomolecule, as shown Figure 9(b), results in generation of more electrons (nevirapine 2e−), causing Ret values to decrease markedly, consequently enhancing the electrode kinetics. Under the optimized conditions, the electrochemical sensor gave parameters as shown in Table 2. The LODs are comparable to both single detections of analytes when one is constant and in equal concentrations of the analytes. The results obtained clearly elucidate possible impedimetric detection of nevirapine using GNR and TiO2 in the composite.


### 3.8. Other Interferent Ions

For demonstrating selectivity of TiO2/GNR/GCE, EIS was obtained before and after interactions with each interferent and values for ΔRet were obtained as Ret(after interaction) − Ret(before interaction). The fabricated sensor was incubated in the presence of some interferent ions such as Na+, K+, Cl−, Ca2+, Mg2+, and PO43- and possible biomolecules (sucrose, ascorbic acid, dopamine, tyrosine, glycine, uric acid, lopinavir, stavudine, zidovudine, lamivudine, and efavirenz) to a solution containing nevirapine (0.2 µM). It should be noted that biomolecules that normally coexist with nevirapine were selected. From the Nyquist plots, the results showed that 200-fold excess of Na+, K+, Ca2+, Mg2+, PO43-, Cl−, and sucrose and 100-fold excess of glycine, tyrosine, and uric acid showed no major influences on changes in Ret. For nevirapine alone, negligible changes in Ret in the presence of lopinavir, stavudine, zidovudine, lamivudine, and efavirenz were observed. By looking at all obtained small values of ΔRet, practical applicability of proposed electrode for determination of nevirapine is possible. Similar results have been reported in literature based on different methods [3, 6].


### 3.9. Reproducibility Studies

When developing sensors, reproducibility is a significant process to be investigated. The TiO2/GNR sensor was investigated through five repetitive measurements of 0.1 mM of nevirapine in PBS (pH 11) by CV (Figure 10). The relative standard deviation of ipa responses to nevirapine sensing was less than 3.5%, indicating good reproducibility.Figure 10
CVs for 0.1 mM nevirapine obtained after 5 test runs. Scan rate = 100 mV/s in PBS (pH 11).


### 3.10. Nevirapine in Pharmaceutical Formulations

The results obtained from the developed sensor for determining amount of nevirapine in commercially available tablets are shown in Table4. These were calculated after running working solutions prepared after suitable dilution with DPV. The results of analysis were found to be satisfactory as shown by high recovery values.Table 4
Results of analysis of nevirapine in pharmaceutical formulations by the DPV method.
Nevirapine
Labelled amount (mg)
200
Amount found (mg)
198.5 ± 0.20
Recovery (%)
99.25
Pure nevirapine added to tablet solution (mg)
15
Amount found (mg)
15.12 ± 0.01
Recovery (%)
100.8


## 3.1. Characterization

FT-IR spectra of TiO2, GNR, and TiO2/GNR composite are shown in Figure 1(a). The characteristic peaks in (b) were assigned as follows: 1654 cm−1 attributed to the stretching vibrations of carboxyl (-COOH) and at 1130 cm−1 corresponding to C-O-H bending and C-OH. The presence of such peaks can be used to explain the hybrid structure of graphene nanoribbons. After making the composite, the small peaks observed in (a) disappeared and peak intensity of C = O in GNR/TiO2 shows some changes as a result of incorporation of nanoparticles (Figure 1(c)). The wavenumber region 3400 cm−1 is the stretching vibration of the hydroxyl group in all spectra. The morphology of prepared materials was observed using SEM (Figure 1(b)). As shown in Figure 1(a) TiO2 nanospheres are aggregated in larger agglomerates. Figure 2(b) shows long curved rod-like structures of GNR [26]. The TiO2 structures can be seen to be closely and homogeneously grown on the GNR (Figure 2(c)). For comparison, the GCE was included (Figure 1(d)).Figure 1
(a) FT-IR; (b) SEM images for (a) TiO2 and (b) GNR; (c) TiO2/GNR nanocomposite; (d) GCE.
(a)
(b)Figure 2
(a) EIS behavior of modified GCE in the presence of 1 mM [Fe(CN)6]3−/4− in 0.1 M KCl; (b) CVs of the modified GCE in the presence of 1 mM [Fe(CN)6]3−/4− in 0.1 M KCl at a scan rate of 100 mV s−1. (c) Plot of ipa and Retversus modified GCEs; (a) GCE, (b) TiO2/GCE, (c) GNR/GCE, and (d) TiO2/GNR/GCE.
(a)
(b)
(c)EIS studies were done in the [Fe(CN)6]3−/4− redox system to deduce the resistance to electron transfer. Nyquist plots (Figure 2) are shown by semicircle portions at high frequencies, while diffusion-limiting steps of the electrochemical process are shown by linear parts at lower frequencies. The equivalent circuit model used to fit impedance data into Ret values is shown as inset in Figure 2. From Table 1, it can be seen that modification of the GCE showed lowering of Ret except for TiO2 with a larger diameter (2.2 kΩ) suggesting that TiO2 acted as an insulating layer and barrier [18]. The incorporation of GNRs significantly lowered Ret values of GCE by 33.3% and that of TiO2 in composite by 77.8%. The order as deduced from impedance values is TiO2/GCE > GCE > GNR/GCE > TiO2/GNR/GCE. The changes in Ret suggested proper modification of modified electrodes; hence, the electrode based on TiO2/GNR composite was used throughout the study.Table 1
Parameters for modified electrodes.
Electrodes
R
et/kΩ [Fe(CN)6]3−/4−(0.1 M KCl)
ΔEP [Fe(CN)6]3 −/4−(0.1 M KCl)
Nevirapine detection current (ipa, μA)
Nevirapine detection potential (Epa, mV)
GCE
0.9
100
6.6
742
TiO2
2.2
120
14.0
779
GNR
0.6
85
14.4
490
TiO2/GNR
0.20
83
18.9
390CV involving modified electrodes was performed in 1 mM [Fe(CN)6]3−/4− and 0.1 M KCl electrolyte at a scan rate of 100 mV/s (Figure 2(b)). Redox peaks were observed on all tested electrodes with different peak current (ip) and change in peak potential separation (ΔEP) (Table 1). The effect of different modifiers was shown by ΔEP, with lower ΔEP showing better electron transfer ability. After drop-casting TiO2/GNR on surface of GCE, ipa, increased ipc, and reduced Ep were observed compared to ipa, ipc, and Ep of GCE. The observation is attributed to TiO2/GNR particles offering a large surface and good conductivity. Furthermore, the GNR acted as a suitable pathway to shuttle electrons; hence, improved peak currents were displayed. TiO2/GCE showed a sluggish electron transfer process. The electrodes gave peak potential differences with the following trend: TiO2<GCE < GNR/GCE < TiO2/GNR/GCE. The obtained results are in good agreement with Ret values obtained from EIS results (Figure 2(c)). To see the change in area of modified electrode, electrode surface area of the TiO2/GNR/GCE was determined in 1 mM K3[Fe(CN)6]3−/4− by applying the Randles–Sevcik equation:(1)ip=2.69×105n3/2AeffCv1/2,where ip is the peak current, n is the number of electrons transferred (n = 1) during the redox couple Fe (II)/Fe (III), Dis the diffusion coefficient of the analyte in solution (7.6 × 10−6 cm2 s−1), C is the solution concentration in mol/cm3, Aeff is the effective surface area, and v is the scan rate (V/s). Voltammograms at different scan rates (50–300 mV/s) were run and gave a linear plot for ipversusv1/2. From the slope, TiO2/GNR/GCE had an effective surface area of 0.208 cm2 relative to the GCE area of 0.0712 cm2 [27].


## 3.2. Effect of pH

The electrochemical reaction of a biomolecule is usually affected by pH; hence, influence of pH onipa and Epa was examined by cyclic voltammetry in the pH range of 7–12 in phosphate-buffered solutions at 100 mV/s (Figure 3). From the voltammograms in Figure 3, it can be seen that increasing pH results in biomolecule being easily oxidized, causing peak current and peak sharpness to increase as well. It should be noted that voltammograms in acidic media were not included, since no oxidation could be observed as a result of protonation on biomolecule. The repulsive forces between the biomolecule and the electrode surface of TiO2/GNR/GCE slowed down arrival of biomolecules to the surface. As depicted in the inset, the current increases up to maximum of pH 11 and then decreases. Therefore, pH 11 was chosen as optimum pH for further studies. The peak potential was affected by pH as shown in the inset (Figure 3). The Epa values of nevirapine were shifted to less positive values with increasing pH, showing deprotonation in the oxidation process at higher pHs [18]. A linear relationship was observed between Epa and pH for the biomolecule with the following regression equations:Figure 3
CVs for pH studies using TiO2/GNR/GCE for nevirapine in 0.1 M PBS; 100 mV/s. Insets: plots of Ipa and Epaversus pH.E
pa (mV) = −63.9 pH + 1123; R2 = 0.9508 for nevirapine.The slope for nevirapine (63.9 mV per pH)≈59.6 mV per pH indicates involvement of equal number of protons and electrons during oxidation reactions. This is in agreement with previous reports [5, 6, 28].


## 3.3. Cyclic Voltammetry Behavior of Nevirapine

It is pertinent to study the behavior of modified electrodes in PBS alone before any determinations (Figure4(a)).Figure 4
CVs in (a) 0.1 M PBS alone and (b) 1 mM nevirapine in 0.1 M PBS (pH 11.0): (a) GCE, (b) TiO2/GCE, (c) GNR/GCE, and (d) TiO2/GNR/GCE. (c) Simultaneous analysis of nevirapine and glucose at TiO2/GNR/GCE, at a scan rate of 100 mV/s.
(a)
(b)
(c)There was an increase in the peak background corrected current from GCE to TiO2/GNR/GCE.Graphene nanoribbons assisted in faster electron transport as well as providing a large surface area compared to TiO2 alone. The electrochemical oxidation behavior of nevirapine was investigated by CV in 0.1 M PBS (pH 11.0) at modified electrodes (Figure 4(b)). The obtained voltammograms indicated electroactiveness of the compound and irreversible behavior. As shown in Figure 4(b), ipa increased for nevirapine following modification on GCE; TiO2/GCE < GNR/GCE < TiO2/GNR (Table 1). The GCE exhibits very low ipa at different potentials, whereas with the TiO2 modified glassy carbon electrode under same conditions, ipa increases compared to that of GCE (Figure 4(b)). On the other hand, when GNR was deposited on GCE, ipa of nevirapine was found to be 2.2 times greater compared to GCE (Figure 4(c)). This may be attributed to fast electron transfer as well as high surface area available on the GNR for electrooxidation of nevirapine. The electrocatalytic oxidation of nevirapine with TiO2/GNR composite modified GCE showed improved behaviors. An increase of ipa, 186% for nevirapine, was deduced (Figure 4(d)), accompanied with a decrease in overpotential (352 mV) compared to glassy carbon electrode. The decrease in overpotential is caused by the synergetic effect of GNRs and TiO2. TiO2 nanoparticles help in electrocatalytic oxidation, whereas the GNRs provide larger surface area for TiO2 as well as for nevirapine and then allow faster electrode kinetics. Hence, the TiO2/GNR/GCE was used for the determination of nevirapine in the presence of interferent glucose in 0.1 M PBS (pH 11.0) using CV (Figure 4(c)). CV shows that it is possible for the biomolecules to be determined simultaneously with no overlap as shown by ΔEpa of 410 mV.In order to study nature of the electrode process, the influence of scan rate (v) on ipa and Epa for a mixture of 0.1 mM glucose (interferent) and nevirapine in a 0.1 M PBS (pH 11.0) was examined by CV with scan rate ranging from 50 to 400 mV/s (Figure 5). From Figure 5 inset (a, b), ipa is directly proportional to scan rate (v) with a correlation coefficient of 0.9991 (nevirapine) and 0.9948 (glucose) showing electrochemical behavior of biomolecules on TiO2/GNR modified electrode as adsorption controlled processes [4, 29, 30]. The adsorption mechanism process has also been reported for nevirapine [5, 6]. The presence of functional groups at edges of GNR in the composite might have facilitated adsorption of nevirapine by π–π stacking, hydrogen bonding, and covalent interactions [31]. Furthermore, plot of log ipa against log v (Figure 5(c), inset) gave a linear plot with the following equation:Figure 5
CVs of 0.1 mM nevirapine (1) and 0.1 mM glucose (2) in 0.1 M PBS (pH 11.0) at TiO2/GNR/GCE (d) at various scan rates (50–400 mV/s). Inset: (a) plot ipa (μA) versus v(mV/s); (b) log ipa (μA) versus log v (mV/s) (nevirapine); (c) plot ipa (μA) versusv (mV/s) (glucose).
(a)
(b)
(c)logipa (µA) = 0.804 log v (mV/s) + 0.493 for nevirapine.The slope of nevirapine (0.804) is greater than the theoretical value of 0.5 V/s, which further confirms that electrochemical oxidation exhibits mixed behavior [32]. Additionally, from voltammograms, both biomolecules show ipa increasing and Epa shifting in the less positive value with increase in scan rate. The obtained results depict a totally irreversible electrochemical process.


## 3.4. Catalytic Rate Constants

Catalytic rate constants for nevirapine at TiO2/GNR/GCE (Figure 6) were determined by chronoamperometry based on favorable oxidation results from voltammetry. The rate constants were evaluated using the following equation [33]:(2)IcatIbuf=γ1/2π1/2erfγ1/2+exp−yγ1/2,where Icat and Ibuf are currents on TiO2/GNR/GCE in presence and absence of nevirapine, respectively, γ = kCot (Co is the bulk concentration of nevirapine), and erf is the error function. The error function is almost equal to 1 when γ exceeds 2 and hence equation (2) reduces to the following equation:(3)IcatIbuf=γ1/2π1/2=π1/2kCot1/2,where k is the catalytic rate constant (cm3/mol/s) and t is the time elapsed in (s). The catalytic rate constants for nevirapine were calculated based on information obtained from chronoamperometry (Figure 6). A plot of Icat/Ibuf versus t1/2 for oxidation of nevirapine gave a linear plot. The calculated values of catalytic constants were 7.9 × 105 cm3/mol/s. The calculated values further elucidate sharp features of catalytic ipa of nevirapine at the surface of TiO2/GNR/GCE.Figure 6
Chronoamperograms for different nevirapine concentrations in 0.1 M PBS (pH 11), curves 2–9 : 0.1–0.7µM. Insets: (a) plots of icat/ibuffer versus t1/2; (b) Slope2versus [nevirapine].
(a)
(b)


## 3.5. Nevirapine Determination by Differential Pulse Voltammetry

Determination of nevirapine in the absence of interferents was investigated (Figure7(a)). In all cases, ipa increased with increase in concentration of analyte as shown by voltammograms and the analytical parameters shown in Table 2.Figure 7
DPVs of TiO2/GNR/GCE in different concentrations of nevirapine. Inset plot of ipa (μA) versus [nevirapine]/μM in 0.1 M PBS solution, pH 11.0.Table 2
Analytical parameters for electrochemical determination of nevirapine in 0.1 M PBS at pH 11.0.
Analyte
E(mV)
LDR (μM)
Linear regression equation (y ↔i/µA; x ↔  [analyte], µM)
R2
LOD (μM)
RSD (%)
Nevirapinea
+400
0.020–0.14
y = 25.36 x + 1.73
0.9912
0.041
2.65
Glucoseb
+800
0.020–0.14
y = 321.43x + 7.94
0.9914
0.012
1.42
Nevirapineb
+400
0.020–0.14
y = 269.52x + 1.13
0.9872
0.043
2.60
Glucosec
+800
0.020–0.14
y = 402.91x + 6.12
0.9971
0.012
1.48
Nevirapinec
+400
0.020–0.14
y = 75.00x + 6.07
0.9911
0.043
2.65
Nevirapined
−
Log (0.20–1.2)
R
et (kΩ) = −2.28 log x + 1.06
0.9842
0.070
2. 75
aStatistical data for DPV single analyte determination. bStatistical data from DPV for single analyte when the concentration of the other analyte is kept constant. cStatistical data from DPV for both analytes simultaneously determined. dImpedimetric determination.


## 3.6. Behavior of Nevirapine in the Presence of Interferent

The effect of different concentrations of analyte on electrochemical response of nevirapine and interferent glucose on TiO2/GNR modified electrode was investigated (Figures 8(a)–8(c)). Different cases were studied, where the concentration of only one compound was varied, while the concentration of the other compound was kept constant. In the first case, the concentration of nevirapine was changed, while the concentration of glucose was kept constant (Figure 8(a)). As shown in Figure 8(a), DPP was obtained with different concentrations (0.20–1.4 µM) of nevirapine in presence of 0.20 µM glucose on TiO2/GNR modified electrode. The oxidation peak currents increased linearly when the bulk concentration of nevirapine was also increased. Furthermore, the oxidation peak currents of glucose remained the same as the numbers of cycles were increased. The inset (Figure 8(a)) shows a plot of ipaversus different concentrations of nevirapine.Figure 8
DPVs of TiO2/GNR/GCE for (a) 0.2 μM glucose (kept constant) and different concentrations of nevirapine. Inset plot of ipa (μA) versus [nevirapine]/μM; (b) 0.2 μM nevirapine (kept constant) and different concentrations of glucose. Inset: plot of ipa (μA) versus [glucose]/μM; (c) DPV for different concentrations of nevirapine and glucose, all in 0.1 M PBS solution, pH 11.0. Different concentrations ranged from 0.02 to 0.14 μM.
(a)
(b)
(c)On the other hand, Figure8(b) shows DPVs obtained in different concentrations (0.20–1.4 µM) of glucose in presence of 0.20 µM nevirapine. The same trend was observed with the glucose molecule, where ipa corresponding to oxidation of glucose showed linearity with an increase in the bulk concentration of glucose, and ipa for the oxidation of nevirapine remained the same as the number of cycles increased. A plot of ipaversus different concentrations of glucose is shown as inset (Figure 8(b)). The analytical parameters are shown in Table 2. Figure 8(c) shows DPVs obtained at TiO2/GNR modified electrode when equal concentrations of nevirapine and glucose were simultaneously changed. As shown in Figure 8(c), clearly resolved voltammetric signals were observed for addition of increasing concentrations with calibration curves as insets. The analytical parameters of the calibration plots are listed in Table 2. From the use of TiO2/GNR modified electrode, it is interesting to note the detection of glucose and nevirapine is independent as shown by detection limits and clear peak potential separation. A comparison of the detection limits of the present method with those reported in recent years at other surfaces is tabulated in Table 3. The values of limit of detection (LOD) were calculated using 3s/k (where s is the standard deviation of the blank and k is the slope of the calibration curve) and using Figures 7, 8(a)–8(c), and 9. It can be clearly seen that the LOD observed using TiO2/GNR is much lower. The synergic effects between TiO2 and excellent physicochemical properties of GNR make the composite highly suitable for nevirapine and glucose sensing.Table 3
A comparison of analysis results of nevirapine at TiO2/GNR sensor with recently reported sensors.
Modifier
Analysis technique
Electrolyte type, pH
Type of analysis
LOD (µM)
LR (µM)
Reference
AuNPs/p (MB)/f-MWCNTs/GE)
DPASV
PBS, 11
Single
53
0.1–50
[6]
CuO-CNP-GCE
LSV
BR buffer 7
Single
66
0.1–100
[3]
Ura/CPE
DPV
0.1 M NaOH, 13
Single
0.05
0.1–70
[1]
CP-Bi2O3
DPV
PBS, 8
Single
0.110
0.05–50
[29]
Pd@rGO/MoS2 QDs
DPV
PBS, 10
Single
0.05
0.1–80
34
TiO2/GNR
DPV
PBS, 11
Simultaneous
0.043
0.020–0.14
Present work
Au NPs, gold nanoparticles; DPASV, differential pulse anodic stripping voltammetry, Ura/CPE, uracil-modified carbon paste electrode; CuO-CNP-GCE, copper oxide carbon nanoparticles glassy carbon electrode; LSV, linear sweep voltammetry; CP-Bi2O3, carbon paste electrode modified with Bi2O3.


## 3.7. Impedimetric Determination

EIS was also employed to study the oxidation behavior of nevirapine at TiO2/GNR/GCE (Figure 9(a)). The results showed that, in absence of nevirapine, Nyquist diagram comprises a large semicircle at high frequencies due to large charge transfer resistance (2.5 kΩ). In the presence of interferent glucose and biomolecule nevirapine, diameter of the semicircle decreases, confirming the electrocatalytic capability of composite. However, glucose showed a smaller Ret value of 0.5 kΩ, while nevirapine had a large value of 0.75 kΩ. The lower Ret in glucose showed that TiO2/GNR provided more favorable orientation and conductive pathway for transfer of electrons. Overall, it can be concluded that TiO2/GNR sensor was specific towards oxidation of glucose and nevirapine.Figure 9
Nyquist diagrams of TiO2/GNR/GCE (a) in the absence (a) and in the presence of 0.2 µM nevirapine and (b) in the presence of 0.2 µM glucose (c). Different concentrations of (b) nevirapine in 0.1 M PBS (pH 11.0). Insets: calibration plots.
(a)
(b)The EIS responses of fabricated sensors to different concentrations of nevirapine were carried out further (Figure9(b)). It can be seen in Figure 9(a) that Ret decreased with increasing nevirapine concentration. The observations can be attributed to TiO2/GNR offering a suitable platform for oxidation of the biomolecule. Increasing the concentrations of biomolecule, as shown Figure 9(b), results in generation of more electrons (nevirapine 2e−), causing Ret values to decrease markedly, consequently enhancing the electrode kinetics. Under the optimized conditions, the electrochemical sensor gave parameters as shown in Table 2. The LODs are comparable to both single detections of analytes when one is constant and in equal concentrations of the analytes. The results obtained clearly elucidate possible impedimetric detection of nevirapine using GNR and TiO2 in the composite.


## 3.8. Other Interferent Ions

For demonstrating selectivity of TiO2/GNR/GCE, EIS was obtained before and after interactions with each interferent and values for ΔRet were obtained as Ret(after interaction) − Ret(before interaction). The fabricated sensor was incubated in the presence of some interferent ions such as Na+, K+, Cl−, Ca2+, Mg2+, and PO43- and possible biomolecules (sucrose, ascorbic acid, dopamine, tyrosine, glycine, uric acid, lopinavir, stavudine, zidovudine, lamivudine, and efavirenz) to a solution containing nevirapine (0.2 µM). It should be noted that biomolecules that normally coexist with nevirapine were selected. From the Nyquist plots, the results showed that 200-fold excess of Na+, K+, Ca2+, Mg2+, PO43-, Cl−, and sucrose and 100-fold excess of glycine, tyrosine, and uric acid showed no major influences on changes in Ret. For nevirapine alone, negligible changes in Ret in the presence of lopinavir, stavudine, zidovudine, lamivudine, and efavirenz were observed. By looking at all obtained small values of ΔRet, practical applicability of proposed electrode for determination of nevirapine is possible. Similar results have been reported in literature based on different methods [3, 6].


## 3.9. Reproducibility Studies

When developing sensors, reproducibility is a significant process to be investigated. The TiO2/GNR sensor was investigated through five repetitive measurements of 0.1 mM of nevirapine in PBS (pH 11) by CV (Figure 10). The relative standard deviation of ipa responses to nevirapine sensing was less than 3.5%, indicating good reproducibility.Figure 10
CVs for 0.1 mM nevirapine obtained after 5 test runs. Scan rate = 100 mV/s in PBS (pH 11).


## 3.10. Nevirapine in Pharmaceutical Formulations

The results obtained from the developed sensor for determining amount of nevirapine in commercially available tablets are shown in Table4. These were calculated after running working solutions prepared after suitable dilution with DPV. The results of analysis were found to be satisfactory as shown by high recovery values.Table 4
Results of analysis of nevirapine in pharmaceutical formulations by the DPV method.
Nevirapine
Labelled amount (mg)
200
Amount found (mg)
198.5 ± 0.20
Recovery (%)
99.25
Pure nevirapine added to tablet solution (mg)
15
Amount found (mg)
15.12 ± 0.01
Recovery (%)
100.8


## 4. Conclusions

A selective nevirapine electrochemical sensor was developed based on the modification of a GCE with metal oxide and graphene nanoribbons nanocomposite. The successful preparation of GNR and TiO2 was confirmed by FT-IR and SEM. EIS confirmed superior electrochemical properties of the prepared TiO2/GNR/GCE in comparison to bare GCE. It was also illustrated that TiO2/GNR/GCE offered a low potential during detection by cyclic voltammetry. The obtained TiO2/GNR/GCE composite exhibited good reproducibility during analysis. The developed method was successfully applied to the quantification of nevirapine in a pharmaceutical formulation. The present study provides a general strategy for monitoring drug-food behavior during electrochemical applications.



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