Electrochemical sensor based on graphene oxide and ionic liquid for ofloxacin determination at nanomolar levels

Talanta 161 (2016) 333–341

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Electrochemical sensor based on graphene oxide and ionic liquid for ofloxacin determination at nanomolar levels Ademar Wong a, Tiago Almeida Silva a, Fernando Campanhã Vicentini b, Orlando Fatibello-Filho a,n a b

Department of Chemistry, Federal University of São Carlos, 13560-970 São Carlos, SP, Brazil Center of Nature Sciences, Federal University of São Carlos, 18290-000 Buri, SP, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 9 July 2016 Received in revised form 12 August 2016 Accepted 14 August 2016 Available online 15 August 2016

New insights into the design of highly sensitive, carbon-based electrochemical sensors are presented in this work by exploring the interesting properties of graphene oxide (GO) and ionic liquids (ILs). An electrochemical sensor based on the carbon paste electrode (CPE) modified with GO and IL was developed for the sensitive detection of ofloxacin using square-wave adsorptive anodic stripping voltammetry (SWAdASV). GO sheets were obtained from the acid treatment of graphene and characterized by scanning and transmission electronic microscopy (SEM and TEM) and selected area electron diffraction (SAED), and the electrochemical behavior of the modified GO-IL/CPE was explored by electrochemical impedance spectroscopy studies. The CPE modification with GO and IL allowed an 8.2 fold increase in the analytical sensitivity for ofloxacin sensing compared to the unmodified CPE. Under the optimized experimental conditions using the SWAdASV technique, the GO-IL/CPE sensor provided an analytical curve for ofloxacin in the concentration range of 7.0
10  9 to 7.0
10  7 mol L  1, with a sensitivity of 7.7
106 μA L mol  1 and limit of detection of 2.8
10  10 mol L  1 (0.28 nmol L  1). The proposed sensor was successfully applied for the ofloxacin determination in human urine and ophthalmic samples, with recoveries near 100%. The results were similar those obtained by a spectrophotometric comparative method. & 2016 Elsevier B.V. All rights reserved.

Keywords: Graphene oxide Ionic liquid Ofloxacin Nanomolar concentration Carbon paste electrode

1. Introduction Ofloxacin is a synthetic antibiotic used against gram-negative and gram-positive bacteria [1,2]. Its white crystalline powder form is generally recommended for people with urinary tract infections, prostatitis, lower respiratory tract infection and skin structure infections [3]. The extensive use of this drug can cause adverse effects such as tendon damage, peripheral neuropathy (which may be irreversible) and, in severe cases, it can result in lifelong disabilities [4]. The analytical determination of ofloxacin is very important for minimizing the risks of human intoxication and prevents serious environmental impacts, such as the development of resistant bacteria [5,6]. Analytical methods such as chemiluminescence [7], spectrofluorimetry [8], high performance liquid chromatography (HPLC) [9], capillary electrophoresis [10], spectrophotometry [11], and mass spectrometry [12] are utilized for the determination of ofloxacin. However, there are some disadvantages associated to n

Corresponding author. E-mail address: [email protected] (O. Fatibello-Filho).

http://dx.doi.org/10.1016/j.talanta.2016.08.035 0039-9140/& 2016 Elsevier B.V. All rights reserved.

the use of these methods, such as the large consumption of reagents, high cost of acquisition and maintenance of equipment, leading to a high cost of analysis. A way to overcome these disadvantages is the electrochemical monitoring of ofloxacin using chemically modified electrodes (CMEs). Several conducting or semiconducting materials are used as modifiers to increase the performance of CMEs. The materials widely used as electrode modifiers are: nanoparticles [13], ions [14], polymers [15], phthalocyanine and porphyrin complexes [16], carbon nanomaterials (e. g., graphene and carbon nanotubes) [17,18], ionic liquids [19], and others. Recently, CMEs based on graphene oxide (GO) and/or ionic liquids (ILs) have shown immense potential for the sensitive determination of target analytes. GO is a hydrophilic nanostructured carbon material obtained from graphene, dispersible in aqueous media owing to the presence of oxygen groups in its structure. It has received great attention due to its excellent physical and chemical properties such as high surface area, excellent conductivity, and high mechanical strength. Thus, GO has been used in a wide range of applications, including electronics [20], energy storage [21], batteries [22], fuel cells [23], biological systems [24] and electrochemical sensors and biosensors [25–28]. Ionic liquids

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are important materials that have also attracted much attention due to their unique physical and chemical properties such as high conductivity, wide potential window, high viscosity, low volatility, and chemical and thermal stability. Ionic liquids are formed by positively (cation) and negatively (anion) charged ions in the liquid phase [29]. The use of ILs as modifiers to electrochemical sensor surfaces increases the stability, sensitivity and selectivity of the oxidation and/or reduction of chemical compounds [30,31]. The modification of electrodes with ILs associated with nanomaterials such as graphene can be used to improve the catalytic and/ or electrocatalytic activity and the heterogeneous electron transfer [32,33]. The numerous advantageous that can be achieved from the use of CMEs constructed using GO and ILs include accuracy, stability, reproducibility, high sensitivity and selectivity [34,35]. In the literature, there are some works employing CMEs based on nanomaterials to enhance detection, such as the study performed by Zhou et al. [33]. In that study, a glassy carbon electrode modified with graphene oxide-ionic liquid composites and gold nanoparticles was applied to the ultrasensitive electrochemical detection of Hg2 þ [33]. Shan et al. [36] determined NADH and ethanol using a modified glassy carbon electrode based on an ionic liquid and functionalized graphene within a chitosan film . In another work, Sun et al. [37]. proposed a novel sensing platform based on graphene oxide, 1-ethyl-3-methylimidazolium tetrafluoroborate ionic liquid and Nafion for the immobilization of hemoglobin (Hb). This sensor exhibited excellent electrocatalytic activity towards trichloroacetic acid and H2O2. The aim of this work it was to develop an electrochemical sensor based on a carbon paste electrode modified with GO and IL for the sensitive voltammetric determination of ofloxacin at nanomolar levels.

2. Experimental 2.1. Reagents and solutions Ofloxacin standard and 1-buthyl-3-methylimidazolium tetrafluoroborate (IL) were purchased from Sigma-Aldrich. Graphene was acquired from Graphene Supermarket (New York, USA). All reagents used in this work were of analytical grade and all aqueous solutions were prepared with ultrapure water with resistivity not less than 18 MΩ cm obtained from a Milli-Q Direct-0.3 (Millipore) purification system. A stock solution of 1.0
10  3 mol L  1 ofloxacin was prepared by dissolving 3.6 mg of the compound in 10.0 mL of supporting electrolyte solution prepared in ultrapure water. The mixture was sonicated for 2 min to ensure the complete dissolution of ofloxacin. 2.2. Apparatus Electrochemical experiments were performed using a model PGSTAT-30 potentiostat/galvanostat (Metrohm-Autolab, Utrecht, Netherlands) fitted with an electrochemical cell containing three electrodes: Ag/AgCl (3.0 mol L  1 KCl) reference electrode (Analion), a platinum wire as the counter electrode and a modified carbon paste electrode (CPE) as the working electrode (2.5 mm diameter). The apparatus was controlled by the GPES 4.9 software (Eco Chemie). The pH measurements were performed using an Orion Expandable Ion Analyzer (model EA-940, USA) employing a combined glass electrode with an Ag/AgCl (3.0 mol L  1 KCl) external reference electrode. The morphologic features of the graphene oxide were evaluated using images acquired by field-emission gun scanning

electron microscopy (FEG/SEM, Supra 35-VP, Carl Zeiss, Germany) with an electron beam energy of 25 keV and by transmission electron microcopy (TEM, FEI Tecnai G2F20) 200 kV. A Shimadzu UV-2550 UV/Vis spectrometer with a quartz cuvette (optical path length of 1 cm) was employed for analytical determination of ofloxacin by the comparative method [35]. 2.3. Synthesis of GO Graphene oxide (GO) was obtained by functionalization of graphene using an acid treatment procedure as reported previously [38]. Initially, 100 mg of graphene was treated with a total of 120 mL of conc. H2SO4/conc. HNO3 in the following volumetric ratios: 1:1, 1:3, and 3:1 (v/v). This mixture was stirred for 12 h and the obtained suspension was washed with deionized water until pH 6.5–7.0. Then, the suspension was dried at 100 °C for 12 h. The obtained electrodes were tested using a 2.44
10  3 mol L  1 [Fe (CN)6]3  in 0.1 mol L  1 KCl solution and, the higher magnitude of analytical signal was achieved for 1:1 (v/v) acids ratio (not shown). 2.4. Preparation of the modified carbon paste electrode The modified carbon paste electrode was prepared using a mixture of 94 mg of graphite powder, 5.0 mg of GO and 1.0 mg of ionic liquid (1-buthyl-3-methylimidazolium tetrafluoroborate). The mixture of these materials was carefully homogenized for 30 min using a mortar and pestle. Afterwards, 80 mL (65 mg) of mineral oil was added to obtain a paste. The prepared paste was then packed into the cavity of the Teflon working electrode (2.5 mm i.d., 1 mm depth), where a Pt disk was used to provide the electrical contact. The obtained modified electrode was named as GO-IL/CPE. Partially modified CPEs were prepared for the comparative studies. Thus, an IL/CPE was made using 1.0 mg of ionic liquid and 99 mg of graphite and a bare CPE was made using 100 mg of graphite. The same amount of mineral oil (Nujol) was used for preparation of the partially modified CPEs. 2.5. Preparation of samples The voltammetric procedure developed using the GO-IL/CPE as an electrochemical sensor was evaluated in the ofloxacin determination in different matrice samples. The analyzed samples were commercial ophthalmic formulations and biological urine samples. In the following sections, the preparation procedures adopted for these samples are described in detail. 2.5.1. Preparation of ophthalmic samples Two commercial ophthalmic formulation samples containing ofloxacin at a concentration of 3 mg mL  1 were purchased in local drugstores and subjected to a simple sample preparation procedure: 12 mL of each ophthalmic sample was diluted in 10 mL of ultrapure water (dilution of 830 times) resulting in sample stock solution containing ofloxacin at 1.0
10  5 mol L  1. Next, an adequate volume of this stock solution was added in the electrochemical cell containing 10 mL of supporting electrolyte solution and the voltammetric measurement collect. 2.5.2. Preparation of biological urine samples The biological urine sample was collected from one healthy individual. The urine sample was spiked with two different concentration levels of ofloxacin (5.0
10  8 and 1.0
10  7 mol L  1) and analyzed using the voltammetric procedure. For this, an adequate volume of the urine sample stock solution (1.0
10  5 mol L  1) was added in the electrochemical cell containing 10 mL of supporting electrolyte solution and the voltammetric measurement collect.

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3. Results and discussion 3.1. Morphological analysis of graphene oxide Graphene oxide was dispersed in isopropanol and morphologically characterized by SEM and TEM. Fig. 1(a) shows the SEM image of GO with a wrinkled sheet structure disposed in stacked blocks of different sizes. The TEM images recorded for GO (Fig. 1 (b) and inset of Fig. 1(b)) showed that GO consisted of thin wrinkled sheets with a transparent appearance. In the inset of Fig. 1 (b) is presented the selected area electron diffraction (SAED) pattern obtained for GO, composed of an array of diffraction peaks indicative of single GO sheets [39]. 3.2. Electrochemical characterization of the modified electrodes Electrochemical impedance spectroscopy (EIS) analysis was explored to provide information about the electronic transfer kinetics of the different modified electrodes. The measurements were performed for a 0.1 mol L  1 KCl solution containing 2.44
10  3 mol L  1 K3[Fe(CN)6]. The Nyquist plots obtained for CPE, IL/CPE, GO/CPE and GO-IL/CPE are shown in Fig. 2. As can be seen, a semi-circle was verified at the region of high frequencies and, as is commonly known, the diameter of this semi-circle is equivalent to the charge-transfer resistance across the electrode interface. Thus, as a preliminary qualitative analysis of the Nyquist plots, the decrease of the semi-circle diameter for the modified CPEs with IL, GO or GO-IL is observed, indicating an improvement of the charge-transfer in these electrodes compared to the unmodified CPE. In order to extract quantitative information from the impedance data, a modified Randles equivalent circuit was fitted to the Nyquist plots. The Randles equivalent circuit models the electrochemical system using the following electrical components: the supporting electrolyte resistance (Rs) in series with the parallel combination of the double-layer capacitance (Cdl), charge-transfer resistance (Rct) and a constant phase element (CPE) for the CPE, IL/ CPE and GO-IL/CPE cases and the supporting electrolyte resistance (Rs) in series with the parallel combination of the charge-transfer resistance (Rct), Warburg impedance (ZW) and a constant phase element (CPE) for the GO/CPE case [40,41]. From this fitting, the following Rct were obtained: 2.90 kΩ for CPE, 1.85 kΩ for GO/CPE, 0.83 kΩ for IL/CPE and 0.29 kΩ for GO-IL/CPE. Thus, the decrease of the charge transfer resistance across the CPE interface modified with GO and IL is clear. The Rct can be used to calculate the heterogeneous electron transfer rate constant (k0) across the electronic interface under study. The Rct is inversely proportional to k0

Fig. 2. Nyquist plot obtained for 2.44
10  3 mol L  1 K3[Fe(CN)6] in 0.1 mol L  1 KCl solution with working electrodes: CPE, IL/CPE, GO/CPE and GO-IL/CPE. Randles circuit (1) used for fitting the Nyquist plots of CPE, IL/CPE and GO-IL/CPE and (2) GO/CPE.

constant, in according to the Eq. (1) [42]:

k 0=

RT F2R ct AC

(1)

where R is the gas universal constant, T is the thermodynamic temperature (E 298.15 K), F is the Faraday's constant (96,485 C mol  1), A is the geometric area and C is the concentration of the electroactive species at the supporting electrolyte solution. Using Eq. (1), the k0 constants were calculated and the obtained values were: 7.7
10  4 cm s  1 for CPE, 1.2
10  3 cm s  1 for GO/CPE, 2.7
10  3 cm s  1 for IL/CPE and 7.7
10  3 cm s  1 for GO-IL/CPE. From the comparison of the k0 constants, it is evident the increase of the electron transfer kinetics for the modified electrodes containing IL and GO, demonstrating the contribution of both modifiers for the improvement of the CPE electrochemical behavior. 3.3. Electrochemical behavior of ofloxacin The electrochemical behavior of ofloxacin molecule was investigated by cyclic voltammetry in the potential range of 0.0 to þ1.2 V. In Fig. 3, the cyclic voltammograms obtained using the CPE, IL/CPE and GO-IL/CPE at 0.1 mol L  1 phosphate buffer solution (pH 6.0) in the presence of 5.0
10  5 mol L  1 ofloxacin are

Fig. 1. FEG-SEM (a) and TEM (b) image obtained for GO. Insets of (b) are TEM images collected in a lower magnification and SAED pattern recorded for GO.

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solution (pH 6.0) containing ofloxacin at different concentrations ranging from 7.9
10  9 to 5.3
10  7 mol L  1 using GO-IL/CPE and IL-CPE and from 1.0
10  7 to 5.3
10  7 mol L  L using CPE. In the inset of Fig. 4, the respective analytical curves constructed for each electrode material are shown. Analyzing the obtained analytical curves, a significant increase of analytical sensitivity was achieved using the GO-IL/CPE. The analytical sensitivities obtained 2.1
106, and for the electrodes were 9.4
105, 6 L 7.7
10 μA L mol for CPE, IL-CPE, and GO-IL/CPE, respectively. Therefore, the combined use of GO and IL increased the sensitivity of the analytical curve toward ofloxacin by 8.2 fold.

Fig. 3. Cyclic voltammograms recorded for a 5.0
10  5 mol L  1 ofloxacin solution prepared in 0.1 mol L  1 phosphate buffer solution (pH 6.0) with working electrodes: (a) CPE, (b) IL/CPE and (c) GO-IL/CPE. v¼ 50 mV s  1.

3.3.1. Effect of pH The influence of supporting electrolyte pH on the ofloxacin response was evaluated using 0.1 mol L  1 phosphate buffer solutions prepared at different pHs in the range 3.0–10.0. In Fig. 5 (a) are shown the SWAdAS voltammograms obtained using the GO-IL/CPE sensor for the different supporting electrolyte solutions containing 5.0
10  7 mol L  1 ofloxacin. From the respective values of peak potential (Ep) and current (Δip) the graphics of Ep vs. pH and Δip vs. pH were constructed, as presented in Fig. 5(b). From the Δip vs. pH graphic, it was possible to verify that the higher anodic peak current was achieved at pH ¼6.0; therefore, this pH condition was selected for the further studies. In addition, the relationship between Ep and pH provided information about the ofloxacin electrooxidation reaction. As can be seen from the Ep vs. pH graphic, the Ep decreased linearly with the increase of pH in the range of 3.0–8.0. The following linear regression equation was obtained for Ep as a function of pH (Eq. (2)):

Ep = 1.17 − 0.053pH, r = 0.998

(2)

1

The slope of  0.053 V pH obtained for the Ep vs. pH curve was close to the theoretical Nernstian value of  0.0592 V pH  1 expected for a redox process involving an equal number of protons and electrons [42]. Therefore, an equal number of electrons and protons are released during the ofloxacin electrooxidation, in accordance with previous reports [43–45]. The loss of linearity of the Ep vs. pH curve at pH higher than 8.0 is in accordance with the pKa of piperazine group of the ofloxacin molecule (located between 8.0 and 9.0) [46]. Fig. 4. SWAdAS voltammograms recorded for a 5.0
10  7 mol L  1 ofloxacin solution prepared in 0.1 mol L  1 phosphate buffer solution (pH 6.0) using: (a) CPE, (b) IL/CPE and (c) GO-IL/CPE. Analysis conditions: f ¼15 Hz, a¼ 75 mV, ΔEs ¼5 mV, Eacc ¼ 0.2 V and tacc ¼ 60 s. Inset: Analytical curves obtained for ofloxacin using CPE, IL/CPE and GO-IL/CPE as sensor.

shown. In all the cases, an oxidation process was observed during the anodic potential scanning, with the absence of an equivalent reduction process during the cathodic potential scanning. This behavior demonstrated that ofloxacin underwent an irreversible oxidation reaction. From the results shown in Fig. 3, the ofloxacin voltammetric response on all the electrodes were evaluated and verified that the modified GO-IL/CPE provided an anodic peak current 8.0 times higher than that obtained on the unmodified CPE. Moreover, the same measurements performed by CV were conducted by square-wave adsorptive anodic stripping voltammetry (SWAdASV), and the obtained SWAdAS voltammograms are presented in Fig. 4. Similarly, the modified GO-IL/CPE showed the higher anodic peak current for ofloxacin oxidation. This significant increase of analytical signal (peak current) was very important for the development of a highly sensitive voltammetric procedure. In order to explore how the increase of peak current can improve the analytical sensitivity toward ofloxacin determination, preliminary analytical curves were constructed by SWAdASV using CPE, IL/CPE and GO-IL/CPE for a comparative study. Thus, SWAdAS voltammograms were recorded for 0.1 mol L  1 phosphate buffer

3.3.2. Number of electrons for ofloxacin oxidation The number of electrons transferred in the ofloxacin electrooxidation reaction was determined by varying the frequency parameter in the SWAdASV technique. The square-wave voltammetry theory establishes that the peak potential (Ep) is linearly dependent on the logarithm of frequency (logf) in an irreversible electrode process, according to Eq. (3) [47]:

⎛ 2.3RT ⎞ ⎟logf E p=⎜ ⎝ αnF ⎠

(3)

where α is the charge-transfer coefficient, f is the frequency and the other terms have been previously described. The graphic of Ep vs. logf (Fig. S1 of Supplementary Material) was constructed in the frequency range of 8.0–30.0 Hz, following the linear regression equation, Eq. (4):

Ep = 0.78 + 0.059logf , r = 0.990

(4)

Comparing the experimental slope (Eq. (4)) with the theoretical slope (Eq. (3)), the value αn ¼1.0 was obtained. Considering that α ¼0.5, which is a charge-transfer coefficient typical of organic molecules, the number of electrons transferred for ofloxacin was calculated as 2.0. As was shown in the pH study, the number of protons and electrons transferred in the ofloxacin oxidation is equal. Thus, a possible electrochemical reaction was proposed for

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337

Fig. 5. (a) SWAdAS voltammograms recorded using the GO-IL/CPE for a 5.0
10  7 mol L  1 ofloxacin solution prepared in 0.1 mol L  1 phosphate buffer solution at different pHs: 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 9.0 and 10.0. Analysis conditions: f¼ 15 Hz, a¼ 75 mV, ΔEs ¼ 5 mV, Eacc ¼ 0.2 V and tacc ¼60 s (b) Δip vs. pH and Ep vs. pH curves.

Fig. 6. Possible electrooxidation reaction proposed for ofloxacin.

ofloxacin oxidation involving the piperazine motion oxidation with the release of two electrons and two protons [43–45], as represented in Fig. 6.

Fig. 7. Cyclic voltammograms recorded at different scan rates (a: 20 mV s  1 to h: 300 mV s  1) using the GO-IL/CPE for a 5.0
10  5 mol L  1 ofloxacin solution prepared in 0.1 mol L  1 phosphate buffer solution (pH 6.0). Insets: (i) logΔip vs. log v; (ii) Δip vs. v1/2. Table 1 Optimized parameters of SWAdASV.

3.3.3. Effect of potential scan rate The effect of potential scan rate on the cyclic voltammetry results for a 5.0
10  5 mol L  1 ofloxacin solution was explored in order to extract additional electrochemical features of the ofloxacin redox process. Thus, cyclic voltammetry measurements were performed at different potential scan rates for 0.1 mol L  1 phosphate buffer solution (pH 6.0) in the presence of 5.0
10  5 mol L  1 ofloxacin using the GO-IL/CPE. In Fig. 7, the cyclic voltammograms recorded in the potential scan rate range of 20–300 mV s  1 are shown. From these, the graphic of log Δip vs. log v was constructed (inset (i) of Fig. 7). The slope obtained for the log Δip vs. log v curve was equal to 0.51, which is close to the theoretical value of 0.5 for a diffusion-controlled process. The diffusional control of the ofloxacin oxidation process was confirmed by the linear relationship between the peak current and the square root of the potential scan rate (v1/2) (inset (ii) of Fig. 7). From this, the apparent diffusion coefficient (Dapp) for the ofloxacin molecule was determined using the Randles-Sevcik equation for an irreversible process controlled by diffusion, Eq. (5):

(

)

Δip = ± 2.99 × 10 5 α1/2n3/2Dapp1/2Cv1/2

Parameter

Range

Optimum value

Frequency (Hz) Amplitude (mV) Step potential (mV)

8–30 25–100 2–10

15 75 5

(5)

where Δip is the anodic or cathodic peak current, C is the electroactive species concentration (in mol cm  3), and the other terms have been previously defined. Comparing the slope of the Δip vs. v1/2 graphic (inset (ii) of Fig. 7) with the slope of the RandlesSevcik equation (Eq. (5)), Dapp ¼ 2.73
10  6 cm2 s  1 was obtained

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A. Wong et al. / Talanta 161 (2016) 333–341

Fig. 8. Influence of the accumulation potential (Eacc) (a), accumulation time (tacc) (b), amount of GO (c) and IL (d) on the sensor response. Analysis conditions: 0.1 mol L  1 phosphate buffer solution (pH 6.0) containing 5.0
10  7 mol L  1 ofloxacin, f¼ 15 Hz, a¼ 75 mV and ΔEs ¼5 mV.

for ofloxacin, which is closed to previous values reported for molecules structurally similar to ofloxacin, namely ciprofloxacin (1.54
10  6 cm2 s  1) and levofloxacin (2.28
10  6 cm2 s  1) [48]. Considering the diffusional control of ofloxacin electrooxidation, the heterogeneous electron-transfer rate constant (ks) was determined using the Nicholson and Shain's approach [49], specifically for the case of an irreversible anodic process controlled by diffusion. In this equation, the peak current (Δip) is linearly dependent on the difference between the peak potential (Ep) and the formal potential (Eo′) for different scan rates, Eq. (6):

⎡ ⎛ αnF ⎞ ⎤ ⎟ E –Eo' ⎥ Δip=0.227nFAC k sexp⎢ ⎜ ⎣ ⎝ RT ⎠ p ⎦

(

)

(6)

where all the terms have been previously described. Applying the natural logarithm and rearranging Eq. (6), one may obtain Eq. (7):

⎛ αnF ⎞ ⎟(E –Eo') lnΔip=ln(0.227nFACk s) +⎜ ⎝ RT ⎠ p Fig. 9. SWAdAS voltammograms obtained using the GO-IL/CPE for a 0.1 mol L  1 phosphate buffer solution (pH 6.0) containing different concentrations of ofloxacin: (a) 7.0
10  9; (b) 5.0
10  8, (c) 9.9
10  8, (d) 2.0
10  7, (e) 2.9
10  7, (f) 3.9
10  7, (g) 4.8
10  7 and (h) 7.0
10  7 mol L  1 and analytical curve (inserted). Analysis conditions: f¼ 15 Hz, a¼ 75 mV, ΔEs ¼5 mV, Eacc ¼ 0.2 V and tacc ¼ 60 s.

(7)

The value of Eo′ was determined at each current, calculated as

Δi¼0.82Δip [50] for the different scan rates, and the average va-

lue found was Eo′ ¼0.92 7 0.02 V. Thus, comparing the linear coefficient of ln Δip vs. ln (Ep  Eo′) graphic (Fig. S2 of

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339

Table 2 Electrochemical sensors proposed for ofloxacin detection. Electrode

Linear range/mol L  1

LOD/mol L  1

Ref.

MWCNTs/PLL film and multi-HRP–AuNFs–Ab2 label/GCEa F3O4-MWCNT/GCEb MWCNTs-CR/GCEc HPMαFP/Ppy/GCEd multi-HRP-GNR-Ab2/GCEe Cu/GCEf Cysteic acid/CPEg MWCNTs-Nafion/GCEh HDMEi GO-IL/CPE

1.0
10  9–3.5
10  8 – 5.0
10  8–3.0
10  5 2.0
10  6–1.0
10  4 2.2
10  10–1.1
10  6 2.2
10  7–1.1
10  4 6.0
10  8–1.0
10  6 1.0
10  8–1.0
10  6 1.3
10  8–1.2
10  7 7.0
10  9–7.0
10  7

8.3
10  10 6.0
10  8 9.0
10  9 6.5
10  8 8.3
10  11 8.2
10  8 2.0
10  8 8.0
10  9 4.1
10  9 2.8
10  10

[52] [53] [43] [54] [51] [55] [44] [45] [56] This work

a

MWCNTs/PLL film and multi-HRP–AuNFs–Ab2 label on GCE; Fe3O4 nanoparticles and MWCNTs on GCE; c Solubilization of MWCNTs in water by congo red resulting water-soluble MWCNTs (MWCNTs-CR) on GCE; d 1-phenyl-3-methyl-4-(2-furoyl)-5-pyrazolone, Ppy: polypyrrole on GCE; e Gold nanorod (GNR) was synthesized to load horseradish peroxidase (HRP) and horseradish peroxidase-secondary antibody (HRP-Ab2); f Cupric ion (Cu2 þ ) on GCE; g Cysteic acid modified CPE; h MWCNTs Nafion film coated on GCE; i Hanging mercury drop electrode (HDME). b

Table 3 Results obtained from analysis of ophthalmic samples. Ophthalmic samples

1 2

Value obtained (mg mL  1)

Nominal value

3.0 3.0 a b c

Proposed methoda

Comparative methoda

3.0 7 0.1 2.9 7 0.1

3.02 7 0.02 2.98 7 0.01

Urine samples

[Ofloxacin]/mol L  1 Proposed method

a b

RSDc (%)

100 96.7

 0.66  2.68

Average of 3 measured concentrations; Recovery percentage ¼(Proposed method)/(Nominal value)
100; RSD ¼[(Proposed method)  (Comparative method)]/(Comparative method)
100.

Table 4 Results obtained from analysis of biological urine samples.

1 2

Recoveryb (sensor,%)

Added

Founda

5.0
10  8 1.0
10  7

(4.87 0.2)
10  8 (9.47 0.5)
10  7

Recoveryb (sensor, %)

96 94

Average of 3 measured concentrations; Recovery percentage ¼[Found]/[Added]
100.

Supplementary Material) with the linear coefficient of the Nicholson and Shain's equation (Eq. (7)), the ks constant was determined as being 1.46
10  3 cm s  1. 3.4. Optimizations The experimental conditions related to the ofloxacin determination using the GO-IL/CPE and the SWAdASV technique were subjected to systematic optimization. Thus, the influence of potential and time of analyte accumulation, amount of modifiers (IL and GO) used for preparation of the modified sensor, as well as the SWAdASV technical parameters were evaluated. In the optimization study, an ofloxacin concentration of 5.0
10  7 mol L  L was employed. The optimum conditions were selected taking into account the experimental conditions for which the higher analytical signal was obtained. First, the optimization of the SWAdASV technical parameters (amplitude, frequency and potential increment) were performed, as shown in Table 1. The optimum values were f¼ 15 Hz, a ¼75 mV,

and ΔEs ¼5 mV. Next, the feasibility of the use of an accumulation step of ofloxacin on the electrode surface was also investigated. Thus, the influence of the accumulation potential (Eacc) and time (tacc) was investigated in the ranges of  0.1 to þ0.5 V and 0–100 s. The peak currents (Δipa) recorded at each of these conditions were plotted as a function of Eacc and tacc, as shown in Fig. 8(a) and (b). Regarding the Eacc effect, the higher peak current was obtained using Eacc ¼ 0.2 V. The peak current increased until a tacc of 60 s then suffered slight suppression, which can be related with the electrode saturation; therefore, 60 s was selected as the optimum accumulation time for the further assays. Fig. 8(c) and (d) contain the graphics of peak current as a function of the amounts of IL and GO, respectively, incorporated in the carbon paste. Analyzing the effect of the amounts of IL and GO, the higher anodic peak currents were obtained using GO¼ 5 mg and IL ¼1.0 mg (20 mg mL  1). Additionally, electrochemical studies to evaluate the influence of the type of IL, as well as studies on the use of reduced graphene oxide (RGO) were performed. The experimental details and the obtained results of these studies are presented in the Supplementary Material. 3.5. SWAdASV determination of ofloxacin Employing the optimum experimental conditions found in the different optimization assays, the analytical determination of ofloxacin drug was performed by SWAdASV using the proposed GO-IL/CPE sensor. Thus, the analytical curve for ofloxacin was constructed from SWAdAS voltammograms recorded in supporting electrolyte solution containing different ofloxacin concentration levels. In Fig. 9, the SWAdAS voltammograms obtained in the different ofloxacin concentrations are presented, and in the inset of Fig. 9 the correspondent analytical curve is shown. The

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analytical curve was linear over a wide concentration range from 7.0
10  9 to 7.0
10  7 mol L  1, with an excellent linear correlation coefficient (0.9991) and a very high analytical sensitivity of (7.707 0.05)
106 μA L mol  1. The limits of detection (LOD) and quantification (LOQ) for ofloxacin were 2.8
10  10 mol L  1 (0.28 nmol L  1) and 9.3
10  10 mol L  1 (0.93 nmol L  1), respectively. A comparison between the results obtained in this study using the GO-IL/CPE sensor and SWAdASV with those reported in the literature for ofloxacin determination is presented in Table 2. As can be seen, the proposed sensor provided a wider dynamic range and a significantly lower LOD than reported by most of the studies published to date, with an exception of the work published by Zang et al. [51], in which a slightly inferior LOD was obtained. However, in that work, an electrochemical immunosensor was used for ofloxacin determination, involving high cost reagents/ materials, and laborious and time-consuming analytical steps. In contrast, the electrochemical sensor proposed in this work was prepared using a simple and highly reproductive technique, lowercost materials, and the electrode surface was renewable, i.e., a fresh electrode surface can be obtained from the simple electrode polishing on a clean paper sheet. Moreover, a single GO-IL/CPE can be used for a great number of determinations. The possible interference of some compounds typically found in ofloxacin pharmaceutical formulations or biological fluid samples on the voltammetric determination of ofloxacin was investigated. To fulfill this objective, SWAdASV measurements were performed for a 0.1 mol L  1 phosphate buffer solution (pH 6.0) containing 5.0
10  7 mol L  1 ofloxacin in the presence and absence of each potential interferent. The chemical compounds tested in these studies were: NaCl, NaOH, HCl, KCl, CaCl2, NaI, glucose, ascorbic acid, uric acid, urea and drugs such as dopamine, captopril and metronidazole at concentration ratios of 1:1 and 1:10 (analyte: potential interferent). Among those compounds analyzed, only ofloxacin showed a measurable electrochemical response, with an anodic peak current (Δipa) at a potential of 860 mV, thus indicating the reliability of the proposed electroanalytical method.

The electrochemical determination was carried out in triplicate (n¼ 3) by SWAdASV and the results obtained were compared with those provided by a comparative procedure (spectrophotometric method). The results obtained for ofloxacin quantification in the ophthalmic products and urine samples are displayed on Tables 3 and 4. In both the cases, excellent recovery percentages were achieved for the analyzed samples, ranging from 94% to 102%. By comparing the results of the proposed and comparative procedures for the ophthalmic products, low RSDs were observed between the results of both the methods. Moreover, by applying the paired t-test at a confidence level of 95%, the value of texperimental (2.23) was lower than the theoretical t value (ttheoretical ¼12.71), indicating the statistical equivalence of the results of ofloxacin quantification in the different samples. Thus, the accuracy of the developed voltammetric procedure and the reliability of the results provided by this technique were demonstrated.

4. Conclusions In this work, a novel and highly sensitive electrochemical method was designed for ofloxacin determination using graphene oxide and ionic liquid as modifiers. Specifically, a carbon paste electrode modified with graphene oxide and the 1-buthyl-3-methylimidazolium tetrafluoroborate ionic liquid was constructed. The GO-IL/CPE sensor provided a considerable improvement in the analytical sensitivity toward ofloxacin voltammetric sensing, resulting in a low limit of detection of 0.28 nmol L  1. Finally, the proposed SWAdASV procedure was successfully applied in the ofloxacin quantification in human urine and commercial ophthalmic samples.

Acknowledgements The authors gratefully acknowledge the financial support granted by CNPq (Proc. 165064/2014-5, 561071/2010-1 and 444150/2014-5).

3.6. Repeatability and stability of response Appendix A. Supporting information The precision of measurements using the GO-IL/CPE sensor was investigated from intra- and inter-day repeatability studies. For evaluation of the intra-day repeatability, six analytical curves were obtained in the same working day. Doing this, a relative standard deviation (RSD) value of 3.8% was determined for the analytical sensitivity. The inter-day repeatability study was performed by comparing the analytical sensitivities obtained from analytical curves constructed during three different days and an RSD of 2.9% was obtained. This set of results indicates a high precision and reproducibility of the measurements obtained using the proposed electrode. Besides being important to ensure a high sensitivity for the voltammetric detection of ofloxacin, the incorporation of the ionic liquid during the preparation of the graphene-modified CPE was found to be important in maintaining the high stability of response of the electrode. For comparison, the GO/CPE sensor prepared without ionic liquid showed a RSD of 13% for the analytical sensitivity of four analytical curves (n ¼4), in contrast with the RSD of 3.8% obtained for the analytical sensitivity of six analytical curves (n ¼6) constructed using the GO-IL/CPE. 3.7. Applications The proposed SWAdASV procedure using the GO-IL/CPE as an electrochemical sensor was applied for ofloxacin determination in two different ophthalmic products and biological urine samples.

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2016.08.035.

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