Square-wave voltammetric determination of clindamycin using a glassy carbon electrode modified with graphene oxide and gold nanoparticles within a crosslinked chitosan film

Sensors and Actuators B 231 (2016) 183–193

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Square-wave voltammetric determination of clindamycin using a glassy carbon electrode modified with graphene oxide and gold nanoparticles within a crosslinked chitosan film Ademar Wong, Claudia A. Razzino, Tiago A. Silva, Orlando Fatibello-Filho ∗ Departamento de Química, Centro de Ciências Exatas e de Tecnologia, Universidade Federal de São Carlos, Rod. Washington Luís km 235, São Carlos CEP: 13560-970, SP, Brazil

a r t i c l e

i n f o

Article history: Received 17 November 2015 Received in revised form 23 February 2016 Accepted 4 March 2016 Available online 6 March 2016 Keywords: Clindamycin Graphene oxide Gold nanoparticles Electrochemical sensor Crosslinked chitosan

a b s t r a c t An electrochemical method for the determination of clindamycin using a glassy carbon electrode modified with graphene oxide and gold nanoparticles within a film of crosslinked chitosan with epichlorohydrin (AuNPs-GO-CTS-ECH/GCE) is proposed. The electrochemical behavior of clindamycin was studied using cyclic voltammetry, and square-wave voltammetry (SWV). Under the optimized working conditions using 0.1 mol L−1 phosphate buffer (pH 7.0) solution as the supporting electrolyte, at a potential of +0.8 V vs. Ag/AgCl (3.0 mol L−1 KCl), using the SWV technique with the proposed AuNPs-GO-CTS-ECH/GCE electrode, the analytical curve showed a wide linear concentration range from 9.5 × 10−7 to 1.4 × 10−4 mol L−1 , with a limit of detection of 2.9 × 10−7 mol L−1 . The voltammetric method was successfully applied in the quantification of clindamycin in pharmaceutical formulations, as well as synthetic urine and river water samples, with results similar to those obtained by a comparative method (HPLC). © 2016 Elsevier B.V. All rights reserved.

1. Introduction Clindamycin (Chart 1) is an antibiotic of the lincosamide group commonly used to treat acne, vaginal, streptococcal, staphylococcal and malarial infections, and other bacterial infections in the lungs, skin, blood and internal organs [1–3]. The typical side effects of clindamycin use include drying, peeling, and irritation of the skin, nausea, vomiting, constipation, rash and, in some cases, anaphylactic reaction, erythema multiforme, Stevens–Johnson syndrome, leukopenia, neutropenia, eosinophilia and jaundice. Another problem with clindamycin is that excessive use promotes the emergence of resistance to this drug over the course of treatment, which is a major concern [1–5]. Various methods for clindamycin detection have been reported in the literature, such as high-performance liquid chromatography (HPLC) [6], spectrophotometry [7], capillary electrophoresis [8], chemiluminescence [9], electrochemiluminescence [10] and electrochemistry [11]. Among the analysis methodologies mentioned, electrochemical techniques have gained considerable attention as an alternative method of analysis [12–14]. The advantages obtained from the use of these methodologies are the versatility of instrumentation, high sensitivity and selectivity, the stability of the

∗ Corresponding author. Fax: +55 16 33518350. E-mail address: [email protected] (O. Fatibello-Filho). http://dx.doi.org/10.1016/j.snb.2016.03.014 0925-4005/© 2016 Elsevier B.V. All rights reserved.

response, low consumption of reagents and low cost [12–14]. In this context, the application of chemically modified electrodes as electrochemical sensors is an important approach, as they provide the characteristics of electroanalytical methods with improved electronic transfer and detection properties of the modified working electrode, allowing for sensing the analyte of interest with high sensitivity and a lower working potential. A number of compounds have been reported in the literature as electrode modifiers, such as porphyrins, metallic nanoparticles, inorganic materials, surfactants, ionic liquids, polymers (e.g., polypyrrole, polyaniline and molecularly imprinted polymers), and carbon nanostructures (e.g., carbon nanotubes and graphene) [15–18]. Graphene oxide (GO) is a carbon material widely used for electroanalysis and electrocatalysis applications. The set of GO properties which justify the scientific interest in its application in electrochemical sensors area include its large surface area, excellent conductivity and high performance in electronic transfer [19,20]. GO is a hydrophilic material based on a single graphite foil composed of a hexagonal carbon structure containing carbon atoms with sp2 and sp3 hybridizations. The oxygenated functional groups present in the GO structure are hydroxyl, epoxy, carboxyl and carbonyl groups, located at the basal and edge planes. GO can be employed directly as an electrode modifier or in combination with another modifier to improve the sensor performance [20–22]. Gold nanoparticles (AuNPs) have attracted considerable attention from a fundamental and practical viewpoint in studies devoted

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Chart 1. Chemical structure of clindamycin.

to the development of electrochemical sensors and biosensors [23,24]. AuNPs, usually with diameter of 1–100 nm, have the ability to perform fast direct electron transfer between a wide range of electroactive species; moreover, this material can be immobilized on the electrode surface [25,26]. This nanomaterial has many advantageous features for use in electrochemical sensors, such as a large surface area, good electrical properties, strong adsorption ability and high surface reaction activity [25,26]. The aim of this work was to develop a highly sensitive electrochemical sensor for the determination of clindamycin (Chart 1) in pharmaceutical, urine and river water samples. The proposed sensor architecture was based on a glassy carbon electrode modified with a film of chitosan crosslinked with epichlorohydrin containing graphene oxide and gold nanoparticles. The combined use of crosslinked chitosan and nanomaterials generated a sensor with increased response stability.

of 173◦ using a Nano ZS zetasizer system (Malvern Instruments) equipped with a laser of wavelength 633 nm and digital autocorrelation. Zeta-potential (␨) measurements, performed using a Zetasizer Nano Series (Malvern Instruments) and an MPT-2 titrator, were used to determine the surface potential of the AuNPs-GOCTS-ECH composite. For such, the sample was stirred for 10 min; then the pH was recorded and the zeta-potential of the nanocomposite was measured. Morphologic analysis of the nanomaterials were carried by field emission gun scanning electron microscopy (SEM) using a Supra 35-VP Carl Zeiss microscope with electron beam energy of 25 keV and by field emission gun transmission electron microscopy (TEM) using a FEI Tecnai G2F20 microscope with an acceleration voltage of 200 kV. Chemical analysis of the nanomaterials was explored by energy dispersive X-ray analysis (EDAX) using an EDX detector in the FEI Tecnai G2F20 microscope. For SEM and TEM analysis, GO and AuNPs-GO were suspended in isopropyl alcohol using an ultrasound bath. In the SEM case, an aliquot of each solution was dropped onto a GCE plate, and then left to dry at room temperature. For the TEM analyses, an aliquot of each solution was dropped onto a porous carbon film supported on a copper grid, and then left to dry at room temperature. X-ray fluorescence (XRF) analyses of the AuNPs-GO-CTS-ECH/GCE surface were carried using an EDX-720/800HS energy dispersive X-ray fluorescence spectrometer (Shimadzu). The chromatographic analyses were performed using a Shimadzu Model 20A liquid chromatography, coupled to an SPD-20A UV/Vis detector, a SIL-20A autosampler and a DGU-20A5 degasser. The chromatographic conditions used were: mobile phase composed of acetonitrile and phosphoric acid (40:60 v:v), flow rate of 1.0 mL min−1 , sample injection volume of 10 ␮L, C18 column and, wavelength of 205 nm [6].

2. Experimental 2.3. Preparation of gold nanoparticles 2.1. Reagents and solutions Clindamycin hydrochloride, epichlorohydrin (ECH), H2 SO4 , HNO3 , HAuCl4 and chitosan (CTS, low molar mass and degree deacetylation of 80%) were purchased from Sigma–Aldrich. NaOH, reagents for phosphate buffer (H3 PO4 , KH2 PO4 , K2 HPO4 , and K3 PO4 ) and KCl were acquired from Acros. Graphene (GN) 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 M cm obtained from a Milli-Q Direct-0.3 (Millipore) purification system. A 1.0% m/m CTS stock solution was prepared in 1.0% m/v acetic acid solution and stored in a refrigerator. A 2.5% v/v ECH stock solution and a 2.5 × 10−3 mol L−1 clindamycin hydrochloride solution were prepared using ultrapure water and stored in a refrigerator. 2.2. Apparatus Electrochemical experiments were performed using a model PGSTAT-30 potentiostat/galvanostat (Metrohm-Autolab, Utrecht, Netherlands) controlled by the GPES 4.9 software (Eco Chemie) fitted with an electrochemical cell containing three electrodes: a commercial Ag/AgCl (3.0 mol L−1 KCl) reference electrode (Analion), a platinum wire as the counter electrode and a modified glassy carbon electrode (GCE) as the working electrode (3.0 mm diameter). The pH measurements were performed using 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. Dynamic light scattering (DLS) was employed to evaluate the particle size distribution in the colloids. These measurements were carried out at room temperature and fixed angle

Gold nanoparticles (AuNPs) were synthesized by stirring a 1.0 × 10−3 mol L−1 HAuCl4 solution prepared in 200 mL of deionized water at 85 ◦ C. Then, 2 mL of a 0.3 mol L−1 sodium citric solution was added to the reaction mixture and stirring was maintained for 4 min. Next, the solution was placed in an ice bath until it reached room temperature. During cooling, it was possible to observe the color of the solution change from yellow to red. This color solution change is a physical indicator of AuNPs formation. The AuNPs colloidal suspension was stored in a dark bottle in the refrigerator [27]. 2.4. Preparation of the modified working electrode Initially, graphene functionalized with oxygenated functional groups (GO) was prepared by stirring 10 mg of graphene (GN) in a concentrated solution of H2 SO4 :HNO3 (1:1 v/v) for 12 h at 25 ◦ C. After this step, the suspension was filtered and the GO was carefully washed with deionized water until the pH was around 7.0, and then dried at 100 ◦ C for 12 h. The GCE surface was carefully polished with 1.0 and 0.5 ␮m alumina slurry on a polishing cloth and subsequently ultrasonically cleaned with deionized water. The best dispersion was prepared as follows: 1.5 mg of GO, 400 ␮L of 0.1% m/m CTS solution (obtained from dilution of the 1.0% m/m stock solution), 600 ␮L of 0.25% v/v ECH solution (obtained from dilution of the 2.5% v/v stock solution in 0.01 mol L−1 NaOH), 500 ␮L of the AuNPs colloidal suspension and 500 ␮L of deionized water. A 0.01 mol L−1 NaOH solution was used during film preparation to deprotonate the amino groups of the chitosan chain [28]. The mixture of compounds was subjected to ultrasonic agitation for 50 min in order to obtain a homogeneous dispersion. Next, an aliquot of 15 ␮L of the obtained dispersion was dropped onto the GCE surface, then left to dry at room temperature for 4 h. Thus, a GCE modified with a crosslinked chitosan

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film containing GO and AuNPs was obtained. This architecture was designated as AuNPs-GO-CTS-ECH/GCE. 2.5. Analytical steps and preparation of samples First, the electrochemical properties of the modified GCE were evaluated and contrasted with those registered for the bare GCE. Thus, cyclic voltammetric studies were conducted using as electrochemical probe the redox pair [Fe(CN)6 ]3-/4- . Next, the voltammetric behavior of clindamycin was investigated using cyclic voltammetry and square-wave voltammetry (SWV). The effect of pH and the composition of the supporting electrolyte solution on the clindamycin response was investigated, as well as of the amount of modifiers (GO and AuNPs) used for electrode preparation. SWV was employed for the clindamycin determination and, therefore, all SWV experimental parameters were optimized. Under the optimum experimental conditions, the analytical curve was constructed, and the analytical parameters for the proposed voltammetric procedure were assessed: linear concentration range, analytical sensitivity, limit of detection and quantification. The precision of the electrochemical sensor was investigated through repeatability studies. Finally, the developed voltammetric procedure was applied for clindamycin determinations in different matrix samples, such as pharmaceutical formulations, urine and environmental water samples. In the next sections, the steps used to collect and/or prepare each type of sample are described in detail. 2.5.1. Preparation of pharmaceutical formulation samples Two commercial pharmaceutical formulations of clindamycin were purchased in local drugstores and subjected to a simple sample preparation procedure. Thus, two tablets of each sample were macerated into a powder using a mortar and pestle, and then a suitable amount of sample was weighed and dissolved in deionized water. Conventional filtration was used to remove insoluble substances. The solution was transferred to a 10 mL volumetric flask, and the volume completed with deionized water. The standard addition method was used for clindamycin determination by the voltammetric procedure, and the results obtained were compared with those provided by a comparative method (HPLC). 2.5.2. Preparation of synthetic urine samples The synthetic urine sample was prepared using the following reagents proposed by Laube et al. [29]: 0.73 g of NaCl, 0.40 g of KCl, 0.28 g of CaCl2 ·2H2 O, 0.56 g of Na2 SO4 , 0.35 g of KH2 PO4 , 0.25 g of NH4 Cl and 6.25 g of urea. These reagents were added in a 250 mL volumetric flask and the volume was completed with water. The urine sample was spiked with two different concentration levels of clindamycin (8.0 × 10−6 and 1.5 × 10−5 mol L−1 ) and directly analyzed by the proposed voltammetric method. The results are reported as the recovery percentage of clindamycin added to the urine sample. 2.5.3. Preparation of river water samples River water samples were collected in the region of AraraquaraSão Carlos, in the interior of São Paulo State (Brazil) and enriched with 1.5 × 10−5 mol L−1 clindamycin. The samples did not require pre-treatment before of the electrochemical analysis and the recoveries were assessed by the standard addition method. 3. Results and discussion 3.1. Characterization of graphene oxide and gold nanoparticles The nanomaterials used for preparation of the electrochemical sensor were characterized by DLS, TEM, SEM, EDX, and XRF analysis.

Fig. 1. DLS measurement recorded for (A) AuNPs and (B) AuNPs-GO-CTS-ECH.

The DLS measurements recorded for the AuNPs and AuNPs-GOCTS-ECH dispersions are presented in Fig. 1. From the data in Fig. 1A, an average particle size of 14.9 nm was obtained for the AuNPs. A second DLS peak was verified for the AuNPs, centered at 180.9 nm. However, this second peak presented a low relative intensity, and can be associated with a small quantity of agglomerates present in the colloidal suspension. The DLS measurement performed for the AuNPs-GO-CTS-ECH dispersion used for the GCE modification (Fig. 1B) demonstrated a considerable increase of the average particle size to 254.4 nm. This average particle size obtained for the suspension containing GO and the crosslinked CTS was expected and it is in accordance with previous reports for graphene oxide-chitosan based composites [30–33]. Zeta-potential (␨) was determined for the AuNPs-GO-CTS-ECH dispersion as (13 ± 4) mV. GO is known for the presence of carboxylic and hydroxyl functional groups, and CTS by the presence of hydroxyl and amino groups in its polymeric chain. At acid solution, ␨ = 40 mV has been observed for GO–CTS dispersions [32,33]. However, at pH ≈ 7.0, as in the case of this work, Bao et al. [32,33] observed a decrease of ␨, which was an effect caused by the deprotonation of the previously mentioned functional groups. Thus, we can conclude that a positive surface charge dominated the net surface charge of the particles in the AuNPs-GO-CTS-ECH dispersion, however with an influence of negatively charged deprotonated functional groups. Similar behavior was also obtained by Fang et al. [34]. The TEM results obtained for AuNPs, GO and AuNPs-GO are shown in Fig. 2. From the TEM images obtained for the AuNPs shown in Fig. 2A and B, it is possible to verify the almost spherical morphology and excellent dispersibility of the nanoparticles, with absence of agglomerates. The synthesised AuNPs presented an average diameter of 10.62 nm (Fig. 2C). TEM images recorded at different magnifications for GO are provided in Fig. 2D–F. GO consisted of thin wrinkled sheets with a transparent appearance. TEM images obtained for GO in the presence of AuNPs at two different magnifications are shown in Fig. 2G and H. As can be seen, the AuNPs were homogeneously distributed on the GO sheets. An HRTEM image obtained for the AuNPs-GO nanocomposite is presented in Fig. 2I. A crystalline structure is observed for the AuNPs, with a fringe space of 0.21 nm in accordance with the spacing between (1 1 1) planes of face-centered-cubic metallic gold [35,36]. Moreover, the amorphous nature of the GO nanosheet can be observed. The surface morphology of GO also was evaluated using SEM images, as shown in Fig. 3A. It can be seen from this figure that the shapes of the GO look like pieces of leaves or wrinkled paper, which is characteristic of this material. The morphology of GO in presence of AuNPs is presented in Fig. 3B and C, which shows

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Fig. 2. TEM image of (A, B) AuNPs and (C) the corresponding histogram of AuNPs diameters. TEM image of (D-F) GO and (G, H) AuNPs-GO. (I) HRTEM image obtained for AuNPs-GO.

highly dispersed AuNPs (white spheres) on the graphene sheets. The AuNPs deposited on the surface of graphene have an average diameter of 10.46 nm (Fig. 3D), which is very close to the diameter observed for the as-synthesised AuNPs (Fig. 2C). The morphological profile obtained is in accordance with previous reports [37,38]. Electrochemical analysis showed that the combination of GO sheets and AuNPs promoted an increase in the electroactive surface area, which is an important improvement for use of this architecture as an electrochemical sensor. EDX spectra recorded for GO and AuNPsGO can be observed in Fig. 4A and B, respectively. Analysis of the EDX spectra demonstrated that the Au mass percentage increases from 0.12% (0.00% atomic percentage) in the GO case to 15.62% (1.11% atomic percentage) in the AuNPs-GO case, definitely proving the presence of the AuNPs on the GO sheets. The gold content in the modified AuNPs-GO-CTS-ECH/GCE surface was also quantified using XRF measurements, when a gold content of 15.98% was obtained, in accordance with the EDX analysis.

3.2. Electrochemical characterization of the modified electrodes The effect of the GCE modified with GO and AuNPs within a crosslinked chitosan film was investigated employing the redox probe [Fe(CN)6 ]4− . Fig. 5 shows the cyclic voltammograms obtained for a 1.22 × 10−3 mol L−1 [Fe(CN)6 ]4− in 0.1 mol L−1 KCl. Comparatively, the use of GO and AuNPs provided a considerable increase in the anodic and cathodic peak currents, i.e. by 2.8 times, suggesting an increase in the electroactive surface area. Regarding electron

transfer kinetics, preliminary information about this aspect may be found from the respective values of the peak potential separation (Ep ): 90 mV (GCE), 90 mV (GN-CTS-ECH/GCE), 90 mV (GO-CTSECH/GCE) and 70 mV (AuNPs-GO-CTS-ECH/GCE). As can be seen, there was better electron transfer for the modified electrode containing AuNPs and GO. The previous results are indicative of considerable improvements to the electroactive surface area and electron transfer kinetics of the bare GCE. Thus, cyclic voltammetry assays at different scan rates were carried out in order to estimate the electrochemical features of the different modified electrodes. In Fig. 6A–D are presented the cyclic voltammograms obtained for a 1.22 × 10−3 mol L−1 [Fe(CN)6 ]4− in 0.1 mol L−1 KCl solution at potential scan rates ranged from 10 to 400 mV s−1 using GCE, GN-CTS-ECH/GCE, GO-CTS-ECH/GCE and AuNPs-GO-CTS-ECH/GCE. Fig. 6E shows the plots of ipa vs. v1/2 and ipc vs. v1/2 . The graphs of ipa and ipc vs. v1/2 were linear in all cases, as expected for a diffusion-controlled process. From the slopes of these relationships, the respective electroactive surface areas were estimated using the Randles–Sevcik equation Eq. (1) [39,40]: ip = ±2.69 × 105 n3/2 DA1/2 C1/2

(1)

Where ip is the anodic or cathodic peak current, C is the [Fe(CN)6 ]4− concentration in bulk solution (1.22 × 10−6 mol cm−3 ), D is the diffusion coefficient of [Fe(CN)6 ]4− in solution (6.2 × 10−6 cm2 s−1 at 0.1 mol L−1 KCl [35]), v1/2 is the square root of potential scan rate and A is the electroactive surface area

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Fig. 3. SEM image of (A) graphene oxide, (B, C) AuNPs-GO and (D) the corresponding histogram of AuNPs diameters.

Fig. 4. EDX spectrum recorded for (A) GO and (B) AuNPs-GO.

(cm2 ). The values were 0.071, 0.093, 0.19 and 0.32 cm2 for GCE, GN-CTS-ECH/GCE, GO-CTS-ECH/GCE and AuNPs-GO-CTS-ECH/GCE, respectively. Thus, the combined use of GO and AuNPs increased the electroactive surface area by a factor of 4.5 compared to GCE. Next, the heterogeneous electron transfer rate constant (k0 ) for each modified electrode was estimated using the Nicholson equation Eq. (2) [41].  = k0 [

DnvF −1/2 ] RT

(2)

where  is a kinetic parameter obtained from Eq. (3) proposed by Lavagnini et al. [42]; the other terms have their usual meanings.  =

(−0.6288 + 0.0021 nEp ) (1 − 0.017 nEp )

The k0

(3)

values were obtained from the slopes of the  vs. 36.31 v−1/2

graphics. The 36.31 factor is equivalent to the term [␲DnF/(RT)]−1/2 ,

Fig. 5. Cyclic voltammograms obtained for a 1.22 × 10−3 mol L−1 [Fe(CN)6 ]4− solution in 0.1 mol L−1 KCl using GCE, GN-CTS-ECH/GCE, GO-CTS-ECH/GCE and AuNPs-GO-CTS-ECH/GCE. v = 50 mV s−1 .

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Fig. 6. Cyclic voltammograms obtained for a 1.22 × 10−3 mol L−1 [Fe(CN)6 ]4− solution in 0.1 mol L−1 KCl at different scan rates (v: 10–400 mV s−1 ) using (A) GCE, (B) GN-CTS) and ipc vs. v1/2 ( ) obtained for GCE (䊉), GN-CTS-ECH/GCE ( ), ECH/GCE, (C) GO-CTS-ECH/GCE and (D) AuNPs-GO-CTS-ECH/GCE. (E) Graphics of ipa vs. v1/2 ( GO-CTS-ECH/GCE () and AuNPs-GO-CTS-ECH/GCE (). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

calculated considering D = 6.2 × 10−6 cm2 s−1 , F = 96485C mol−1 , R = 8.314 J K−1 mol−1 and T = 298.15 K. The following k0 values were obtained for GCE, GN-CTS-ECH/GCE, GO-CTS-ECH/GCE and AuNPsGO-CTS-ECH/GCE, respectively: 2.9 × 10−3 , 4.2 × 10−3 , 4.2 × 10−3 and 1.1 × 10−2 cm s−1 . It can be observed that the electron transfer constants were similar for bare GCE, GN-CTS-ECH/GCE and GO-CTS-ECH/GCE and better for AuNPs-GO-CTS-ECH/GCE, with an increase of 2.6 and 3.8 fold regarding GO-CTS-ECH/GCE and bare GCE, respectively. 3.3. Electrochemical behavior of clindamycin An electrochemical study applying cyclic voltammetry was performed with the purpose of evaluating the response profile of the clindamycin compound on the GCE surface and modified GCE surfaces. In Fig. 7 are shown the cyclic voltammograms collected in the absence and in the presence of clindamycin using the unmodified GCE (A), CTS-ECH/GCE (B), GO-CTS-ECH/GCE (C), and AuNPs-GO-CTS-ECH/GCE (D) at the potential range of 0.0 to +1.2 V. The electrochemical response of clindamycin on the bare

GCE was verified using the variation of anodic current at a potential of 950 mV (Fig. 7A), featuring as an irreversible oxidation process, because a non-equivalent reduction peak was registered after the inversion of the potential scanning direction. When the sensor was modified only with the crosslinked chitosan film (CTS–ECH), the electrochemical signal decreased because of blockage of electron transfer across the electrode surface, which was caused by the insulating feature of the chitosan film (Fig. 7B). GO was added to the film and provided a well-defined anodic peak at 800 mV (Fig. 7C). After the incorporation of AuNPs in the film, the current signal for clindamycin improved significantly, as can be seen by the cyclic voltammograms presented in Fig. 7D. The anodic peak current registered for AuNPs-GO-CTS-ECH/GCE was 2.4 times higher than for GO-CTS-ECH/GCE, demonstrating the favorable voltammetric response of clindamycin on the GCE surface modified with GO and AuNPs. In addition to the cyclic voltammetry results, square-wave voltammograms (Fig. 8) were also collected for clindamycin solutions using the different modified electrodes. Once again, the GCE modified with GO and AuNPs provided the most intense peak cur-

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Fig. 7. Cyclic voltammograms obtained for a 0.1 mol L−1 phosphate buffer solution (pH 7.0) in the absence and presence of 1.0 × 10−4 mol L−1 clindamycin using (A) GCE, (B) CTS-ECH/GCE, (C) GO-CTS-ECH/GCE and (D) AuNPs-GO-CTS-ECH/GCE. v = 50 mV s−1 .

the graphene electrochemical activity is associated with the high heterogeneous electron transfer through the edge-plane sites. Moreover, Pumera and collaborators [49,50] proved that the electrochemical response observed for the graphene-based materials can be affected by the presence of electrochemically active metallic impurities. From these, it is clear that the proposed architecture sensor combines the known electrocatalytic activities of the AuNPs and GO sheets with a great number of edge-plane sites exposed (see Fig. 3B), resulting in a synergistic effect that considerably improved the electron transfer kinetics across the electrode interface. 3.4. Clindamycin determination by square-wave voltammetry (SWV) Fig. 8. Square-wave voltammograms recorded for a 0.1 mol L−1 phosphate buffer solution (pH 7.0) containing 1.4 × 10−4 mol L−1 clindamycin using (A) GCE, (B) CTS-ECH/GCE, (C) GN-CTS-ECH/GCE, (D) GO-CTS-ECH/GCE and (E) AuNPs-GO-CTSECH/GCE. Analysis conditions: f = 10 Hz, a = 50 mV and Es = 5 mV.

rent and, regarding the anodic peak potential, a decrease from 1.0 V on the bare GCE (Fig. 8A) to 0.8 V using the AuNPs-GO-CTSECH/GCE (Fig. 8E) was observed. Therefore, clindamycin can be detected at a less positive potential, so potential interferents can be diminished. Moreover, another effect can be observed from this square-wave voltammetry study. In Fig. 8, the voltammograms registered using the GCE modified with untreated graphene (Fig. 8C) and GO (Fig. 8D) are presented. A comparison of these results shows that the insertion of oxygenated functional groups on the graphene sheets also favoured the voltammetric response for clindamycin. These results demonstrated the electrocatalytic effect of GO and AuNPs on the clindamycin oxidation. AuNPs and graphene have shown excellent electrocatalytic activity toward redox processes of a number of organic targets, such as dopamine [43], hydroquinone [44], bisphenol A [45], levofloxacin [46], and diethylstilboestrol [47], among others. Kampouris and Banks [48] demonstrated that

Square-wave voltammetry was employed as an electroanalytical technique in this work, and all experimental parameters related to clindamycin determination by this technique were systematically optimized. Thus, the influence of electrolyte composition, pH and the concentration of modifiers (GO and AuNPs) was studied within the film as well as the SWV technical parameters (amplitude, frequency and potential increment). In these studies, a clindamycin concentration of 1.4 × 10−4 mol L−1 was used and the best experimental conditions were chosen in order to obtain a well-defined oxidation peak and higher anodic peak current. 3.4.1. Optimization The influence of the analytical parameters on the analytical response of the modified electrode was investigated by SWV using previously different supporting electrolyte compositions, such as 0.1 mol L−1 Britton–Robinson buffer (pH = 7.0), 0.1 mol L−1 phosphate buffer (pH = 7.0) and 0.1 mol L−1 TRIS buffer (pH = 7.0). In Fig. 9A, the anodic peak currents obtained using each supporting electrolyte condition are shown. As can be seen, the phosphate buffer solution provided the higher peak current and was therefore selected for further studies. Next, the pH effect was evaluated

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Fig. 9. Optimization of parameters for the proposed sensor: (A) buffer composition, (B) pH, (C) Amount of GO and (D) Amount of AuNPs. The graphics were constructed using the analytical signals (ip ) recorded by SWV measurements performed for a 0.1 mol L−1 phosphate buffer solution (pH 7.0) containing 1.4 × 10−4 mol L−1 clindamycin employing the following analysis conditions: f = 10 Hz, a = 50 mV and Es = 5 mV.

using 0.1 mol L−1 phosphate buffer with the pH ranging between 6.0 and 10.0. Based on the graph showed in Fig. 9B, the maximum value of the anodic peak current was achieved at pH 7.0 and, thus, this pH was selected for the next steps. The amounts of GO and AuNPs used for the construction of the proposed architecture electrode were also optimized. The best results were obtained using 1.5 mg of GO and 500 ␮L of the AuNPs suspension (Fig. 9C and D). Using the previous optimum conditions regarding the supporting electrolyte (composition and pH) and modifier amount (GO and AuNPs), the SWV parameters were evaluated. The evaluated parameters and respective ranges were: frequence, f (8–80 Hz); amplitude, a (25–100 mV); step potential, Es (2–8 mV). The optimum values for each parameter were f = 10 Hz, a = 50 mV, and Es = 4 mV, respectively.

3.4.2. Analytical parameters In Fig. 10, the SWVs obtained for different clindamycin concentration levels are presented; the insert is the respective analytical curve, which was constructed in triplicate. Using AuNPs-CTS-GOECH/GCE, the analytical curve presented a linear response in the concentration range of 9.5 × 10−7 to 1.4 × 10−4 mol L−1 , with a limit of detection (LOD) of 2.9 × 10−7 mol L−1 and limit of quantification (LOQ) of 9.5 × 10−7 mol L−1 . The LOD was experimentally determined as being the lowest clindamycin concentration detectable by the sensor under repeatability analytical conditions. The SW voltammogram obtained for the LOD determination is shown in the inset of Fig. 10. The linear correlation coefficient and sensitivity were 0.9992 and 4.80 × 104 ␮A L mol−1 , respectively. As discussed, the analytical curve was constructed in triplicate; thus the intercept (n) and analytical sensitivity (m, slope of the analytical curve) presented relative standard deviations (RSD) of 1.6 and 3.5%, respectively.

Fig. 10. Square-wave voltammograms obtained using the AuNPs-GO-CTS-ECH/GCE for a 0.1 mol L−1 phosphate buffer solution (pH 7.0) containing different concentrations of clindamycin: (a) blank (b) 9.5 × 10−7 ; (c) 7.0 × 10−6 , (d) 2.5 × 10−5 , (e) 4.9 × 10−5 , (f) 7.3 × 10−5 , (g) 9.6 × 10−5 , (h) 1.2 × 10−4 and (i) 1.4 × 10−4 mol L−1 . Insets: analytical curve and square-wave voltammogram obtained in the LOD determination. Analysis conditions: f = 10 Hz, a = 50 mV and Es = 4 mV.

The precision of the proposed electrochemical sensor was evaluated with an intra-day repeatability study, when three analytical curves were independently constructed using the same modified electrode. The RSD for the thus obtained analytical sensitivity was 4.2%, evidencing the high measurement precision of the proposed electrode. The construction reproducibility of the proposed architecture sensor was assessed by constructing analytical curves using three different modified electrodes. The RSD obtained for the analytical sensitivity using these different electrodes was 5.1%, evidencing the good preparation reproducibility of the modified electrode.

A. Wong et al. / Sensors and Actuators B 231 (2016) 183–193

191

Fig. 11. Crosslinking reaction for chitosan using ECH and GO. Table 1 Research into electrochemical sensors used for the detection of clindamycin. Electrode

Linear range/mol L−1

Sensitivity

Detection limit/mol L−1

Response time/s

Stability of response (RSD, %)a

Reproducibility References (RSD, %)

MIP/sol–gel/MWNT/Au electrode Au ultra- microelectrode CPE AuNPs-GO-CTS-ECH/GCE

5.0 × 10−7 –8.0 × 10−5 9.4 × 10−12 –9.9 × 10−11 2.1 × 10−7 –1.0 × 10−6 9.5 × 10−7 –1.4 × 10−4

4.24 × 104 ␮A L mol−1 36.85 S/N [log (mol L−1 )] −1 4,9 × 105 ␮A L mol−1 4.8 × 104 ␮A L mol−1

2.4 × 10−8 3.0 × 10−12 7.7 × 10−8 2.9 × 10−7

Not reported 0.3 30 <10

Not reported 0.41–3.76 2.2 4.2

Not reported 1.0–3.8 0.3 5.1

a

[53] [54] [55] This work

Based on the results of intra-day repeatability study.

Table 2 Application of sensor for assessing pharmaceutical samples. Pharmaceutical

Nominal

Value obtained (mg tablet−1 ) a

formulation

value

Comparative method

1 2

300 300

312 ± 7 300 ± 3

a b

RSDb (%) a

Proposed method 298 ± 10 291 ± 3

−4.5 −3.0

Average of 3 measured concentrations. RSD = [(Proposed method)−(Comparative method)]/(Comparative method) × 100.

The proposed architecture sensor was designed using a crosslinked chitosan film as polymeric matrix for the incorporation of the nanomaterials (AuNPs and GO). This natural polymer presented good homogeneity and a very strong adherence on the GCE surface, achieved by using epichlorohydrin as crosslinking agent. Epichlorohydrin is an organochlorine and epoxide compound, and formed covalent bonds with the hydroxyl groups of chitosan. Fig. 11 illustrates the bonding between ECH and the hydroxyl groups of chitosan, resulting in the rupture of the epoxide ring and the removal of a chlorine atom from the ECH monomer [28,51]. In this illustration, it is possible to show that ECH is connected to two molecules of chitosan. Additionally, there is a crosslinking reaction between the amino group of chitosan and the hydroxyl group of GO [51,52]. Therefore, the modified electrode has high long-term stability. In a typical working day, a same electrode was used to perform the construction of six analytical curves, when the analytical sensitivity presented a RSD of 12.3%; more than 70 measurements were performed with stable voltammetric response for the construction of the analytical curves, demonstrating that the proposed electrode indeed has good long-term stability. Additionally, a study was performed on interferents typically found in pharmaceutical formulations, such as starch, talc, magnesium stearate and lactose monohydrate. Using the SWV method,

the results show that only clindamycin had an electrochemical response at +0.8 V vs. Ag/AgCl (3.0 mol L−1 KCl). Table 1 contains the analytical characteristics of the proposed electrode used for the determination of clindamycin. The developed electroanalytical method for the determination of the analyte is simple and does not require analyte pre-concentration; this method also presents excellent repeatability and accuracy and a similar sensitivity and limit of detection to those of other analytical methods described in the literature. It should be noted that the results obtained with this electrode in the monitoring of clindamycin are very important, since there are few electrochemical electrodes reported in the literature for the determination of this analyte.

3.5. Application of AuNPs-GO-CTS-ECH/GCE for clindamycin determination in commercial pharmaceutical, synthetic urine and river water samples Clindamycin was determined in two different pharmaceutical products, synthetic urine and river water samples, by applying the proposed voltammetric method using the AuNPs-GO-CTS-ECH/GCE electrode.

192

A. Wong et al. / Sensors and Actuators B 231 (2016) 183–193

Table 3 Results obtained from the analysis of artificial urine samples. Samples

1 2 a b c

[Clindamycin]/mol L−1 a

a

Recoveryb

RSDc

Added

Comparative method

Proposed method

(sensor, %)

(%)

8.0 × 10−6 1.5 × 10−5

(7.91 ± 0.06) × 10−6 (1.58 ± 0.02) × 10−5

(7.8 ± 0.2) × 10−6 (1.42 ± 0.09) × 10−5

97.5 94.7

−1.9 −10.1

Average of 3 measured concentrations. Recovery percentage = [Found]/[Added] × 100. RSD = [(Proposed method)−(Comparative method)]/(Comparative method) × 100.

Table 4 Results obtained from the analysis of river water samples. River

[Clindamycin]/mol L−1

Recoveryb a

a

RSDc

water samples

Added

Comparative method

Proposed method

(sensor, %)

(%)

Chibarro Gregorio

1.5 × 10−5 1.5 × 10−5

(1.50 ± 0.02) × 10−5 (1.52 ± 0.02) × 10−5

(1.55 ± 0.05) × 10−5 (1.43 ± 0.08) × 10−5

103 95.3

+3.3 −5.9

a b c

Average of 3 measured concentrations. Recovery percentage = [Found]/[Added] × 100. RSD = [(Proposed method)−(Comparative method)]/(Comparative method) × 100.

All samples were analyzed employing the standard addition method and analyzed in triplicate. The samples were subjected to analysis by the proposed voltammetric method and by a comparative HPLC method to verify the accuracy of the developed method for clindamycin quantification in different matrix samples. The results obtained for the commercial pharmaceutical samples using the proposed method and the comparative method (HPLC method) are presented in Table 2. As can be seen from this table, the results provided by both the procedures are very consistent, with RSD values of only −3.0% and −4.5%. In addition, by applying the paired t-test at a confidence level of 95%, it was verified that texper (4.6) was lower than ttheoretical (12.71), demonstrating that the results of both methods were not statistically different. The synthetic urine and river water samples were spiked with a known clindamycin concentration and analyzed directly. Tables 3 and 4 present the results obtained for the clindamycin determination in these matrix samples employing the proposed and comparative methods. As can be seen, excellent results were obtained for the recovery percentage in both cases (close to 100%), showing that the proposed voltammetric method did not suffer any matrix effect from the analyzed samples. The results obtained using both methods were in good agreement, with RSDs ranging between −10.1% and 3.3%. Moreover, the application of the paired t-test demonstrated the statistical equivalence of the results, since the values of texper (1.21 for the urine samples, and 0.29 for the river water samples) were lower than the ttheoretical one (12.71) at a 95% confidence level. These results demonstrate the excellent accuracy, reliability, and effectiveness of the proposed electrode for the quantification of clindamycin in different matrix samples.

4. Conclusions The results presented in this work show that the use of gold nanoparticles and graphene oxide incorporated within a crosslinked chitosan film was essential to the increased sensitivity of the modified electrode. This device is a good alternative to others already described in the literature, as it offers advantages including high sensitivity, repeatability, fast response, long-term stability, applicability over a wide range of concentrations and samples, and low cost. The AuNPs-GO-CTS-ECH/GCE electrode shows excellent results in the recovery tests for different pharmaceutical products, synthetic urine and river water samples.

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

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Biographies Ademar Wong received his PhD degree in Analytical Chemistry (2014) from Universidade Estadual Paulista (UNESP), Araraquara (Brazil). He is currently performing his post-doctoral work at the São Carlos Federal University (UFSCar), São Carlos, SP (Brazil). His research interests include biosensors and biomimetic sensors (porphyrin, phthalocyanine compounds and molecularly imprinted polymers), nanostructured materials (carbon nanotube, graphene oxide) and gold nanoparticles. Claudia A. Razzino received her PhD degree in Analytical Chemistry (2013) from the São Carlos Institute of Chemistry, University of São Paulo (IQSC-USP), São Carlos, SP, Brazil. She is currently performing her post-doctoral work at the Department of Chemistry, São Carlos Federal University (DQ-UFSCar), São Carlos, SP, Brazil. Her research interests include the development and characterization of modified electrodes with carbon nanotubes, noble metal nanoparticles encapsulated in dendrimers, carbon black, ionic liquids, chitosan, self-assembly and layer-by-layer techniques and enzyme immobilization for construction and applications in sensors, biosensors and biofuel cells. Tiago A. Silva received his BSc degree in Chemistry from the Faculty of Integrated Sciences of Pontal, Uberlândia Federal University, MG (Brazil) in 2012, with an academic mobility period (2010–2011) at the University of Lisbon (Portugal) and MS degree in Analytical Chemistry from the São Carlos Federal University, SP (Brazil) in 2015. Currently, he is a graduate student (PhD degree, Analytical Chemistry) at the São Carlos Federal University under the supervision of Professor Orlando Fatibello-Filho. His research interests include the development of novel electroanalytical methods using different nanoarchitectures as electrode materials. Orlando Fatibello-Filho is a full Professor in Analytical Chemistry at the São Carlos Federal University-UFSCar, Department of Chemistry and, head of the LABBES (http://www.labbes.ufscar.br). He received his PhD degree in Analytical Chemistry from the University of São Paulo, São Paulo, SP (Brazil) in 1985 and held a postdoctoral position from 1987 to 1989 at the University of New Orleans (USA), Department of Chemistry. He was a visiting professor at University of Coimbra in the Christopher and Ana Maria Brett Group. His research interests include the development of analytical procedures employing chemical and electrochemical sensors, biosensors, solid-phase reactors, and flow-injection systems and their applications in the determination of analytes in pharmaceutical formulations as well as environmental and food samples. He has published over 350 papers with a h-index of 35 (Web of Science, November 2015) and has written 5 books and contributed 08 book chapters.