Development of non-enzymatic glucose sensor based on efficient loading Ag nanoparticles on functionalized carbon nanotubes

Accepted Manuscript Title: Development of non-enzymatic glucose sensor based on efficient loading Ag nanoparticles on functionalized carbon nanotubes Author: Mehdi Baghayeri Amirhassan Amiri Samaneh Farhadi PII: DOI: Reference:

S0925-4005(15)30587-6 http://dx.doi.org/doi:10.1016/j.snb.2015.11.003 SNB 19261

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

9-6-2015 14-10-2015 2-11-2015

Please cite this article as: M. Baghayeri, A. Amiri, S. Farhadi, Development of non-enzymatic glucose sensor based on efficient loading Ag nanoparticles on functionalized carbon nanotubes, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.11.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development of non-enzymatic glucose sensor based on efficient loading Ag nanoparticles on functionalized carbon nanotubes

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Mehdi Baghayeri,* Amirhassan Amiri, Samaneh Farhadi

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Department of Chemistry, Faculty of Science, Hakim Sabzevari University, P.O. Box 397, Sabzevar, Iran.

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*Corresponding author. Tel: +98 5144013325; fax: +98 5144013170

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E-mail address: [email protected]

Abstract

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A facile strategy has been developed to fabricate silver nanoparticles (Ag NPs) through an electrochemical method with the assistance of metformin functionalized MWCNTs (Ag@MH/MWCNT nanocomposite). Investigations by field emission scanning electron microscopy (FESEM) confirmed that the prepared nanocomposite have a porous structure that is constructed by interconnecting functionalized MWCNTs framework. Electrochemical studies show that the nanocomposite exhibits high stability and excellent activity for electrocatalytic oxidation of glucose in alkaline solutions, which allows the Ag@MH/MWCNT to be used in enzyme-free amperometric sensors for glucose determination. It was confirmed that the Ag@MH/MWCNT based glucose biosensor presents wide response window for glucose concentrations of 1.0 nM–350 µM, short amperometric response time (4 s), low detection limit of 0.0003 µM (S/N=3), high sensitivity as well as good selectivity.

Keywords: Silver nanoparticles; Non-enzymatic sensor; Electrodeposition; Electrocatalysis

1. Introduction Diabetes mellitus is a serious and growing health care problem worldwide and is associated with acute and chronic complications [1]. Substantial evidences have been demonstrated the glucose concentration increases in blood serum of diabetic patients [2]. On the other hand, urinary excretion of glucose can reflect serious kidney disease [3]. With careful management, these complications can be delayed and even prevented. For many patients, this involves the regular measurement of blood glucose levels. Therefore, an assay that is able to determine 1 Page 1 of 31

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directly the level of glucose in biological fluids can better reflect the extent of disease in various people. Direct glucose sensing by portable electronic devices is recognized to be a promising power source for distinguish of disease in initial steps. However, since biological fluids such as urine and blood serum have a complex matrix, measurement of their glucose is an analytical challenge [4]. Hence, the quantification of glucose in biological fluids requires sensitive approach. Moreover, method should be selective, as major metabolites such as uric acid and ascorbic acid may also interfere the glucose determination [5]. Literature survey reveals that the glucose sensors can be categorized into two classifications: enzyme-based sensor [6-9] and non-enzymatic glucose sensor [10-13]. Although various enzyme-based sensors have been widely reported as amperometric biosensors but some disadvantages such as instability, the high cost of enzymes, complicated immobilization procedures, and critical operating conditions restrict their use. Therefore, considerable attention has been paid to developing the electrochemical nonenzymatic techniques to detect glucose with the advantages of high sensitivity and reliability, fast response, good selectivity, and low detection limit. Particularly, glucose sensing depending on electrocatalysis over various nanomaterials has been involved great consideration with the advantage of avoiding the problems in traditional enzyme-based sensors.

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Among various nanomaterials, the metal nanoparticles (NPs) such as Au NPs [14], Ni NPs [15], Cu NPs [16] and Co NPs [17], were operated to construct non-enzymatic glucose sensors. Silver NPs, as a typical noble metal NPs, have attracted great attention in recent years, because they not only have public characteristics of noble metal nanoparticles but also have unique properties of biocompatibility, excellent catalytic activity, low toxicity and antibacterial properties [18]. Current investigations found that performance of the sensors strongly depended on the distribution, size and shape of Ag NPs on the electrode surface [19]. Hence, the appropriate matrix for the preparation of vastly dispersed Ag NPs is very significant. Recently, the support-catalyst composites have received considerable attention at fabrication of sensors due to their influences on general sensor performance [20-22]. One of the factors that are crucial to the activity of the catalyst is the properties of the support used to anchor the catalyst. The support properties are very important since they affect catalyst dispersion, electrical conductivity, and stability of the overlying catalyst; all of which are needed for the stable and effective operation of a sensor [21]. Carbon nanotubes (CNTs) exhibit a favorable combination of the aforementioned properties as heterogeneous catalyst supports [23]. They have stimulated growing interest in nonenzymatic sensors since they possess relatively large surface area, high chemical stability, strong adsorptive ability and excellent electric conductivity. In order to take full advantage of the two kinds of nanomaterials, more attempts have been made on utilizing CNTs as the templates for supporting metal nanoparticle catalysts [24]. Actually, without the modification of surface, most of CNTs lack adequate binding sites for anchoring precursor metal ions or metal nanoparticles, which usually lead to poor dispersion and aggregation of metal nanoparticles, especially at high loading conditions. In principle, metal nanoparticles involuntarily are formed at the defect sites on the surface not on sidewall of CNTs [25]. Therefore the surface of CNTs must be functionalized via a 2 Page 2 of 31

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suitable covalent or non-covalent interaction, to obtain the homogeneously distributed nanoparticles [26-28]. For example, Li et al. fabricated an electrochemical H2O2 sensors based on electrodeposition of Pt NPs on multiwall carbon nanotube (MWCNTs) functionalized with sodium dodecyl sulfate (SDS), which PtNPs/MWCNTs–SDS composite yielded good performance toward the detection of H2O2 [29]. Li et al. fabricate a novel nonenzymatic sensor of H2O2 by utilizing ionic liquid functionalized MWCNTs as the matrix for electrodepositing of AgNPs [30]. Chen et al. discussed the synthesis of Pd NPs on differently functionalized MWCNTs and study the effects of MWCNT surface functionalization on the Pd-catalyzed glucose oxidation reaction [31]. Nevertheless, biocompatibility of functionalized CNTs is critical challenges that must be met to successfully produce such high property materials that can be used at fabrication of sensors. Therefore, it is strongly desired to find pathways for overcoming above obstacle. Functionalization of CNTs with biocompatible materials may be a way of obtaining unique structures and applications via combination of characteristics of the components that are not available with their single component counterparts [32].

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Metformin hydrochloride (MH), chemically [1,1-dimethylbiguanidehydrochloride], is an oral biguanide antihyperglycemic drug, which has been used for several decades in the treatment of type 2 diabetes [33]. Because of its biocompatibility, biodegradability, multiple functional groups, as well as its solubility in aqueous medium, MH appears to be an attractive candidate for functionalization of CNTs [34]. However, to our knowledge there is little report about the use of MH to functionalization the CNTs.

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In this paper, a facile path has been described for the electrodeposition of Ag NPs through covalent functionalization of MWNTs. The present work seeks to describe a new approach at the intersection between pharmacology and nanotechnology to develop the next promising biocompatible benign nanocomposites. The strategy we chose to achieve improved nanocomposite consisted in preparing functionalized MWCNTs with MH (MH/MWCNT). Since MH contains amino groups, MH-functionalized MWCNTs can act as the matrix for electrodepositing of Ag NPs. By combining the advantages of MWCNTs and Ag NPs, a novel nonenzymatic glucose sensor was fabricated. In fact, this cooperation of MHfunctionalized MWCNTs and Ag NPs in a unit network imparts electrocatalytic activity of the film and increases operational and long-term stability of designed sensor. Also, Ag NPs assembled on the MH/MWCNT (Ag@MH/MWCNT) enhanced the electrode conductivity, facilitate the electron transfer and improve the analytical sensitivity of sensor. The sensor exhibited excellent performance towards glucose with low detection limit, wide linear range, excellent selectivity and reproducibility, and could be applied to real samples analysis.

2. Experimental 2.1 Materials Glucose (Sigma, 99%) was purchased from USA and used without further purification. MWCNTs (>95%, O.D.: 10-15 nm, I.D.: 2-6 nm, length: 0.1-10 µm) were purchased from 3 Page 3 of 31

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Sigma–Aldrich. The carboxylated MWCNTs (denoted as MWCNTs-COOH) were prepared from pristine MWCNTs according to the literature reported in ever before [35]. SOCl2, HCl, H2SO4, HNO3, H2O2 (30 wt.%, aq), deionized water, NaH (80%), anhydrous dimethylformamide (DMF) and CaH2 were obtained from Sigma Aldrich. Metformin hydrochloride was prepared from Sigma Aldrich and treated before use as follow: 1.65 g of MH (10 mmol) and 0.40 g of NaOH (10 mmol) were added to 100 ml of ethanol and the resulting suspension was mixed for 5 h. Then, the resulted suspension was filtered and ethanol was removed with rotary evaporation leading to free metformin in 99% yield. The obtained free metformin was used in the whole of next experiments. The glucose stock solution was prepared by 0.1 M NaOH. All other chemicals were of analytical grade and were used as received without any purification process. All the supplementary chemicals were of analytical grades and solutions were prepared with deionized water.

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2.2 Apparatus and instrumentations

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Autolab Electrochemistry Instruments (Autolab, Eco Chemie, Netherlands) was used for amperometry measurements and electrochemical impedance spectroscopy (EIS). Cyclic voltammetry measurements were carried out on a Metrohm (797 VA Computrace, Switzerland) controlled by personal computer. An Ag|AgCl|KCl (3M) as reference electrode and a platinum wire as auxiliary electrode were used. A glassy carbon electrode (GCE), Azar electrode, Iran, with a geometrical area of 0.0314 cm2, bare or modified, was used as working electrode. EIS was performed in 1.0 mM K3Fe(CN)6/K4Fe(CN)6 (1/1) mixture with 0.1 M KCl as supporting electrolyte, using an alternating current voltage of 5 mV, within the frequency range of 0.1–105 Hz. FT-IR spectra were recorded on a Perkin Elmer GX FT-IR spectrometer. X-ray diffraction (XRD, Rigaku Corporation, Tokyo, Japan) patterns were obtained at room temperature on a Riga kuD/Max-2550 powder diffractometer. Field emission scanning electron microscopy, FESEM, (Hitachi S4160 instrument, Tokyo, Japan) was used to obtain information on the morphology of nanocomposite. All experiments were performed at room temperature (25 ± 2 °C).

2.3. MH-functionalization of MWCNTs The procedure for the chemical functionalization of MWCNTs using MH is as follows [34]: The generated carboxylic groups at MWCNTs-COOH were converted to acyl chlorides by a treatment with thionyl chloride. After this, the appropriate amount of acyl chloridefunctionalized MWCNT (MWCNTs-COCl) was dispersed in 20 mL DMF. In a separate preparation, free metformine were mixed with 1 mL solution of DMF and NaH (80%) and then stirred for 1 hour. Then, MWCNTs-COCl was added to this solution (metformine-toMWCNTs weight ratio was 10:1) and kept at 120 °C for 3 days to complete the reaction. The prepared black solid (donated as MH/MWCNT) was filtered, washed (with CH2Cl2 and deionized water) and dried in vacuum at ambient temperature.

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2.4 Preparation of the modified electrodes

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GCE was polished before each experiment with 1.0 and 0.3 µm alumina powder in sequence, rinsed thoroughly with distilled water between each polishing step, ultrasonicated in ethanol and distilled water, and allowed to dry at room temperature. Then 1.0 mg MH/MWCNT was dispersed in 2.0 ml of ethanol. GCEs were amended by a 6.0 µl drop of MH/MWCNT (MH/MWCNT/GCE) or MWCNT (MWCNT/GCE) solution and dried in air. The Ag NPs were electrochemically deposited on the electrodes using chronoamperometry in 1.0 mM AgNO3 solution, deaerated by bubbling with nitrogen at potential of 0.0 V (vs. Ag|AgCl|KCl (3M)) for 180 s. The applied rote for the construction of Ag@MH/MWCNT nanocomposite is shown schematically in Scheme 1. The final resulting modified electrodes were denoted by Ag/GCE, Ag/MWCNT/GCE and Ag@MH/MWCNT/GCE. The electrodes were rinsed with distilled water and dried and stored at 4°C in a refrigerator.

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"Here please Scheme 1"

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3. Results and Discussion 3.1. Characterization of materials

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The chemical structure of pristine MWCNT, MWCNT-COOH, MH/MWCNT and Ag@MH/MWCNT was characterized by FT-IR spectroscopy (Fig. 1A). In contrast to the spectrum of the pristine MWCNTs, MWCNT-COOH showed new absorption band at 1728 cm-1, apparently corresponding to the stretching vibrations of the COOH groups [36]. Also, the analysis of the chemical structure of both MWCNT-COOH and MH/MWCNT showed a peak at 1658 cm-1 corresponding to the carbonyl stretching of the amide groups (–CONH–) and a 1671 cm-1 peak corresponding to the imine (C=NH) bond of the attached metformin. These results confirm that the metformin bonded onto MWCNTs through an amidation reaction [34]. Compared to the spectrum of MH/MWCNT, the intensities of the presentative peaks at 1658 cm-1 (amide stretching vibration) and 1671 cm-1 (imine stretching vibration) almost disappeared or decreased significantly in the spectrum of Ag@MH/MWCNT, suggesting the strong interactions between the Ag nanoparticles and the surface of MH/MWCNT. The redox behavior of Ag NPs on the surface of the Ag@MH/MWCNT/GCE was evaluated by cyclic voltammetry to obtain definite evidence for the formation of these nanoparticles. The cyclic voltammogram of the Ag@MH/MWCNT/GCE after the holding of the potential constant during the electrodeposition process was shown in Fig. 1B. As shown in Fig. 1B, the electrodeposited Ag showed a clear anodic peak at 0.4 V and a small cathodic peak at -0.1 V when the potential scanned from -0.9 to 0.9 V vs. Ag|AgCl|KCl (3 M) in a solution containing 0.1 M NaOH. The anodic peak was due to the oxidation of electrodeposited Ag NPs and the cathodic peak was associated with the reduction of Ag+ cations [37]. The surface coverage (Γ) can be calculated from the integration of the anodic peak charge, Q, in CV by 5 Page 5 of 31

using the equation of Γ=Q/nFA, where n is the number of electron transferred, F is Faraday constant, A is the surface area of the electrode. After the background correction, Γ was calculated as 1.68 × 10-7 mol cm-2.

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"Here please Figure 1"

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The morphology of various surfaces was presented by FESEM images. Fig. 2A shows the FESEM image of the MH/MWCNT on GCE. It can be seen from FESEM image that the MH/MWCNT formed the network-like structure. After Ag electrodeposited on MH/MWCNT (Fig. 2B), it can be seen that a large numbers of Ag NPs are embedded onto functionalized MWCNT without any aggregation. As anticipated the direct electrodeposition of Ag+ on the bare GCE cause to aggregation of Ag particles on the electrode surface (Fig. 2C). It is seen clearly by the FESEM image that the synthesized Ag NPs at Ag@MH/MWCNT nanocomposite have advantages such as: identical particle size, uniform distribution on the surface of MH/MWCNT without any aggregation, and stable adsorption. Furthermore, crystallinity of the Ag@MH/MWCNT was investigated by XRD, and the result is shown in Fig. 2D. The 2θ peak at ca. 26º was signal from graphite-like CNTs. Also, three peaks in the XRD pattern of Ag@MH/MWCNT are characteristic of face-centered cubic (fcc) crystalline Ag metal, corresponding to the (111), (200) and (220) planes at 2θ values of 39.7º, 46.3º, 67.6º, respectively (JCPDS 04-0783). The average particle size of the deposited Ag nanoparticles was calculated to be 46.6 nm, in terms of the Scherrer’s equation, which are in agreement with the result presented by FESEM. No peaks from other phases were detected, indicating high purity of the products. All of these advantages are beneficial for improving the performance of the Ag@MH/MWCNT/GCE as sensor. Also, these results confirm that the chemical functionalization of MWCNT is an excellent way to immobilization of Ag NPs and increase of their stability on the surface of electrode [30]. "Here please Figure 2"

3.2. Optimization of the sensor

As Ag NPs could particularly enhance the electrode response toward glucose detection, therefore, the amount of the electrodeposited AgNPs on MH/MWCNT in the nanocomposite may be an important aspect to fabricate a sensor with the best electrocatalytic property. Fig. 3 shows the glucose oxidation current intensity as a function of the AgNO3 concentration (panel A) and electrodeposition time (panel B). As it can be seen in panels A in Fig. 3, the addition of AgNO3 concentration in the range of 0.025-1 mM resulted in a significant increase in the glucose current responses, indicating the appropriate formation of Ag NPs on MH/MWCNT in the nanocomposite films. However, at a concentration range of up to 1mM in the electrochemical synthetic process, the obtained Ag@MH/MWCNT nanocomposite has lower analytical performance instead. This may be because that great number of Ag NPs have been aggregated, and decreased the catalytic performance of the nanocomposite. 6 Page 6 of 31

"Here please Figure 3"

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3.3. Electrochemical behavior of glucose at different electrodes

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In addition to these, the performance of the sensor was evaluated by the variation of the electrodeposition time in the range of 100-250 s. As seen in panels B in Fig. 3, the nanocomposites deposited for 180 s have shown a better performance; in contrast, nanocomposites deposited for longer or shorter times. It seems that too many Ag NPs have been aggregated in the longer deposition times, and decreased the catalytic efficiency of the nanocomposite. Also, lower glucose current responses in the shorter electrodeposited times could be explained in terms of the time needed to effective immobilization of Ag NPs at the surface of nanocomposite.

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Fig. 4 describes the cyclic voltammograms of glucose on the bare GCE, MH/MWCNT/GCE and Ag@MH/MWCNT/GCE in 0.1 M NaOH, respectively. The bare GCE (curve a) exhibit no electrochemical response in the presence of glucose. In contrast, MH/MWCNT/GCE (curve b) shows no significant current response, whereas Ag@MH/MWCNT/GCE (curve c) shows a notable catalytic current peak about 176 µA in intensity at 0.69 V. The excellent anodic peak current of glucose at surface of Ag@MH/MWCNT nanocomposite may be attributed to the synergistic effect of MH/MWCNT and Ag NPs. The network-like structure of MH/MWCNT provide extra proper surface for adsorption, and then more adsorbent Ag NPs improve the catalytic performance. All the above explanations prove that AgNPs contained in the nanocomposites present a remarkable catalytic performance for glucose oxidation.

Electrochemical impedance spectroscopy (EIS) is a powerful tool for studying the interface properties of surface-modified electrodes [38]. Thus, the electron transfer ability of different modified electrodes was studied by electrochemical impedance technique. Fig. 5 shows Nyquist plots, obtained at an open circuit potential in 1.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) as redox probe, for bare GCE (a), MH/MWCNT/GCE (b) and Ag@MH/MWCNT/GCE (c). The charge transfer resistance (Rct) is a key parameter for the kinetics of a redox probe at the electrode interface. The Rct value decreased from 890 Ω for the bare GCE (curve a) to about 524 Ω for MH/MWCNT/GCE (curve b). This behavior can be related to improving electron transfer at the electrode surface by MH/MWCNT. On the other hand, the Rct value decreased to 160 Ω upon deposition of Ag NPs on the MH/MWCNT layer (curve c), showing that the assembly containing Ag NPs facilitates electron transfer of the redox probe. Hence, MH/MWCNT played an important role in fabrication of the proposed sensor, not only in supplying more sites for immobilization of Ag NPs, but also it acted as template to prevent aggregation of Ag NPs for construction of a sensor with improved electron transfer properties. "Here please Figure 5" 7 Page 7 of 31

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The effect of potential scan rate in 0.1 M NaOH solution with 100.0 µM glucose at the Ag@MH/MWCNT/GCE was investigated using cyclic voltammetry. As seen from Fig. 6A, the anodic peak potential shifted to more positive potentials with scan rate in the range of 20300 mVs-1, presenting the kinetic limitation in the electrochemical reaction. Also, the anodic peak currents rise linearly with increasing scan rate (inset at Fig. 6A), which indicates a surface-controlled electrochemical process [39]. Fig. 6B offered the cyclic voltammograms responses obtained at Ag@MH/MWCNT/GCE in 0.1 M NaOH solution containing different concentrations of glucose at a scan rate of 50 mVs-1. As shown, an anodic oxidation peak corresponding to glucose oxidation increases proportionally with increasing of glucose concentration. The plot of the Ip against glucose concentration exhibits a linear correlation in the range of 1.0-100.0 µM which can be fitted into the equation; Ip(µA) = 0.755 [glucose] (µM) + 102.12 (µA).

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3.4. Amperometric detection of glucose at Ag@MH/MWCNT/GCE

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To evaluate the sensitivity and quantitative range of the fabricated sensor, amperometry is preferred over other electrochemical techniques such as cyclic voltammetry and differential pulse voltammetry [40]. The performance of the sensor at amperometric detection of an analyte is depends on the applied potential. Therefore, the effect of applied potential was investigated on the response of proposed sensor to glucose. Fig. 7A shows the effect of applied potential on the electrochemical oxidation of glucose. With the increase of the applied potential from +0.50 V to +0.90 V, the response current increased consequently. This implies that the response of the modified electrode is resulted from the electrochemical oxidation of glucose. When the applied potential was higher than +0.70 V, the response current began to level off. Hence, a potential of +0.70 V was selected for glucose detection in the subsequent experiments as working potential. Fig. 7B shows the performance of Ag@MH/MWCNT/GCE by recording the amperometric response of sensor during the successive additions of glucose into stirred solution at an applied potential of +0.70 V. As can be seen, shortly after the addition of glucose to solution, the oxidation current increases and reaches a steady state within 4 s. The oxidation of current scales linearly with concentration of glucose in the range of 1.0 nM to 350.0 µM. Calibration curve for glucose concentrations from 1.0 nM to 500.0 nM with a linear regression equation of Ip(µA) = 0.0285 [glucose] (nM) + 4.1707 (µA) was presented in inset a of Fig. 7B, suggesting the excellent sensitivity of the proposed sensor. The notable current sensitivity of the sensor can be correlated to the efficiency of the electron transfer between Ag@MH/MWCNT nanocomposite and glucose due to the catalytic effect resulting from the synergism coupling influence of MH/MWCNT and Ag NPs. The limit of detection for glucose in the studied region was calculated to be 0.0003 µM based on signal to noise ratio of 3. Table 1 exhibits the advantages of the proposed Ag@MH/MWCNT/GCE sensor compared with other reported non-enzymatic glucose sensors. As presented in Table 1, the 8 Page 8 of 31

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response time, linear calibration range and detection limit for glucose determination at the Ag@MH/MWCNT/GCE are comparable or even better than those obtained at several reported electrodes. In addition, the relative standard deviation is 5.2% for 10 repetitive measurements of 100 µM glucose solution. After measurement, the sensor was rinsed with doubly distilled water and stored at room temperature, and it was observed that the response reduced about 12% in 6 days, and 20% after more than two weeks. Hence, the Ag@MH/MWCNT nanocomposite acts as a superior electrocatalytic material for sensitive and stable amperometric determination of glucose.

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3.5. Selectivity of the glucose sensor

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Investigation about selectivity of the sensor is very important in applied usages. Therefore, selectivity of the Ag@MH/MWCNT/GCE sensor was evaluated with some biological interfering species such as (acetic acid, ethanol, ascorbic acid, uric acid, dopamine, L-Dopa, epinephrine, tryptophan and L-tyrosine) by using amperometic method. Fig. 8 shows the amperometric responses obtained at the Ag@MH/MWCNT/GCE sensor for each successive addition of 400.0 µM mentioned interfering species at regular intervals (10 s once) into experimental solution (after twice addition of 100.0 µM glucose). As shown in Fig. 8, no significant responses were attained for each 4-fold excessive addition. These results revealed that the proposed sensor shows high selectivity toward glucose, and it has the ability to reduce the interference from the electroactive species, which is more helpful for practical applications. "Here please Figure 8"

3.6. Analytical applications

In order to investigate the ability of the sensor, Ag@MH/MWCNT/GCE was used to determination of glucose in human blood serum and urine samples of diabetic and healthy people. The samples were purchased from Dr. Abed Medical Diagnostic Laboratory, Sabzevar, Iran. The blood serum (20 µL) and urine samples (10 µL) were added to 10 mL 0.1 M NaOH solution. The obtained concentration was in the linear range of the sensor. Amperometric method was carried out to evaluate the current response by using the standard addition method [47]. The obtained results were compared with those achieved by a spectrophotometric method in medical diagnostic laboratory. The experimental results were accessible in Table 2 and confirmed that the prepared sensor can be sufficient for practical applications. "Here please Table 2"

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4. Conclusion

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Fabrication of a facile nanocomposite was reported based on electrodeposition of Ag NPs on MH functionalized MWCNTs. The MH functionalized MWCNTs have been identified to act as a three-dimensional backbone that form effective interactions with Ag NP active sites to promote the operation of nanocomposite. The fabricated Ag@MH/MWCNT nanocomposite was successfully applied for electrode modification in the fabrication of a none-enzymatic glucose sensor, which showed advantages such as high sensitivity, fast current response, and good stability for determination of glucose in experimental and real samples. The simple preparation of nanocomposite, low cost of used materials, and improved electrocatalytic operation of sensor can potentially pave the way for fabrication of an applicable glucose sensor. Acknowledgement

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We would like to thank the Hakim Sabzevari University for its financial support.

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[21] S. Prakash, T. Chakrabarty, A.K. Singh, V.K. Shahi, Polymer thin films embedded with metal nanoparticles for electrochemical biosensors applications, Biosens. Bioelectron. 41 (2013) 43-53.

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[22] J. Zhao, L. Wei, C. Peng, Y. Su, Z. Yang, A non-enzymatic glucose sensor based on the composite of cubic Cu nanoparticles and arc-synthesized multi-walled carbon nanotubes, Biosens. Bioelectron. 47 (2013) 86-91.

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[23] C.E. Banks, A. Crossley, C. Salter, S.J. Wilkins, R.G. Compton, Carbon nanotubes contain metal impurities which are responsible for the “electrocatalysis” seen at some nanotube-modified electrodes, Angewandte Chemie. 45 (2006) 2533–2537.

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[24] Z.-P. Sun, X.-G. Zhang, Y.-Y. Liang, H.-L. Li, Highly dispersed Pd nanoparticles on covalent functional MWNT surfaces for methanol oxidation in alkaline solution, Electrochem. Commun. 11 (2009) 557-561.

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[25] Y.-P. Sun, K. Fu, Y. Lin, W. Huang, Functionalized carbon nanotubes: properties and applications, Acc. Chem. Res. 35 (2002) 1096-1104.

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[26] B. Kim, W.M. Sigmund, Functionalized multiwall carbon nanotube/gold nanoparticle composites, Langmuir. 20 (2004) 8239-8242. [27] R. Zanella, E.V. Basiuk, P. Santiago, V.A Basiuk, E. Mireles, I. PuenteLee, J.M. Saniger, Deposition of gold nanoparticles onto thiol-functionalized multiwalled carbon nanotubes, J. Phys. Chem. B 109 (2005) 16290-162955. [28] C. Miao, A. Zhang, Y. Xu, S. Chen, F. Ma, C. Huang, N. Jia, An ultrasensitive electrochemiluminescence sensor for detecting diphenhydramine hydrochloride based on lcysteine-functionalized multiwalled carbon nanotubes/gold nanoparticles nanocomposites, Sensors and Actuators B 213 (2015) 5-11. [29] X. Li, X. Liu, W. Wang, L. Li, X. Lu, High loading Pt nanoparticles on functionalization of carbon nanotubes for fabricating nonenzyme hydrogen peroxide sensor, Biosens. Bioelectron. 59 (2014) 221-226. [30] X. Li, Y. Liu, L. Zheng, M. Dong, Z. Xue, X. Lu, X. Liu, A novel nonenzymatic hydrogen peroxide sensor based on silver nanoparticles and ionic liquid functionalized multiwalled carbon nanotube composite modified electrode, Electrochim. Acta 113 (2013) 170-175.

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[31] C. Chen, C. Lin, L. Chen, Functionalized carbon nanomaterial supported palladium nano-catalysts for electrocatalytic glucose oxidation reaction, Electrochim. Acta 152 (2015) 408-416. [32] N. Yang, X. Chen, T. Ren, P. Zhang, D. Yang, Carbon nanotube based biosensors, Sens. Actuators B: Chem. 207 (2015) 690-715.

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[33] T. Huo, S. Cai, X. Lu, Y. Sha, M. Yu, F. Li, Metabonomic study of biochemical changes in the serum of type 2 diabetes mellitus patients after the treatment of metformin hydrochloride, J. Pharm. Biomed. Anal. 49 (2009) 976-982.

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[34] H. Veisi, A. Khazaei, M. Safaei, D. Kordestani, Synthesis of biguanidefunctionalized single-walled carbon nanotubes (SWCNTs) hybrid materials to immobilized palladium as new recyclable heterogeneous nanocatalyst for Suzuki-Miyaura coupling reaction, J. Mol. Catal. A: Chem. 382 (2014) 106-113.

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[35] M. Baghayeri, E.N. Zare, M. Namadchian, Direct electrochemistry and electrocatalysis of hemoglobin immobilized on biocompatible poly (styrene-alternative-maleic acid)/functionalized multi-wall carbon nanotubes blends, Sens. Actuators B: Chem. 188 (2013) 227-234. [36] P. Kar, A. Choudhury, Carboxylic acid functionalized multi-walled carbon nanotube doped polyaniline for chloroform sensors, Sens. Actuators B: Chem. 183 (2013) 25-33.

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[37] M. Baghayeri, M. Namadchian, H. Karimi-maleh, H. Beitollahi, Determination of nifedipine using nanostructured electrochemical sensor based on simple synthesis of Ag nanoparticles at the surface of glassy carbon electrode: Application to the analysis of some real samples, J. Electroanal. Chem. 697 (2013) 53-59.

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[38] M. Baghayeri, E.N. Zare, M.M. Lakouraj, A simple hydrogen peroxide biosensor based on a novel electro-magnetic poly(pphenylenediamine)@Fe3O4 nanocomposite, Biosens. Bioelectron. 55 (2014) 259-265. [39] M. Baghayeri, M. Namadchian, Fabrication of a nanostructured luteolin biosensor for simultaneous determination of levodopa in the presence of acetaminophen and tyramine: Application to the analysis of some real samples, Electrochim. Acta 108 (2013) 22-31. [40] M. Baghayeri, E.N. Zare, M.M. Lakouraj, Novel superparamagnetic PFu@Fe3O4 conductive nanocomposite as a suitable host for hemoglobin immobilization, Sens. Actuators B: Chem. 202 (2014) 1200-1208. [41] R.M.A. Tehrani, S.A. Ghani, MWCNT-ruthenium oxide composite paste electrode as non-enzymatic glucose sensor, Biosens. Bioelectron. 38 (2012) 278-283. [42] J. Song, L. Xu, R. Xing, W. Qin, Q. Dai, H. Song, Ag nanoparticles coated NiO nanowires hierarchical nanocomposites electrode for nonenzymatic glucose biosensing, Sens. Actuators B: Chem.182 (2013) 675-681.

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[43] T. Marimuthu, S. Mohamad, Y. Alias, Needle-like polypyrrole-NiO composite for nonenzymatic detection of glucose, Synth. Met. 207 (2015) 35-41. [44] W. Lu, X. Qin, A.M. Asiri, A.O. Al-Youbi, X. Sun, Ni foam: a novel three-dimensional porous sensing platform for sensitive and selective nonenzymatic glucose detection, Analyst 138 (2013) 417-420.

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[47] M. Baghayeri, Glucose sensing by a glassy carbon electrode modified with glucose oxidase and a magnetic polymeric nanocomposite, RSC Advances. 5 (2015) 18267-18274.

Biographies

Mehdi Baghayeri is a professor in the Department of Science, Hakim Sabzevari University, Sabzevar, Iran. He received his Ph.D. from University of Mazandaran, Babolsar, Iran in 2012. His main research interests are focused on bioelectrochemistry and nanobiotechnology.

Amirhassan Amiri Chemistry from the

received his BSc and PhD in Analytical Ferdowsi University of Mashhad (Iran) in 2007 14 Page 14 of 31

and 2012, respectively. In September of 2012 he joined the Faculty at the Hakim Sabzevari University at Sabzevar (Iran) as an Assistant Professor of Analytical Chemistry. Currently, his group is focused on development and evaluation of new coatings and nanostructured coatings for SPME and SPE.

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Samaneh Farhadi, received the B.S. degree in pure chemistry from Hakim Sabzevari University, Sabzevar, Iran, in 2012. Now, she is working towards the M.S. degree in analytical chemistry at Hakim Sabzevari University. Her interests are electrochemistry, composites and nanomaterial application.

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Figure and scheme captions

d

Scheme 1. Scheme for the preparation of Ag@MH/MWCNT nanocomposite on the electrode surface.

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Fig. 1. (A) FT-IR spectra of pristine MWCNT, MWCNT-COOH, MH/MWCNT and Ag@MH/MWCNT nanocomposite. (B) Cyclic voltammogram of bare GCE (a) and Ag NPs immobilized on MH/MWCNT/GCE (b) in 0.1 M NaOH at scan rate 50 mV s-1.

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Fig. 2. The SEM images of (A) MH/MWCNT/GCE, (B) Ag@MH/MWCNT/GCE and (C) Ag/GCE. (D) XRD patterns of Ag@MH/MWCNT nanocomposite. Fig. 3. Glucose oxidation current intensity as a function of the AgNO3 concentration (A) and electrodeposition time (B). Fig. 4. Cyclic voltammograms of 100.0 µM glucose in 0.1 M NaOH at the surface of different electrodes: bare GCE (a), MH/MWCNT/GCE (b) and Ag@MH/MWCNT/GCE (c) at scan rate of 50 mV s -1. Fig. 5. Electrochemical impedance spectra of (a) bare GCE, (b) MH/MWCNT/GCE and (c) Ag@MH/MWCNT/GCE recorded at the open circuit potential, containing 1.0 mM K3[Fe(CN)6]/K4[Fe(CN)6]. Fig. 6 (A) Cyclic voltammograms of Ag@MH/MWCNT/GCE in 0.1 M NaOH at different scan rates of (1) 20, (2) 40, (3) 60, (4) 80, (5) 100, (6) 140, (7) 160, (8) 180, (9) 200, (10) 240, (11) 280 and (12) 300, mVs-1. Inset: plot of the anodic peak current against the potential scan rate. (B) Cyclic voltammograms of Ag@MH/MWCNT/GCE in 0.1 M NaOH solution

15 Page 15 of 31

with 0 (a), 1.0 (b), 5.0 (c), 10.0 (d), 50.0 (e), and 100.0 µM (f) of glucose at the scan rate of 50 mV s-1. Inset: plot of the anodic peak current against the glucose concentration.

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Fig. 7. (A) Influence of applied potential on steady-state response current of the fabricated sensor to 100 µM glucose. (B) Amperometric response of the sensor to successive addition of different concentration of glucose in 0.1 M NaOH at the working potential of +0.70 V. Inset a: plot of electrocatalytic peak current vs. concentration of glucose (from 1.0 nM to 500.0 nM).

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Fig. 8. Amperometric response of the Ag@MH/MWCNT/GCE in 0.1 M NaOH upon successive additions of 100 µM glucose (a) and 400 µM of each of acetic acid (b), ethanol (c), ascorbic acid (d), uric acid (e), dopamine (f), L-Dopa (g), epinephrine (h), and L-tyrosine (i). Applied potential is +0.70 V.

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ip t cr us an M d te Ac ce p Scheme 1

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ip t cr us an M d te

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Figure 1A

18 Page 18 of 31

ip t cr us an M

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Figure 1B

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ip t cr us an M d te Ac ce p

Figure 2

20 Page 20 of 31

ip t cr us an M d te Ac ce p Figure 3

21 Page 21 of 31

ip t cr us an M d

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te

Figure 4

22

b

a

Page 22 of 31

ip t cr us an M

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te

d

Figure 5

23 Page 23 of 31

ip t cr us an M d te Ac ce p

Figure 6A

24 Page 24 of 31

ip t cr us an M d te Ac ce p

Figure 6B

25 Page 25 of 31

ip t cr us an M d te

Ac ce p

Figure 7A

26 Page 26 of 31

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Figure 7B

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Figure 8

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Reference

300-3300

(µM) 3.3

[16]

-

500-50000

33

[41]

Amperometry

7

0-9680

1.01

[42]

PPy –NiO composite

Amperometry

6

10-500

0.33

[43]

NF4

Amperometry

5

50-7350

2.2

AuNP/GONR5/CS6

Voltammetry

-

0.5-10000

cr

[44]

0.5

[20]

ZnO-NiO

Amperometry

3

0.5-6400

0.5

[45]

Cu–Ag/NF

Amperometry

2

5-3500

0.08

[46]

PtAu alloy

Amperometry

2

1.6-5400

0.5

[13]

Ag@MH/MWCNT

Amperometry

4

0.001-350

0.0003

This work

Detection methode

Cu2O/GNs1

Amperometry

response time 9

MWCNT–RuO2

Voltammetry

5% NiO@Ag NWs2

Linear range (µM)

M

3

Amperometric

an

Modifier

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LOD

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Table 1. The performance of the various reported modified electrodes for determination of glucose.

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1- Graphene nanosheets 2- Nanowires 3- Polypyrrole 4- Nickel foam 5- Graphene oxide nanoribbons 6- Carbon sheet

29 Page 29 of 31

Table 2. Results of the glucose detection and the recovery test for real samples analysis (n=5). Added (µM)

Found (µM)

Determined by spectrophotometry

A

0

254.4 ±0.05

253.6

30.0

284.9±0.04

50.0

303.6±0.02

0

173.7±0.06

20

192.1±0.05

60

234.4±0.04

0

-

170 190

D

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M

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172.6

99.7

99.1 100.2

-

168.9±0.06

168.1

100.4

191.2±0.04

190.2

100.6

204.6±0.04

203.8

-

20

223.7±0.05

0.295

99.5

40

245.2±0.04

0.51

100.2

d

-

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C

100.1

0

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B

Recovery (%)

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Serum sample

A and B samples of glucose in human serum. C and D samples of glucose in human urine.

Biographies Mehdi Baghayeri is a professor in the Department of Science, Hakim Sabzevari University, Sabzevar, Iran. He received his Ph.D. from University of Mazandaran, Babolsar, Iran in 2012. His main research interests are focused on bioelectrochemistry and nanobiotechnology.

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Amirhassan Amiri received his BSc and PhD in Analytical Chemistry from the Ferdowsi University of Mashhad (Iran) in 2007 and 2012, respectively. In September of 2012 he joined the Faculty at the Hakim Sabzevari University at Sabzevar (Iran) as an Assistant Professor of Analytical Chemistry. Currently, his group is focused on development and evaluation of new coatings and nanostructured coatings for SPME and SPE.

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Samaneh Farhadi, received the B.S. degree in pure chemistry from Hakim Sabzevari University, Sabzevar, Iran, in 2012. Now, she is working towards the M.S. degree in analytical chemistry at Hakim Sabzevari University. Her interests are electrochemistry, composites and nanomaterial application.

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