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Electrodeposition of copper nanoparticles using pectin scaffold at graphene nanosheets for electrochemical sensing of glucose and hydrogen peroxide
Electrochimica Acta 176 (2015) 804–810
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Electrodeposition of copper nanoparticles using pectin scaffold at graphene nanosheets for electrochemical sensing of glucose and hydrogen peroxide Veerappan Mania,** , Rajkumar Devasenathipathya , Shen-Ming Chena,* , Sea-Fue Wangb , Parvathy Devic , Yian Taic a Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No. 1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan, ROC b Department of Materials and Mineral Resources Engineering, National Taipei University of Technology, No. 1, Sec. 3, Chung-Hsiao E. Road, Taipei, Taiwan, ROC c Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan, ROC
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 April 2015 Received in revised form 17 July 2015 Accepted 18 July 2015 Available online 26 July 2015
A simple electrodeposition approach has been described for the preparation of copper nanoparticles (CuNPs) using biopolymer pectin as a scaffold and graphene as a support. The formation of graphene/ pectin-CuNPs was confirmed by scanning electron microscopy, UV-Visible spectroscopy and X-ray diffraction studies. The graphene/pectin-CuNPs film modified electrode was prepared and its electrocatalytic applications to the oxidation of glucose and reduction of H2O2 have been explored. An amperometric glucose sensor was fabricated which exhibited excellent sensor performance in terms of wide linear range (10 mM–5.5 mM), low detection limit (2.1 mM) and high sensitivity (0.0457 mAmM 1 cm 2). Likewise, an amperometric sensor has been fabricated for the determination of H2O2 which displayed linear range of 1 mM–1 mM, detection limit of 0.35 mM and sensitivity of 0.391 mAmM 1 cm 2. The sensor displayed appreciable repeatability, reproducibity and stability. Furthermore, practical feasibility of the sensor has been demonstrated in human serum and contact lens cleaning solution to determine glucose and H2O2, respectively. The main advantages of sensor include simple and green fabrication approach, roughed and stable electrode matrix, high sensitivity and stability, fast in sensing and highly reproducible. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: copper nanoparticles Electrodeposition glucose hydrogen peroxide electrochemical sensor
1. Introduction The development of sensitive glucose biosensors for the accurate and reliable determination of blood glucose level is of great significance to control diabetics [1–3]. Numerous enzymatic glucose biosensors have been reported based on glucose oxidase (GOx) immobilized at various modified electrodes [3–8]. However, enzyme electrodes often suffer from serious drawbacks, such as, instability of immobilized enzyme, lack of long term stability, enzyme leaching and poor electrical communication to the highly buried active sites of enzyme [1]. Also, activity of the GOx has been affected by pH, temperature and immobilization methods [9]. Nonenzymatic glucose sensors are attractive alternative approach
* Corresponding author. Tel.: +886 2270 17147; fax: +886 2270 25238. ** Corresponding author. Tel.: +886 2271 2171 2525; fax: +886 02 2731 7117. E-mail address: [email protected] (S.-M. Chen). http://dx.doi.org/10.1016/j.electacta.2015.07.098 0013-4686/ ã 2015 Elsevier Ltd. All rights reserved.
for the detection of glucose to avoid enzyme related issues [10] and therefore, many efforts have been focused on the fabrication of non-enzymatic glucose sensor using different electrode modifiers [11–13]. Electrodes modified with nanomaterials are widely used for the construction of glucose sensors attributed to their extraordinary physiochemical properties such as, large surfaceto-volume ratio, high conductivity, and excellent electrocatalytic ability [12,14]. In recent times, numerous copper nanoparticles (CuNPs) based electrodes are reported for the determination of glucose [9,10,15–17]. However, most of the methods reported in the literature are based on chemical methods which require the use of reduction agents [18], thermal condition [19], microemulsions [20], and micelles [19] etc., These methods also involved with tedious procedures and the as-prepared CuNPs has the possibility of easily oxidized to copper oxide during the course of preparation in the aforementioned methods [21]. On the other hand, green agents such as biopolymers stabilized preparation methods have the ability to overcome the difficulties encountered by the
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chemical synthesis methods. Therefore, herein, we established a simple green method to synthesis biopolymer stabilized CuNPs using pectin as a scaffold. The method involves simple and efficient electrodeposition methods without using any reducing agent and complex methods. Pectin (polygalacturonic acid) is a naturally occurring polysaccharide occurring at the cell walls of the plants and highly biodegradable [22,23]. The use of carbon based materials such as carbon nanotubes and graphene as supports is well established approach to improve the stability and conductivity of the nanoparticles, which also aid to overcome surface fouling issues [24,25]. Therefore, we deposited pectin stabilized CuNPs at graphene, where graphene prepared on the electrode surface through electrochemical reduction method. The as-prepared graphene/pectin-CuNPs has been well characterized by analytical, spectral and electrochemical methods. The graphene/pectinCuNPs film exhibited excellent electrocatalytic ability to the oxidation of glucose and displayed excellent sensor performance with the reported glucose sensors in terms of wide linear range, high sensitivity and selectivity, fast response time, high stability and appreciable practicality. Additionally, the graphene/pectinCuNPs film also exhibited excellent electrocatalytic ability to the reduction of hydrogen peroxide (H2O2) and therefore we also developed a nonenzymatic H2O2 sensor. H2O2 is a vital constituent of plant tissues, which regulates the plant metabolism, acclamatory processes and gene expression [26,27]. In addition, it possesses good antibacterial and antiseptic properties and extensively used in industries as an oxidizing agent, antibacterial agent and bleaching agent [28,29]. Therefore, an accurate and reliable method for the determination of H2O2 is highly important for clinical and industrial analysis [15,30–32]. The main aim of this method is to develop a biopolymer assisted electrochemical synthetical method for the preparation of CuNPs and for the development of glucose and H2O2 sensor. The described method has advantages over previous methods to prepare high stable, uniform and electrochemically active CuNPs. The preparation is fast, green, simple electrode fabrication procedure and highly reproducible.
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2. Experimental 2.1. Reagents and apparatus Graphite (powder, <20 mm), LM-pectin (DE 35%, pectin (from citrus peel), copper nitrate (purum, 98%, 26% Cu basis), glucose and H2O2 were purchased from Sigma-Aldrich and used as received. The supporting electrolyte used for all the electrochemical studies was 0.1 M phosphate buffer solution, prepared using NaH2PO4 and Na2HPO4, while the pH were adjusted to get desired pH using either using H2SO4 or NaOH. Prior to each experiment, all the solutions were deoxygenated with pre-purified nitrogen gas for 15 min unless otherwise specified. Blood sample used in this study were collected from healthy man (26-year-old male). The blood sample was taken from cubital vein and transferred to a test tube and stored at 20 C before analysis. The collected blood sample was allowed to clot; subsequently the clot was removed through centrifugation for 20 min at the speed of 2000 g. The serum separated as a supernatant was collected and stored at the temperature of –20 C. Contact lens cleaning solution containing 3% H2O2 was purchased from a local drug store in Taipei, Taiwan to demonstrate practicality osf the sensor. The electrochemical measurements were carried out using CHI 611A electrochemical work station. Electrochemical studies were performed in a conventional three electrode cell using BAS glassy carbon electrode (GCE) as a working electrode (area 0.071 cm2), Ag| AgCl (saturated KCl) as a reference electrode and Pt wire as a counter electrode. Amperometric measurements were performed with analytical rotator AFMSRX (PINE instruments, USA) with a rotating disc electrode (RDE) having working area of 0.24 cm2. Scanning electron microscope (SEM) studies were performed using Hitachi S-3000H scanning electron microscope. Ultra violet visible (UV-Vis) spectroscopy studies were performed by U-3300 spectrophotometer. X-ray diffraction (XRD) and Attenuated total reflectance-FT-IR (ATR–FTIR) spectroscopy studies were carried out using XPERT-PRO (PANalytical B.V., The Netherlands) diffractometer (Cu Ka radiation, k = 1.54 Å) and Perkin-Elmer IR spectrometer respectively.
Fig. 1. SEM images of pectin-CuNPs at the potential of –1.20 V (A), – 0.80 V (B) and – 0.60 V (C). SEM image of graphene/pectin-CuNPs.
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2.2. Electrochemical preparation of GCE/graphene/pectin-CuNPs Graphite oxide was prepared from graphite by Hummers method [33] and exfoliated to graphene oxide (GO) through ultrasonic agitation for 1 h and subsequent centrifugation at 3000 RPM for 30 min. Prior to electrode modification, both GCE and rotating disk GCE were polished with 0.05 mm alumina slurry using a Buehler polishing kit and subsequently washed with water and allowed to air-dried. Aliquot (5 mL) of GO dispersion was dropped onto the pre-cleaned GCE surface and dried in over for 30 min. After the electrodes were air-dried for 30 min at ambient temperature, the GO film modified GCE was transferred to electrochemical cell containing nitrogen saturated phosphate buffer (pH 5). 30 consecutive cyclic voltammograms were applied at the potential range between 0 to –1.5 V (Fig. S1A). The scan rate was hold at 50 mV s 1. During electrochemical cycling, GO has been reduced to graphene on the electrode surface and the resulting electrode was named as graphene/GCE. 20 mg mL 1 of Cu(NO3)2 solution prepared in 0.1 M H2SO4 containing 1 mg mL 1 of pectin. The graphene/GCE has been transferred to an electrochemical cell containing the mixture of Cu(NO3)2 and pectin. 10 consecutive cyclic voltammograms were carried out at the scan rate of 50 mV s 1 (Fig. S1B). Different potential windows (0.50 to –1.20 V, 0.5 to –0.80 V and 0.50 to 0.60 V (vs. Ag|AgCl) respectively) have been applied to optimize the formation of stable CuNPs. After completion of electrodeposition process, the electrode was carefully washed with water and dried for 30 min. The final nanocomposite is denoted as graphene/pectin-CuNPs. As a control, graphene/GCE and pectin-CuNPs/GCE also prepared. 3. Results and Discussion 3.1. Characterization of graphene/pectin-CuNPs The SEM images of pectin-CuNPs prepared at different potentials are given as Fig. 1. As can be seen from the SEM images, the CuNPs prepared at the potential ranges of 0.50 to –1.20 V (Fig. 1A) and 0.50 to –0.80 V (Fig. 1B) are showed aggregated formation of CuNPs. However, the CuNPs prepared at the potential range of 0.50 V to –0.60 V (Fig. 1C) has shown uniformly distributed individual nanoparticles with particles size ranges in nanometers. Therefore, we choose this optimized potential window for the preparation of CuNPs at the graphene nanosheets. The SEM image of graphene/pectin-CuNPs is depicts the uniform decoration of CuNPs at the graphene nanosheets (Fig. 1D). The observations of thin sheets like morphology evident the presents of graphene nanosheets. 200 successive cyclic voltammograms (CVs) were recorded at graphene/pectin-CuNPs/GCE in 0.1 M NaOH (Figure not shown) in order to evaluate stability of the CuNPs. Only 7.1% of the initial peak currents were decreased even after cycling for 200 cycles which revealing the high stability of the graphene/ pectin-CuNPs. The UV-Visible spectrum of pectin (Curve a, Fig. 2A) exhibited a sharp absorption peak at 290 nm which arose due to the free carboxyl group of pectin. However, this absorption peak is disappeared in the spectrum of graphene/pectin-CuNPs (Curve b, Fig. 2A). Perhaps, the carboxyl groups of pectin might commit to accommodate CuNPs which makes the disappearance of its absorption peak in the spectrum. Additionally, a new absorption peak has been observed at the wavelength of 570 nm which was assigned to the CuNPs. Fig. 2B displays the ATR-FTIR spectra of pectin (curve a) and graphene/pectin-CuNPs (curve b). The peaks observed at 3600 cm 1 and 2800 cm 1 were assigned to the hydroxyl and carboxylic hydroxyl groups respectively. The peak observed at 1026 cm 1 is assigned to the glycosidic bonds linking two galacturonic sugar units, while the peak obtained at 1608 cm 1
Fig. 2. UV-Visible (A) and AT–FTIR (B) spectra of pectin (a) and graphene/pectinCuNPs (b)
is assigned to the carbonyl group of the esterified pendant of pectin [22,34]. However, all these peaks were disappeared in the ATR-FTIR spectrum of graphene/pectin-CuNPs which clearly indicating that these functional groups are devoted to accommodate CuNPs. Fig. 3A displays the XRD pattern of graphene/pectin-CuNPs. The
Fig. 3. (A) XRD pattern and cyclic voltammogram (B) of graphene/pectin-CuNPs. Cyclic voltammogram is obtained in 0.1 M NaOH at the scan rate of 50 mV s 1.
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observation of three diffraction peaks at 2u angles of 43.28 , 50.36 and 73.88 can be manifested to the (111), (2 0 0) and (2 2 0) reflections of CuNPs, respectively. Electrochemical behavior of graphene/pectin-CuNPs has been investigated by cyclic voltammetry in 0.1 M NaOH which exhibited typical voltammogram of CuNPs in alkaline medium (Fig. 3B) [18,21]. The anodic and cathodic scans have shown two obvious oxidation and reduction peaks, respectively. The anodic peaks I and II are assigned to the formation of Cu2O and CuO + Cu(OH)2. The cathodic peak III was assigned to the reduction of CuO or Cu(OH)2 to Cu2O. Then, Cu2O is reduced to metallic Cu as represented by peak IV. Thus, the CV studies revealed the successful formation of CuNPs and also validated that the CuNPs exhibited their characteristic electrochemical behaviour.
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3.2. Electrocatalysis of glucose at GCE/graphene/pectin-CuNPs Fig. 4A displays the CVs of bare (a), pectin-CuNPs (b), graphene (c) and graphene/pectin-CuNPs (d) films modified electrodes recorded in 0.1 M NaOH containing 1 mM glucose. The scan rate was hold at 50 mV s 1. The electrocatalytic ability of these modified electrodes towards oxidation of glucose are in the following order: graphene/pectin-CuNPs > graphene > pectin-Cu NPs > bare GCE. The bare GCE does not exhibited electrocatalytic ability to the oxidation of glucose. The graphene/GCE and pectin-CuNPs/GCE were exhibited sigmoidal type curves which indicating sluggish electron transfers kinetic process of glucose at these electrodes. However, GCE/graphene/pectin-CuNPs exhibited maximum electrocatalytic ability to the oxidation of glucose which is evident form the observation of obvious anodic peak at the potential of 0.45 V. Highly enhanced anodic peak current (Ipa) and low overpotential observed for the oxidation of glucose at graphene/ pectin-CuNPs indicates the fast electron transfer kinetics and promising electrocatalytic ability of the modified electrode. Interestingly, the peak current observed at graphene and pectinCu NPs films were considerably lower that of graphene/pectinCuNPs. The outstanding electrocatalytic ability of the graphene/ pectin-CuNPs can be manifested to the great synergetic effect between large surface area and high electrical conductivity of graphene nanosheets and good electrocatalytic ability of CuNPs. 3.3. Determination of glucose
Fig. 4. (A) Cyclic voltammograms obtained at bare (a), pectin-CuNPs (b), graphene (c) and graphene/pectin-CuNPs (d) films modified GCEs in 0.1 M NaOH containing 1 mM glucose at the scan rate of 50 mV s 1. (B) Cyclic voltammograms obtained at GCE/graphene/pectin-CuNPs in the absence (a) and presence of glucose (curves b to f; each addition of 1 mM glucose) in 0.1 M NaOH. (C) Amperometric i-t response of graphene/pectin-CuNPs nanocomposite film modified rotating GCE upon addition of 10 mM, 100 mM and 500 mM glucose into 0.1 M NaOH at the rotation speed of 1500 RPM. Eapp = + 0.40 V. Inset: Plot of Ip vs [glucose].
Fig. 4B shows the CVs obtained at GCE/graphene/pectin-CuNPs in the absence (curve a) and presence of glucose (b = 1 mM, c = 2 mM, d = 3 mM, e = 4 mM and f = 5 mM) in 0.1 M NaOH. The Ipa corresponding the the oxidation of glucose increases linearly for the each addition of glucose from 1 mM to 5 mM. The linear concentration range is observed between 1 and 5 mM. In order to improve the sensitivity and detection limit, we choose amperometry for the determination of glucose. Fig. 4C displays the amperometric i-t response of graphene/pectin-CuNPs film modified rotating disc GCE upon sequential injection of different concentration of glucose (10, 100 and 500 mM) into continuously stirred 0.1 M NaOH at regular interval of 50s. The rotation speed of the electrode was 1500 RPM, while the applied potential (Eapp) was + 0.40 V. For every addition of glucose, quick and stable amperometric responses were observed. The amperometric response current reaches its 95% steady-state current within 5s indicating fast electrocatalytic oxidation of glucose at the graphene/pectin-CuNPs film modified electrode. A plot between concentration of glucose and response current exhibits linear relationship and linear range is found from 10 mM and 5.5 mM (Inset to Fig. 4C). The respective linear regression equation was expressed as Ip/mA = 0.0096 [glucose]/mA mM 1 + 0.8956; R2 = 0.99. Sensitivity and limit of detection (LOD) were calculated to be 0.0457 mAmM 1 cm 2 and 2.1 mM respectively. The LOD of the sensor was calculated by using the formula, LOD = 3 sb/S (where, sb = standard deviation of blank signal and S = sensitivity) [3]. Selectivity of the sensor to the determination of glucose has been investigated in the presence of common interfering agents. Fig. S2 displays the amperometric responses of the sensor to the detection of 100 mM of H2O2 (a) and each 1 mM of dopamine (b), ascorbic acid (c), uric acid (d), fructose (e) lactose (f) and galactose (g). Well defined response was observed for glucose; however, no notable responses were obtained for the interfering species tested. In addition, well defined response was observed for the addition of 100 mM H2O2 into the electrolyte solution coexisting with the aforementioned interferences. Therefore, the selectivity studies revealing the excellent selectivity of the sensor to the determination of glucose even in the presence of interfering species.The
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analytical performance of the graphene/pectin-CuNPs film modified electrode is quite comparable with the previous reports in terms of wide linear ranges, high sensitivity and low LOD (Table 1). The storage stability of the sensor is the crucial parameter to evaulated the perfromance of any sensor. In order to determine the storage stability of the graphene/pectin-CuNPs film modified electrode, its electrocatalytic response to the oxidation of glucose was monitored every day. During one month storage period, the sensor presented stable response. 94.23% of the initial Ipa was retained over one month of its continuous use, revealing good storage stability. Besides, operational stability of the modified electrode was investigated upon continuous rotation of graphene/ pectin-CuNPs film modified electrode at the rotation speed of 1500 rpm in 0.1 M NaOH. Stable amperomerometric response was observed for the addition of 100 mM glucose. Only 5.3% of the initial response current is decreased even after continously rotated for 3000 s revealing good operational stability of the sensor. Repeatability and reproducibility of the proposed sensor was evaluated in 0.1 M NaOH containing 10 mM glucose. The sensor exhibits appreciable repeatability with relative standard deviation (R.S.D) of 2.35% for 6 repeatitive measurements which were carried out using single graphene/pectin-CuNPs film modified electrode. Moreover, the sensor exhibits apprecible reproducibility of 2.82% for five independent measurments carried out in five different graphene/pectin-CuNPs film modified electrodes. In order to evaluate practicality of the fabricated sensor, 2 ml of the serum sample was diluted to 10 ml by the addition of 0.1 M NaOH. Then experiments were carried out with the diluted serum sample following similar experimental conditions used for the lab samples. The amount of glucose present in the serum sample was found to be 4.95 0.15 mM adopting standard addition method. The concentration of glucose present in the blood serum was predetermined to be 4.80 mM by the standard methods (photometric sensor, Roche Cobas c 111 analyzer). Accordingly, the concentration measured by our graphene/pectin-CuNPs based sensor was in good agreement with the standard method which validated the practical feasibility of our sensor to detect the glucose present in clinical samples. 3.4. Determination of H2O2 at graphene/pectin-CuNPs film modified electrode Fig. 5A displays the CVs obtained at unmodified GCE (a), pectinCuNPs (b), graphene (c) and graphene/pectin-CuNPs (d) films modified electrodes in 0.1 M NaOH containing H2O2. The unmodified GCE does not exhibit scarcely reduce H2O2. However, graphene/pectin-CuNPs and graphene films have shown little electrocatalytic behaviour with presence of reduction peak at the potential of –0.33 V and –0.28 V respectively. The graphene/pectinCuNPs film modified electrode exhibited highly enhanced cathodic peak at less overpotential of –0.24 V (onset potential at –0.15 V) which is assigned to the reduction of H2O2. From the CV results, we inferred that graphene/pectin-CuNPs film modified electrode displayed significantly improved electrocatalytic ability compared with other electrodes. The excellent synergy between graphene and CuNPs facilitated the electrocatalytic ability of the composite. Graphene act as support for CuNPs and also renders numerous
Fig. 5. (A) Cyclic voltammograms obtained at unmodified (a), pectin-CuNPs (b), graphene (c) and graphene/pectin-CuNPs (d) in 0.1 M NaOH containing 1 mM H2O2 at the scan rate of 50 mV s 1. (B) Cyclic voltammograms obtained at GCE/graphene/ pectin-CuNPs in the absence (a) and presence of each 1 mM H2O2 addition (b-f) in 0.1 M NaOH. (C) Amperometric i–t response of graphene/pectin-CuNPs nanocomposites film modified rotating GCE upon addition of various concentrations of H2O2 in to 0.1 M NaOH at the rotation speed of 1500 RPM. Eapp = – 0.20 V. Inset: Plot of Ip vs [H2O2].
edge planes like defects to provide additional catalytic sites to access H2O2. Fig. 5B shows the CVs obtained at GCE/graphene/pectin-CuNPs in the presence of H2O2 (b = 1 mM, c = 2 mM, d = 3 mM, e = 4 mM
Table 1 Comparison of analytical parameters for the determination of glucose at graphene/pectin-CuNPs film modified electrode with other films modified electrodes. Electrode
Linear range
Limit of detection
Sensitivity
Ref.
Colloidal gold-carbon paste electrode/GOx Chitosan–GOx–gold nanoparticles Nafion–GOx– singlewalled carbon nanohorns Graphene/pectin-CuNPs
0.04–0.28 5.0–2.4 0–6.0 10–5.5 mM
10 mM 2.7 6 mM 2.1 mM
8.4 mA mM 1 – 1.06 mA mM 1 0.0457 mA mM
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Table 2 Comparison of analytical parameters for the determination of H2O2 at graphene/pectin-CuNPs film modified electrode with other films modified electrodes. Electrode
Linear range
Limit of detection
Sensitivity
Reduced graphene oxide/ferroferric oxide poly(p-aminobenzene sulfonic acid) Carbon nanotube decorated with silver nanoparticles graphene/pectin-CuNPs
0.1–6 mM 50–550 mM 50 mM–9 mM 1 mM–1 mM
3.2 mM 10 mM 1.6 mM 0.35 mM
0.688 mA mM – – 0.391 mA mM
and f = 5 mM) in 0.1 M NaOH. The cathodic peak current corresponding the the reduction of H2O2 increases linearly for the each addition of H2O2. The linear concentration range is observed from 1–5 mM. Fig. 5C displays the amperometric response of graphene/pectin-CuNPs film modified rotating disc GCE upon sequential injection of H2O2 into continuously stirred 0.1 M NaOH. The rotation speed was hold at 1500 RPM and the Eapp was –0.20 V. For every addition of H2O2, quick amperometric responses were observed. A plot between concentration of H2O2 and response current exhibits linear relationship (Inset to Fig. 5C) and the respective linear regression equation was expressed as, Ip/ mA = 0.0821 [H2O2]/mA mM 1 + 1.521; R2 = 0.997. The working concentration range was from 1 mM and 1 mM. The sensitivity and LOD were calculated to be 0.391 mAmM 1 cm 2 and 0.35 mM respectively. The sensor performance is quite comparable with the previous reports (Table 2). Selectivity of the sensor to determine H2O2 was investigated in the presence of common interfering agents (Fig. S3). The electrode did not respond for usual intereference speies (dopamine, ascorbic acid, uric acid, acetaminophen, glucose and L-cysteine), even at high concentration; however quickly responded to H2O2. During one month storage period, the sensor presented stable response; only 6.83% of the initial current response was loosed over one month of its continuous use. The sensor also possessed good operational stability; only 6.2% of the initial response current is decreased even after continously rotated for 3000 s in 0.1 M NaOH containing 2 mM H2O2. The sensor exhibits repeatability (5 measurmentments) and reproducibility (5 different electrodes) with R.S.D of 2.52% and 2.92% respectively. Repeatability and reproducibility of the sensor was evaluated in 0.1 M NaOH containing 2 mM H2O2. Practical applicability of the sensor was demonstrated in contact lens cleaning solution containing 3% H2O2. The required dilutions were made using 0.1 M NaOH and amperometry experiments were performed. The sensor displayed well defined amperometric responses to each addition of H2O2 in the linear range of 50 mM–0.75 mM (Fig. S4). 4. Conclusions We described a simple electrochemical method for the preparation of pectin scaffold stabilized CuNPs at graphene nanosheets. The successful formation of the composite was confirmed by CV, SEM, UV-Visible spectroscopy and XRD methods. GCE/graphene/pectin-CuNPs exhibited excellent electrocatalytic ability to glucose and H2O2. The fabricated glucose sensor displayed excellent analytical parameters for the determination of glucose in terms of linear range (10 mM–5.5 mM), low detection limit (2.1 mM) and sensitivity (0.0457 mAmM 1 cm 2). The sensor exhibited wide linear range (1 mM–1 mM), low detection limit (0.35 mM) and high sensitivity (0.391 mAmM 1 cm 2) for the determination of H2O2. The sensor showed appreciable stability, repeatability, reproducibity and practicality for the determination of glucose and H2O2. The graphene/pectin-CuNPs hold great potential for the fabrication of electrochemical sensors attributed to its advantages such as, easy preparation protocols, good stability and excellent electrocatalytic activity.
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Acknowledgement This work was supported by the National Science Council and the Ministry of Education of Taiwan (Republic of China). Dr. Veerappan Mani greatfully acknowledge the National Science Council, Taiwan for the postdoctoral fellowship (NSC 103-2811-M027-002). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. electacta.2015.07.098. References [1] J. Wang, Electrochemical glucose biosensors, Chemical reviews 108 (2008) 814–825. [2] J. Wang, Glucose biosensors: 40 years of advances and challenges, Electroanalysis 13 (2001) 983. [3] V. Mani, B. Devadas, S.-M. Chen, Direct electrochemistry of glucose oxidase at electrochemically reduced graphene oxide-multiwalled carbon nanotubes hybrid material modified electrode for glucose biosensor, Biosensors and Bioelectronics 41 (2013) 309–315. [4] B. Unnikrishnan, S. Palanisamy, S.-M. Chen, A simple electrochemical approach to fabricate a glucose biosensor based on graphene–glucose oxidase biocomposite, Biosensors and Bioelectronics 39 (2013) 70–75. [5] S. Palanisamy, S. Cheemalapati, S.-M. Chen, Amperometric glucose biosensor based on glucose oxidase dispersed in multiwalled carbon nanotubes/ graphene oxide hybrid biocomposite, Materials Science and Engineering: C 34 (2014) 207–213. [6] Y. Yan, W. Zheng, L. Su, L. Mao, Carbon-Nanotube-Based Glucose/O2 Biofuel Cells, Advanced Materials 18 (2006) 2639–2643. [7] V. Mani, R. Devasenathipathy, S.-M. Chen, S.-T. Huang, V. Vasantha, Immobilization of glucose oxidase on graphene and cobalt phthalocyanine composite and its application for the determination of glucose, Enzyme and microbial technology 66 (2014) 60–66. [8] K. Gong, Vertically-aligned Prussian blue/carbon nanotube nanocomposites on a carbon microfiber as a biosensing scaffold for ultrasensitively detecting glucose, Journal of colloid and interface science 410 (2013) 152–157. [9] H.-X. Wu, W.-M. Cao, Y. Li, G. Liu, Y. Wen, H.-F. Yang, S.-P. Yang, In situ growth of copper nanoparticles on multiwalled carbon nanotubes and their application as non-enzymatic glucose sensor materials, Electrochimica Acta 55 (2010) 3734–3740. [10] A.A. Ensafi, M.M. Abarghoui, B. Rezaei, A new non-enzymatic glucose sensor based on copper/porous silicon nanocomposite, Electrochimica Acta 123 (2014) 219–226. [11] J. Wang, D.F. Thomas, A. Chen, Nonenzymatic electrochemical glucose sensor based on nanoporous PtPb networks, Analytical chemistry 80 (2008) 997–1004. [12] L.-C. Jiang, W.-D. Zhang, A highly sensitive nonenzymatic glucose sensor based on CuO nanoparticles-modified carbon nanotube electrode, Biosensors and Bioelectronics 25 (2010) 1402–1407. [13] D. Zhao, C. Xu, A nanoporous palladium-nickel alloy with high sensing performance towards hydrogen peroxide and glucose, Journal of colloid and interface science 447 (2015) 50–57. [14] X. Ren, D. Chen, X. Meng, F. Tang, X. Hou, D. Han, L. Zhang, Zinc oxide nanoparticles/glucose oxidase photoelectrochemical system for the fabrication of biosensor, Journal of colloid and interface science 334 (2009) 183–187. [15] A.A. Ensafi, M. Jafari-Asl, N. Dorostkar, M. Ghiaci, M.V. Martínez-Huerta, J. Fierro, The fabrication and characterization of Cu-nanoparticle immobilization on a hybrid chitosan derivative-carbon support as a novel electrochemical sensor: application for the sensitive enzymeless oxidation of glucose and reduction of hydrogen peroxide, Journal of Materials Chemistry B 2 (2014) 706–717. [16] J. Luo, S. Jiang, H. Zhang, J. Jiang, X. Liu, A novel non-enzymatic glucose sensor based on Cu nanoparticle modified graphene sheets electrode, Analytica chimica acta 709 (2012) 47–53.
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Electrodeposition of copper nanoparticles using pectin scaffold at graphene nanosheets for electrochemical sensing of glucose and hydrogen peroxide Veerappan Mania,** , Rajkumar Devasenathipathya , Shen-Ming Chena,* , Sea-Fue Wangb , Parvathy Devic , Yian Taic a Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No. 1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan, ROC b Department of Materials and Mineral Resources Engineering, National Taipei University of Technology, No. 1, Sec. 3, Chung-Hsiao E. Road, Taipei, Taiwan, ROC c Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan, ROC
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 April 2015 Received in revised form 17 July 2015 Accepted 18 July 2015 Available online 26 July 2015
A simple electrodeposition approach has been described for the preparation of copper nanoparticles (CuNPs) using biopolymer pectin as a scaffold and graphene as a support. The formation of graphene/ pectin-CuNPs was confirmed by scanning electron microscopy, UV-Visible spectroscopy and X-ray diffraction studies. The graphene/pectin-CuNPs film modified electrode was prepared and its electrocatalytic applications to the oxidation of glucose and reduction of H2O2 have been explored. An amperometric glucose sensor was fabricated which exhibited excellent sensor performance in terms of wide linear range (10 mM–5.5 mM), low detection limit (2.1 mM) and high sensitivity (0.0457 mAmM 1 cm 2). Likewise, an amperometric sensor has been fabricated for the determination of H2O2 which displayed linear range of 1 mM–1 mM, detection limit of 0.35 mM and sensitivity of 0.391 mAmM 1 cm 2. The sensor displayed appreciable repeatability, reproducibity and stability. Furthermore, practical feasibility of the sensor has been demonstrated in human serum and contact lens cleaning solution to determine glucose and H2O2, respectively. The main advantages of sensor include simple and green fabrication approach, roughed and stable electrode matrix, high sensitivity and stability, fast in sensing and highly reproducible. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: copper nanoparticles Electrodeposition glucose hydrogen peroxide electrochemical sensor
1. Introduction The development of sensitive glucose biosensors for the accurate and reliable determination of blood glucose level is of great significance to control diabetics [1–3]. Numerous enzymatic glucose biosensors have been reported based on glucose oxidase (GOx) immobilized at various modified electrodes [3–8]. However, enzyme electrodes often suffer from serious drawbacks, such as, instability of immobilized enzyme, lack of long term stability, enzyme leaching and poor electrical communication to the highly buried active sites of enzyme [1]. Also, activity of the GOx has been affected by pH, temperature and immobilization methods [9]. Nonenzymatic glucose sensors are attractive alternative approach
* Corresponding author. Tel.: +886 2270 17147; fax: +886 2270 25238. ** Corresponding author. Tel.: +886 2271 2171 2525; fax: +886 02 2731 7117. E-mail address: [email protected] (S.-M. Chen). http://dx.doi.org/10.1016/j.electacta.2015.07.098 0013-4686/ ã 2015 Elsevier Ltd. All rights reserved.
for the detection of glucose to avoid enzyme related issues [10] and therefore, many efforts have been focused on the fabrication of non-enzymatic glucose sensor using different electrode modifiers [11–13]. Electrodes modified with nanomaterials are widely used for the construction of glucose sensors attributed to their extraordinary physiochemical properties such as, large surfaceto-volume ratio, high conductivity, and excellent electrocatalytic ability [12,14]. In recent times, numerous copper nanoparticles (CuNPs) based electrodes are reported for the determination of glucose [9,10,15–17]. However, most of the methods reported in the literature are based on chemical methods which require the use of reduction agents [18], thermal condition [19], microemulsions [20], and micelles [19] etc., These methods also involved with tedious procedures and the as-prepared CuNPs has the possibility of easily oxidized to copper oxide during the course of preparation in the aforementioned methods [21]. On the other hand, green agents such as biopolymers stabilized preparation methods have the ability to overcome the difficulties encountered by the
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chemical synthesis methods. Therefore, herein, we established a simple green method to synthesis biopolymer stabilized CuNPs using pectin as a scaffold. The method involves simple and efficient electrodeposition methods without using any reducing agent and complex methods. Pectin (polygalacturonic acid) is a naturally occurring polysaccharide occurring at the cell walls of the plants and highly biodegradable [22,23]. The use of carbon based materials such as carbon nanotubes and graphene as supports is well established approach to improve the stability and conductivity of the nanoparticles, which also aid to overcome surface fouling issues [24,25]. Therefore, we deposited pectin stabilized CuNPs at graphene, where graphene prepared on the electrode surface through electrochemical reduction method. The as-prepared graphene/pectin-CuNPs has been well characterized by analytical, spectral and electrochemical methods. The graphene/pectinCuNPs film exhibited excellent electrocatalytic ability to the oxidation of glucose and displayed excellent sensor performance with the reported glucose sensors in terms of wide linear range, high sensitivity and selectivity, fast response time, high stability and appreciable practicality. Additionally, the graphene/pectinCuNPs film also exhibited excellent electrocatalytic ability to the reduction of hydrogen peroxide (H2O2) and therefore we also developed a nonenzymatic H2O2 sensor. H2O2 is a vital constituent of plant tissues, which regulates the plant metabolism, acclamatory processes and gene expression [26,27]. In addition, it possesses good antibacterial and antiseptic properties and extensively used in industries as an oxidizing agent, antibacterial agent and bleaching agent [28,29]. Therefore, an accurate and reliable method for the determination of H2O2 is highly important for clinical and industrial analysis [15,30–32]. The main aim of this method is to develop a biopolymer assisted electrochemical synthetical method for the preparation of CuNPs and for the development of glucose and H2O2 sensor. The described method has advantages over previous methods to prepare high stable, uniform and electrochemically active CuNPs. The preparation is fast, green, simple electrode fabrication procedure and highly reproducible.
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2. Experimental 2.1. Reagents and apparatus Graphite (powder, <20 mm), LM-pectin (DE 35%, pectin (from citrus peel), copper nitrate (purum, 98%, 26% Cu basis), glucose and H2O2 were purchased from Sigma-Aldrich and used as received. The supporting electrolyte used for all the electrochemical studies was 0.1 M phosphate buffer solution, prepared using NaH2PO4 and Na2HPO4, while the pH were adjusted to get desired pH using either using H2SO4 or NaOH. Prior to each experiment, all the solutions were deoxygenated with pre-purified nitrogen gas for 15 min unless otherwise specified. Blood sample used in this study were collected from healthy man (26-year-old male). The blood sample was taken from cubital vein and transferred to a test tube and stored at 20 C before analysis. The collected blood sample was allowed to clot; subsequently the clot was removed through centrifugation for 20 min at the speed of 2000 g. The serum separated as a supernatant was collected and stored at the temperature of –20 C. Contact lens cleaning solution containing 3% H2O2 was purchased from a local drug store in Taipei, Taiwan to demonstrate practicality osf the sensor. The electrochemical measurements were carried out using CHI 611A electrochemical work station. Electrochemical studies were performed in a conventional three electrode cell using BAS glassy carbon electrode (GCE) as a working electrode (area 0.071 cm2), Ag| AgCl (saturated KCl) as a reference electrode and Pt wire as a counter electrode. Amperometric measurements were performed with analytical rotator AFMSRX (PINE instruments, USA) with a rotating disc electrode (RDE) having working area of 0.24 cm2. Scanning electron microscope (SEM) studies were performed using Hitachi S-3000H scanning electron microscope. Ultra violet visible (UV-Vis) spectroscopy studies were performed by U-3300 spectrophotometer. X-ray diffraction (XRD) and Attenuated total reflectance-FT-IR (ATR–FTIR) spectroscopy studies were carried out using XPERT-PRO (PANalytical B.V., The Netherlands) diffractometer (Cu Ka radiation, k = 1.54 Å) and Perkin-Elmer IR spectrometer respectively.
Fig. 1. SEM images of pectin-CuNPs at the potential of –1.20 V (A), – 0.80 V (B) and – 0.60 V (C). SEM image of graphene/pectin-CuNPs.
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2.2. Electrochemical preparation of GCE/graphene/pectin-CuNPs Graphite oxide was prepared from graphite by Hummers method [33] and exfoliated to graphene oxide (GO) through ultrasonic agitation for 1 h and subsequent centrifugation at 3000 RPM for 30 min. Prior to electrode modification, both GCE and rotating disk GCE were polished with 0.05 mm alumina slurry using a Buehler polishing kit and subsequently washed with water and allowed to air-dried. Aliquot (5 mL) of GO dispersion was dropped onto the pre-cleaned GCE surface and dried in over for 30 min. After the electrodes were air-dried for 30 min at ambient temperature, the GO film modified GCE was transferred to electrochemical cell containing nitrogen saturated phosphate buffer (pH 5). 30 consecutive cyclic voltammograms were applied at the potential range between 0 to –1.5 V (Fig. S1A). The scan rate was hold at 50 mV s 1. During electrochemical cycling, GO has been reduced to graphene on the electrode surface and the resulting electrode was named as graphene/GCE. 20 mg mL 1 of Cu(NO3)2 solution prepared in 0.1 M H2SO4 containing 1 mg mL 1 of pectin. The graphene/GCE has been transferred to an electrochemical cell containing the mixture of Cu(NO3)2 and pectin. 10 consecutive cyclic voltammograms were carried out at the scan rate of 50 mV s 1 (Fig. S1B). Different potential windows (0.50 to –1.20 V, 0.5 to –0.80 V and 0.50 to 0.60 V (vs. Ag|AgCl) respectively) have been applied to optimize the formation of stable CuNPs. After completion of electrodeposition process, the electrode was carefully washed with water and dried for 30 min. The final nanocomposite is denoted as graphene/pectin-CuNPs. As a control, graphene/GCE and pectin-CuNPs/GCE also prepared. 3. Results and Discussion 3.1. Characterization of graphene/pectin-CuNPs The SEM images of pectin-CuNPs prepared at different potentials are given as Fig. 1. As can be seen from the SEM images, the CuNPs prepared at the potential ranges of 0.50 to –1.20 V (Fig. 1A) and 0.50 to –0.80 V (Fig. 1B) are showed aggregated formation of CuNPs. However, the CuNPs prepared at the potential range of 0.50 V to –0.60 V (Fig. 1C) has shown uniformly distributed individual nanoparticles with particles size ranges in nanometers. Therefore, we choose this optimized potential window for the preparation of CuNPs at the graphene nanosheets. The SEM image of graphene/pectin-CuNPs is depicts the uniform decoration of CuNPs at the graphene nanosheets (Fig. 1D). The observations of thin sheets like morphology evident the presents of graphene nanosheets. 200 successive cyclic voltammograms (CVs) were recorded at graphene/pectin-CuNPs/GCE in 0.1 M NaOH (Figure not shown) in order to evaluate stability of the CuNPs. Only 7.1% of the initial peak currents were decreased even after cycling for 200 cycles which revealing the high stability of the graphene/ pectin-CuNPs. The UV-Visible spectrum of pectin (Curve a, Fig. 2A) exhibited a sharp absorption peak at 290 nm which arose due to the free carboxyl group of pectin. However, this absorption peak is disappeared in the spectrum of graphene/pectin-CuNPs (Curve b, Fig. 2A). Perhaps, the carboxyl groups of pectin might commit to accommodate CuNPs which makes the disappearance of its absorption peak in the spectrum. Additionally, a new absorption peak has been observed at the wavelength of 570 nm which was assigned to the CuNPs. Fig. 2B displays the ATR-FTIR spectra of pectin (curve a) and graphene/pectin-CuNPs (curve b). The peaks observed at 3600 cm 1 and 2800 cm 1 were assigned to the hydroxyl and carboxylic hydroxyl groups respectively. The peak observed at 1026 cm 1 is assigned to the glycosidic bonds linking two galacturonic sugar units, while the peak obtained at 1608 cm 1
Fig. 2. UV-Visible (A) and AT–FTIR (B) spectra of pectin (a) and graphene/pectinCuNPs (b)
is assigned to the carbonyl group of the esterified pendant of pectin [22,34]. However, all these peaks were disappeared in the ATR-FTIR spectrum of graphene/pectin-CuNPs which clearly indicating that these functional groups are devoted to accommodate CuNPs. Fig. 3A displays the XRD pattern of graphene/pectin-CuNPs. The
Fig. 3. (A) XRD pattern and cyclic voltammogram (B) of graphene/pectin-CuNPs. Cyclic voltammogram is obtained in 0.1 M NaOH at the scan rate of 50 mV s 1.
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observation of three diffraction peaks at 2u angles of 43.28 , 50.36 and 73.88 can be manifested to the (111), (2 0 0) and (2 2 0) reflections of CuNPs, respectively. Electrochemical behavior of graphene/pectin-CuNPs has been investigated by cyclic voltammetry in 0.1 M NaOH which exhibited typical voltammogram of CuNPs in alkaline medium (Fig. 3B) [18,21]. The anodic and cathodic scans have shown two obvious oxidation and reduction peaks, respectively. The anodic peaks I and II are assigned to the formation of Cu2O and CuO + Cu(OH)2. The cathodic peak III was assigned to the reduction of CuO or Cu(OH)2 to Cu2O. Then, Cu2O is reduced to metallic Cu as represented by peak IV. Thus, the CV studies revealed the successful formation of CuNPs and also validated that the CuNPs exhibited their characteristic electrochemical behaviour.
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3.2. Electrocatalysis of glucose at GCE/graphene/pectin-CuNPs Fig. 4A displays the CVs of bare (a), pectin-CuNPs (b), graphene (c) and graphene/pectin-CuNPs (d) films modified electrodes recorded in 0.1 M NaOH containing 1 mM glucose. The scan rate was hold at 50 mV s 1. The electrocatalytic ability of these modified electrodes towards oxidation of glucose are in the following order: graphene/pectin-CuNPs > graphene > pectin-Cu NPs > bare GCE. The bare GCE does not exhibited electrocatalytic ability to the oxidation of glucose. The graphene/GCE and pectin-CuNPs/GCE were exhibited sigmoidal type curves which indicating sluggish electron transfers kinetic process of glucose at these electrodes. However, GCE/graphene/pectin-CuNPs exhibited maximum electrocatalytic ability to the oxidation of glucose which is evident form the observation of obvious anodic peak at the potential of 0.45 V. Highly enhanced anodic peak current (Ipa) and low overpotential observed for the oxidation of glucose at graphene/ pectin-CuNPs indicates the fast electron transfer kinetics and promising electrocatalytic ability of the modified electrode. Interestingly, the peak current observed at graphene and pectinCu NPs films were considerably lower that of graphene/pectinCuNPs. The outstanding electrocatalytic ability of the graphene/ pectin-CuNPs can be manifested to the great synergetic effect between large surface area and high electrical conductivity of graphene nanosheets and good electrocatalytic ability of CuNPs. 3.3. Determination of glucose
Fig. 4. (A) Cyclic voltammograms obtained at bare (a), pectin-CuNPs (b), graphene (c) and graphene/pectin-CuNPs (d) films modified GCEs in 0.1 M NaOH containing 1 mM glucose at the scan rate of 50 mV s 1. (B) Cyclic voltammograms obtained at GCE/graphene/pectin-CuNPs in the absence (a) and presence of glucose (curves b to f; each addition of 1 mM glucose) in 0.1 M NaOH. (C) Amperometric i-t response of graphene/pectin-CuNPs nanocomposite film modified rotating GCE upon addition of 10 mM, 100 mM and 500 mM glucose into 0.1 M NaOH at the rotation speed of 1500 RPM. Eapp = + 0.40 V. Inset: Plot of Ip vs [glucose].
Fig. 4B shows the CVs obtained at GCE/graphene/pectin-CuNPs in the absence (curve a) and presence of glucose (b = 1 mM, c = 2 mM, d = 3 mM, e = 4 mM and f = 5 mM) in 0.1 M NaOH. The Ipa corresponding the the oxidation of glucose increases linearly for the each addition of glucose from 1 mM to 5 mM. The linear concentration range is observed between 1 and 5 mM. In order to improve the sensitivity and detection limit, we choose amperometry for the determination of glucose. Fig. 4C displays the amperometric i-t response of graphene/pectin-CuNPs film modified rotating disc GCE upon sequential injection of different concentration of glucose (10, 100 and 500 mM) into continuously stirred 0.1 M NaOH at regular interval of 50s. The rotation speed of the electrode was 1500 RPM, while the applied potential (Eapp) was + 0.40 V. For every addition of glucose, quick and stable amperometric responses were observed. The amperometric response current reaches its 95% steady-state current within 5s indicating fast electrocatalytic oxidation of glucose at the graphene/pectin-CuNPs film modified electrode. A plot between concentration of glucose and response current exhibits linear relationship and linear range is found from 10 mM and 5.5 mM (Inset to Fig. 4C). The respective linear regression equation was expressed as Ip/mA = 0.0096 [glucose]/mA mM 1 + 0.8956; R2 = 0.99. Sensitivity and limit of detection (LOD) were calculated to be 0.0457 mAmM 1 cm 2 and 2.1 mM respectively. The LOD of the sensor was calculated by using the formula, LOD = 3 sb/S (where, sb = standard deviation of blank signal and S = sensitivity) [3]. Selectivity of the sensor to the determination of glucose has been investigated in the presence of common interfering agents. Fig. S2 displays the amperometric responses of the sensor to the detection of 100 mM of H2O2 (a) and each 1 mM of dopamine (b), ascorbic acid (c), uric acid (d), fructose (e) lactose (f) and galactose (g). Well defined response was observed for glucose; however, no notable responses were obtained for the interfering species tested. In addition, well defined response was observed for the addition of 100 mM H2O2 into the electrolyte solution coexisting with the aforementioned interferences. Therefore, the selectivity studies revealing the excellent selectivity of the sensor to the determination of glucose even in the presence of interfering species.The
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analytical performance of the graphene/pectin-CuNPs film modified electrode is quite comparable with the previous reports in terms of wide linear ranges, high sensitivity and low LOD (Table 1). The storage stability of the sensor is the crucial parameter to evaulated the perfromance of any sensor. In order to determine the storage stability of the graphene/pectin-CuNPs film modified electrode, its electrocatalytic response to the oxidation of glucose was monitored every day. During one month storage period, the sensor presented stable response. 94.23% of the initial Ipa was retained over one month of its continuous use, revealing good storage stability. Besides, operational stability of the modified electrode was investigated upon continuous rotation of graphene/ pectin-CuNPs film modified electrode at the rotation speed of 1500 rpm in 0.1 M NaOH. Stable amperomerometric response was observed for the addition of 100 mM glucose. Only 5.3% of the initial response current is decreased even after continously rotated for 3000 s revealing good operational stability of the sensor. Repeatability and reproducibility of the proposed sensor was evaluated in 0.1 M NaOH containing 10 mM glucose. The sensor exhibits appreciable repeatability with relative standard deviation (R.S.D) of 2.35% for 6 repeatitive measurements which were carried out using single graphene/pectin-CuNPs film modified electrode. Moreover, the sensor exhibits apprecible reproducibility of 2.82% for five independent measurments carried out in five different graphene/pectin-CuNPs film modified electrodes. In order to evaluate practicality of the fabricated sensor, 2 ml of the serum sample was diluted to 10 ml by the addition of 0.1 M NaOH. Then experiments were carried out with the diluted serum sample following similar experimental conditions used for the lab samples. The amount of glucose present in the serum sample was found to be 4.95 0.15 mM adopting standard addition method. The concentration of glucose present in the blood serum was predetermined to be 4.80 mM by the standard methods (photometric sensor, Roche Cobas c 111 analyzer). Accordingly, the concentration measured by our graphene/pectin-CuNPs based sensor was in good agreement with the standard method which validated the practical feasibility of our sensor to detect the glucose present in clinical samples. 3.4. Determination of H2O2 at graphene/pectin-CuNPs film modified electrode Fig. 5A displays the CVs obtained at unmodified GCE (a), pectinCuNPs (b), graphene (c) and graphene/pectin-CuNPs (d) films modified electrodes in 0.1 M NaOH containing H2O2. The unmodified GCE does not exhibit scarcely reduce H2O2. However, graphene/pectin-CuNPs and graphene films have shown little electrocatalytic behaviour with presence of reduction peak at the potential of –0.33 V and –0.28 V respectively. The graphene/pectinCuNPs film modified electrode exhibited highly enhanced cathodic peak at less overpotential of –0.24 V (onset potential at –0.15 V) which is assigned to the reduction of H2O2. From the CV results, we inferred that graphene/pectin-CuNPs film modified electrode displayed significantly improved electrocatalytic ability compared with other electrodes. The excellent synergy between graphene and CuNPs facilitated the electrocatalytic ability of the composite. Graphene act as support for CuNPs and also renders numerous
Fig. 5. (A) Cyclic voltammograms obtained at unmodified (a), pectin-CuNPs (b), graphene (c) and graphene/pectin-CuNPs (d) in 0.1 M NaOH containing 1 mM H2O2 at the scan rate of 50 mV s 1. (B) Cyclic voltammograms obtained at GCE/graphene/ pectin-CuNPs in the absence (a) and presence of each 1 mM H2O2 addition (b-f) in 0.1 M NaOH. (C) Amperometric i–t response of graphene/pectin-CuNPs nanocomposites film modified rotating GCE upon addition of various concentrations of H2O2 in to 0.1 M NaOH at the rotation speed of 1500 RPM. Eapp = – 0.20 V. Inset: Plot of Ip vs [H2O2].
edge planes like defects to provide additional catalytic sites to access H2O2. Fig. 5B shows the CVs obtained at GCE/graphene/pectin-CuNPs in the presence of H2O2 (b = 1 mM, c = 2 mM, d = 3 mM, e = 4 mM
Table 1 Comparison of analytical parameters for the determination of glucose at graphene/pectin-CuNPs film modified electrode with other films modified electrodes. Electrode
Linear range
Limit of detection
Sensitivity
Ref.
Colloidal gold-carbon paste electrode/GOx Chitosan–GOx–gold nanoparticles Nafion–GOx– singlewalled carbon nanohorns Graphene/pectin-CuNPs
0.04–0.28 5.0–2.4 0–6.0 10–5.5 mM
10 mM 2.7 6 mM 2.1 mM
8.4 mA mM 1 – 1.06 mA mM 1 0.0457 mA mM
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Table 2 Comparison of analytical parameters for the determination of H2O2 at graphene/pectin-CuNPs film modified electrode with other films modified electrodes. Electrode
Linear range
Limit of detection
Sensitivity
Reduced graphene oxide/ferroferric oxide poly(p-aminobenzene sulfonic acid) Carbon nanotube decorated with silver nanoparticles graphene/pectin-CuNPs
0.1–6 mM 50–550 mM 50 mM–9 mM 1 mM–1 mM
3.2 mM 10 mM 1.6 mM 0.35 mM
0.688 mA mM – – 0.391 mA mM
and f = 5 mM) in 0.1 M NaOH. The cathodic peak current corresponding the the reduction of H2O2 increases linearly for the each addition of H2O2. The linear concentration range is observed from 1–5 mM. Fig. 5C displays the amperometric response of graphene/pectin-CuNPs film modified rotating disc GCE upon sequential injection of H2O2 into continuously stirred 0.1 M NaOH. The rotation speed was hold at 1500 RPM and the Eapp was –0.20 V. For every addition of H2O2, quick amperometric responses were observed. A plot between concentration of H2O2 and response current exhibits linear relationship (Inset to Fig. 5C) and the respective linear regression equation was expressed as, Ip/ mA = 0.0821 [H2O2]/mA mM 1 + 1.521; R2 = 0.997. The working concentration range was from 1 mM and 1 mM. The sensitivity and LOD were calculated to be 0.391 mAmM 1 cm 2 and 0.35 mM respectively. The sensor performance is quite comparable with the previous reports (Table 2). Selectivity of the sensor to determine H2O2 was investigated in the presence of common interfering agents (Fig. S3). The electrode did not respond for usual intereference speies (dopamine, ascorbic acid, uric acid, acetaminophen, glucose and L-cysteine), even at high concentration; however quickly responded to H2O2. During one month storage period, the sensor presented stable response; only 6.83% of the initial current response was loosed over one month of its continuous use. The sensor also possessed good operational stability; only 6.2% of the initial response current is decreased even after continously rotated for 3000 s in 0.1 M NaOH containing 2 mM H2O2. The sensor exhibits repeatability (5 measurmentments) and reproducibility (5 different electrodes) with R.S.D of 2.52% and 2.92% respectively. Repeatability and reproducibility of the sensor was evaluated in 0.1 M NaOH containing 2 mM H2O2. Practical applicability of the sensor was demonstrated in contact lens cleaning solution containing 3% H2O2. The required dilutions were made using 0.1 M NaOH and amperometry experiments were performed. The sensor displayed well defined amperometric responses to each addition of H2O2 in the linear range of 50 mM–0.75 mM (Fig. S4). 4. Conclusions We described a simple electrochemical method for the preparation of pectin scaffold stabilized CuNPs at graphene nanosheets. The successful formation of the composite was confirmed by CV, SEM, UV-Visible spectroscopy and XRD methods. GCE/graphene/pectin-CuNPs exhibited excellent electrocatalytic ability to glucose and H2O2. The fabricated glucose sensor displayed excellent analytical parameters for the determination of glucose in terms of linear range (10 mM–5.5 mM), low detection limit (2.1 mM) and sensitivity (0.0457 mAmM 1 cm 2). The sensor exhibited wide linear range (1 mM–1 mM), low detection limit (0.35 mM) and high sensitivity (0.391 mAmM 1 cm 2) for the determination of H2O2. The sensor showed appreciable stability, repeatability, reproducibity and practicality for the determination of glucose and H2O2. The graphene/pectin-CuNPs hold great potential for the fabrication of electrochemical sensors attributed to its advantages such as, easy preparation protocols, good stability and excellent electrocatalytic activity.
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