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Green synthesis of reduced graphene oxide decorated with gold nanoparticles and its glucose sensing application
Accepted Manuscript Title: Green synthesis of reduced graphene oxide decorated with gold nanoparticles and its glucose sensing application Author: Mahmoud Amouzadeh Tabrizi Javad Nadali Varkani PII: DOI: Reference:
S0925-4005(14)00638-8 http://dx.doi.org/doi:10.1016/j.snb.2014.05.099 SNB 16973
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
15-4-2014 23-5-2014 23-5-2014
Please cite this article as: M.A. Tabrizi, J.N. Varkani, Green synthesis of reduced graphene oxide decorated with gold nanoparticles and its glucose sensing application, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.05.099 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.
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Mahmoud Amouzadeh Tabrizia*, Javad Nadali Varkanib
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Department of Chemistry, Razi University, Kermanshah, Iran
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Department of Physics, Mazandaran University, Mazandaran, Iran
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* Corresponding author. Tel/fax: (+98) 21-44042955, Email:[email protected]
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Green synthesis of reduced graphene oxide decorated with gold nanoparticles and its glucose sensing application
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We report green synthesis of rGO-Aunano composite by rose water as
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reducing agent.
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UV-vis , XRD spectra, SEM and AFM images indicate preparation of rGO-
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Aunano composite.
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A direct electron transfer reaction of glucose oxidase was observed on rGO-
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Aunano /GC electrode.
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This composite film was also successfully applied in preparation of glucose
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biosensor.
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Abstract
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Here, we report an eco-friendly method for synthesis of reduced graphene oxide decorated with
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gold nanoparticles (rGO-Aunano) by using rose water as reducing agent. The prepared materials
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were characterized using UV-visible absorption spectroscopy, Raman spectroscopy, atomic force
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microscopy (AFM), scanning electron microscopy (SEM) and X-ray diffraction (XRD).
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Furthermore, the direct electrochemistry of glucose oxidase (GOD) was achieved at a glassy
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carbon electrode modified with rGO-Aunano. The resulting biosensor exhibited good response to
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glucose with linear range from 1 to 8 mM with a low detection limit of 10µM.
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Keywords: Green chemistry; Rose water; Reduced graphene oxide decorated with gold
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nanoparticles; Glucose oxidase; Biosensor
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1. Introduction
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Diabetes is a major public health problem in the worldwide which classed as a metabolism
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disorder. A new report has established that there are now nearly 350 million people on earth who
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suffer from diabetes [1]. Therefore, the determination and controlling of blood glucose is very
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important, which if not controlled, it can cause retinopathy, nephropathy, neuropathy,
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hypertension, heart disease, stroke, gastroparesis, peripheral arterial disease, cellulitis and
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depression [2]. Up to now, several analytical methods, such as UV fluorescence [3, 4]
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chemiluminescence [5] and titrimetry [6] have been proposed for the determination of glucose.
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However, these methods are generally time-consuming, lack of sensitivity, susceptibility to
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interference by other substances in analyte samples and difficult for an automated detection. To
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overcome all these shortcomings, the electrochemical biosensor based on the direct
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electrochemistry between an electrode and the immobilized glucose oxidase is especially
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promising because of its simplicity, high sensitivity and selectivity [7, 8]. Recently, several
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materials for the fabrication of glucose biosensor based on direct electron transfer have been
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reported in the literature [7, 9-12]. Among them, the carbon based nanomaterials such as
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graphene nano sheet provides not only a large microscopic surface area Immobilization of
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enzymes, but also it provide a desirable biocompatible microenvironment [13-17]. Graphene, a
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single aromatic sheet of sp2 bonded carbon, has been shown to possess unique electronic, optical,
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thermal, mechanical and catalytic properties which are attractive for widely varied potential
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applications in many fields of science ranging from nanoelectronics to biomedical devices [18,
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19]. This nanosheet can be synthesized by various methods [20]. Among them, the chemical
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reduction of exfoliated GO with green reducing agents have become a favorable topic for
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researchers. In recent years, increasing numbers of researches in field of nanoscience have been
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also devoted to green synthesis of reduced graphene oxide (rGO) decorated with novel metal
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nanoparticles such as rGO/Pt [21], rGO/Ag [22] and rGO/Au [23], opening the door to
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application in nanobiotechnology. The decoration of graphene with nanoparticles might be led to
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new composite with different properties via cooperative interaction. In this paper, we report a
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green method for synthesis of reduced graphene oxide decorated with gold nanoparticles (rGO-
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Aunano) by using rose water as reducing agent. Also, we show that the prepared modified
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electrode with rGO-Aunano nanocomposite would enhance the direct electron transfer capability
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of glucose oxidase (GOD) on the electrode surfaces. The prepared biosensor exhibited high
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sensitivity, anti-fouling properties, lower detection limit and high stability for glucose sensing.
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2.1. Materials
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All chemicals were of analytical reagent grade and used without further purification. Double
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distilled water was used throughout. GOD from Aspergillus niger, 221U mg-1) and D-(+)-
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glucose (97%) were obtained from Sigma (St. Louis, MO, USA). 10 mg of GOD was dissolved
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in 1mL of phosphate buffer solution (0.05 M, pH=7.0) to prepare GOD working solution. A
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stock solution of glucose (0.5 M) was prepared with doubly distilled water and stored at 4 °C
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when not in use. The glucose stock solution was allowed to mutarotate at room temperature for
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at least 24 h before use. Rose water (12 mg natural essence per 100 mL (so called 12%), Rabee
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Golab, Iran) was obtained from a local market.
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2.2 Apparatus
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UV-visible absorption spectra were recorded using a single beam Pharmacia UV-vis
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spectrophotometer (Ultrospec, model 4000). Raman scattering was performed on a Almega
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Thermo Nicolet Dispersive Raman spectrometer using the second harmonic (532 nm) of a
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Nd:YLF laser source. Scanning electron microscopy (SEM) was performed with a Philips
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instrument, Model XL-30. Atomic force microscopy (AFM) was conducted with a DME
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DualScope Scanner DS 95-200. X-ray diffraction (XRD) was performed with a D8ADVANCE
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(Bruker) X-ray diffractometer equipped with a Cu Kα (1.5406 Å) radiation source. Cyclic
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voltammetry (CV) was performed using an Autolab potentiostat-galvanostat model PGSTAT30
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with a conventional three-electrode setup, in which a bare glassy carbon (GC) electrode or
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modified GC electrode, an Ag|AgCl|KClsat and a platinum rod served as the working, reference
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and auxiliary electrodes, respectively. Electrochemical impedance spectroscopy (EIS)
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experiments were carried out using a Zahner Zennium workstation in the presence of 5.0 mM
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Fe(CN)63-/4- couple (1:1) as the redox probe. The oscillation potential was 5 mV and the applied
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potential was 0.23 V. Also, a frequency range of 100 kHz to 0.1 Hz was applied and the output
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signal was acquired with the Thales z (Zennium release) software.
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118 2.3. Preparation of the rGO-Aunano solution
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Graphite oxide was synthesized using graphite powders by the modified Hummers method [24].
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Briefly, 5.0 g graphite powder was dispersed in a 120 mL concentrated H2SO4 kept at 0 °C under
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stirring. Then, 15.0 g of KMnO4 was added gradually to the mixture kept in an ice bath to ensure
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that the temperature remained around 0 °C. After that, the temperature was raised to 0 °C and the
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mixture was stirred for 30 min and then diluted gradually with 225 mL deionized water. The
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mixture was re-diluted with 700 mL deionized water and treated with 3% hydrogen peroxide.
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The color of mixture changed to yellow-brown during the drop wise addition of H2O2. The
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mixture was filtered and washed with HCl solution (5%) and then repeatedly washed with water
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until neutral pH was obtained for filtrate. This solution was centrifuged at 3000 rpm for 10 min
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and then the filtrate was re-dispersed in water and centrifuged for several times. Finally, the dark
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brown GO powder was obtained through drying at 50 °C in a vacuum oven for a day. The
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graphite oxide was then exfoliated by ultrasonication. For this purpose, GO powder dispersed in
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a known volume of water was subjected to ultrasonication for 60 min to give a stable suspension
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of GO and then centrifuged at 3000 rpm for 30 min to remove any aggregates remained in the
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transparent light brown exfoliated GO suspension. Then, 10 mL of rose water and 100 µL
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NaAuCl4 (0.01 mM) were added into 10 mL above solution and the solution was mixed by
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ultrasonication for 30 min. The mixture was transferred into a Teflon-lined stainless steel
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autoclave and reacted at 95° C for 5 h. The resulting rGO-Aunano suspension was filtered and
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washed with plenty of water. The scheme 1 illustrates the mechanism of rGO-Aunano synthesis by
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phenolic compounds in rose water. The proposed mechanism is based on the reducing capability
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of phenolic compounds that contained in rose water [25].
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3. Results and discussion
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3.1. Characterization of the composites
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We monitored UV-visible absorption spectroscopy GO and rGO-Aunano, firstly. As shown in
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Figure 1A, the absorption peak at 230 nm shifted to 263 nm, indicating that the electronic
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conjugation within the reduced graphene sheet was revived upon reduction of graphene oxide
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[26, 27]. Also, the absorption was obtained at wavelength 530 nm showing the formation of gold
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nanoparticles. Raman spectroscopy is a powerful nondestructive tool to distinguish ordered and
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disordered crystal structures of carbon. G band is usually assigned to the E2g phonon of C sp2
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atoms, while D band is a breathing mode of κ-point phonons of A1g symmetry [28, 29]. The
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Raman spectrum of the prepared GO, rGO and rGO-Aunano showed two absorption bands (for D
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and G bands respectively) (Figure 1.B). The increase of D/G intensity ratio is predicted for rGO
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after reduction of the GO because of the restoration of sp2 domain [30-32], contrary to that
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observed in Figure 1.B. Also, Compared with D/G intensity ratio (1.24) of rGO, the D/G
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intensity ratio (1.05) of rGO-Aunano, which indicates that Au nanoparticles aggregated on rGO
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can decrease the defects of rGO to some extent in the process of reduction with rose water [16].
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The obtained composites are also characterized by XRD (Figure 1.C). After chemical reduction
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with rose water, the diffraction peak of exfoliated GO at 2θ = 13.01° disappeared and a very
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broad peak around 23° (b) was observed in the rGO sample, indicating that most oxygen
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functional groups had been removed [33]. It should be noted that the diffraction peak in rGO
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sheets were rather broad and significantly different from that in graphite (c). Also, the diffraction
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peaks were obtained at wave at 38.1 (111), 44.4 (200), 64.8 (220) and 77.6 (33.1) showing the
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formation of gold nanoparticles [34].
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The representative SEM image is displayed in Figure 2, which shows that the surface of rGO
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sheet was decorated by gold nanoparticles, forming the rGO-Aunano. The average size of Au nano
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particles were approximately 25 nm.
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Figure S1 (supporting information) shows the typical AFM image of exfoliated GO (a), rGO (b)
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and rGO-Aunano on a clean mica substrate, where the cross-sectional view indicates that the
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average thickness of exfoliated GO nanosheet is about 2.9 nm (Figure S1a, supporting
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information). After the final reduction step, this value decreased to about 1.3 nm (Figure S1b,
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supporting information), which could be explained by the removal of the surface oxide groups.
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Also, the height profile diagram of the AFM image showed that the typical thickness of rGO-
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Aunano nanocomposite shows a 2.69 nm increase in reduced graphene oxide thickness (1.3 nm) 8 Page 8 of 33
due to the presence of gold nanoparticles on both sides of the surface (Figure. S1c, supporting
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information).
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3.2. Preparation of GOD/rGO-Aunano GC electrode
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The surface of a glassy carbon (GC) electrode was polished successively with 0.3, 0.1 and 0.05
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µm alumina slurry and then cleaned in ethanol and water, respectively under ultra-sonication.
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Then, 6 µL of the rGO-Aunano solution was cast on the surface of GC electrode and allowed to
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dry at ambient temperature. The prepared rGO-Aunano/GC electrode was immersed in a GOD
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working solution (10 mg mL-1 in phosphate buffer solution (0.05 M, pH 7.0)) for about 24 h at 4
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°C to immobilize GOD on the electrode surface. Finally, the fabricated glucose biosensor
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(GOD/rGO-Aunano/GC electrode) was rinsed thoroughly with water to wash away the loosely
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adsorbed enzyme molecules. This glucose biosensor was stored at 4 °C in refrigerator when not
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in use.
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3.3. Electrochemical properties of the electrodes
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EIS has been used to characterize the interface properties of surface-modified electrodes. The
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typical impedance spectrum (presented in the form of the Nyquist plot) includes a semicircle
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portion at higher frequencies corresponding to the electron-transfer-limited process and a linear
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part at lower frequency range representing the diffusion limited process. The semicircle diameter
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in the impedance spectrum equals the electron-transfer resistance (Ret). This resistance controls
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the electron-transfer kinetics of the redox probe at the electrode interface. Figure 3.A displays the
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Nyquist plots obtained for a rGO-Aunano /GC (a), rGO/GC (b), GO/GC (c) and GC (d) electrodes
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in a solution containing 5.0 mM Fe(CN)6
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0.22 V, respectively. This figure shows that the Nyquist diameter (Ret=45Ω) of rGO-Aunano/GC
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-3/-4
couple (1:1) and 0.1 M KCl at a bias potential of
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electrode was much less than these observed for GO/GC electrode (Ret=28kΩ), rGO/GC
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(Ret=185Ω) and GC electrode (Ret=294Ω), suggesting that rGO-Aunano nanocomposite facilitates
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the rate of electron transfer. However, when GOD was immobilized on rGO-Aunano/GC
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electrode, the diameter of the semicircle increased (Ret=3.6 kΩ) (Figure S2.A), indicating that the
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immobilized GOD led to a great increase in resistance. The values of the electron-transfer
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resistance (Ret) obtained by fitting the experimental data. CV is also an efficient method for
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studying the interface properties of electrodes. CV studies showed (Figure 3.B) that the observed
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peak to peak separation for the Fe(CN)6-3/-4 redox couple on rGO-Aunano/GC electrode (23 mV)
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was less than those observed for rGO/GC (50 mV), GO/GC (73 mV) and GC electrodes (152
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mV) at a scan rate of 100 mV s−1. The peak current intensity on rGO-Aunano/GC electrode was
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also higher than that observed for the other electrodes. However, after the Immobilization of
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GOD on the surface of rGO-Aunano/GC electrode, the peak to peak separation for the Fe(CN)6-3/-4
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redox couple increased (Figure S2.B). The results were consistent with the EIS results. All these
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results demonstrated that rGO-Aunano nanocomposites provide higher electron conduction
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pathways.
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3.4. Electrochemical behaviour of GOD /rGO-Aunano/GC electrode
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Figure 4.A shows cyclic voltammograms (CVs) of a GOD /rGO-Aunano/GC electrode (solid line),
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rGO-Aunano/GC electrode (dotted line) and Nafion/GOD/GC electrode (dashed line), in a N2-
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saturated phosphate buffer (PB) solution (0.1 M, pH=7.0) at a scan rate of 50 mV s−1. No redox
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peaks were observed at Nafion/GOD/GC electrode (Figure 4.A, dashed line), indicating that GC 10 Page 10 of 33
electrode was not a suitable medium to observe GOD direct electron transfer. But, a couple of
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well-defined and reversible redox peaks were observed for the immobilized GOD in rGO-
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Aunano/GC electrode (Figure 4.A, solid line). The result indicates that the active redox center of
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GOD (flavin adenine dinucleotide (FAD)), which is deeply embedded in the protective protein
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shell of GOD is able to communicate with electrode, directly. The anodic and cathodic peak
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potentials were observed at about -440 and -464 mV, respectively. The formal
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potential (E°’) was about -452 mV (versus Ag|AgCl|KClsat). The peak-to-peak potential
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separation (ΔEp) and the ratio of cathodic to anodic current intensities were about 24 mV and 1,
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respectively.
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Figure S3 shows that the peak currents of GOD/rGO-Aunano/GC electrode are higher than those
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of Nafion/GOD/rGO electrode and Nafion/Aunano/GOD/GC electrode. This may be attributed to
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the good conductivity and biocompatibility of rGO-Aunano composite. The surface concentration
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(Γ) of the electroactive GOD on the film can be calculated from the charge integration of the
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cathodic peak in the cyclic voltammogram at a scan rate of 50 mV s−1according to the formula,
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Q= nFAΓ, where Q is the charge consumed in C, A is the electrode area (cm2), F is the Faraday
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constant and n is the number of electrons transferred. The value of Γ was 3.52×10−10 mol/cm2 (n
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=2). This value is two orders of magnitude higher than that the theoretical value (2.86×10−12 mol
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cm−2) for the monolayer of GOD on the bare electrode surface [35], suggesting that the
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nanostructured rGO-Aunano provide a large surface area and a higher capability of rGO-Aunano
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nanocomposite for enzyme immobilization.
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Figure 4.B shows the cyclic voltammograms of GOD/rGO-Aunano/GC electrode in pH 7.0 PB
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solution at different scan rates. Both the cathodic and anodic peaks currents are linearly 11 Page 11 of 33
proportional to the scan rate in the range from 10 to 700 mVs-1, as shown in the inset of Figure
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4.B, which indicate that the electrode reaction corresponds to a surface-controlled quasi-
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reversible process. According to the Laviron’s formula for a surface controlled electrochemical
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system (ΔEp < 200 mV), Ks =mnFν/RT where the apparent heterogeneous electron transfer rate
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constant (Ks) was estimated to be 5.35s-1, suggesting that direct electron transfer of GOD
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adsorbed onto rGO-AunanoGC/electrode had good reversibility. This Ks is higher than those
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reported previously for GOD at on MWCNT chitosan (1.08 s−1) [36, 37] , on boron-doped
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MWCNTs (1.56 s−1) [37], on MWCNTs-CTAB (1.53 s−1) [38], on SWCNTs-chitosan (3.0 s−1)
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[39] and CNTs-poly(diallyldimethylammonium chloride) (PDDA) modified electrode (2.76 s−1)
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[40]. The cyclic voltammetric measurements of the present film modified electrode also show a
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strong dependence on solution pH. From Figure 4.C, both anodic and cathodic peak potentials of
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GOD shift to negative direction and the formal potential (E0') versus pH give a straight line with
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the slope of 57.0 mV pH -1 (inset of Figure 4.C), which is close to the theoretical value (59.0 mV
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pH −1) for a two-proton coupled with two-electron redox reaction process shown in equation (1)
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[35].
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GOD-FAD+2e-+2H+→GOD-FADH2 (1)
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Figure 5.A shows the CVs of the GOD/rGO-Aunano nano-bio-composite on the GC electrode in a
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solution containing different concentrations of glucose under the condition of oxygen saturation
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on scan rate of 20 mV/s. It can be seen from this figure that the baseline of the reduction
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decreased with the increase in glucose concentration indicating the oxygen consumption. It was
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found that the oxygen consumption is linearly increased with the increase in glucose
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concentration ranging from 1 to 8 mM with a correlation coefficient (R2) of 0.9885 and a high
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sensitivity of about 0.0835µAmM−1 (Figure 5.B). Therefore, this GOD/rGO-Aunano/GC electrode
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can be served as a glucose sensor. The detection limit of the biosensor was estimated to be 10
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µM at a signal-to-noise ratio of 3.
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By using the electrochemical version of the Lineweaver–Burk equation [29] 1/Im =1/Imax +
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KMapp/(C × Imax), where Im is the steady-state current after the addition of substrate, Imax is the
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maximum current and C is the glucose concentration, a linear plot of 1/Im versus1/C (Figure S4,
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supporting information) is obtained to calculate Michaelis-Menten constant
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GOD/rGO-Aunano/GC electrode from its intercept and slope. The value of KMapp was 0.144 mM.
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This value is smaller than those reported for biosensor [41-44]. The smaller KMapp of GOD on
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rGO-Aunano nanocomposites means that the enzyme electrode possesses higher enzymatic
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activity to glucose oxidation and higher affinity towards the substrate.
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With a series of 5 experiments, the relative standard deviation (R.S.D.) of 3.5% was achieved.
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These results indicated that the immobilized GOD possesses high enzymatic activity, and the
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rGO-Aunano film provides favorable microenvironment for GOD to perform direct electron
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transfer at the modified electrode. The further measurement indicated that after 100 cycles at
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scan rate of 100 mV/s the anodic peak current of GOD is only decreased by 6.4%, illustrating
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that GOD immobilized on rGO-Aunano possesses the excellent stability. To evaluate the
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selectivity of the proposed biosensor, the effect of the presence of two possible interfering
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substances, uric acid 2.0 mM (UA) and ascorbic acid 2.0 mM (AA) on the cyclic voltammetry
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response of the proposed biosensor towards 0.4 mM glucose was investigated. A 8.43 % error
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criterion was adopted. This was probably due to the smaller size of ascorbic acid, which could
(KMapp) of
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diffuse through the porous film to the electrode surface and be oxidized. Thus, the response was
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not interfered by co-oxidizable substances such as AA and UA. The proposed glucose biosensor
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was employed for the determination of glucose in normal human serum to examine its
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applicability for real sample analysis. A 400 μL of normal human serum was mixed with 3.6 mL
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O2-saturated PB solution (0.1 M and pH = 7.0) and then analyzed using CVs and applying the
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proposed biosensor. The concentration of glucose in normal human serum was determined to be
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5.41 mM. The recovery of the analysis was about 98.3%, considering the value determined by a
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local hospital (5.50 mM). The comparison of the analytical performance of the proposed
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glucose biosensor with other graphene-based glucose biosensors are summarized in Table1. It
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can be seen that the analytical performance GOD/rGO-Aunano/GC electrode are comparable with
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other graphene-based glucose biosensors.
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309 4. Conclusions
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In this study, we proposed the green synthesis of rGO-Aunano nanocomposite by using rose water
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as an efficient reducing agent. UV-Vis, Raman and XRD spectra indicate preperating rGO-
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Aunano nanocomposite via eco-friendly chemical reduction method. We also investigated the
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direct electrochemistry of GOD at rGO-Aunano/GC electrode. The apparent heterogeneous
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electron transfer rate constant (ks) of GOD at the rGO-Aunano/GC electrode surface is estimated
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to be 5.35 s−1. The dependence of the formal potential solution pH indicated that the direct
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electron transfer reaction of GOD was a two-proton coupled with two-electron redox reaction
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process. The proposed biosensor can catalyze the reduction of dissolved oxygen and glucose
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determination was achieved based on the decrease of peak currents due to the reduction of
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dissolved oxygen. The constructed biosensor shows a good reproducibility and stability.
321 Acknowledgement
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The author thanks Dr P. Marashi and Miss somayeh jalilzadeh from Maharfan Abzar Co
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(Tehran, Iran) for the support with the AFM system.
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Supporting Information Available:
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Supplementary data associated with this article can be found in the online version.
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Biography
484
Mahmoud Amouzadeh Tabrizi received BSc degree in chemistry from the department of
485
chemistry, Urmia University, Urmia, Iran, in 2005. He received his MSc in chemistry in 2008
486
from the Department of Chemistry Kharazmi University, Tehran, Iran in the field of analytical
487
chemistry. He received his PhD in analytical chemistry in 2012 from the Institute for Advanced
488
Studies in Basic Sciences, Zanjan, Iran. He joined to the professor mojtaba shamsipur research
489
group in the Department of Chemistry in 2013 at Razi University, Kermanshah, Iran. His main
490
research interests are in electrochemical sensors and biosensors, green synthesis of graphene
491
nanocomposite and conducting polymers.
d
M
an
us
cr
ip t
479
te
492
Javad Nadali Varkani received BSc degree in chemistry from the department of physics,
494
Kashan University, Kashan, Iran, in 2000. He received his MSc in from the department of
495
physics, Urmia University, Urmia, Iran, in 2004. He is currently working toward the PhD degree
496
at the Mazandaran University. His main research interest is carbon based nanocomposits.
497 498
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499 500 501 502 503
Captions figure Scheme .1. The mechanism of rGO-Aunano synthesis.
22 Page 22 of 33
Figure. 1. (A) UV-vis absorption spectra obtained for exfoliated GO (a) and rGO-Aunano (b)
505
solutions respectively. (B) Raman spectra obtained for exfoliated GO (a), rGO-Aunano (b) and
506
rGO (c), (C) Typical XRD patterns obtained for exfoliated GO (a) and rGO-Aunano (b) and
507
pristine graphite(c), respectively.
508 509
Figure. 2. The SEM image of the prepared rGO-Aunano nanocomposite.
510
Figure. 3. Nyquist plots (A) and cyclic voltammograms (B) for the rGO-Aunano/GC (a),
511
rGO/GC (b), GO/GC (c) and GC (d) electrodes in a solution containing 5.0 mM Fe(CN)6-3/-
512
4
513
bias potential for EIS is 0.23 V. Inset is the equivalent electric circuit compatible with the
514
Nyquist diagrams. Rs: solution resistance, Ret: electron transfer resistance, Cdl: double layer
515
capacitance, Zw: Warburg impedance.
516
Figure. 4. (A) Cyclic voltammograms of the GOD/rGO-Aunano/GC electrode (solid line) the
517
rGO-Aunano/GC electrode (dotted line) and Nafion/GOD /GC electrode (dashed line) in a N2-
518
saturated phosphate buffer solution (0.1 M, pH = 7.0) at a scan rate of 50 mV s−1. (B) Cyclic
519
voltammograms of the GOD/rGO-Aunano/GC electrode in a N2-saturated phosphate buffer
520
solution (0.1 M, pH = 7.0) at various scan rates from 10 to 700 mV s-1 (10, 50, 100, 150, 200,
521
250, 300, 350, 400, 450, 500, 550, 600, 650 and 700 from inner to outer). Inset: plot of the peak
522
current (Ip) vs. scan rate (ν). (C) Cyclic voltammograms of the GOD/rGO-Aunano/GC electrode in
523
a N2-saturated phosphate buffer solution (0.1 M, pH = 7.0) at different pH values from 3.0 to 9 at
524
a scan rate of 50 mVs−1. Inset: plot of the formal potential (E°’) vs. pH.
525
Figure. 5. (A) Cyclic voltammograms of the GOD/rGO-Aunano/GC electrode in an O2-saturated
526
phosphate buffer solution (0.1 M, pH = 7.0) containing different amounts of glucose at a scan
527
rate of 20 mV s-1. (B) The corresponding calibration plots of CV response toward glucose.
us
cr
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couple (1:1) and 0.1 M KCl. Scan rate for cyclic voltammetry studies was 100 mVs−1. The
23 Page 23 of 33
528 529 530
ip t
531 532
cr
533
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534 535
an
536 537
M
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d
539
te
540
542 543 544 545 546
Ac ce p
541
547
Table.1. Comparison of the analytical performance of the proposed glucose biosensor with
548
other graphene-based glucose biosensors. biosensors GOD/MGF/GC electrode
Linear range
Detection
Ks
KMapp
(mM)
limit (µM)
(s-1)
(mM)
1-12
250
4.8
3.2
References [45]
24 Page 24 of 33
GOD/rGO–AuNPs/GC
0.02-2.26
4.1
3.25
0.038
[14]
0.1-10
10
2.68
-
[46]
2-14
180
-
-
[47]
0.004-1.12
0.6
4.8
0.6
[17]
0.2-20
17
-
0.5-12.5
160
electrode GOD self assembledGOD/rGO/Aunano/chit/GC GOD/rGO/PAN/Aunano/GC
cr
electrode
electrode GOD/rGO/Agnano/GC
GOD/GQD/GC electrode +
-
GOD/poly(ViBuIm Br )-
2-16 0.005-1.27 1-20
d
rGO/GC electrode 2-16
te
SGN/Au/GC electrode GOD/ptnanoflowers/GO/GC
0.005-1
Ac ce p
electrode
GOD/rGO-Aunano/GC
1-8
-
[48]
-
[49]
700
5.9
1.6
[50]
1.73
1.12
0.76
[51]
267
-
2.4
[52]
200
-
3.25
[53]
2.8
-
-
[54]
10
5.35
0.144
This work
M
GOD/rGO-CdS/GC electrode
5.27
an
electrode
us
electrode GOD/rGO-Aunano/GC
ip t
rGO/GC electrode
electrode 549
MGF: mesocellular graphene foam, rGO: reduced graphene oxide, PAN: polyaniline, GQD:
550
graphene
551
butylimidazolium bromide)-reduced graphene oxide, SGN: Sulfonated graphene nanosheet,GO:
552
Graphene oxide
quantum
dots,
chit:
chitosan,
poly(ViBuIm+Br−)-rGO:
poly(1-vinyl-3-
553 554
25 Page 25 of 33
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575
576
an
577 578
Scheme.1.
M
579
583 584 585 586 587
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582
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d
580
588
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A
D
B
G
b
ip t
c b
a
a
an
us
cr
a
C
c
589 590 591 592 593 594 595 596 597 598 599 600 601 602 603
Ac ce p
te
d
M
b
Figure.1.
28 Page 27 of 33
M te
d
Figure.2.
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607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635
an
us
cr
ip t
604 605 606
29 Page 28 of 33
636 637 638 639 640 A
a
ip t
c b
B
d
cr
d
c
us
a
c
d te
Figure.3.
Ac ce p
641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666
M
an
b
30 Page 29 of 33
667 668 669 670
an
us
cr
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A
671
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672
673 674 675 676 677 678 679 680 681 682 683 684
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C
Figure.4.
31 Page 30 of 33
685 686 A
M te
d
Figure.5.
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687 688 689 690 691 692 693
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B
32 Page 31 of 33
Biography
694
Mahmoud Amouzadeh Tabrizi received BSc degree in chemistry from the department of
695
chemistry, Urmia University, Urmia, Iran, in 2005. He received his MSc in chemistry in 2008
696
from the Department of Chemistry Kharazmi University, Tehran, Iran in the field of analytical
697
chemistry. He received his PhD in analytical chemistry in 2012 from the Institute for Advanced
698
Studies in Basic Sciences, Zanjan, Iran. He joined to the professor mojtaba shamsipur research
699
group in the Department of Chemistry in 2013 at Razi University, Kermanshah, Iran. His main
700
research interests are in electrochemical sensors and biosensors, green synthesis of graphene
701
nanocomposite and conducting polymers.
an
us
cr
ip t
693
702
Javad Nadali Varkani received BSc degree in chemistry from the department of physics,
704
Kashan University, Kashan, Iran, in 2000. He received his MSc in from the department of
705
physics, Urmia University, Urmia, Iran, in 2004. He is currently working toward the PhD degree
706
at the Mazandaran University. His main research interest is carbon base nanocomposits.
d
te
Ac ce p
707 708
M
703
33 Page 32 of 33
709
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cr
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708
710 711
an
Ac ce p
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M
712
34 Page 33 of 33
S0925-4005(14)00638-8 http://dx.doi.org/doi:10.1016/j.snb.2014.05.099 SNB 16973
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
15-4-2014 23-5-2014 23-5-2014
Please cite this article as: M.A. Tabrizi, J.N. Varkani, Green synthesis of reduced graphene oxide decorated with gold nanoparticles and its glucose sensing application, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.05.099 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.
3 4
Mahmoud Amouzadeh Tabrizia*, Javad Nadali Varkanib
5
a
6
b
Department of Chemistry, Razi University, Kermanshah, Iran
cr
Department of Physics, Mazandaran University, Mazandaran, Iran
7
* Corresponding author. Tel/fax: (+98) 21-44042955, Email:[email protected]
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Green synthesis of reduced graphene oxide decorated with gold nanoparticles and its glucose sensing application
1
21 22
Highlight
1 Page 1 of 33
We report green synthesis of rGO-Aunano composite by rose water as
24
reducing agent.
25
UV-vis , XRD spectra, SEM and AFM images indicate preparation of rGO-
26
Aunano composite.
27
A direct electron transfer reaction of glucose oxidase was observed on rGO-
28
Aunano /GC electrode.
29
This composite film was also successfully applied in preparation of glucose
30
biosensor.
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Abstract
36
Here, we report an eco-friendly method for synthesis of reduced graphene oxide decorated with
37
gold nanoparticles (rGO-Aunano) by using rose water as reducing agent. The prepared materials
38
were characterized using UV-visible absorption spectroscopy, Raman spectroscopy, atomic force
39
microscopy (AFM), scanning electron microscopy (SEM) and X-ray diffraction (XRD).
40
Furthermore, the direct electrochemistry of glucose oxidase (GOD) was achieved at a glassy
41
carbon electrode modified with rGO-Aunano. The resulting biosensor exhibited good response to
42
glucose with linear range from 1 to 8 mM with a low detection limit of 10µM.
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2 Page 2 of 33
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Keywords: Green chemistry; Rose water; Reduced graphene oxide decorated with gold
44
nanoparticles; Glucose oxidase; Biosensor
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1. Introduction
59
Diabetes is a major public health problem in the worldwide which classed as a metabolism
60
disorder. A new report has established that there are now nearly 350 million people on earth who
61
suffer from diabetes [1]. Therefore, the determination and controlling of blood glucose is very
62
important, which if not controlled, it can cause retinopathy, nephropathy, neuropathy,
63
hypertension, heart disease, stroke, gastroparesis, peripheral arterial disease, cellulitis and
64
depression [2]. Up to now, several analytical methods, such as UV fluorescence [3, 4]
65
chemiluminescence [5] and titrimetry [6] have been proposed for the determination of glucose.
3 Page 3 of 33
However, these methods are generally time-consuming, lack of sensitivity, susceptibility to
67
interference by other substances in analyte samples and difficult for an automated detection. To
68
overcome all these shortcomings, the electrochemical biosensor based on the direct
69
electrochemistry between an electrode and the immobilized glucose oxidase is especially
70
promising because of its simplicity, high sensitivity and selectivity [7, 8]. Recently, several
71
materials for the fabrication of glucose biosensor based on direct electron transfer have been
72
reported in the literature [7, 9-12]. Among them, the carbon based nanomaterials such as
73
graphene nano sheet provides not only a large microscopic surface area Immobilization of
74
enzymes, but also it provide a desirable biocompatible microenvironment [13-17]. Graphene, a
75
single aromatic sheet of sp2 bonded carbon, has been shown to possess unique electronic, optical,
76
thermal, mechanical and catalytic properties which are attractive for widely varied potential
77
applications in many fields of science ranging from nanoelectronics to biomedical devices [18,
78
19]. This nanosheet can be synthesized by various methods [20]. Among them, the chemical
79
reduction of exfoliated GO with green reducing agents have become a favorable topic for
80
researchers. In recent years, increasing numbers of researches in field of nanoscience have been
81
also devoted to green synthesis of reduced graphene oxide (rGO) decorated with novel metal
82
nanoparticles such as rGO/Pt [21], rGO/Ag [22] and rGO/Au [23], opening the door to
83
application in nanobiotechnology. The decoration of graphene with nanoparticles might be led to
84
new composite with different properties via cooperative interaction. In this paper, we report a
85
green method for synthesis of reduced graphene oxide decorated with gold nanoparticles (rGO-
86
Aunano) by using rose water as reducing agent. Also, we show that the prepared modified
87
electrode with rGO-Aunano nanocomposite would enhance the direct electron transfer capability
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of glucose oxidase (GOD) on the electrode surfaces. The prepared biosensor exhibited high
89
sensitivity, anti-fouling properties, lower detection limit and high stability for glucose sensing.
90 2. Experimental
92
2.1. Materials
93
All chemicals were of analytical reagent grade and used without further purification. Double
94
distilled water was used throughout. GOD from Aspergillus niger, 221U mg-1) and D-(+)-
95
glucose (97%) were obtained from Sigma (St. Louis, MO, USA). 10 mg of GOD was dissolved
96
in 1mL of phosphate buffer solution (0.05 M, pH=7.0) to prepare GOD working solution. A
97
stock solution of glucose (0.5 M) was prepared with doubly distilled water and stored at 4 °C
98
when not in use. The glucose stock solution was allowed to mutarotate at room temperature for
99
at least 24 h before use. Rose water (12 mg natural essence per 100 mL (so called 12%), Rabee
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Golab, Iran) was obtained from a local market.
101 102
2.2 Apparatus
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UV-visible absorption spectra were recorded using a single beam Pharmacia UV-vis
104
spectrophotometer (Ultrospec, model 4000). Raman scattering was performed on a Almega
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Thermo Nicolet Dispersive Raman spectrometer using the second harmonic (532 nm) of a
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Nd:YLF laser source. Scanning electron microscopy (SEM) was performed with a Philips
107
instrument, Model XL-30. Atomic force microscopy (AFM) was conducted with a DME
108
DualScope Scanner DS 95-200. X-ray diffraction (XRD) was performed with a D8ADVANCE
109
(Bruker) X-ray diffractometer equipped with a Cu Kα (1.5406 Å) radiation source. Cyclic
110
voltammetry (CV) was performed using an Autolab potentiostat-galvanostat model PGSTAT30
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with a conventional three-electrode setup, in which a bare glassy carbon (GC) electrode or
112
modified GC electrode, an Ag|AgCl|KClsat and a platinum rod served as the working, reference
113
and auxiliary electrodes, respectively. Electrochemical impedance spectroscopy (EIS)
114
experiments were carried out using a Zahner Zennium workstation in the presence of 5.0 mM
115
Fe(CN)63-/4- couple (1:1) as the redox probe. The oscillation potential was 5 mV and the applied
116
potential was 0.23 V. Also, a frequency range of 100 kHz to 0.1 Hz was applied and the output
117
signal was acquired with the Thales z (Zennium release) software.
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118 2.3. Preparation of the rGO-Aunano solution
120
Graphite oxide was synthesized using graphite powders by the modified Hummers method [24].
121
Briefly, 5.0 g graphite powder was dispersed in a 120 mL concentrated H2SO4 kept at 0 °C under
122
stirring. Then, 15.0 g of KMnO4 was added gradually to the mixture kept in an ice bath to ensure
123
that the temperature remained around 0 °C. After that, the temperature was raised to 0 °C and the
124
mixture was stirred for 30 min and then diluted gradually with 225 mL deionized water. The
125
mixture was re-diluted with 700 mL deionized water and treated with 3% hydrogen peroxide.
126
The color of mixture changed to yellow-brown during the drop wise addition of H2O2. The
127
mixture was filtered and washed with HCl solution (5%) and then repeatedly washed with water
128
until neutral pH was obtained for filtrate. This solution was centrifuged at 3000 rpm for 10 min
129
and then the filtrate was re-dispersed in water and centrifuged for several times. Finally, the dark
130
brown GO powder was obtained through drying at 50 °C in a vacuum oven for a day. The
131
graphite oxide was then exfoliated by ultrasonication. For this purpose, GO powder dispersed in
132
a known volume of water was subjected to ultrasonication for 60 min to give a stable suspension
133
of GO and then centrifuged at 3000 rpm for 30 min to remove any aggregates remained in the
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6 Page 6 of 33
transparent light brown exfoliated GO suspension. Then, 10 mL of rose water and 100 µL
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NaAuCl4 (0.01 mM) were added into 10 mL above solution and the solution was mixed by
136
ultrasonication for 30 min. The mixture was transferred into a Teflon-lined stainless steel
137
autoclave and reacted at 95° C for 5 h. The resulting rGO-Aunano suspension was filtered and
138
washed with plenty of water. The scheme 1 illustrates the mechanism of rGO-Aunano synthesis by
139
phenolic compounds in rose water. The proposed mechanism is based on the reducing capability
140
of phenolic compounds that contained in rose water [25].
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3. Results and discussion
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3.1. Characterization of the composites
145
We monitored UV-visible absorption spectroscopy GO and rGO-Aunano, firstly. As shown in
146
Figure 1A, the absorption peak at 230 nm shifted to 263 nm, indicating that the electronic
147
conjugation within the reduced graphene sheet was revived upon reduction of graphene oxide
148
[26, 27]. Also, the absorption was obtained at wavelength 530 nm showing the formation of gold
149
nanoparticles. Raman spectroscopy is a powerful nondestructive tool to distinguish ordered and
150
disordered crystal structures of carbon. G band is usually assigned to the E2g phonon of C sp2
151
atoms, while D band is a breathing mode of κ-point phonons of A1g symmetry [28, 29]. The
152
Raman spectrum of the prepared GO, rGO and rGO-Aunano showed two absorption bands (for D
153
and G bands respectively) (Figure 1.B). The increase of D/G intensity ratio is predicted for rGO
154
after reduction of the GO because of the restoration of sp2 domain [30-32], contrary to that
155
observed in Figure 1.B. Also, Compared with D/G intensity ratio (1.24) of rGO, the D/G
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7 Page 7 of 33
intensity ratio (1.05) of rGO-Aunano, which indicates that Au nanoparticles aggregated on rGO
157
can decrease the defects of rGO to some extent in the process of reduction with rose water [16].
158
The obtained composites are also characterized by XRD (Figure 1.C). After chemical reduction
159
with rose water, the diffraction peak of exfoliated GO at 2θ = 13.01° disappeared and a very
160
broad peak around 23° (b) was observed in the rGO sample, indicating that most oxygen
161
functional groups had been removed [33]. It should be noted that the diffraction peak in rGO
162
sheets were rather broad and significantly different from that in graphite (c). Also, the diffraction
163
peaks were obtained at wave at 38.1 (111), 44.4 (200), 64.8 (220) and 77.6 (33.1) showing the
164
formation of gold nanoparticles [34].
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The representative SEM image is displayed in Figure 2, which shows that the surface of rGO
168
sheet was decorated by gold nanoparticles, forming the rGO-Aunano. The average size of Au nano
169
particles were approximately 25 nm.
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Figure S1 (supporting information) shows the typical AFM image of exfoliated GO (a), rGO (b)
173
and rGO-Aunano on a clean mica substrate, where the cross-sectional view indicates that the
174
average thickness of exfoliated GO nanosheet is about 2.9 nm (Figure S1a, supporting
175
information). After the final reduction step, this value decreased to about 1.3 nm (Figure S1b,
176
supporting information), which could be explained by the removal of the surface oxide groups.
177
Also, the height profile diagram of the AFM image showed that the typical thickness of rGO-
178
Aunano nanocomposite shows a 2.69 nm increase in reduced graphene oxide thickness (1.3 nm) 8 Page 8 of 33
due to the presence of gold nanoparticles on both sides of the surface (Figure. S1c, supporting
180
information).
181 182
3.2. Preparation of GOD/rGO-Aunano GC electrode
183
The surface of a glassy carbon (GC) electrode was polished successively with 0.3, 0.1 and 0.05
184
µm alumina slurry and then cleaned in ethanol and water, respectively under ultra-sonication.
185
Then, 6 µL of the rGO-Aunano solution was cast on the surface of GC electrode and allowed to
186
dry at ambient temperature. The prepared rGO-Aunano/GC electrode was immersed in a GOD
187
working solution (10 mg mL-1 in phosphate buffer solution (0.05 M, pH 7.0)) for about 24 h at 4
188
°C to immobilize GOD on the electrode surface. Finally, the fabricated glucose biosensor
189
(GOD/rGO-Aunano/GC electrode) was rinsed thoroughly with water to wash away the loosely
190
adsorbed enzyme molecules. This glucose biosensor was stored at 4 °C in refrigerator when not
191
in use.
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3.3. Electrochemical properties of the electrodes
194
EIS has been used to characterize the interface properties of surface-modified electrodes. The
195
typical impedance spectrum (presented in the form of the Nyquist plot) includes a semicircle
196
portion at higher frequencies corresponding to the electron-transfer-limited process and a linear
197
part at lower frequency range representing the diffusion limited process. The semicircle diameter
198
in the impedance spectrum equals the electron-transfer resistance (Ret). This resistance controls
199
the electron-transfer kinetics of the redox probe at the electrode interface. Figure 3.A displays the
200
Nyquist plots obtained for a rGO-Aunano /GC (a), rGO/GC (b), GO/GC (c) and GC (d) electrodes
201
in a solution containing 5.0 mM Fe(CN)6
202
0.22 V, respectively. This figure shows that the Nyquist diameter (Ret=45Ω) of rGO-Aunano/GC
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-3/-4
couple (1:1) and 0.1 M KCl at a bias potential of
9 Page 9 of 33
electrode was much less than these observed for GO/GC electrode (Ret=28kΩ), rGO/GC
204
(Ret=185Ω) and GC electrode (Ret=294Ω), suggesting that rGO-Aunano nanocomposite facilitates
205
the rate of electron transfer. However, when GOD was immobilized on rGO-Aunano/GC
206
electrode, the diameter of the semicircle increased (Ret=3.6 kΩ) (Figure S2.A), indicating that the
207
immobilized GOD led to a great increase in resistance. The values of the electron-transfer
208
resistance (Ret) obtained by fitting the experimental data. CV is also an efficient method for
209
studying the interface properties of electrodes. CV studies showed (Figure 3.B) that the observed
210
peak to peak separation for the Fe(CN)6-3/-4 redox couple on rGO-Aunano/GC electrode (23 mV)
211
was less than those observed for rGO/GC (50 mV), GO/GC (73 mV) and GC electrodes (152
212
mV) at a scan rate of 100 mV s−1. The peak current intensity on rGO-Aunano/GC electrode was
213
also higher than that observed for the other electrodes. However, after the Immobilization of
214
GOD on the surface of rGO-Aunano/GC electrode, the peak to peak separation for the Fe(CN)6-3/-4
215
redox couple increased (Figure S2.B). The results were consistent with the EIS results. All these
216
results demonstrated that rGO-Aunano nanocomposites provide higher electron conduction
217
pathways.
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3.4. Electrochemical behaviour of GOD /rGO-Aunano/GC electrode
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Figure 4.A shows cyclic voltammograms (CVs) of a GOD /rGO-Aunano/GC electrode (solid line),
223
rGO-Aunano/GC electrode (dotted line) and Nafion/GOD/GC electrode (dashed line), in a N2-
224
saturated phosphate buffer (PB) solution (0.1 M, pH=7.0) at a scan rate of 50 mV s−1. No redox
225
peaks were observed at Nafion/GOD/GC electrode (Figure 4.A, dashed line), indicating that GC 10 Page 10 of 33
electrode was not a suitable medium to observe GOD direct electron transfer. But, a couple of
227
well-defined and reversible redox peaks were observed for the immobilized GOD in rGO-
228
Aunano/GC electrode (Figure 4.A, solid line). The result indicates that the active redox center of
229
GOD (flavin adenine dinucleotide (FAD)), which is deeply embedded in the protective protein
230
shell of GOD is able to communicate with electrode, directly. The anodic and cathodic peak
231
potentials were observed at about -440 and -464 mV, respectively. The formal
232
potential (E°’) was about -452 mV (versus Ag|AgCl|KClsat). The peak-to-peak potential
233
separation (ΔEp) and the ratio of cathodic to anodic current intensities were about 24 mV and 1,
234
respectively.
235
Figure S3 shows that the peak currents of GOD/rGO-Aunano/GC electrode are higher than those
236
of Nafion/GOD/rGO electrode and Nafion/Aunano/GOD/GC electrode. This may be attributed to
237
the good conductivity and biocompatibility of rGO-Aunano composite. The surface concentration
238
(Γ) of the electroactive GOD on the film can be calculated from the charge integration of the
239
cathodic peak in the cyclic voltammogram at a scan rate of 50 mV s−1according to the formula,
240
Q= nFAΓ, where Q is the charge consumed in C, A is the electrode area (cm2), F is the Faraday
241
constant and n is the number of electrons transferred. The value of Γ was 3.52×10−10 mol/cm2 (n
242
=2). This value is two orders of magnitude higher than that the theoretical value (2.86×10−12 mol
243
cm−2) for the monolayer of GOD on the bare electrode surface [35], suggesting that the
244
nanostructured rGO-Aunano provide a large surface area and a higher capability of rGO-Aunano
245
nanocomposite for enzyme immobilization.
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Figure 4.B shows the cyclic voltammograms of GOD/rGO-Aunano/GC electrode in pH 7.0 PB
248
solution at different scan rates. Both the cathodic and anodic peaks currents are linearly 11 Page 11 of 33
proportional to the scan rate in the range from 10 to 700 mVs-1, as shown in the inset of Figure
250
4.B, which indicate that the electrode reaction corresponds to a surface-controlled quasi-
251
reversible process. According to the Laviron’s formula for a surface controlled electrochemical
252
system (ΔEp < 200 mV), Ks =mnFν/RT where the apparent heterogeneous electron transfer rate
253
constant (Ks) was estimated to be 5.35s-1, suggesting that direct electron transfer of GOD
254
adsorbed onto rGO-AunanoGC/electrode had good reversibility. This Ks is higher than those
255
reported previously for GOD at on MWCNT chitosan (1.08 s−1) [36, 37] , on boron-doped
256
MWCNTs (1.56 s−1) [37], on MWCNTs-CTAB (1.53 s−1) [38], on SWCNTs-chitosan (3.0 s−1)
257
[39] and CNTs-poly(diallyldimethylammonium chloride) (PDDA) modified electrode (2.76 s−1)
258
[40]. The cyclic voltammetric measurements of the present film modified electrode also show a
259
strong dependence on solution pH. From Figure 4.C, both anodic and cathodic peak potentials of
260
GOD shift to negative direction and the formal potential (E0') versus pH give a straight line with
261
the slope of 57.0 mV pH -1 (inset of Figure 4.C), which is close to the theoretical value (59.0 mV
262
pH −1) for a two-proton coupled with two-electron redox reaction process shown in equation (1)
263
[35].
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GOD-FAD+2e-+2H+→GOD-FADH2 (1)
265 266 267 268 269
Figure 5.A shows the CVs of the GOD/rGO-Aunano nano-bio-composite on the GC electrode in a
270
solution containing different concentrations of glucose under the condition of oxygen saturation
271
on scan rate of 20 mV/s. It can be seen from this figure that the baseline of the reduction
272
decreased with the increase in glucose concentration indicating the oxygen consumption. It was
273
found that the oxygen consumption is linearly increased with the increase in glucose
12 Page 12 of 33
concentration ranging from 1 to 8 mM with a correlation coefficient (R2) of 0.9885 and a high
275
sensitivity of about 0.0835µAmM−1 (Figure 5.B). Therefore, this GOD/rGO-Aunano/GC electrode
276
can be served as a glucose sensor. The detection limit of the biosensor was estimated to be 10
277
µM at a signal-to-noise ratio of 3.
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By using the electrochemical version of the Lineweaver–Burk equation [29] 1/Im =1/Imax +
280
KMapp/(C × Imax), where Im is the steady-state current after the addition of substrate, Imax is the
281
maximum current and C is the glucose concentration, a linear plot of 1/Im versus1/C (Figure S4,
282
supporting information) is obtained to calculate Michaelis-Menten constant
283
GOD/rGO-Aunano/GC electrode from its intercept and slope. The value of KMapp was 0.144 mM.
284
This value is smaller than those reported for biosensor [41-44]. The smaller KMapp of GOD on
285
rGO-Aunano nanocomposites means that the enzyme electrode possesses higher enzymatic
286
activity to glucose oxidation and higher affinity towards the substrate.
287
With a series of 5 experiments, the relative standard deviation (R.S.D.) of 3.5% was achieved.
288
These results indicated that the immobilized GOD possesses high enzymatic activity, and the
289
rGO-Aunano film provides favorable microenvironment for GOD to perform direct electron
290
transfer at the modified electrode. The further measurement indicated that after 100 cycles at
291
scan rate of 100 mV/s the anodic peak current of GOD is only decreased by 6.4%, illustrating
292
that GOD immobilized on rGO-Aunano possesses the excellent stability. To evaluate the
293
selectivity of the proposed biosensor, the effect of the presence of two possible interfering
294
substances, uric acid 2.0 mM (UA) and ascorbic acid 2.0 mM (AA) on the cyclic voltammetry
295
response of the proposed biosensor towards 0.4 mM glucose was investigated. A 8.43 % error
296
criterion was adopted. This was probably due to the smaller size of ascorbic acid, which could
(KMapp) of
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diffuse through the porous film to the electrode surface and be oxidized. Thus, the response was
298
not interfered by co-oxidizable substances such as AA and UA. The proposed glucose biosensor
299
was employed for the determination of glucose in normal human serum to examine its
300
applicability for real sample analysis. A 400 μL of normal human serum was mixed with 3.6 mL
301
O2-saturated PB solution (0.1 M and pH = 7.0) and then analyzed using CVs and applying the
302
proposed biosensor. The concentration of glucose in normal human serum was determined to be
303
5.41 mM. The recovery of the analysis was about 98.3%, considering the value determined by a
304
local hospital (5.50 mM). The comparison of the analytical performance of the proposed
305
glucose biosensor with other graphene-based glucose biosensors are summarized in Table1. It
306
can be seen that the analytical performance GOD/rGO-Aunano/GC electrode are comparable with
307
other graphene-based glucose biosensors.
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309 4. Conclusions
311
In this study, we proposed the green synthesis of rGO-Aunano nanocomposite by using rose water
312
as an efficient reducing agent. UV-Vis, Raman and XRD spectra indicate preperating rGO-
313
Aunano nanocomposite via eco-friendly chemical reduction method. We also investigated the
314
direct electrochemistry of GOD at rGO-Aunano/GC electrode. The apparent heterogeneous
315
electron transfer rate constant (ks) of GOD at the rGO-Aunano/GC electrode surface is estimated
316
to be 5.35 s−1. The dependence of the formal potential solution pH indicated that the direct
317
electron transfer reaction of GOD was a two-proton coupled with two-electron redox reaction
318
process. The proposed biosensor can catalyze the reduction of dissolved oxygen and glucose
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319
determination was achieved based on the decrease of peak currents due to the reduction of
320
dissolved oxygen. The constructed biosensor shows a good reproducibility and stability.
321 Acknowledgement
323
The author thanks Dr P. Marashi and Miss somayeh jalilzadeh from Maharfan Abzar Co
324
(Tehran, Iran) for the support with the AFM system.
325
Supporting Information Available:
326
Supplementary data associated with this article can be found in the online version.
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Analytical Biochemistry, 332(2004) 75-83.
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carbon nanotube wrapped by polyelectrolyte, Electrochimica Acta, 52(2007) 5312-7.
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nanotubes in poly(diallyldimethylammonium chloride) for preparation of a glucose biosensor,
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[42] H.N. Choi, M.A. Kim, W.-Y. Lee, Amperometric glucose biosensor based on sol–gel-
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derived metal oxide/Nafion composite films, Analytica Chimica Acta, 537(2005) 179-87.
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a CdS nanoparticles modified electrode, Biosensors and Bioelectronics, 21(2005) 817-21.
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encapsulated sol-gel and carbon-nanotube-modified basal plane pyrolytic graphite electrode,
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nanocomposites film for glucose biosensing, Biosensors & bioelectronics, 25(2010) 1070-4.
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Biography
484
Mahmoud Amouzadeh Tabrizi received BSc degree in chemistry from the department of
485
chemistry, Urmia University, Urmia, Iran, in 2005. He received his MSc in chemistry in 2008
486
from the Department of Chemistry Kharazmi University, Tehran, Iran in the field of analytical
487
chemistry. He received his PhD in analytical chemistry in 2012 from the Institute for Advanced
488
Studies in Basic Sciences, Zanjan, Iran. He joined to the professor mojtaba shamsipur research
489
group in the Department of Chemistry in 2013 at Razi University, Kermanshah, Iran. His main
490
research interests are in electrochemical sensors and biosensors, green synthesis of graphene
491
nanocomposite and conducting polymers.
d
M
an
us
cr
ip t
479
te
492
Javad Nadali Varkani received BSc degree in chemistry from the department of physics,
494
Kashan University, Kashan, Iran, in 2000. He received his MSc in from the department of
495
physics, Urmia University, Urmia, Iran, in 2004. He is currently working toward the PhD degree
496
at the Mazandaran University. His main research interest is carbon based nanocomposits.
497 498
Ac ce p
493
499 500 501 502 503
Captions figure Scheme .1. The mechanism of rGO-Aunano synthesis.
22 Page 22 of 33
Figure. 1. (A) UV-vis absorption spectra obtained for exfoliated GO (a) and rGO-Aunano (b)
505
solutions respectively. (B) Raman spectra obtained for exfoliated GO (a), rGO-Aunano (b) and
506
rGO (c), (C) Typical XRD patterns obtained for exfoliated GO (a) and rGO-Aunano (b) and
507
pristine graphite(c), respectively.
508 509
Figure. 2. The SEM image of the prepared rGO-Aunano nanocomposite.
510
Figure. 3. Nyquist plots (A) and cyclic voltammograms (B) for the rGO-Aunano/GC (a),
511
rGO/GC (b), GO/GC (c) and GC (d) electrodes in a solution containing 5.0 mM Fe(CN)6-3/-
512
4
513
bias potential for EIS is 0.23 V. Inset is the equivalent electric circuit compatible with the
514
Nyquist diagrams. Rs: solution resistance, Ret: electron transfer resistance, Cdl: double layer
515
capacitance, Zw: Warburg impedance.
516
Figure. 4. (A) Cyclic voltammograms of the GOD/rGO-Aunano/GC electrode (solid line) the
517
rGO-Aunano/GC electrode (dotted line) and Nafion/GOD /GC electrode (dashed line) in a N2-
518
saturated phosphate buffer solution (0.1 M, pH = 7.0) at a scan rate of 50 mV s−1. (B) Cyclic
519
voltammograms of the GOD/rGO-Aunano/GC electrode in a N2-saturated phosphate buffer
520
solution (0.1 M, pH = 7.0) at various scan rates from 10 to 700 mV s-1 (10, 50, 100, 150, 200,
521
250, 300, 350, 400, 450, 500, 550, 600, 650 and 700 from inner to outer). Inset: plot of the peak
522
current (Ip) vs. scan rate (ν). (C) Cyclic voltammograms of the GOD/rGO-Aunano/GC electrode in
523
a N2-saturated phosphate buffer solution (0.1 M, pH = 7.0) at different pH values from 3.0 to 9 at
524
a scan rate of 50 mVs−1. Inset: plot of the formal potential (E°’) vs. pH.
525
Figure. 5. (A) Cyclic voltammograms of the GOD/rGO-Aunano/GC electrode in an O2-saturated
526
phosphate buffer solution (0.1 M, pH = 7.0) containing different amounts of glucose at a scan
527
rate of 20 mV s-1. (B) The corresponding calibration plots of CV response toward glucose.
us
cr
ip t
504
Ac ce p
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M
an
couple (1:1) and 0.1 M KCl. Scan rate for cyclic voltammetry studies was 100 mVs−1. The
23 Page 23 of 33
528 529 530
ip t
531 532
cr
533
us
534 535
an
536 537
M
538
d
539
te
540
542 543 544 545 546
Ac ce p
541
547
Table.1. Comparison of the analytical performance of the proposed glucose biosensor with
548
other graphene-based glucose biosensors. biosensors GOD/MGF/GC electrode
Linear range
Detection
Ks
KMapp
(mM)
limit (µM)
(s-1)
(mM)
1-12
250
4.8
3.2
References [45]
24 Page 24 of 33
GOD/rGO–AuNPs/GC
0.02-2.26
4.1
3.25
0.038
[14]
0.1-10
10
2.68
-
[46]
2-14
180
-
-
[47]
0.004-1.12
0.6
4.8
0.6
[17]
0.2-20
17
-
0.5-12.5
160
electrode GOD self assembledGOD/rGO/Aunano/chit/GC GOD/rGO/PAN/Aunano/GC
cr
electrode
electrode GOD/rGO/Agnano/GC
GOD/GQD/GC electrode +
-
GOD/poly(ViBuIm Br )-
2-16 0.005-1.27 1-20
d
rGO/GC electrode 2-16
te
SGN/Au/GC electrode GOD/ptnanoflowers/GO/GC
0.005-1
Ac ce p
electrode
GOD/rGO-Aunano/GC
1-8
-
[48]
-
[49]
700
5.9
1.6
[50]
1.73
1.12
0.76
[51]
267
-
2.4
[52]
200
-
3.25
[53]
2.8
-
-
[54]
10
5.35
0.144
This work
M
GOD/rGO-CdS/GC electrode
5.27
an
electrode
us
electrode GOD/rGO-Aunano/GC
ip t
rGO/GC electrode
electrode 549
MGF: mesocellular graphene foam, rGO: reduced graphene oxide, PAN: polyaniline, GQD:
550
graphene
551
butylimidazolium bromide)-reduced graphene oxide, SGN: Sulfonated graphene nanosheet,GO:
552
Graphene oxide
quantum
dots,
chit:
chitosan,
poly(ViBuIm+Br−)-rGO:
poly(1-vinyl-3-
553 554
25 Page 25 of 33
us
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ip t
575
576
an
577 578
Scheme.1.
M
579
583 584 585 586 587
te
582
Ac ce p
581
d
580
588
27 Page 26 of 33
A
D
B
G
b
ip t
c b
a
a
an
us
cr
a
C
c
589 590 591 592 593 594 595 596 597 598 599 600 601 602 603
Ac ce p
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d
M
b
Figure.1.
28 Page 27 of 33
M te
d
Figure.2.
Ac ce p
607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635
an
us
cr
ip t
604 605 606
29 Page 28 of 33
636 637 638 639 640 A
a
ip t
c b
B
d
cr
d
c
us
a
c
d te
Figure.3.
Ac ce p
641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666
M
an
b
30 Page 29 of 33
667 668 669 670
an
us
cr
ip t
A
671
M
672
673 674 675 676 677 678 679 680 681 682 683 684
Ac ce p
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C
Figure.4.
31 Page 30 of 33
685 686 A
M te
d
Figure.5.
Ac ce p
687 688 689 690 691 692 693
an
us
cr
ip t
B
32 Page 31 of 33
Biography
694
Mahmoud Amouzadeh Tabrizi received BSc degree in chemistry from the department of
695
chemistry, Urmia University, Urmia, Iran, in 2005. He received his MSc in chemistry in 2008
696
from the Department of Chemistry Kharazmi University, Tehran, Iran in the field of analytical
697
chemistry. He received his PhD in analytical chemistry in 2012 from the Institute for Advanced
698
Studies in Basic Sciences, Zanjan, Iran. He joined to the professor mojtaba shamsipur research
699
group in the Department of Chemistry in 2013 at Razi University, Kermanshah, Iran. His main
700
research interests are in electrochemical sensors and biosensors, green synthesis of graphene
701
nanocomposite and conducting polymers.
an
us
cr
ip t
693
702
Javad Nadali Varkani received BSc degree in chemistry from the department of physics,
704
Kashan University, Kashan, Iran, in 2000. He received his MSc in from the department of
705
physics, Urmia University, Urmia, Iran, in 2004. He is currently working toward the PhD degree
706
at the Mazandaran University. His main research interest is carbon base nanocomposits.
d
te
Ac ce p
707 708
M
703
33 Page 32 of 33
709
us
cr
ip t
708
710 711
an
Ac ce p
te
d
M
712
34 Page 33 of 33