Microchim Acta DOI 10.1007/s00604-015-1545-1
ORIGINAL PAPER
Enzymatic glucose biosensor based on bismuth nanoribbons electrochemically deposited on reduced graphene oxide Rajkumar Devasenathipathy 1 & Raj Karthik 1 & Shen-Ming Chen 1 & Mohammad Ajmal Ali 2 & Veerappan Mani 1 & Bih-Show Lou 3 & Fahad Mohammed Abdullrahman Al-Hemaid 2
Received: 12 March 2015 / Accepted: 14 June 2015 # Springer-Verlag Wien 2015
Abstract We describe the electrochemical preparation of bismuth nanoribbons (Bi-NRs) with an average length of 100± 50 nm and a width of 10±5 μm by a potentiostatic method. The process occurs on the surface of a glassy carbon electrode (GCE) in the presence of disodium ethylene diamine tetraacetate that acts as a scaffold for the growth of the BiNRs and also renders them more stable. The method was applied to the preparation of Bi-NRs incorporated into reduced graphene oxide. This nanocomposite was loaded with the enzyme glucose oxidase onto a glassy carbon electrode. The resulting biosensor displays an enhanced redox peak for the enzyme with a peak-to-peak separation of about 28 mV, revealing a fast electron transfer at the modified electrode. The loading of the GCE with electroactive GOx was calculated to be 8.54×10−10 mol cm−2, and the electron transfer rate constant is 4.40 s−1. Glucose can be determined (in the presence of oxygen) at a relatively working potential of −0.46 V (vs. Ag|AgCl) in the 0.5 to 6 mM concentration range, with a Electronic supplementary material The online version of this article (doi:10.1007/s00604-015-1545-1) contains supplementary material, which is available to authorized users. * Shen-Ming Chen
[email protected] * Bih-Show Lou
[email protected] 1
Electroanalysis and Bioelectrochemistry Lab, Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No.1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan, Republic of China
2
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
3
Chemistry Division, Center for General Education, Chang Gung University, Tao-Yuan 333, Taiwan
104 μM lower detection limit. The sensor also displays appreciable repeatability, reproducibility and remarkable stability. It was successfully applied to the determination of glucose in human serum samples. Keywords Potentiostatic method . Bismuth nanoribbons . Glucose oxidase . Biosensor . Reduced graphene oxide
Introduction Bismuth with its unusual electrical, optical and structural features has been extensively used in applications such as physical and chemical sensors, biosensor, lithium ion batteries, vanadium redox flow battery, drug delivery, and in solar cells [1–6]. Relative to other film electrodes such as mercury and carbon dots electrode, bismuth film electrode have shown an attractive electrode material because of their low cost, environmentally friendly, biocompatible and have high mechanical stability [7]. Various chemical, electrochemical, electron– beam irradiation and hydrothermal reduction methods for synthesis have been demonstrated [8–11] and electrochemical methods are preferred because they are simple, easily scalable, and inexpensive. They yield particles of uniform size whose shape can be controlled by varying the concentration of the electrolyte, current densities and potential [12]. Jiang et al. [13] reported on a potentiodynamic method that provides a simple strategy for the preparation of various morphologies of bismuth including prickly rod, bench skeleton and striplike, however all in μm-dimensions. Zhou et al. [14] demonstrated a method for the preparation of rhombohedral and smooth bismuth structures of high purity. The deposition time is 1 h. Here, we report on the synthesis of bismuth nanoribbons (Bi–NRs) by a time-controlled potentiostatic method using disodium ethylenediaminetetraacetate dihydrate
R. Devasenathipathy et al.
(Na2EDTA) as the capping agent. Ethylene diamine (EDA) and its derivative such as ethylene diamine tetra acetic acid (EDTA) and Na2EDTA have been used as a capping or chelating agent for the growth of one dimensional metal nanomaterials in the recent years [13, 15, 16]. Relative to EDA and EDTA, Na2EDTA is highly hydrophilic and acts as a strong ligand for the formation of stable complex with metal ions in aqueous medium because it has two molecule of water and therefore, it is easy to dissolve in aqueous medium than that of other anhydrous form. Owing to this reason we have taken Na2EDTA as a scaffold for the growth of one dimensional bismuth nanomaterials through electrochemical potentiostatic method. The method was further applied to the deposition of Bi-NRs incorporated reduced graphene oxide to design an enzymatic glucose biosensor. Numerous chemically and electrochemically reduced graphene oxide (RGO) based nanomaterials have been prepared for the fabrication of electrode materials in many fields such as sensor, biosensor, super capacitor etc., owing to their low cost, high surface area, good conductivity and chemical stability [17, 18]. Moreover, the reduced graphene based nanomaterials help the enzyme to improve its stability and to increase the direct electron transfer between the electrode and active site of the enzyme [19]. Thus, several kind of reduced graphene oxide based metal and metal oxide includes, Au, Pt, Pd, ZnO, Co3O4 and NiO sensor have been fabricated and used in the detection of glucose [20–25]. The enzymatic electrochemical glucose biosensors are most extensively used for measuring blood glucose levels in diabetic patients and for constructing the biofuel cell application because of their low cost, high selectivity and sensitivity [26–28]. However, the immobilization of the enzyme and prevention of the leaching enzyme from the electrode surface, are important task in the fabrication of glucose biosensor. To solve this key issues, several kind of nanomaterials and methods along with good immobilization and retain the stability of enzyme have been developed in the fabrication of glucose biosensor [29]. To date, finding a suitable stabilizing and a capping agent for the preparation of metal and metal oxide nanomaterials are continuous research interests in our research group. We reported pectin stabilized gold nanoparticles on graphene through simple electrochemical potentiodynamic method for the determination of hydrazine sensor quite recently [30]. Herein, we utilized RGO and Bi-NRs to immobilize GOx for the development of a voltammetric glucose biosensor. RGO/Bi–NRs composite was deposited by a simple electrochemical potentiostatic approach. The successful formation of the RGO/Bi–NRs were confirmed by scanning electron microscopy (SEM), energy dispersive electron X-ray spectroscopy (EDX), X-ray diffraction studies (XRD) and cyclic voltammetry (CV). The GOx was adsorbed on the RGO/Bi–NRs nanocomposite without using any covalent linkage and it was
employed in the detection of glucose. The practicality of the proposed RGO/BiNRs/GOx sensor was successfully demonstrated in human serum sample.
Experimental Reagents and apparatus Graphite (powder, 20 μm), Bismuth (III) nitrate pentahydrate (Bi(NO 3 ) 3 .5H 2 O) and ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA.2H2O) were purchased from Sigma Aldrich (http://www.sigmaaldrich.com/taiwan. html). The supporting electrolyte used for all the electrochemical studies was 0.05 M Phosphate buffer (PB) solution, prepared using NaH2PO4 and Na2HPO4, while the pH were adjusted to get desired pH using either using H2SO4 or NaOH. Glucose oxidase from Aspergillus Niger was obtained from Sigma Aldrich (http://www.sigmaaldrich.com/ taiwan.html). Prior to each experiment, all the solutions were deoxygenated with pre-purified N2 gas for 15 min unless otherwise specified. Double distilled water with conductivity of ≥18 MΩ cm−1 was used for all the experiments. The human blood serum sample was collected from the valley biomedical, Taiwan product & services, Inc. (www.valleybiomedical.com/ ce_humanab.phtml). This study was reviewed and approved by the ethics committee of Chang-Gung memorial hospital through the contract no. IRB101-5042A3. The electrochemical measurements were carried out using CHI 611A electrochemical work station. Electrochemical studies were performed in a conventional three electrode cell using screen printed carbon electrode (SPCE) as a working electrode (area=0.071 cm2), Ag|AgCl (saturated KCl) as a reference electrode and Pt wire as a counter electrode. Scanning electron microscope (SEM) and energy dispersive X-ray (EDX) spectra were performed using Hitachi S-3000H scanning electron microscope and HORIBA EMAX X-ACT, respectively. X-ray diffraction (XRD) studies were carried out using XPERT-PRO diffractometer using Cu Kα radiation (k= 1.54 Å). Electrodeposition of Bi-NRs on graphene oxide and fabrication of biosensor Hummer’s method was used to prepare the graphite oxide and suspended in water (1 mg mL−1). Thereafter, the as prepared graphite oxide was exfoliated to attain a stable suspension of graphene oxide (GO) through ultra-sonication for 2 h. The potentiostatic co-electrochemical reduction of RGO/Bi–NRs was performed in a conventional three electrode system. The electrochemical cell was filled with a mixture of 5 mM Bi(NO3)3 and 10 mM aqueous solution of Na2EDTA. An 8 μl of aqueous GO was drop cast on the pre-cleaned glassy
Electrochemically deposited Bi-NRs on RGO
carbon electrode (GCE) surface and dried at ambient conditions. Subsequently, the modified GO/GCE was carefully transferred to an electrochemical cell containing the solution of 5 mM Bi(NO 3 ) 3 and 10 mM aqueous solution of Na2EDTA. Before performing the potentiostatic experiment, the electrodes (GO/GCE, Platinum and Ag|AgCl) were dipped in electrolytic solution for 2 min to obtain equilibrium condition. Then, electrochemical deposition was carried out at a constant applied potential of −1.20 V (vs. Ag|AgCl) on GO/GCE for 15 min. eventually, an 8 μL of GOx (10 mg/mL) was drop cast onto the RGO/Bi–NRs modified GCE and dried. The fabricated RGO/Bi–NRs/GOx modified GCE was cleaned with double distilled water to remove the loosely bounded GOx and dried at 40 °C in air oven. For the control electrodes, the similar procedure was used to prepare Bi–NRs/GOx/GCE, RGO/GOx/GCE and GO/GOx/GCE (Scheme 1).
Results and discussion Surface morphological study of RGO/Bi–NRs composite Figure 1 displays the SEM images of Bi-NRs (a) and RGO/ Bi–NRs (b). The SEM images of Bi-NRs clearly show ribbon like structure. Na2EDTA acts as a capping or chelating agent for the formation of Bi-EDTA complex and provides the uniform growth of Bi-NRs. Moreover, we noted that the formation of nanoribbons is depending on the concentration of Na2EDTA. Therefore, the concentration of Na2EDTA was optimized and fixed 10 mM Na2EDTA in 5 mM Bi(NO3)3. SEM images of RGO/Bi–NRs, revealing that the Bi-NRs are highly incorporated on the graphene networks. Moreover, the
average length and width of the Bi-NRs were calculated to be 10±5 μm and 150±50 nm, respectively. Elemental analysis and spectroscopic characterization of RGO/Bi–NRs nanocomposite Energy dispersive X-ray (EDX) spectroscopic measurement was used to analyze the elemental composition of Bi-NRs (Fig. 1c) and RGO/Bi–NRs (Fig. 1d). The EDX spectra of Bi-NRs show signals for C, O and Bi with a weight percentage of 5.96, 9.93 and 84.11 %, respectively. Whereas, EDX spectra of RGO/Bi–NRs portray signals for C, O and Bi with a weight percentage of 16.01, 8.03 and 75.96 %, respectively. From these EDX spectra measurements, the formation of nanocomposite can be pure bismuth, however the small percentage of carbon and oxygen might be coming from Na2EDTA. In addition, the decreasing in bismuth content of RGO/Bi–NRs from 84.11 to 75.96 % confirms the incorporation of Bi-NRs with RGO. The crystal structures of the RGO and RGO/Bi–NRs were confirmed by X-ray diffraction (XRD) spectroscopy (Fig. 2a). The XRD pattern of RGO (a) shows a diffraction peaks at 2θ angle of 23.10°, revealing that the successful electrochemical reduction of GO at the electrode. While, the XRD pattern of RGO/Bi–NRs (b) displays the efficient incorporation of the Bi-NRs on the RGO. Furthermore, the XRD pattern of RGO/ Bi–NRs is exactly concordance with the previously reported XRD pattern of rhomohedral bismuth (JCPDS no. 05-0519) which confirmed the successful formation of Bi-NRs on the RGO network. Fig. S1 (Electronic Supplementary Material, ESM) shows the Raman spectra of GO (a) and RGO (b), respectively. Compared to GO, the D band and G band of RGO shifted significantly towards lower frequencies (D band=1342 to 1336 cm−1; G band=1600 to 1586 cm−1). Furthermore, the intensity ratio (ID/IG) of D band to G band increased from 0.96 to 1.10, revealing a decrease in the average size of sp2 domain upon electrochemical reduction of GO [31]. Electrochemical properties of RGO/Bi–NRs nanocomposites
Scheme 1 Schematic representation for the fabrication of RGO/Bi–NRs/ GOx/GCE biosensor
The inset of Fig. 2b displays the cyclic voltammograms Cyclic voltammograms obtained at RGO/Bi–NRs/GCE in pH 7 PB solution exhibited two redox couples in which anodic and cathodic peaks are located at the potentials of +0.05 V (vs. Ag/AgCl) , +0.33 V (vs. Ag/AgCl) and –0.42 V (vs. Ag/ AgCl), –0.72 V (vs. Ag/AgCl), respectively. These peaks are assignable to the oxidation of bismuth (Bi0/Bi+ and Bi+/Bi3+) and reduction of Bi species (Bi3+/Bi+ and Bi+/Bi0) [9]. In addition, cyclic voltammetry further applied to study the effect of scan rate at RGO/Bi–NRs modified GCE in pH 7 PB solution. At the various scan rates from 0.05 to 0.2 Vs−1, the redox
R. Devasenathipathy et al. Fig. 1 SEM images of Bi-NRs (a) and RGO/Bi–NRs (b). EDX spectra of Bi-NRs (c) and RGO/ Bi–NRs (d)
peaks current increases with scan rate increases. It can be observed that cyclic voltammograms of RGO/Bi–NRs hold a similar shape even at high scan rate which indicates that RGO/Bi–NRs possessed excellent electrochemical behavior.
Direct electrochemistry of GOx on RGO/Bi–NRs Figure 3a presents the cyclic voltammograms obtained at BiNRs/GOx (a), GO/GOx (b), RGO/GOx (c) and RGO/Bi– NRs/GOx (d) modified glassy carbon electrode (GCEs) in pH 7 (PB) at a scan rate of 50 mV/s. For the sake of clarity, electrochemical parameters of GOx such as anodic (Epa), cathodic potential (Epc), formal potential (E°), peak to peak separation (ΔEp), anodic peak current (Ipa) and cathodic peak current (Ipc) are given in Table 1. The electrochemical redox peak of the GOx indicates to the direct electron transfer (DET) of cofactor flavin adenine dinucleotide (FAD) at the modified GCEs. The DET ability of GOx at these modified electrodes is
Fig. 2 a XRD spectra of RGO and RGO/Bi–NRs. b Cyclic voltammograms obtained at RGO/Bi–NRs/GCE in 0.05 M PB solution (pH 7) at various scan rate from 50 to 200 mV s−1. Inset: Cyclic voltammograms of RGO/Bi–NRs
in the order of Bi-NRs/GOx < GO/GOx < RGO/GOx < RGO/ Bi–NRs/GOx. The Cyclic voltammograms of RGO/Bi–NRs/ GOx GCE exhibited well defined and highly enriched redox peak at E° of −0.4220 V corresponds to the direct electron transfer of GOx (FAD/FADH2). The peak-to-peak separation value (ΔEp) of the redox pair at the RGO/Bi–NRs/GOx/GCE was calculated to be 28 mV and highly enhanced redox Ipa, pc and low ΔEp revealed that RGO/Bi-NRs/GOx composite is an excellent electrode material for the immobilization of GOx. This can be attributed to the large surface area, high conductivity, good porosity and good biocompatibility of the RGOBi-NRs composite.
The effect of scan rate and pH at RGO-Bi–NRs/GOx/GCE Figure 3b displays the cyclic voltammograms of RGO-BiNRs/GOx modified GCE in pH 7 PB solution at various scan
Fig. 3 a Cyclic voltammograms obtained at Bi-NRs/GOx, GO/GOx, RGO/GOx and RGO/Bi–NRs/GOx film modified GCEs in PB solution of pH 7 at the scan rate of 50 mVs−1. b Cyclic voltammograms of RGO/ Bi–NRs/GOx film modified GCEs in PB solution (pH 7) at various scan rates from inner to outer are 100 to 1000 mV s−1
Electrochemically deposited Bi-NRs on RGO Table 1 Electrochemical parameters for the redox reaction of GOx at various modified electrodes
Electrode/GCE
Epa (V)
Epc (V)
E°′/V
ΔEp/mV
Ipa/μA
Ipc/μA
Bi-NRs/GOx GO/GOx RGO/GOx RGO/Bi–NRs/GOx
– −0.421 −0.4094 −0.4079
– −0.4611 −0.4421 −0.4365
– −0.4415 −0.4274 −0.4222
– 40.1 32.4 28.25
– 3.044 12.68 17.71
– –4.308 −11.30 −16.95
rates (ν) from 0.1 to 1 Vs−1 (curve a to j). Both the oxidation and reduction peaks current increase as scan rate increases. A plot of scan rates versus anodic (Ipa) and cathodic peak current (Ipc) exhibited linear relationship which indicates that both the oxidation and reduction of RGO-Bi-NRs/GOx are surface confined process (Fig. S2, ESM). The corresponding linear regression equations can be expressed as Eqs. 1 and 2
is Epa (V)=0.0556–0.0091 pH with a correlation coefficient of R2 =0.9933. Besides, the slope value of 55.6 mV is in close agreement with the theoretical value of 58.6 mV pH 1 for a reversible process, involving an equal number of protons and electrons during the electrochemical reaction [33].
For anodic process : I pa ðμAÞ ¼ 25:7ν V s−1 −9:28ðμAÞ : R2 ¼ 99:6
Electrocatalytic determination of glucose
For cathodic process : I pc ðμAÞ ¼ 23:07ν V s−1 −13:02ðμAÞ : R2 ¼ 99:3
ð1Þ
ð2Þ
The surface coverage ( ) of the electroactive GOx ( ) at the RGO-Bi-NRs composite film modified electrode surface was calculated by substituting the slope values of various scan rate in Eq. (3) (3)
Where, ν (V s−1) is the scan rate, A (cm2) is the electrode surface area and the constants R, T and F have their usual meanings (R=8.314 J k−1 mol−1, T=298 K, F=96,485 C mol−1). Assuming, the number of electrons (n) transferred as 2, the amount of electroactive GOx is calculated to be 8.54×10−10 mol cm−2 which is higher than the theoretical monolayer coverage of GOx [32]. The high surface coverage of GOx at the composite film modified electrode may be due to the presence of large number of active sites present on the surface of RGO-Bi-NRs/GCE. The apparent heterogeneous electron transfer rate constant (ks) for the direct electron transfer of GOx was calculated to be 4.407 s−1 by adopting the following Eq. (4) [32] LogKS ¼ αlogð1−αÞ þ ð1−αÞlogα−log
RT nFν
The cyclic voltammograms of RGO-Bi-NRs/GOx modified GCE in the presence of N2 (curve a) and O2 (curve b) are presented in the Fig. S3 (ESM). The Ipc of the RGO-Bi-NRs/GOx modified GCE increased dramatically in the presence of O2 PB solution (pH 7). Whereas, in the presence of N2, there is no changes observed at the RGO-Bi-NRs/GOx, which indicates that GOx is highly reactive in the presence of O2. The determination of glucose at RGO-Bi-NRs/GOx was obtained by the linear sweep voltammograms in O2 saturated PB solution (pH 7). A sharp cathodic peak at −0.484 V (vs. Ag/AgCl) was observed in oxygen saturated PB solution (Fig. 5a). The cathodic current of RGO-Bi-NRs/GOx linearly decreased for the each addition of glucose in the linear range from 0.5 to 6.0 mM because the decrease in oxygen content upon addition of glucose [34]. Upon increasing the concentration of glucose, the cathodic peak potential was shifted towards the positive direction, which is attributed to the direct electron transfer of GOx being effectively participating in the glucose oxidation. A calibration plot between the glucose and I pc gave the linear response with the correlation
ð4Þ
The redox couple of GOx at the RGO-Bi-NRs/GOx modified film exhibit stable and well defined redox peaks over the pH range of 3 to 11 (Fig. 4a). The plot of pH versus E° (Fig. 4b) exhibits a linear dependence over the entire pH range. The linear regression equation
Fig. 4 a Cyclic voltammograms obtained at RGO/Bi–NRs/GOx film modified GCE in various PB buffer solutions (pH 3–11) at Scan rate 50 mV s−1. b Calibration Plot of pH vs. E°
R. Devasenathipathy et al.
bismuth has high stable electrochemical behavior in the negative potential region than the positive potential region, since bismuth is deposited as nanoribbons, the active surface are of the materials must be higher and therefore, it provides an excellent platform for immobilization of glucose oxidase and shows the higher performance towards the determination of glucose than the other modified electrodes. Fig. 5 a Linear sweep voltammograms obtained at RGO/Bi–NRs/GOx modified GCE in the absence (a) and presence of each addition 0.5 mM glucose in oxygen saturated PB solution (pH 7) at a scan rate of 50 mVs−1. b The calibration plot for the linear dependence of [glucose] vs. Ipc
coefficient of 0.995 (Fig. 5b) and the respective linear regression equation can be expressed as Eq. 5 I p ðμAÞ ¼ 2:77½glucose μA mM −1 þ 0:7329ðμAÞ; R2 ¼ 0:995
ð5Þ
Sensitivity of the sensor was calculated to be 39.57 μA mM−1 cm−2 from the slope of the calibration plot. Limit of detection (LOD) was calculated to be 0.104 μM using the formula, LOD=3 sb/S. Here, sb is the standard deviation of blank signal and S is the slope of the sensor. The sensor possesses excellent analytical parameters such as low limit of detection, high sensitivity and linear range towards determination of glucose. Table 2 lists the important electroanalytical parameters compared with other glucose sensors. The Table 2 shows that the GR/Bi–NRs/GOx composite modified electrode exhibits quite comparable or better performance than previously reported other modified electrodes. Because the
Table 2 Comparison of the analytical performance of the GR/ Bi–NRs/GOx modified electrode with other GOx modified electrodes reported previously
Selectivity, stability, repeatability and reproducibility The selectivity of the proposed biosensor towards determination of glucose has been investigated in the presence of 1 mM biologically active molecule such as dopamine, ascorbic acid, uric acid and acetaminophen. For the sake of simplicity, interference compound, relative error and relative standard deviation are given in Table S1 (ESM). It is evident that the potentially interfering biologically active molecules show low effect (
