The Immobilization of Glucose Oxidase at Manganese Dioxide

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The Immobilization of Glucose Oxidase at Manganese Dioxide Particles-Decorated Reduced Graphene Oxide Sheets for the Fabrication of a Glucose Biosensor A. T. Ezhil Vilian,† Veerappan Mani,† Shen-Ming Chen,*,† Bose Dinesh,‡ and Sheng-Tung Huang*,† †

Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No.1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan, R.O.C ‡ Department of Materials Science, School of Chemistry, Madurai Kamaraj University, Madurai, Tamilnadu 625 021, India S Supporting Information *

ABSTRACT: Here, we describe a simple approach for the efficient immobilization of poly-L-lysine (PLL) impregnated glucose oxidase (GOx) at a manganese dioxide (MnO2)-decorated chemically reduced graphene oxide (CRGO) composite film modified electrode. The MnO2/CRGO film was analyzed by various techniques such as scanning electron microscopy, UV−visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and electrochemical impedance spectroscopy. MnO2 particles are uniformly deposited at the CRGO surface through an easily adoptable electropolymerization method. The asprepared (GOx-PLL/MnO2/CRGO) composite film exhibited a surface coverage concentration (Γ) of 2.68 × 10−10 mol/cm−2, indicative of high GOx loading. The heterogeneous rate constant for the redox reaction of GOx has been calculated to be 4.92 s−1. A glucose biosensor was fabricated which exhibits a wide linear range from 0.04 to 10 mM with a limit of detection of 0.02 mM (S/N = 3). The proposed inexpensive, simple, and sensitive sensing approach based on the MnO2/CRGO nanocomposite electrode can be a promising method for the determination of glucose present in the clinical and environmental food analysis. sensors.12−14 The positively charged biomolecules improve the microenvironment to provide a suitable enzyme immobilization matrix. This method can be adapted to find better enzyme immobilization strategies.15 Graphene oxide (GO), an oxygenated derivative of graphene is the adoptable precursor for the preparation of graphenebased electrodes owing to its significant advantages such as inexpensive production from graphite, easy processing in aqueous dispersion, and available sites for functionalization.16,17 GO can be prepared by the oxidation of graphite with subsequent exfoliation through ultrasonication.18 Manganese dioxide (MnO2) and its derivatives have attracted enormous attention because of their excellent physicochemical properties, and they find widespread applications in diverse research areas.19,20 Different preparation methods could produce different morphologies which are highly useful for various kinds of electrochemical applications.21 The development of graphene and MnO2 composites was reported recently for highperformance electrochemical capacitors22 and supercapacitors23−25 applications. However, very few reports are available about the electrodeposition of MnO2 particles at the graphene surface. Nevertheless, electrodeposition is an attractive and easy strategy for the preparation of highly stable and uniformly arranged particles on the electrode surface. Herein, we have used a simple electrodeposition approach for the preparation of MnO2 particles at the chemically reduced graphene oxide

1. INTRODUCTION In the past few years, the incidence of diabetes mellitus has been rising, and it has now become a serious public health problem worldwide. To facilitate rapid response to variations in the body’s glucose levels and detection of this condition, highly sensitive and selective biosensors are being developed.1 Recent interest has been focused toward development of glucose oxidase (GOx) incorporated biosensors for the determination of glucose concentration, and this continues to be the chief model for the fabrication of next generation sensors.2 However, at the present time, the development of amperometric biosensors remains challenging because of the issues associated with enzyme immobilization and ensuring efficient electrical communication between the redox sites of enzymes and electrodes.3,4 Electron transport between the active sites of GOx and the electrode is difficult since the flavin adenine dinucleotide (FAD) group is deeply buried within the interior of the protein backbone5 The direct electrocatalytic oxidation of glucose that occurs in enzyme free glucose sensors is thus stimulating keen interest.6 Recently, efforts have been focused on the use of poly-Llysine (PLL) as electrode material for various applications.7 PLL is a polycationic homopolymer and it has been shown to be a good support for the protection of biomolecules.7,8 The successful attachment of PLL with the graphene sheets is possible via a covalent approach. PLL-based nanocomposites are considered to be excellent alternatives to conventional materials for the manufacture of electrochemical sensors attributed to the presence of various functional groups.10 They have also been widely utilized to modify the working electrode used for electrochemical detection11 and in DNA © 2014 American Chemical Society

Received: Revised: Accepted: Published: 15582

June 16, 2014 August 13, 2014 September 12, 2014 September 12, 2014 dx.doi.org/10.1021/ie502430d | Ind. Eng. Chem. Res. 2014, 53, 15582−15589

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wt %). Finally, the solid suspension consigned as graphite oxide was washed with a 2 M HCl solution and then washed 3−4 times with ethanol and dried overnight. The graphite oxide dispersion (1 mg/mL in water) was exfoliated to GO through ultrasonication for 1 h. The unexfoliated graphite oxide was removed by centrifugation at 3000 rpm for 30 min. Then 400 mg of NaBH4 was slowly added to the GO dispersion (20 mL, 1 mg/mL) with vigorous stirring, and then the reaction mixture remained stirred for 20 h. Thereafter, the CRGO was separated through filtration, washed with water and ethanol, and dried at 60 °C for 48 h in a vacuum oven. Finally, the as-prepared CRGO powder was redispersed in DMF (1 mg/mL) through ultrasonic agitation. 2.4. Fabrication of the Glucose Biosensors. Before starting each experiment, GCE was carefully polished utilizing 0.05 mm alumina slurry, and then subjected to ultrasonication in ethanol, and water for 5 min. A 5 μL aliquot of CRGO (1 mg/mL) was spread at the GCE surface and dried at room temperature. The electrochemical deposition of MnO2 particles onto the CRGO-modified electrode surface was also performed following a previously reported procedure.27 In a typical preparation process, MnO2 was deposited at the CRGO/GCE from 10 mM of KMnO4 (5 mL) present in the 0.04 M H2SO4 aqueous solution. The scan was performed between the potentials of 0.5 V and −0.3 V at the scan rate of 2 mV s−1 for eight cycles. After electrodeposition, a 5 μL dispersion of GOx (10 mg/mL in PBS, pH 7) was vigorously mixed together with PLL in PBS (pH 7) solution and stored in a refrigerator for at least 24 h. Then, 5 μL from the mixture of GOx and PLL (1:10) was dropped onto the MnO2/CRGO/GCE and allowed to dry at ambient conditions. Finally, 5 μL (0.5%) of nafion solution was drop casted on the GOx-PLL/MnO2/CRGO film modified electrode and allowed to dry. The control electrodes such as bare GCE, GOx-PLL/MnO2/GCE, GOx-PLL/CRGO/ GCE, CRGO/GCE, and CRGO/MnO2 /GCE are also prepared accordingly. A schematic illustration of the entire electrode fabrication procedure has been given as Scheme 1.

(CRGO) surface. The electrodeposition method reported involves very simple, fast, and easily adoptable protocols for the preparation of highly ordered and uniform MnO2 particles and does not require high temperature, any chemical agents, difficult protocols, or additional equipment. In this present study, for the first time, we have prepared MnO2/CRGO film modified GCEs, where GOx-PLL was adsorbed on the nanocomposite film. We found that the MnO2/CRGO/GCE nanocomposite film provided a greatly favorable microenvironment for GOx to retain its redox activity. The GOx-PLL/MnO2/CRGO/GCE film modified electrode has the advantage of facilitating electrochemical detection for glucose sensing as demonstrated by the improved linear range, low detection limit, high sensitivity, rapid response time, reproducibility, selectivity, and stability, which were all investigated in detail.

2. EXPERIMENTAL SECTION 2.1. Materials. GOx (type x-s from Aspergillus niger), Na2HPO4, and NaH2PO4, N, N-dimethylformamide (DMF), and PLL were purchased from Sigma and used without further purification. KMnO4 reagent was acquired from Shimakyu, Osaka, Japan. The GOx stock solution was prepared by using a 0.05 M phosphate buffer solution (PBS, pH 7) and stored at 4 °C. Phosphate buffer solution (PBS, 0.05 M) was prepared from Na2HPO4 and NaH2PO4 and used as supporting electrolyte. Double-distilled water was used for the preparation of all the solutions. 2.2. Equipment. Cyclic voltammetry and differential pulse voltammetry experiments were performed using CHI electrochemical workstation (CHI 405A). A three-electrode system comprised modified GCE (area 0.07 cm2) as working electrode, Pt as a counter electrode, Ag/AgCl (saturated KCl) as a reference electrode. Electrochemical impedance spectroscopy (EIS) analysis was performed at a frequency range of 0.1 Hz to 1 MHz with a ZAHNER instrument (Kroanch, Germany). The structures of the samples were characterized by SEM measurements with a Hitachi S-3000 H, and energy-dispersive X-ray spectroscopy (EDX) was recorded using a HORIBA EMAX X-ACT model 51-ADD0009. XRD analysis was performed with an XPERT-PRO diffractometer (PANalytical B.V., The Netherlands) using Cu Kα radiation (k = 1.54 Å). Fourier transform infrared (FT-IR) measurements were performed using a Perkin Elmer spectrophotometer RXI. Hitachi U-3300 spectrophotometer was used to carry out UV−visible absorption spectroscopy measurements. 2.3. Preparation of GO. The GO was prepared by following the modified Hummer’s method26 whereby 1 g of graphite (graphite powder, < 20 μm, Aldrich) was suspended in 2.5 g of K2S2O8 and 46 mL of H2SO4 and then stirred in a round-bottom flask at 0 °C for 15 min. Next, 2.5 g of P2O5 was added into the mixture over 15 min in order to avoid a temperature spike, and the mixture was left to be stirred vigorously for 6 h at 20 °C. On completion, the mixture was poured, diluted by adding it to 1000 mL of water, and filtered. Afterward, 6 g of KMnO4 and 1 g of NaNO3 in 31.2 mL of water was added gradually, while the temperature was managed to be less than 20 °C. The reaction occurred as the mixture was stirred at 35 °C for 2 h. Then, 500 mL of distilled water was added, and the temperature was maintained to be less than 50 °C. The solution was kept at this temperature for 2 h. The color of the resulting solution was changed to brilliant yellow upon the addition of 250 mL of water and 6 mL of H2O2 (30

3. RESULTS AND DISCUSSION 3.1. Structural Characterization of the MnO2/CRGO and GOx-PLL/MnO2/CRGO Composites Film. The XRD Scheme 1. Schematic Representation of the Preparation of the GOx-PLL/MnO2/CRGO/GCE and Its Application for the Determination of Glucose

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Figure 1. (A) XRD patterns of (a) MnO2/ITO, (b) MnO2/CRGO/ITO composite; (B) FTIR spectra of (a) GO, (b) MnO2/CRGO composite; and (C) FTIR spectra of (a) GOx, (b) GOx-PLL/MnO2/CRGO modified electrode; (D) UV−vis absorption spectra of (a) GOx, (b) GOx-PLL, (c) GOx-PLL/MnO2/CRGO composite.

for N−H stretching, and two peaks centered at 1650 and 1544 cm−1 are assigned to the characteristic amide I and II absorption bands of GOx. The peak appearing around 1082 cm−1 was assigned to the stretching mode of C−O of GOx (Figure 1Ca). The FT-IR spectrum of GOx-PLL/MnO2/ CRGO nanocomposites (see Figure 1 Cb) also presents two typical adsorption bands of GOx at the wavenumber of 1648 and 1558 cm−1 revealing the successful immobilization of GOx.30 UV−visible and FT-IR spectroscopy were utilized to examine the structural information about the GOx. Figure 1D shows the typical UV−visible absorption spectra for GOx (a), GOx-PLL (b), and GOx-PLL/MnO2/CRGO (c). There are sharp peaks at 380 nm and 453 nm indicating the presence of GOx.31,32 It can be seen in Figure 1Dc that the positively charged GOx-PLL was adsorbed on the MnO2/CRGO surface, suggesting that the GOx-PLL was immobilized in the MnO2/CRGO film and retained its native structure. 3.2. Surface Morphological Characterization of MnO2/ CRGO Composite. The morphology and microstructure were examined through scanning electron microscope (SEM) experiments. The SEM image of CRGO shows the characteristic crumpled and wrinkled sheet-like morphology of CRGO (Figure 2A). Figure 2B shows typical SEM images of the MnO2 particles showing the spherical shape, which was very consistent with previous results obtained with the electrochemical approach. The inset shows that the results of the EDX analysis confirm that the MnO2 particles consisted of Mn and O. Furthermore, Figure 2C displays a representative SEM image of

patterns of the MnO2 particles are shown in Figure 1A,a. The XRD patterns of the as-prepared samples exhibit three characteristic diffraction peaks around 39.7°, 46.2°, and 67.4° corresponding to (100), (101), and (102), respectively, and the (110) of MnO2 (JCPDS Card No. 44-0141), which is confirmed by the SEM micrographs (see Figure 1A). This can be clearly seen in the XRD patterns of the as-synthesized MnO2/CRGO composites shown in Figure 1A,b. Graphite showed a very sharp diffraction peak at 22.93° which corresponds to the (002) suggesting that the CRGO was fully reduced. The peaks at 2θ° = 24.4°, 32.2°, 39.7°, 46.2°, and 67.4° can be assigned to the (100), (101), (102), and (110) crystalline plane diffraction peaks, respectively, suggesting that the MnO2 had been successfully deposited on the surface of the CRGO Sheet.28 FT-IR spectroscopy was used to characterize the samples further and confirmed the presence of MnO2 in the MnO2/ CRGO nanocomposites. The results of the analysis of GO, MnO2/CRGO, GOx, and GOx-PLL/MnO2/CRGO nanocomposites by FT-IR are shown in Figure 1B,C. In the FTIR spectrum of GO, the absorption peaks appearing at the wavenumber of 1743 cm−1 and 1637 cm−1 are assigned the CO stretching and CC stretching vibrations, respectively (see curve a).29 In contrast, the peaks at 1731 and 1183 cm−1 are missing from the FTIR spectrum of the MnO2/CRGO nanocomposites, which indicates a reduction of GO and its transformation into CRGO (see curve b). The FT-IR spectrum of GOx exhibits the characteristic peaks of native GOx (see Figure 1C,a). The intense absorption at 3409 cm−1 is assigned 15584

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(d) GOx-PLL/MnO2/CRGO electrodes and (e) GOx-PLL films modified GCEs. The semicircular diameter of the EIS curve corresponds to the electron-transfer resistance (Ret). According to experimental data based on equivalent circuits (Supporting Information, Table S1), the Ret of the MnO2/ CRGO/GCE is lower than that of the bare GCE and CRGO/ GCE (curve b). This means that MnO2 particles were uniformly distributed over the surface of the CRGO surface which could lead to faster charge transfer in the MnO2/CRGO film. After the GOx-PLL was coated on the bare electrode, there was an increase in Ret (curve e) owing to the immobilization of GOx-PLL on the bare GCE surface. After GOx-PLL was coated on the MnO2/CRGO modified electrode, the minimum resistance value changed (curve d), suggesting that the MnO2/CRGO accelerated the electron transfer between electrode surface and the redox probe. The low Ret might also result from the electrostatic repulsion between the negatively charged redox probe and positively charged GOxPLL/MnO2/CRGO/GCE. This result shows the successful fabrication of a modified electrode and confirms immobilization of GOx on PLL/MnO2/CRGO. 3.4. Direct Electrochemistry of GOx at PLL-MnO2/ CRGO Film Modified Electrode. Figure 3B shows the cyclic voltammograms (CVs) obtained at GOx-PLL/MnO2/CRGO and control electrodes in nitrogen saturated PBS. The absence of observable voltammetric peaks at the bare GCE, CRGO, and MnO2/CRGO indicating that no redox reaction took place. The background current of GOx-PLL/MnO2/CRGO film modified electrode shows good conductive performance which assists in faster transportation than that of the MnO2/ CRGO/GCE. This could be attributed to the large surface area of MnO2/CRGO. In fact, the high electroactive area of MnO2/ CRGO induces a high background current, and MnO2 may enable the accumulation of negatively charged GOx. GOx-PLL immobilized on the surface of the MnO2/CRGO displays a

Figure 2. SEM images of (A) CRGO, (B) MnO2, (C) MnO2/CRGO and (D) GOx-PLL/MnO2/CRGO composite.

the MnO2/CRGO/ITO film. The spherical morphology intrinsic to the group can be observed. The surface of the CRGO sheets is uniformly decorated with MnO2 particles. This clearly indicates the suitability of the MnO2/CRGO modified electrode for electroanalysis. The corresponding EDX patterns for the MnO2 deposited on the CRGO/ITO surface for each case are shown in Figure 2C (see inset image). From these results, we can conclude that the MnO2 particles completely covered and were uniformly distributed on the surface of the CRGO after this process, and they also maintained a high surface area. After the immobilization of GOx-PLL on the MnO2/CRGO there was an obvious change in the corresponding SEM image. The relatively blurry surface, as shown in Figure 2D, suggests that the GOx-PLL has been effectively immobilized on the surface of the MnO2/CRGO film. 3.3. EIS Measurements. Figure 3A displays the Nyquist plots of (a) bare/GCE, (b) CRGO/GCE, (c) MnO2/CRGO,

Figure 3. (A) EIS spectra of (a) bare/GCE, (b) CRGO/GCE, (c) MnO2/CRGO/GCE, (d) GOx-PLL/MnO2/CRGO/GCE electrodes, (e) GOxPLL/GCE electrodes in 5 mM Fe2 (CN)63−/4− in PBS; frequency range, 0.1 Hz to 1 MHz. Inset: Randles equivalence circuit. (B) CVs of (a) bare/ GCE, (b) GOx-PLL/MnO2/GCE, (c) GOx-PLL/CRGO/GCE, (d) CRGO/GCE, (e) CRGO/MnO2/GCE, and (f) GOx-PLL/CRGO-MnO2 electrodes in a 0.05 M deoxygenated PBS (pH 7) at the scan rate of 50 mV s−1. (C) CVs of GOx-PLL/CRGO-MnO2/GCE at different scan rates (from inner to outer: 10−100 mV s−1). (D) Plots of the anodic and cathodic peak currents vs the scan rates. 15585

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Figure 4. (A) CVs of MnO2/CRGO modified GCE in different pH solutions from a to d (3, 5, 7, 9), at a scan rate of 0.5 V s−1. (B) Plot of the formal potential vs value. (C) CVs of MnO2/CRGO-modified GCE electrodes in N2-saturated 0.05 M PBS (pH 7.0) with different concentrations of H2O2 (0, 1, 2, 3, mM). Scan rate 0.5 V s−1. (D) Typical amperometric response of MnO2/CRGO nanostructure modified electrode after successive injections of 0.05 mM and 0.5 mM H2O2 to the stirred 0.05 M deoxygenated PBS (pH 7.0). Applied potential: −0.45 V. Inset: plot of response current versus [H2O2].

either the other modified electrodes or the theoretical monolayer value (2.86 × 10−12 mol cm−2) indicating the effective immobilization of GOx on the MnO2/CRGO film. The electron transfer rate constant (ks) of GOx at the modified electrode can be calculated using Laviron’s equation.34

couple of well-defined quasi-reversible redox peaks with formal potential (E0′) of −0.430 V which revealing the attainment of direct electrochemistry of GOx. Nevertheless, the utilization of GOx-PLL on the surface of the MnO2/CRGO/GCE exhibited a different electrochemical behavior. Eventhough, GOx-PLL/ MnO2 and GOx-PLL/CRGO modified electrode are presented minimal redox peaks, they possess very short period of stability compared to the final GOx-PLL/MnO2/CRGO composite. The enhanced electrochemical responses of GOx at the GOxPLL/MnO2/CRGO should be ascribed to the efficient immobilization of GOx at this film. PLL is a cationic film, which can facilitate the accumulation of negatively charged GOx on the electrode surface. Moreover, MnO2/CRGO contains a negatively charged surface which also help to immobilize GOx at the electrode surface through electrostatic interaction. The CVs obtained at the GOx-PLL/MnO2/CRGO for various scan rates from 0.01 V s−1 to 0.1 V s−1 are shown in Figure 3C. Both anodic peak current (Ipa) and cathodic peak current (Ipc) of the redox peak were linearly increased when increasing the scan rate. In addition, E0′ of the redox peaks was also shifted when scan rates increased. Figure 3C implies that the redox process of the GOx occurring at the GOx-PLL/ MnO2/CRGO/GCE is a surface-controlled process. The surface coverage concentration of GOx (Γ) can be calculated using the following equation:33 Ip = n2F 2vA Γ/4RT

log K s = α log(l − α) + (l − α) log α − log(RT /nFv) − α(l − α)nF ΔEp/2.3RT

(2)

where α (≈ 0.5), R and T are representing the charge transfer coefficient, universal gas constant, and temperature. By substituting all the parameters into eq 2, the value of ks was calculated to be 4.92 s−1. The ks value of 4.92 s−1 for GOx-PLL/ MnO2/CRGO is comparatively larger than that obtained for GOx immobilized on common graphene (2.83 s−1),35 GOx/ RGO (4.8 s−1),36 CNT (2.76 s−1),37 multiwalled carbon nanotubes/electrochemically reduced graphene oxide (ERGO) (3.02 s−1),33 mesoporous carbon (4.09 s−1),38 mesoporous silica (3.89 s−1),39 graphene/nafion (3.42 s−1),40 CdS nanoparticles (1.56 s−1),41 ERGO/poly L-lysine (3.27 s−1),42 or graphene/nafion/Au (1.96 s−1).43 From these results, it can be concluded that the GOx-PLL/MnO2/CRGO film provides an efficient platform for the immobilization of large quantities of GOx. 3.5. Effect of pH. The pH of the electrolyte is one of the crucial parameters affecting the activity and stability of the resulting modified electrode. Therefore, we have investigated the effect of pH on the redox reaction of GOx. The CVs of GOx-PLL/MnO2/CRGO show well-defined redox peaks, and both Ipa and Ipc were shifted negatively with an increase in pH (see Figure 4A). A plot of E0′ versus pH shows a linear relationship as can be seen from Figure 4B. The linear regression equation can be expressed as E0′ = −0.054 pH − 0.057, R2 = 0.994. The obtained slope value is very close to the expected slope value of −0.058 V given by the Nernst equation

(1)

where, n, A, and ν represent the number of electrons, area of the electrode (cm2), and scan rate (V s−1). The constants R, T, and F have their usual meanings. The Γ values for GOx at the GOx-PLL/MnO2/CRGO/GCE and GOx-PLL/CRGO/GCE electrodes were estimated to be 2.68 × 10−10 mol/cm−2 and 1.42 × 10−10 mol/cm−2, respectively. The Γ value of GOx observed at the GOx-PLL/MnO2/CRGO/GCE is higher than 15586

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Figure 5. (A) CVs of GOx-PLL/MnO2/CRGO/GCE in oxygen saturated 0.05 M pH 7.0 PBS containing (a) 0 mM, (b) 1 mM, (c) 2 mM, (d) 3 mM, (e) 4 mM, (f) 5 mM, and (g) 6 mM glucose at a scan rate of 50 mV s−1; (B) DPVs obtained at GOx-PLL/MnO2/CRGO/GCE in oxygen saturated PBS (pH 7) in the absence (a) and presence of various concentrations of glucose (from a to j); (C) [glucose] vs electrocatalytic current.

3.7. Determination of Glucose. The biosensor activity of the fabricated biosensor has been studied through oxygen reductive detection of glucose. Figure 5A presents the CVs obtained at the GOx-PLL/MnO2/CRGO/GCE in oxygen saturated PBS (pH 7) in the absence (a) and presence of glucose (b to g; each 1 mM). When glucose was added to the oxygen saturated PBS, the Ipc was decreased because the reduction of oxygen occurred via oxidation of glucose catalyzed by the GOx as expressed in eq 5−7. Further additions of glucose lead to the linear decrease of Ipc which is suitable for the determination of glucose,

for the quasireversible reaction involving two electrons and two protons.33 We choose pH 7 to carry out electrochemical studies since neutral pH is suitable for the practicality analysis in the biological samples. 3.6. Electrocatalytic Reduction of Hydrogen Peroxide at MnO2/CRGO/GCE. The electrocatalytic behavior of the MnO2/CRGO/GCE toward reduction of hydrogen peroxide (H2O2) has been investigated. The CVs obtained at the MnO2/ CRGO/GCE in the deoxygenated PBS (pH 7) with and without the addition of H2O2 are shown in Figure 4C. With the gradual addition of H2O2, there was a steady increase in the Ipc and decrease in the Ipa of the MnO2/CRGO modified electrode. A large Ipc was observed for the MnO2/CRGO/ GCE in comparison with the bare electrode (Figure 4C). The MnO2/CRGO exhibited significantly enhanced electrocatalytic ability for the reduction of H2O2 which might be attributed to the excellent synergy between MnO2 and CRGO. The electrochemical pathway responsible for catalyzing the H2O2 at the MnO2/CRGO electrode can be described as follows,44 MnO2 + H 2O2 → MnO + H 2O + O2

(3)

MnO → MnO2+2e−

(4)

glucose + GOx(FAD) → gluconolactone + GOx(FADH 2)

(5)

GOx(FADH 2) + O2 → GOx(FAD) + H 2O2

(6)

O2 + 4H+ + 4e− → 2H 2O

(7)

To investigate the role of MnO2 in the determination of glucose, we have fabricated an electrode without incorporating MnO2 (GOx-PLL/CRGO/GCE) and tested its biosensing ability toward determination of glucose (Supporting Information, Figure S1). As evident from the Figure S1, minimal decrease in the Ipc was observed at GOx-PLL/CRGO/GCE for the each addition of glucose, however greatly improved response was observed at GOx-PLL/CRGO/GCE (Figure 5). These discussions are clearly revealing that MnO2 play critical role in both direct electrochemistry of GOx and also in the biosensor performance. Figure 5B illustrates typical differential pulse voltammetry (DPV) curves obtained at the GOx-PLL/ CRGO/GCE upon each sequential addition of glucose to the oxygen saturated PBS (pH 7) in the absence and presence of glucose. The Ipc responsible for the reduction of oxygen was increase for each sequential addition of glucose. A calibration plot was made between concentration of glucose and response current which exhibited linear behavior (Figure 5A), while the

The amperometric performance of the MnO2/CRGO film modified rotating disc electrode (with a rotation speed of 1300 rpm) is measured after each successive addition of H2O2 to PBS (pH 7) at an applied potential (Eapp) of −0.45 V (Figure 4D). The amperometric responses are linearly increased with H2O2 concentration. The linear range was found to be from 0.05 μM to 7 mM and the sensitivity was calculated to be 46.36 mAcm−2 mM−1. The limit of detection (LOD) was calculated to be as low as 0.01 μM (signal-to-noise ratio, S/N = 3). The proposed MnO2/CRGO sensor exhibited higher sensitivity, lower LOD, wide working linear range, and less overpotential. The excellent performance of the MnO2/CRGO/GCE shows promise for the development of electrochemical sensors for H2O2 detection. 15587

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linear regression equation can be expressed as, I (μA) = 12.288 + 0.986Cg (mM), R = 0.9936, where I is the response current and Cg is the concentration of glucose. The linear concentration range was found between 0.04 mM and 10 mM of glucose. The LOD was calculated to be 0.02 μM (S/N = 3), and the sensitivity was calculated to be 28.8 μA/mM cm2. The important analytical parameters were compared with those obtained for other reported sensors available in the literature (Supporting Information, Table S2). As can be seen from Table S2, the sensor performance of the GOx-PLL/CRGO/GCE was quite comparable with previous reports and revealed the good capability of the sensor toward the determination of glucose. This can be attributed to the excellent electrocatalytic ability of the GOx-PLL/CRGO/GCE modified electrode leading to the efficient reduction of oxygen, which indirectly assists in monitoring the concentration of glucose. Selectivity of the GOx-PLL/CRGO-MnO2/GCE has been investigated in the presence of interferences including dopamine, uric acid, ascorbic acid, and acetaminophen. DPV experiments have been carried out in oxygen-saturated PBS containing glucose (1 mM) and the above-mentioned interferences (1 mM). The amount of interference percentage has been calculated from the ratio between response current obtained in the presence of glucose and that obtained in the presence of respective interfering agents. As can be seen from Supporting Information, Table S3, all the interference agents have shown less than 5% interference with the glucose determination at the GOx-PLL/CRGO-MnO2/GCE revealing the appreciable selectivity of the modified electrode. 3.8. Real Sample Analysis. The practicality of the biosensor was tested by determining the amount of glucose present in human blood serum sample. Fresh human serum samples were supplied from the local hospital, Taipei, Taiwan. The real samples were diluted with a known amount of PBS (pH 7), and the DPV experiments were carried out using similar experimental conditions of the lab sample. The glucose content present in the serum sample was calculated from the DPVs, and the results are summarized in Supporting Information, Table S4. The found and recovery results are satisfactory and agree very closely with the hospital data. This indicates that the GOx-PLL/MnO2/CRGO/GCE film is a suitable candidate for the determination of glucose present in clinical samples. 3.9. Reproducibility, Repeatability, and Stability Determination. This biosensor shows acceptable reproducibility with a relative standard deviation (R.S.D.) of 5.2% for the determination of glucose (1 mM) at four different modified electrodes. In addition, the biosensor shows appreciable repeatability with an R.S.D. of 3.1% for the five repetitive measurements. The storage stability of the sensor was studied over 20 days by monitoring the response currents toward the detection of glucose. Only 4.02% of the initial response current was decreased even after continuous scanning for 100 cycles, indicating good stability of the sensor.

mM to 10 mM and low LOD of 0.02 mM. The biosensor possesses appreciable reproducibility, repeatability, and longterm stability. Additionally, a nonenzymatic amperometric sensor was demonstrated at MnO2/CRGO/GCE for the sensitive determination of H2O2. The preparation protocols for the biosensor fabrication are simple and convenient, not requiring toxic reagents and not involving any complex multistep process. The results demonstrate that the GOxPLL/MnO2/CRGO-modified electrode is a promising architecture for further development of microelectronic devices.

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ASSOCIATED CONTENT

S Supporting Information *

The cyclic voltammetry curves for the determination of glucose at GOx-PLL/CRGO/GCE (Figure S1), electrochemical impedance spectroscopy parameters (Table S1), comparison table with references (Table S2), data for the selectivity studies (Table S3), and real sample analysis (Table S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*S.M.C. E-mail: [email protected]. Tel: +886 2270 17147. Fax: +886 2270 25238. *S.-T.H. E-mail: [email protected]. Tel.: +886 2271-2171 2525. Fax: +886-02-2731-7117. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology, Taiwan (Republic of China).

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REFERENCES

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4. CONCLUSION A PLL/MnO2/CRGO film was used as a suitable matrix for the immobilization of GOx which was found to efficiently accelerate the direct electrochemistry of GOx. The GOx immobilized at the MnO2/CRGO composite film exhibited significantly improved electrocatalytic ability toward oxidation of glucose via reduction of oxygen. The proposed GOx-PLL/ MnO2/CRGO sensor exhibited a wide linear range from 0.04 15588

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Industrial & Engineering Chemistry Research

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