chitosan composite film on glassy carbon electrodes and its biosensing application

Bioelectrochemistry 74 (2009) 246–253

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Bioelectrochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o e l e c h e m

Direct electrochemistry of glucose oxidase immobilized on NdPO4 nanoparticles/ chitosan composite film on glassy carbon electrodes and its biosensing application Qinglin Sheng, Kai Luo, Lei Li, Jianbin Zheng ⁎ Institute of Analytical Science/Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi'an, Shaanxi 710069, China

a r t i c l e

i n f o

Article history: Received 15 November 2007 Received in revised form 21 August 2008 Accepted 26 August 2008 Available online 6 September 2008 Keywords: Glucose oxidase Chitosan NdPO4 nanoparticles Direct electrochemistry Biosensor

a b s t r a c t The direct electrochemistry of glucose oxidase (GOx) immobilized on a composite matrix based on chitosan (CHIT) and NdPO4 nanoparticles (NPs) underlying on glassy carbon electrode (GCE) was achieved. The cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the modified electrode. In deaerated buffer solutions, the cyclic voltammetry of the composite films of GOx/NdPO4 NPs/CHIT showed a pair of well-behaved redox peaks that are assigned to the redox reaction of GOx, confirming the effective immobilization of GOx on the composite film. The electron transfer rate constant was estimated to be 5.0 s− 1. The linear dynamic range for the detection of glucose was 0.15–10 mM with a correlation coefficient of 0.999 and the detection limit was estimated at about 0.08 mM (S/N = 3). The calculated apparent Michaelis–Menten constant was 2.5 mM, which suggested a high affinity of the enzyme-substrate. The immobilized GOx in the NdPO4 NPs/CHIT composite film retained its bioactivity. Furthermore, the method presented here can be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins. © 2008 Elsevier B.V. All rights reserved.

1. Introduction In recent years, direct electrochemistry of biologically important proteins has been extensively studied due to its significance in both theoretical and practical application [1,2]. The direct electron transfer of redox proteins can be applied to the study of physiological electron transfer process, the structures and mechanisms of different enzymatic reactions in biological systems as well as for the design of labelfree biosensors, and biofuel cells [3,4]. However, it is difficult to process the electron transfer between redox proteins and electrodes directly, since the three-dimensional structure of proteins hinders interaction with the electrode or due to its denaturation upon adsorption onto the surface of the electrode and subsequent passivation of the electrode surface [5]. Glucose oxidase (GOx), containing a flavin adenine dinucleotide (FAD) redox center that catalyzes the electron transfer from glucose to gluconlactone, has been extensively used to monitor the glucose levels in diabetics. However, direct electron transfer between GOx and electrode can not be achieved easily. Nanoparticles (NPs) have attracted more and more attention because of their biocompatibility [6]. Among various kinds of NPs, Au NPs [7–9] and carbon nanotube (CNT) [2,10–14] are the most favorable materials that were used to promote the electron transfer of redox proteins, since the unique

⁎ Corresponding author. Tel.: +86 29 88302077; fax: +86 29 88303448. E-mail address: [email protected] (J. Zheng). 1567-5394/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2008.08.007

electronic and structure properties of NPs allow good communication between NPs and redox proteins [15,16]. Wu and Hu [17] have reported the direct electrochemistry of GOx immobilized within the Au NPs-dihexadecylphosphate (DHP) composite film and the proposed biosensor was used for glucose determination. Salimi [18] et al. has reported the fabrication of glucose biosensor by immobilizing GOx into a sol–gel composite at the surface of a basal plane pyrolytic graphite electrode modified with CNT. Other nanomaterials, such as nickel oxide [19], titanium oxide [20,21], tungsten oxide [22], iron oxide [23] manganese oxide [24], and zirconium oxide [25] have been used successfully for immobilization of enzymes and proteins. Those studies have showed that the new metal oxide NPs exhibit high biocompatibility, high adsorption ability and little harm to the biological activity of redox proteins. Immobilization of redox proteins is another significant step in constructing biosensors or bioreactors [26]. Biocompatible materials have long been attracting increased attention worldwide to realize direct electrochemistry of redox proteins, because of their desirable properties, such as nontoxic and biocompatible, and potential applications for the fabrication of biosensors [27]. Entrapment or encapsulation of an enzyme or protein within a biocompatible material by using simple procedures, especially physical entrapment of biomolecules without the need of complicated covalently attachment, is certainly desirable. Among various biocompatible materials, chitosan (CHIT), a versatile biopolymer, has been widely used as an immobilization matrix for biosensors and biocatalysis [28–30]. The combination of NPs and CHIT can create unique materials that have open up new opportunities for the study of direct electrochemistry of

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redox proteins and the construction of biosensors [9,31]. Shan et al. [26] have reported the combination between nano-CaCO3 and CHIT for the entrapment of hemoglobin, and the resulting CaCO3/CHIT nanocomponents showed enhanced electron transfer, higher thermal stability and better electrocatalytic activity. Monazite, especially lanthanum phosphate, has long been known as a ceramic material with high-temperature stability [32], chemical inertness/non-reactivity towards other ceramic oxidases, and catalytic property [33,34]. Nanosized NdPO4 shows greater advantages and novel characteristics than regular sized particles, such as the much larger specific surface area. These properties may provide favorable conditions for enzyme or protein immobilization. In this work, we describe the direct electrochemistry of GOx immobilized on NdPO4 NPs/CHIT composite matrix. The electrochemical behaviors of the composite film are thoroughly investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The resulting biosensor can catalyze the reduction of O2, and glucose determination is achieved based on the increase of reduction peak current of O2 with the addition of glucose. The possibilities for further analytical application of such electrodes are also discussed.

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The solid collected was then digested in a 100 ml aqueous solution of phosphoric acid (1 M) for 48 h with intermittent slow shaking at room temperature. The resultant product was filtered, washed with distilled water, dried in air at ambient temperature. Prior to use, a glass carbon electrode (GCE) was polished on a polishing cloth with alumina of successively smaller particles (1.0 μm and 0.05 μm diameter, respectively). Then the electrode was cleaned by ultrasonication in ethanol and ultrapure water, respectively. Typically, 2.0 mg NdPO4 NPs was dispersed in 1.0 ml of CHIT solution (3.0 mg ml− 1) with the help of ultrasonic agitation. Then, 2 mg of GOx was added into the above solution. The mixture was hand-mixed completely and was allowed to be left for overnight at 4 °C in a refrigerator. The glucose biosensor was constructed by coating a drop of 10 μl the resulting solution onto the modified electrode. The biosensor was stored at 4 °C in a refrigerator when not in use. Electrochemical impedance spectra (EIS) were performed in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) containing 0.2 M KCl. The impedance measurements were recorded at a bias potential of +205 mV (vs. SCE) within the frequency range of 10 mHz–10 kHz. 3. Results and discussion

2. Experimental 2.1. Reagents GOx (E.C. 1.1.3.4, 182 U·mg− 1, Type X-S from Aspergillus niger), β-D(+)-glucose, lanthanum nitrate, potassium hexacyanoferrate and potassium hexacyaniferrate were of analytical grades and used without further purification. CHIT (MW 1 × 106, N90% deacetylation) was purchased from Shanghai reagent company (China). All solutions were prepared with ultrapure water (N18 MΩ·cm) obtained from a Millipore Milli-Q water purification system. Ferrocene monocarboxylic acid (FMCA, 97%, Aldrich) was used as received. 2.2. Apparatus A Model CHI660A Electrochemistry Workstation (Chenhua Instruments in Shanghai, China) was employed for all the electrochemical techniques. A three-electrode system, where a standard saturated calomel electrode (SCE) served as reference electrode, a platinum wire electrode as the auxiliary electrode, and the prepared electrodes as the working electrode. All the electrochemical experiments were conducted at room temperature. UV–Vis spectra were recorded on a Shimadzu UV-2501PC spectrophotometry at room temperature. X-ray diffraction (XRD) experiment was performed with a Shimadzu XD-3A X-ray diffractometer (Japan) using Cu-Ka radiation (k = 0.15418 nm). The scan rate was 4°min− 1. Scanning electron microscopic (SEM) measurements were carried out on a scanning electron microscope (JEOL JSM-6700F) at 20 kV. Samples were placed in aluminum stubs and were then coated with a gold layer by sputtering in order to enhance their conductivity. 2.3. Electrode preparation A 1.0 wt.% CHIT solution was prepared by dissolving certain amount of CHIT into a 0.5 wt.% acetic acid (HAc) and diluted with ultrapure water. NdPO4 NPs was synthesized based on a previous report [35]. In a typical synthesis, 2.4 g of surfactant cetyltrimethylammonium bromide (CTAB) was dissolved in 50 ml of distilled water, followed by addition of 50 ml absolute ethanol and aqueous ammonia (12 ml, 32 wt.%). The white mixture was stirred for 10 min at 450 r·min− 1, and to this mixture 9.52 g of lanthanum nitrate was added at once. The stirring was continued for 2 h at room temperature during which the pink slurry turned to a bulky gelatinous precipitate. The resulting solid was collected by filtration, washed with distilled water and dried in air at ambient temperature.

3.1. Characterization of NdPO4 NPs, GOx/NdPO4 NPs/CHIT composite matrix and electrodes Fig. 1A shows the XRD data of the NdPO4 NPs. All of the diffraction peaks could be indexed to the hexagonal phase of NdPO4 (JCPDS, 461439). No obvious impurity phase could be found in the sample. The broaden shape of the diffraction peaks suggests that the sample could be well crystallized with small particle sizes. Fig. 1B, C presents the SEM images of NdPO4 NPs and GOx/NdPO4 NPs/CHIT films. The NdPO4 NPs with the particle size range from 30 to 100 nm (B). Large particles are caused by little agglomeration of NPs. On the other hand, the GOx/ NdPO4 NPs/CHIT film was compact and uniform, suggesting that GOx/ NdPO4 NPs have been successfully incorporated in the CHIT film. UV–Vis spectroscopy is a useful tool for monitoring the possible changes of the absorption band in the GOx group region of the composite film [36]. From the spectra, the absorbance at about 271 (solid line) and 277 nm (dot line) in both the cases is due to the e–e⁎ transitions arising from the tryptophan and tyrosine residues on the enzyme surface [37]. The position and shape of absorption bands (378 and 454 nm) for GOx in GOx/NdPO4 NPs/CHIT composite (Fig. 2) is almost the same as those for free GOx, suggesting that the chromophoric groups of FAD responsible for the GOx visible absorption spectrum should be embedded in the GOx polypeptide matrix and do not come out of the pockets during the process of immobilization of GOx on NdPO4 NPs/CHIT. Fig. 3A shows the cyclic voltammograms of different electrodes in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) containing 0.2 M KCl. From Fig. 3A (curve a), a well-defined redox peaks is observed with the formal potential of 0.205 V (vs. SCE) and the peak to peak separation is 121 mV for the redox reaction of [Fe(CN)6]3−/4− at the CHIT film modified GCE. However, when the GCE is modified with NdPO4 NPs/ CHIT, the peak to peak separation of the electrode (Fig. 3A, curve b) is 189 mV and the peak currents decreased compared with the bare GCE. The peak currents markedly decrease after GOx is immobilized on the NdPO4 NPs/CHIT (Fig. 3A, curve c). The result indicates that GOx is effectively immobilized on the composite film, which inhibits the electron transfer between [Fe(CN)6]3−/4− and the electrode. EIS is an effective method of probing the features of surfacemodified electrodes [38,39], which provide useful information on the impedance changes of the electrode surface during the fabrication process [9]. Nyquist plot commonly include a semicircle region lying on the axis followed by a straight line. The semicircular part at higher frequencies corresponds to the electron-transfer-limited process and its diameter is equal to the electron transfer resistance (Ret), which

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Fig. 1. (A) The XRD spectra of NdPO4 NPs; SEM images of (B) NdPO4 NPs and (C) the electrode surface covered with GOx/NdPO4 NPs/CHIT film.

controls the electron transfer kinetics of the redox probe at the electrode interface. Meanwhile, the linear part at lower frequencies corresponds to the diffusion process. Such spectra can be used for extracting the electron transfer kinetics and diffusional characteristics. The respective semicircles diameters at the high frequencies corresponding to the charge transfer resistance at the electrode

surface. Thus, the charge transfer resistance was used as a sensor signal [40–42]. Fig. 3B displays the EIS of the CHIT/GCE (a), NdPO4 NPs/ CHIT/GCE (b), and GOx/NdPO4 NPs/CHIT/GCE (c) in 5.0 mM K3Fe(CN)6/ K4Fe(CN)6 (1:1) containing 0.2 M KCl. After the GCE surface being modified by a CHIT layer, the EIS of the resulting electrode layer shows higher interfacial electron transfer resistance (curve a), indicating that

Fig. 2. The UV–Vis absorption spectra of GOx (dot line), NdPO4 NPs (dash line) and GOx/ NdPO4 NPs (solid line).

Fig. 3. (A) Cyclic voltammograms of CHIT/GCE (a), NdPO4 NPs/CHIT/GCE (b) and GOx/ NdPO4 NPs/CHIT/GCE (c) in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) containing 0.2 M KCl, scan rate: 100 mV·s− 1; (B) EIS of CHIT/GCE (a), NdPO4 NPs/CHIT/GCE (b) and GOx/NdPO4 NPs/CHIT/GCE (e) in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) containing 0.2 M KCl. Applied potential: +0.205 V (vs. SCE). Frequency range: 10 mHz–10 kHz.

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the CHIT film obstruct the electron transfer of the redox probe. Increased electron transfer resistance is observed when NdPO4 NPs/ CHIT is modified onto the GCE surface (curve b) compared with that of CHIT modified GCE. The reason is the non-conductivity of CHIT component in the film. Similar results are also observed when the GOx is immobilized on NdPO4 NPs/CHIT/GCE (curves c), which further indicates that GOx is effectively immobilized in the composite film. The increase of Ret might have been caused by the hindrance of the macromolecular structure of GOx to the electron transfer. Therefore, the strong adsorption of NdPO4 to GOx is essential in this case. 3.2. Direct electrochemistry of the composite film modified electrode The direct electrochemistry of the composite film modified electrode is carried out in a 0.05 M PBS (pH 6.8) solution. A welldefined redox peaks is observed with the formal potential of −0.48 V (vs. SCE) and the peak to peak separation is 56 mV, indicating a fast and direct electron transfer reaction is occurred. Such peaks can be ascribed to the redox reaction of the prosthetic FAD bound to GOx, not free FAD [43] which may have dissociated away from GOx due to conformational changes during immobilization, as GOx kept its biochemical activity (see Section 3.4). Fig. 4 is the cyclic voltammograms of the composite film modified electrode obtained in 0.05 M PBS (pH 6.8) solution at various scan rates. The values of ΔEp are almost independent on the scan rates in the range of 100 to 1000 mV·s− 1. Inset of Fig. 4a shows that the cathodic and anodic peaks current increases linearly with the increase of scan rates, suggesting the electrochemical reaction of the composite film modified electrode is a surface-controlled process. According to the equation Ip = n2F2vAГ / 4RT [44], the surface coverage (Г) of GOx on the electrode surface is estimated to be 2.95 × 10− 10 mol cm− 2 from the slope of the Ip–v curve. This value is much larger than the theoretical value (2.86 × 10− 12 mol cm− 2) for the monolayer of GOx on the bare

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electrode surface [45], suggesting that the nanostructured NdPO4 NPs provide a large surface area and a higher capability of composite NdPO4 NPs/CHIT matrix for enzyme immobilization. At higher scan rates, the plot of peak currents versus scan rate deviates from linearity and the peak currents become proportional to the square root of the scan rates (Fig. 4b). On the other hand, the oxidation peak shifts to more positive potentials and the reduction peak shifts to more negative potentials (Fig. 4c). The values of cathodic and anodic peak potentials are proportional to the logarithm of the scan rate when v ≥2000 mV s− 1. Based on the Laviron theory, the electron transfer rate constant ks as well as the transfer coefficient (α) can also be calculated. The calculated value of ks and α is about 5.0 s− 1 and 0.42, respectively. The value of ks is much larger than the value obtained for colloidal gold NPs/nafion film 1.3 s− 1 [46], CNTs 1.7 s− 1 [47], but smaller than that of on nickel oxide 25.2 s− 1 [19]. Therefore, NdPO4 NPs can provide a good environment for GOx and facilitate the electron transfer of GOx. Moreover, the content of NdPO4 NPs proved to be a very critical variable for the performance of the biosensor. Meanwhile, GOx cannot exhibit any response either if the enzyme alone is modified onto GCE surface. This can be as a result of the presence of NPs can increase the effective surface area and active point for adsorbing GOx and the strong interaction between GOx molecules and NdPO4 NPs. However, we are not clear about the interaction between the NdPO4 NPs and GOx at present. 3.3. Effects of solution pH, NdPO4 NPs, and CHIT on the direct electron transfer of immobilized GOx Previous reports have shown that the solution pH is essential to the electrochemical behaviors of proteins. Cyclic voltammetric measurements of the present film modified electrode also show a strong dependence on solution pH. From Fig. 5, both anodic and cathodic peak potentials of GOx shift to negative direction and the

Fig. 4. Cyclic voltammograms of the composite film modified electrode in 0.05 M PBS (pH 6.8) solution at various scan rates (from inner to outer curve): 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 mV·s− 1. Insert graph: a) Plots of peak currents vs. scan rates (v ≤ 1000 mV·s− 1); b) Plots of peak currents vs. square root of scan rates; c) Variation of Ep vs. log (v) for the higher scan rates.

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increase the background current, 3.0 mg ml− 1 is chosen as the appropriate amount. In both cases, the concentration of GOx is fixed at 2.0 mg ml− 1 level. 3.4. Electrocatalytic ability of the composite film modified electrode It has been reported that when the GOx modified electrodes in an air-saturated solution with glucose, the cathodic peak current of GOx decreased and the anodic peak current increased [7,14,17]. The reason is that the dissolved oxygen and GOx take part in the oxidation reaction of glucose. This trend can be explained by the enzymatic transformation of glucose with the help of entrapped GOx, which is accompanied by a consumption of molecular oxygen from the solution and production of H2O2. The electrocatalytic mechanism for oxidation of glucose in the presence GOx can be expressed as follows:

Fig. 5. Cyclic voltammograms of the composite film modified electrode in 0.05 M PBS with different pH values (1: 4.0, 2: 4.5, 3: 5.5, 4: 6.0, 5: 6.8, and 6: 7.5), scan rate: 50 mV·s− 1. Inset graph: plot of E0' vs. pH values.

GOx ðFADÞ þ glucose→GOx ðFADH2 Þ þ gluconolactone

ð1Þ

GOx ðFADH2 Þ þ O2 → GOx ðFADÞ þ H2 O2

ð2Þ

formal potential (E0') versus pH give a straight line with the slope of 52.1 mV pH− 1 (inset of Fig. 5), which is close to the theoretical value (59.0 mV pH− 1) for a two-proton coupled with two-electron redox reaction process. The maximum current response is observed when the solution pH is 6.8. The effect of amount of NdPO4 NPs is tested by varying different amount of NdPO4 NPs through preparing composite film modified electrode process (Fig. 6, symbol ●). Results show that the peak current of the electrode is increasing with the increase of NdPO4 NPs and then not increase, when reaching the optimum amount of 2 mg ml− 1. The amount of CHIT is also studied (Fig. 6, symbol ▲). Results show that without CHIT, the peaks are unstable and disappear after successive potential scan for over ten cycles. When varying the CHIT amount from 0.1 mg ml− 1 to 3.0 mg ml− 1, the remained peak currents after successive potential scan are increase with the increase of CHIT concentration up to 2.0 mg ml− 1, then almost not grown. Considering that large amount of CHIT will

This is also observable in our experiments, suggesting that GOx molecules preserved their native structure after the immobilization process. As can be seen from Fig. 7, the composite film modified electrode exhibits an excellent electrocatalytic activity toward the reduction of oxygen, and the anodic peak currents decreases with increasing the exposure time of the anaerobic solution to air. Once immobilized, GOx retain its biochemical properties towards glucose in aerobic conditions. Moreover, with the addition of glucose to airsaturated PBS, the reduction current decreases. The higher glucose concentration is added, the more reduction current decreases. The result further supports that the redox peak of Fig. 4 is the result of the direct electron transfer of native GOx not free FAD. In order to investigate whether GOx immobilized in the composite film retains its bioelectrocatalytic activity for the oxidation of glucose, the cyclic voltammetric experiments were carried out in solution with the presence of FMCA as an electron mediator. Fig. 8 shows the cyclic voltamograms of the composite film modified electrode in a 0.05 M PBS solution (pH 6.8) with various concentrations of glucose. Results show that a couple of well-defined redox peaks (curve a), which correspond to the redox reaction of FMCA, appears when FMCA is present in solution. After addition of glucose, the anodic peak current increases with increasing glucose concentration and the cathodic peak

Fig. 6. Influence of the amount of (symbol ●) NdPO4 NPs and the concentration of (symbol ▲) CHIT for the immobilization of GOx.

Fig. 7. Cyclic voltammograms of the composite film modified electrode recorded in a) air-saturated PBS solution (pH 6.8) and further bumped with N2 to deaerate oxygen for every 10 min (from curve b to i).

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The use of the Eadie–Hofstee form of the Michaelis–Menten equation is quite efficient in the kinetic analysis of the enzymatic reaction. For amperometric biosensors the reaction rates are substituted with steady-state current, app Is ¼ Ismax −Km ðIs =Cs Þ

Fig. 8. Cyclic voltammograms of GOx/NdPO4 NPs/CHIT/GCE in 0.05 M PBS (pH 6.8) containing 0.5 mM FMCA in the absence (a) and presence of (b) 1.0 mM, (c) 2.0 mM, (d) 4.0 mM, (e) 6.0 mM, (f) 8.0 mM, (g) 10.0 mM and (h) 12.0 mM glucose.

current decreases. These results can be explained from the following equations: glucose þ GOx ðFADÞ→gluconolactone þ GOx ðFADH2 Þ

ð3Þ

GOx ðFADH2 Þ þ 2FMCAþ →GOx ðFADÞ þ 2FMCA þ 2Hþ

ð4Þ

2FMCA→2FMCAþ þ 2e−

ð5Þ

where GOx (FAD) and GOx (FADH2) represent oxidized and reduced forms of GOx, FMCA and FMCA+ the reduced and oxidized forms of ferrocene monocarboxylic acid mediator. These results indicate that the GOx immobilized in the composite film cannot only occur in the direct electron transfer reaction but also remain in the bioelectrocatalytic activities and catalyze the oxidation of glucose. The result further supports that the redox peak in Fig. 4 is the result of the direct electron transfer of native GOx not free FAD. The current-time amperometric curve is recorded under the conditions of continuous stirring of the solution and successive step changes of glucose concentration at +400 mV (vs. SCE) (as shown in Fig. 9). When an aliquot of glucose is added into 0.05 M PBS, the oxidative current rises steeply to reach a stable value. The time to reach 90% of the maximum current is within 5 s, which indicates a fast response process. The glucose biosensor displays increasing amperometric responses to glucose with good linear range from 0.15 to 10 mM. The linear regression equation is Ip (µA) = 0.53 + 1.92·C (mM) with the correlation coefficient of 0.999. The detection limit was estimated at about 0.08 mM (S/N = 3). The linear range and detection limit obtained with the biosensor based on the GOx/NdPO4 NPs/CHIT composite film are comparable with that of on CdS NPs [48], nickel oxide [19], and sol– gel-derived metal oxide/nafion [49] composite films. Moreover, the linear response obtained with the biosensor covers the clinical region for a range of 3.5–6.5 mM glucose [50], indicating that the biosensor can be possibly used in the analysis of undiluted real samples. When the glucose concentration was above 12 mM, the calibration curve deviated from the linear relationship can be ascribed to the saturation of glucose to GOx. The sensitivity of GOx/NdPO4 NPs/CHIT to glucose was found to be 1.92 μA mM− 1. This value is higher than that of 1.14 μA mM− 1 at Au NPs/DHP [17], 1.02 μA mM− 1 at Nafion/CNTs/CdTe [51], 0.45 μA mM− 1 at nickel oxide NPs modified film [19], or 0.405 μA mM− 1 at sol–gel-derived titanium oxide/copolymer (PVA-g-PVP) composite matrix [52] film modified electrodes.

ð6Þ

Here Is is the steady-state current, Cs the concentration of substrate, Kapp is the apparent Michaelis–Menten constant, and Imax is the m s intercept on the current axis [53]. The corresponding plot yielded an “apparent” Km of 2.5 mM. The value is smaller than those reported for GOx in solution, 33 mM [54], enzymatic electrodes prepared by immobilizing GOx on gold NPs/nafion, 4.6 mM [22], covalent attachment of GOx on gold NPs, 4.3 mM [55], TiO2 film, 6.08 mM [56], sol–gel-derived metal oxide/nafion composite films, 8.4 and 14 mM [49], copper oxide, 7.8 mM [57], CdS NPs, 5.1 mM [48] or comparable with Km = 2.7 mM on nickel oxide [18] and dendrimers [58,59]. The good microenvironment due to the synergistic effect of the hybrid composites might contribute to the improvement of the affinity and good performances of the biosensor. 3.5. Stability and reproducibility of the composite film modified electrode The stability of the GOx/NdPO4 NPs/CHIT composite film modified electrode is first evaluated by examining the cyclic voltammetric peak currents of GOx after continuously scanning for 30 cycles. No decrease of the voltammetric response is observed, indicating that the enzyme electrode is stable in buffer solution. The stability of the composite film modified electrode is also checked by measuring the current response over a period of 30 days. When not in use, the electrode is stored dry at 4 °C in a refrigerator. It is found that the composite film modified electrode maintain its 90% initial activity after 30 days. The fabrication reproducibility of six electrodes, made independently, shows an acceptable reproducibility with the relative standard deviations of 4.4% and 5.6% for the current determination of 2.0 mM glucose. 3.6. Application for the glucose determination The determination of glucose in human plasma is performed by using the glucose biosensor utilizing standard addition method. After the current response was determined in 10.0 ml of 0.05 M PBS (pH 6.8) solution containing sample of 1.0 ml, 20 μl of 1.0 M glucose was added to the system for standard addition determination. The measurement

Fig. 9. Amperometric responses of the glucose biosensor at +400 mV (vs. SCE) upon successive addition of a) 10, b) 33 and c) 100 μl 150 mM glucose to 10 ml PBS (0.05 M, pH 6.8). Inset graph: plot of peak current vs. glucose concentration.

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Table 1 Determination of glucose in human plasma samples by the proposed biosensor Sample No.

Found (mM)

Added (mM)

Found (mM)

Recovery (%)

1 2 3

5.05 5.22 5.25

2.0 2.0 2.0

6.95 7.31 7.42

98.2 101.7 103.2

results are listed in Table 1 The average glucose level in human plasma is found to be 5.1 mM. The recoveries for the determination of glucose are between 98.2% and 103.2% for three times determination. The interference effects are also tested by SWV responses of 0.1 mM glucose in presence of different concentrations of uric acid or ascorbic acid. Results show that no obvious current changes occurred, even with the concentration of uric acid or ascorbic acid up to 0.1 mM or 0.2 mM, respectively. This is enough to limit the interference effects for real sample analysis. 4. Conclusion In the present work, the direct electrochemistry of GOx immobilized by using a composite matrix based on CHIT and NdPO4 NPs was achieved. The proposed biosensor can catalyze the reduction of dissolved oxygen, and glucose determination was achieved based on the decrease of peak currents due to the reduction of dissolved oxygen. The proposed composite glucose biosensor can be used for the determination of glucose in human plasma. Furthermore, an improved stability, reproducibility and efficiency to exclude the interferences of uric acid and ascorbic acid were also obtained. In summary, the composite film, which combined the advantages of NdPO4 NPs and CHIT, offers new design of sensitive, selective and stable enzymatic biosensors. Acknowledgement The authors gratefully acknowledge the financial support of this project by the National Science Foundation of China (No. 20675062 and 20875076) and the Research Fund for the Doctoral Program of Higher Education (No. 20060697013).

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