Determination of Glucose Using Pseudobienzyme Channeling Based

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J. Phys. Chem. C 2010, 114, 21397–21404

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Determination of Glucose Using Pseudobienzyme Channeling Based on Sugar-Lectin Biospecific Interactions in a Novel Organic-Inorganic Composite Matrix Wenjuan Li, Ruo Yuan,* and Yaqin Chai Chongqing Key Laboratory of Analytical Chemistry, College of Chemistry and Chemical Engineering, Southwest UniVersity, Chongqing 400715, China ReceiVed: May 31, 2010; ReVised Manuscript ReceiVed: NoVember 3, 2010

This work presents the synthesis of gold/platinum hybrid nanostructure supported on silica nanofibers (GPSNFs) and then a novel strategy to fabricate pseudobienzyme channeling sensor based on sugar-lectin biospecific interactions for sensitive determination of glucose. First, prussian blue (PB) was deposited on the glassy carbon electrode as a redox probe. Then, a porous organic conducting polymer containing abundant amino groups (a derivative of 3,4,9,10-perylenetetracarboxylicdianhydride, was abbreviated as PTC-NH2) was coated on the surface of the PB film, which could not only prevent the leakage of the PB efficiently, but also provide an interface of abundant amino-groups to assemble GP-SNFs for the immobilization of concanavalin A (Con A). Finally, the glucose oxidase (GOD) was attached on the electrode surface through the strong biological affinity links between Con A and sugar chains intrinsically. Some important parameters of the sensor such as the catalytic rate constant (ks), the charge transfer coefficient (R), the surface coverage, app and the apparent Michaelis-Menten constant (KM ) were evaluated and discussed in detail. Furthermore, the proposed biosensor possessed high sensitivity and stability for the detection of glucose, and the linear response range from 3.0 × 10-6 to 2.3 × 10-3 M and the detection limit of 1.0 × 10-6 M. 1. Introduction Glucose oxidase (GOD) as an enzymatic catalyst has been widely used for glucose biosensor fabrication. High selectivity, high sensitivity, and a low detection limit have been achieved for quantitative determination of glucose by such enzymatic sensors. However, the redox center of the majority of the enzymes is buried inside the protein matrix, which results in a slow process of direct electron transfer between the electrode and enzyme.1,2 To facilitate superior electron transport, the mediator may be selectively placed in an optimum position between the redox center and the enzyme periphery.3 Prussian blue (PB, ferric hexacyanoferrate), a classical pigment but with a relatively high catalytic activity toward the reduction of hydrogen peroxide (H2O2), was regarded as “artificial peroxidase”.4 The PB-based glucose sensor had seen greatly widened use as it combined the advantages (1) for PB’s good electrochemical behavior, which allowed the detection of H2O2 at much lower potentials, and therefore avoids the interference of coexisting electroactive species,5 and (2) more importantly, as “artificial peroxidase”, the PB modified electrode could couple with GOD to form pseudobienzymatic systems. In such configurations, H2O2 produced by the GOD is subsequently reduced by the PB. The cascade schemes, where an enzyme is catalytically linked to another enzyme, can produce signal amplification and therefore increase the biosensor efficiency.6 A main drawback of a PB-based biosensor is its poor cycling stability when the sensor is used continuously. To overcome the problem, an outer polymer film, like polymer materials (e.g., Nafion, chitosan, etc.), has been coated on the PB surface to serve as a protective layer, but which may cause sensitivity problems since lots of polymer membrane acted as a barrier of the electron propagation.7 So there is a growing demand to look * To whom correspondence should be addressed. Tel: +86-23-68252277. Fax: +86-23-68254000. E-mail: yuanruo@swu. edu.cn..

for a stable membrane material with efficient electrochemical performance and conductivity. In our previous work, we reported a new organic conductive compound that is abbreviated to PTCNH2, possessing well-known certified properties such as being stable, easy to form a porous film and transfer electrons, as well as offering abundant amino groups,8 which will be favored for further modification. And it has been used to suppress successfully the leakage of the mediators (e.g., ferrocene, toluidine blue, thionine) from the electrode surface in our research group.7,9,10 And therefore PTC-NH2 has been proven to be a promising matrix for enzyme immobilization and has been applied in constructing biosensors. For these reasons, this work targets a high performance glucose sensing electrode based on the PTCNH2/PB films. To further improve sensitivity and selectivity of biosensors, efforts have been attempted to modify the substrate electrode with nanoparticles. Recent researches indicate that the properties of catalysts could be modified through changing the grain size, texture, and the surface profile, thus, the search for producing nanosized catalysts to improve its activity is a critical theme in these research areas.11 To date, a large amount of effort toward various nanostructures has been devoted to carbon nanotubes even though carbon nanotubes are very inert, which makes them difficult to functionalize. It has recently been demonstrated that nanotubes, wires, and fibers-shape made of silica other than carbon exhibit interesting physical and chemical properties that are dependent on their constituent materials and morphologies.12 The mesoporous silica nanofibers have high surface areas and highly ordered nanoscale pore channels. The pore channels can be aligned by synthetic control either parallel to or circularly around the fiber axis. Therefore, these nanofibers become excellent candidates for the construction of functional hybrid nanostructures. Furthermore, the nanofibers with nanometerscale diameter and the porous nature allow for easy incorporation of metal nanoparticles and the particular internal pore archi-

10.1021/jp1049638  2010 American Chemical Society Published on Web 11/22/2010

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tecture makes it possible to control the orientations of spherical nanoparticles. In the past few years, silica nanofibers have been functionalized with gold or silver nanomaterials.13 Recent research suggested that metallic alloy nanoparticles often exhibit better catalytic properties than do their monometallic counterparts,14 so the study of alloy composite material is motivated primarily from the anticipation of a synergistic electrocatalytic benefit from the combined properties of the component metals of alloys.15 However, no publications have reported the synthesis of alloy nanoparticle using homogeneous silica as a spacer. In this study, we explored a facile, efficient, and economical route to obtain gold/platinum alloy nanoparticles supported on 1D silica nanofibers (GP-SNFs) which were expected to find interesting applications for ultrasensitive chemical and biological sensing. In the manufacturing process of a biosensor, the immobilization of enzyme is a critical step. Many different methods have been performed in the previous literature, such as entrapment in different matrices,16,17 cross-linking with bifunctional chemical reagents,18 charge interaction with gold nanoparticles,19 etc. Every immobilization protocol has its own advantages and drawbacks. However, most of the procedures are often characterized by conformational changes in the enzyme, simultaneously with a significant loss of enzymatic activity. So, there is still a challenge to look for novel immobilization schemes in order to improve the sensitivity and the stability of the sensor in various conditions. Recently, a lectin-sugar affinity system provided a very attractive and promising tool for enzyme immobilization, which is based on the strong affinity links between the concanavalin A (Con A) and the mannose residues of the enzyme.20 Con A, a lectin protein, is known to contain four binding sites specific for terminal R-mannose residues,21 and presents several interesting aspects. First, it possesses multiple sites with high affinity for the attachment of the glycolenzyme, such as glucose oxidase (GOD), which is favorable for the increase of the immobilization quantity and improves the stability of the enzyme biosensor. Second, the protocol can avoid any chemical modification by the binding of Con A and the native enzymes, which is preferable to retain the enzymatic bioactivity. Presently, we are interested in the study of the possible cooperation of enzyme with GP-SNFs to interference-free determination of glucose. In this biosystem, PB was successfully deposited on the surface of glassy carbon electrodes and then PTC-NH2 was filled in the holes of the PB to prohibit the PB from leaking. Later, GP-SNFs were immobilized on the PTCNH2/PB composite membrane for the capture of Con A, which can reduce the insulating property of the protein and facilitate the electron transfer. Finally, GOD can be attached to it by the lectin-sugar system. Transmission electron microscopy, X-ray photoelectron spectroscopy, UV-vis absorption spectroscopy, and scanning electron microscopy had been adopted to characterize the assembly process, and the electrochemical behavior of the sensor was studied in detail by using cyclic voltammetry and chronoamperometry. 2. Experimental Section 2.1. Reagent and Materials. 3,4,9,10-Perylenetetracarboxylic dianhydride (PTCDA) were purchased from Lian Gang Dyestuff Chem. Co. (Liaoning, China). Glucose oxidase (GOD), Concanavalin A (Con A) from CanaValia ensiformis (jack bean), ascorbic acid (AA), 3-aminopropyltriethoxysilane (APTES), HAuCl4, H2PtCl6, and sodium citrate were obtained from Sigma Chemical Co. (St. Louis, MO, USA). K4Fe(CN)6, FeCl3, HBr,

Li et al. tetraethoxysilane (TEOS), cetyltrimethylammonium bromide (CTAB), and sodium borohydride (NaBH4) were purchased from Chemical Reagent Co., Sichuan, China. All chemicals and solvents used were of analytical grade. Double distilled water was used throughout all experiments. Phosphate-buffered solutions were prepared by mixing the solutions of KH2PO4, Na2HPO4, and KCl. 2.2. Apparatus. Electrochemical measurements were carried out on a CHI 660A electrochemical workstation (CH Instruments, Chenhua Corp., Shanghai, China). The electrochemical cell contained a three-electrode system where bare or modified glassy carbon electrode (4.0 mm in diameter) was used as a working electrode, platinum wire as an auxiliary electrode, and a saturated calomel electrode (SCE) as reference electrode. All potentials were measured and reported versus the SCE; the test solutions were 5 mL of PBS. Transmission electron microscopy (TEM) was carried out on a TECNAI 10 (Philips Fei Co., Hillsboro, OR). The morphologies of the films were investigated with scanning electron microscopy (SEM, S4800, HITACHI Co., Japan). The UV-vis absorption spectra were recorded in the range of 200-800 nm, using a UV-vis spectrometer (UV-vis 8500). X-ray photoelectron spectroscopy (XPS) measurements were carried out with a VG Scientific ESCALAB 250 spectrometer, using Al KR X-ray (1486.6 eV) as the light source. All the electrochemical experiments were carried out at room temperature. 2.3. Preparation of GP-SNFs. Mesostructured silica nanofibers were prepared by using CTAB as the structure-directing agent in aqueous HBr solutions according to the literature12,13,22 with small modification. Briefly, CTAB was first dissolved in an aqueous HBr solution, and TEOS was then added at a proper molar ratio. The reaction mixture was stirred at room temperature for 10 min and then transferred into 100 mL glass bottles, which were closed and kept in an isothermal oven at 65 °C for 2 d. All of the grown products were fiber-like flocculates. Then, the mesostructured silica nanofibers were functionalized via the reaction of silanol groups with APTES. Before functionalization, the silica nanofibers (0.2 g) were first dried at ca. 120 °C under vacuum for 5 h. The dried nanofiber sample was then dispersed in dry toluene (50 mL), and APTES (32 µL) was added under stirring. The reaction mixture was refluxed for 24 h. The resulting functionalized nanofibers were filtered, washed with toluene, and dried under vacuum for ca. 5 h before use. Subsequently, nonspherical Au nanoparticles were attached on silica nanofibers by using a seed-mediated process in CTAB solutions. In a typical synthesis, the APTES-functionalized nanofibers (15 mg) dispersed in ethanol (5 mL) were mixed with the resulting CTAB-stabilized seed solution (130 mL). After the solution was stirred for 5 h, the resulting nanofibers were collected by centrifugation and washed with water 3 times, and then redispersed in water (80 mL) until use. Finally, the obtained nanofibers coated with Au-seed nanoparticles were heated to a boil, and afterward 2 mL of 1% H2PtCl6 and 3 mL of 1% citrate were added to the previous solution, followed by the addition of 0.2 M AA, which acted as a reductant for the reduction of H2PtCl6. After the mixture was heated for 30 min, GP-SNFs were obtained. The resulting solution was centrifuged 4 times and redispersed in water. The typical synthetic process is given in Scheme 1. 2.4. Preparation of Glucose Biosensor. The glassy carbon electrode (GCE, Φ ) 4 mm) was polished with 0.3 and 0.05 µm alumina slurry to obtain a mirror-like surface, and then ultrasonically cleaned in ethanol and water thoroughly. Then it was allowed to dry at room temperature.

Determination of Glucose with Pseudobienzyme Channeling SCHEME 1: Synthesis Scheme for the Preparation of the GP-SNFs

SCHEME 2: Illustration of the Preparation Process of Modified Electrode

Following this pretreatment, the cleaned GCE was immersed in an unstirred fresh aqueous solution consisting of 2.5 mM FeCl3, 2.5 mM K3[Fe(CN)6], 0.1 M KCl, and 0.1 M HCl with a constant working potential of +0.4 V and applied for 60 s to obtain PB/GCE. After that, the PB/GCE was coated with 10 µL of PTC-NH2 ethanol solution and dried in air. Subsequently, it was immersed in GP-SNFs solution with potentiostatic electrodeposition for 40 s at -0.2 V. Afterward, the obtained electrode was incubated in a Con A solution (0.3 mg/mL in phosphate-buffered saline, PBS, containing 1 mmol/L CaCl2 and 1 mmol/L MnCl2, pH 7.0) for 2 h. Finally, the above modified electrode was immersed in GOD solution (2 mg/mL in PBS, pH 7.0) for 2 h to deposit a GOD layer through biospecific Con A-sugar interaction. The resulting electrode, which is denoted here as GOD/Con A/GP-SNFs/PTC-NH2/PB/GCE, was stored at 4 °C in a refrigerator under dry conditions when not in use. The procedure for constructing the modified electrode was schematically shown in Scheme 2. 3. Results and Discussion 3.1. Characterization of GP-SNFs. TEM was used to observe the morphology and distribution of gold or gold/ platinum supported silica nanofibers. Figure 1A shows a TEM image of silica nanofibers, which have an average diameter of 35 nm, with their lengths ranging from micrometers up to millimeters. When gold nanoparticles are modified onto the nanofibers (Figure 1B), a great number of small gold nanoparticles are distributed almost uniformly on the walls of nanofibers. As a comparison, nanoparticles with a larger size than the preformed gold nanoparticles were found in Figure 1C, revealing that gold/platinum hybrid nanostructure was produced. The previous result indicates that the gold/platinum nanoparticles are indeed bound to the surface of silica nanofibers.

J. Phys. Chem. C, Vol. 114, No. 49, 2010 21399 To further confirm the surface composition of the resulting nanomaterials, XPS measurement was performed to gain the information concerning the sample. As shown in Figure 2, panels A-D represent significant O1s signal, C1s signal, N1s signal, and Si2p signals characteristic of silica nanofibers, and panel E is the XPS signature of the Au4f doublet (83.7 and 87.4 eV for 4f7/2 and 4f5/2) corresponding to the binding energy of metallic Au, and the Pt4f region exhibited doublets from the spin-orbit splitting of 70.3 eV for 4f7/2 and 74.2 eV 4f5/2 states (Figure 2F) for the metallic Pt, which firmly affirmed that GP-SNFs can be obtained via the previous method. The formation of GP-SNFs was also monitored using a UV-vis spectrophotometer. For silica nanofibers solution, it was found that no absorption peak is observed (Figure 3a). When gold nanoparticles are bound onto the nanofibers, there is an absorption peak that appears at 521 nm (Figure 3b), as compared with gold nanoparticles at 518 nm (figure not shown); the absorption peaks exhibited a red-shift of the surface-plasmon band, which indicated the interparticle interactions adsorbed on the silica nanofibers. In the case of the mixed solution of nanofibers coated with Au-seed nanoparticles containing H2PtCl6 (Figure 3c), a strong band appears at 261 nm, which can be assigned to the PtCl62- ions. However, the characteristic absorbance peaks at 261 nm disappeared almost completely after adding the AA (Figure 3d), suggesting that the PtCl62- ions were reduced to Pt(0) by AA. On the basis of the above results, we concluded that complex reactions on the prepared GP-SNFs might have occurred successfully as expected. 3.2. Scanning Electron Microscope. The surface morphologies of the PB film, PTC-NH2/PB film, GP-SNFs/PTC-NH2/ PB film, and GOD/Con A/GP-SNFs/PTC-NH2/PB film were investigated with a scanning electronic microscope (SEM) with images shown in Figure 4. As can be observed from the image of Figure 4A, there are many particle-like clusters with a particle diameter of 0.3-3 µm on the PB modified film, which is consistent with reports in previous studies.23 After being coated with PTC-NH2 film, irregularly quadrate-shaped molecular islands can be obtained (Figure 4B). When GP-SNFs were assembled onto the PTC-NH2/PB composite film (Figure 4C), fiber shapes were seen clearly, which indicated the formation of GP-SNFs. Figure 4D shows an SEM image of GOD immobilizing to the GP-SNFs/PB/PTC-NH2 layers, the corresponding image obviously changed, which demonstrated GOD biomolecules immobilized successfully on the surface of electrodes. 3.3. Electrochemical Characteristics of the Modified Electrode. The assembly process of GOD/Con A/GP-SNFs/PTCNH2/PB multilayer films on the GCE is monitored by CVs experiments in the working solution, as shown in Figure 5. No cyclic voltammetric peak was observed at bare GCE in the potential range of -0.15 to 0.5 V in 0.025 M pH 6.0 PBS as a lack of redox mediator (data not shown). When PB was electrodeposited on GCE surface the resulting electrode showed a stable and well-defined redox peaks at 0.11 and 0.19 V at a scan rate of 50 mV/s (Figure 5a), which was attributed to prussian white to PB conversion. In comparison with Figure 5a, the peak currents decreased after PTC-NH2 loading (Figure 5b). However, after attaching GP-SNFs onto the electrode via binding interactions between GP-SNFs and amino groups of PTC-NH2, an increase in peak currents was observed (Figure 5c), suggesting that GP-SNFs were beneficial to the electron transfer. With the immobilization of the Con A layer, an obvious decrease of redox peak was noticed (Figure 5d). The reason is that Con A, acting as the mass transfer blocking layer, hinders

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Figure 1. TEMs of silica nanofibers (a), the as-prepared gold nanoparticle supported silica nanofibers (b), and the GP-SNFs at different magnifications (c, d).

the diffusion of the electron toward the electrode surface. Further to capture the GOD (Figure 5e), a peak current decrease may be contributed to the GOD layer, which had been immobilized on the electrode surface through the strong affinity links between the Con A and the mannose residues of the enzyme. 3.4. Cyclic Voltammetric Response of Biosensor to Glucose. The catalytic activity of the biosensor toward glucose was investigated by CVs. Figure 6 is the CVs recorded for the biosensor in PBS in the absence (a) and in the presence of 1.2 mM glucose (b). With the addition of glucose, the biosensor triggers bioelectrocatalytic reaction, which dramatically changes the cyclic voltammogram with a sharp increase in reduction peak current and a concomitant decrease in the oxidation peak current, indicating the enzyme biosensor exhibited excellent electrocatalytic activity toward glucose. Figure 7 shows recorded CVs at different scan rates. It can be observed the peak current increased and the peak potential shifted slightly. As shown in the inset of Figure 7a in the scan rate range of 10-100 mV/s, both anodic and cathodic peak currents rise linearly with the increased scan rate, indicating that the reaction is a surface-controlled process. At higher sweep rates, the plot of anodic and cathodic peak currents vs scan rate plot deviated from linearity and the peak currents became proportional to the square root of the scan rate (Figure 7b), showing a diffusion-controlled process. An average surface coverage of enzymes was calculated to be 5.8 × 10-10 mol · cm-2. The coverage was much larger than 2.35 × 10-11 mol · cm-2 for a fully packed monolayer of GOD24 and 8.77 × 10-11 mol · cm-2 reported in the Nafion-CNTs-CdTe-GOD film,25 indicating that more enzyme molecules were assembled on the enzyme cocoated GP-SNFs/PTC-NH2/PB film modified electrode. In addition, a couple of stable redox peaks were found at 190 and 130 mV with the formal potential of 160 mV at a scan rate of 50 mV/s. According to the model of the Laviron equation,26 the charge transfer coefficient (R) was calculated as

0.5. The apparent surface electron transfer rate constant (ks) can be estimated from the following formulation when the peak potential separation value (∆Ep) was less than 200 mV:

log ks ) R log(1 - R) + (1 - R) log R log(RT/nFV) - R(1 - R) - log(nF∆Ep /2.303RT) and the calculated value for ks was 6.63 s-1. The above results also show that the integration of GP-SNFs and PTC-NH2/PB can provide a remarkable synergistic augmentation of sensor performance. 3.5. Optimum Conditions of the Experimental Conditions. Deposition time may have some effect on the biosensor, such as linear range, sensitivity, and detection limit. The deposition time was investigated from 10 to 80 s. Figure 8A shows the corresponding sensitivities as determined from the slopes of the calibration curves. As shown in Figure 8A, with the increase of the deposition time from 10 to 60 s, the response sensitivity increased. When the deposition time increased to 80 s, the sensitivity decreased slightly, indicating that the thickness of the PB film increased and the electrochemical redox active area may be decreased. Therefore, the deposition time of 60 s was chosen in this work. The amount of the PTC-NH2 coated on the PB surface is a vital factor affecting the analytical sensitivity and stability of the biosensor. The effect of the amount of PTC-NH2 on the stability was investigated by comparing the current value after the 100 continuous cyclic scans with that of the initial current value, and the corresponding result was shown in Figure 8B. In this figure, it can be observed that the stability of the biosensor increased with the increase of the volume of PTC-NH2, and for higher amounts than 10 µL, the value has started to level off, which corresponded to the saturated station. However, with a further increase of the amount of PTC-NH2, a negative change

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Figure 2. XPS spectra of the as-prepared GP-SNFs: (A) O1s, (B) C1s, (C) N1s, (D) Si2p, (E) Au4f, and (F) Pt4f.

deposited GP-SNFs on the sensitivity of the biosensor was investigated. As seen from Figure 8C, when the GP-SNFs deposition time was more than 40 s, the sensitivity of the biosensor decreased slightly, which might be due to the decrease of the real surface area of the electrode resulting from the deposition of a large amount of GP-SNFs on the electrode surface. So, the deposition time of 40 s was selected in the following investigation.

Figure 3. UV-vis spectra of (a) silica nanofibers, (b) the as-prepared gold nanoparticle supported silica nanofibers, (c) b solution containing H2PtCl6, and (d) c solution after adding AA.

of the biosensor sensitivity is observed due to diffusion limitation. As a result 10 µL was the optimal amount of PTC-NH2 used in this work. Here, GP-SNFs were used to accelerate electron transfer and help the immobilization of protein. The effect of the amount of

To obtain an optimal biosensor for glucose, the influence of pH on the response of the GOD/Con A/GP-SNFs/PTCNH2/PB/GCE was investigated by studying the change of chronoamperometric current. The effect of pH was tested in a series of PBS with pH from 4.5 to 9.0. Figure 8D shows the change of chronoamperometric current with the pH under constant glucose concentration (20.0 µM). As can be seen, the highest response current is obtained at pH 6.0. The reason may be that PB was unstable in neutral and alkaline solutions and prussian white was somewhat soluble in alkaline condition, which would lead to the slow decrease of the signal. Although optimal activity of the GOD exhibits at the

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Figure 4. SEM photos of PB film (A), PTC-NH2/PB film (B), GP-SNFs/PTC-NH2/PB film (C), and GOD/Con A/GP-SNFs/PTC-NH2/PB film (D).

Figure 6. CVs of GOD/Con A/GP-SNFs/PTC-NH2/PB/GCE electrode at a scan rate of 50 mV/s in 0.025 M PBS (pH 6.0) without glucose (a) and with 1.2 mM glucose (b). Figure 5. Cyclic voltammograms of the electrodes at different stages: PB/GCE (a), PTC-NH2/PB/GCE (b), GP-SNFs/PTC-NH2/PB/GCE (c), Con A/GP-SNFs/PTC-NH2/PB/GCE (d), and GOD/Con A/GP-SNFs/ PTC-NH2/PB/GCE (e) in PBS (pH 6.0). The potential scan rate was 50 mV/s.

physiological pH, pH 6.0 was selected so as to ensure higher sensitivity and stability of the biosensor. 3.6. Comparison of Electrochemical Response. The composite nanoparticles were formed by a combination of two or more phases of different natures. It acts not only as a support for the biologic material but also as a transducer. The resulting material is essential in the nanocomposite formation. As a controll experiment, the amperometric responses were also measured at GOD/Con A/GP-SNFs/PTC-NH2/PB/GCE (Figure 9a), GOD/Con A/nano-Pt/PTC-NH2/PB/GCE (Figure 9b), and GOD/Con A/nano-Au/PTC-NH2/PB/GCE (Figure 9c). Compared with the calibration plots of curves a, b, and c in Figure 9, it can be found that curve a illustrated a wider dynamic measurement range and higher current response than curves b and c, which could be attributed to the GP-SNFs distinctive structures, offering a good matrix for capturing more enzyme

protein on the electrode surface and maintaining the bioactivities. Moreover, they reduce the insulating property of the protein and facilitate the electron transfer, therefore exhibiting a high electrocatalytic activity toward glucose and enhancing the sensitivity of the enzyme assay. 3.7. Response of the Biosensor to Glucose. Figure 10 shows a typical current-time curve of the biosensor under the optimized experimental conditions after the addition of successive glucose concentration to the PBS under stirring. With the increase of the concentration of substrate, the biosensor responded rapidly. The inset of Figure 10 displays the calibration curve of the biosensor for glucose determination. The response current increases linearly with the glucose concentration in the range of 3.0 × 10-6 to 2.3 × 10-3 M with a correlation coefficient of 0.996, as shown in the inset of Figure 10. From the slope of the calibration curve, the detection limit of 1.0 × 10-6 M is estimated at a signal-to-noise ratio of three. The sensitivity of 12.28 µA · mM-1 is higher than that of other glucose biosensors based on PB or nanoparticle-derivatized surfaces.27-29 Some possible explanations might contribute to these phenomena: (i) GP-SNFs nanocomposite, with the high

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Figure 7. CVs of GOD/Con A/GP-SNFs/PTC-NH2/PB/GCE electrode in 0.025 M PBS (pH 6.0) at a scan rate of (from inner to outer) 10, 30, 50, 80, 100, 150, 200, 300, 400, 500, and 600 mV/s. Inset a: Plot of Ip vs V. Inset b: Ip vs V1/2.

Figure 8. Optimization of experimental parameters. (A) Effect of deposition time of PB film. (B) Influence of the amount of PTC-NH2 on the sensitivity and stability of the biosensor. (C) Effect of deposition time of GP-SNFs. (D) Influence of pH of the PBS on the sensor response. All potentials are given vs SCE.

surface-to-volume ratio, might greatly enhance the immobilization density of bound protein and provide the necessary conduction pathways and allow efficient electron tunneling; (ii) the sugar-lectin system offered a versatile strategy for the immobilization of enzyme by the strong biospecific affinity links between the Con A and the mannose residues of the enzyme (binding constant, ca. 1015 M-1) without any chemical modification, which possessed high stability and maintained the good bioactivities and high biocompatibility; and (iii) more importantly, this wonderful analytical performance is attributed to the synergistic action of modified materials on the electrode surface. From the slope of the calibration curve, when the concentration of glucose is higher than 2.3 × 10-3 M, a response plateau appears, showing the characteristics of the Michaelis-Menten kinetic mechanism. The apparent Michaelis-Menten constant

(Kapp M ) is calculated to be 1.77 mM according to the LineweaverBurk equation.30 3.8. Precision of Measurements and Stability of Biosensor. The intra-assay precision of the biosensors was evaluated by assaying enzyme electrode for eight replicate determinations in PBS containing 0.1 mM glucose. Similarly, the interassay precision, or fabrication reproducibility, was estimated at eight different electrodes. The relative standard deviation (RSD) of intra-assay and interassay were found to be 4.1% and 5.6%, respectively, indicating acceptable precision and reproducibility. The storage stability of the enzyme electrode was investigated over a period of 30 days. When not in use, the electrode was suspended above 0.025 M PBS at 4 °C in a refrigerator. The response to 0.1 mM glucose was tested every 2-3 days. The enzyme electrode retained 85.8% of its original response after

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Li et al. vantages of the obtained sensors should be highlighted. First, it was found that this resulting nanomaterial exhibited a high electrocatalytic activity toward glucose, and a necessary pathway of electron transfer. Second, the use of porous PTC-NH2 overcame the defect of mediator leakage successfully, which enhanced the stability and sensitivity of the bioassay. Third, the pseudobienzymatic sensors could be operated at low working potentials, and interferences from other electroactive compounds are minimal in biological samples. The proposed method can be extended to a large group of enzymes to provide promising platforms for biosensor and bioelectronics applications.

Figure 9. Typical current-time response curve for successive addition of glucose obtained by GOD/Con A/GP-SNFs/PTC-NH2/PB/GCE (a), GOD/Con A/nano-Pt/PTC-NH2/PB/GCE (b), and GOD/Con A/nanoAu/PTC-NH2/PB/GCE (c) in stirring 0.025 M PBS of pH 6.0 at applied potential -100 mV.

Acknowledgment. This work was supported by the NNSF of China (21075100), the Ministry of Education of China (Project 708073), the Natural Science Foundation of Chongqing City (2009BA1003), High Technology Project Foundation of Southwest University (XSGX02), the Fundamental Research Funds for the Central Universities (Southwest University, XDJK2009C082), and the Postgraduate Science and Technology Innovation Fund of Southwest University (ky2009003), China. References and Notes

Figure 10. Amperometric response of the biosensor to glucose in a pH 6.0 PBS at an applied potential of -100 mV upon successive additions of glucose with different concentrations at time intervals of 40 s. The inset shows linear calibration curves.

over 1 month, showing a longer lifetime. The good stability may be due to the fact that the PTC-NH2/PB composite film was stable and prevented the leakage of the PB. On the other hand, the lectin-sugar affinity system provides an appropriate microenvironment for retaining the bioactivity of the enzyme molecules. 3.9. Interference Determination. Under the optimized experiment conditions described above, the influence of the most common electrochemical interfering species (such as ascorbic acid, uric acid, L-cys, and p-acetaminophenol) to the current response of 0.2 mM glucose was investigated. Addition of 0.1 mM ascorbic acid, uric acid, L-cys, and acetaminophen to 0.2 mM glucose solution had almost no observable interference on the current response of glucose (signal change below 6.8%). The result revealed that the glucose biosensor has good antiinterferent ability, which may be attributed to the cascade schemes taking place at low potentials (-100 mV), and thus, the selectivity of the device is improved considerably. 4. Conclusions In summary, we have demonstrated herein a kind of novel hybrid nanostructure material, GP-SNFs, which was obtained through a seed-mediated growth approach on the nanofibers. On the basis of this nanomaterial film, a scheme to fabricate a mediated biocomposite pseudobienzyme channeling sensor was proposed for sensitive determination of glucose. Several ad-

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