Enzyme Functionalized Single-Walled Carbon Nanotubes and
Its Application for Glucose Biosensor
Yang Xiaoying, Ma Yanfeng, Lu Yanhong, Chen Yongsheng *
Center for Nanoscale Science and Technology, The State Key Laboratory of Functional Polymer
Materials College of Chemistry, Nankai University, Tianjin, PRC (300071)
E-mail：yangxiaoying@tijmu.edu.cn, maggie@nankai.edu.cn, luyanhong_2003@126.com, yschen99@nankai.edu.cn
Abstract
In this paper, glucose oxidase (GOx) was covalently immobilized on the single-walled carbon nanotubes (SWNTs) via carbodiimide bond by forming amide linkages between the residual amine of GOx and carboxylic acid groups on the SWNTs tips. The functionalized SWNTs were successfully immobilized on the surface of a glassy carbon (GC) electrode. The modified electrode with SWNTs and SWNTs-GOx showed excellent quantitative response of reduction current for the determination of H2O2 and glucose, respectively.
Keywords: Single-Walled Carbon Nanotubes, Glucose Biosensor, Electrochemistry
1. Introduction
Since their discovery in 1991, 1 carbon nanotubes (CNTs) have fascinated many scientists in the fields of physics, chemistry and materials science. CNTs, perfect conducting molecular wires, are promising sensor materials because their properties can be tailed to detect a wide range of chemical and biological compounds. Also they have very high specific surface area, and most of this surface area, in principle, is accessible to both electrochemistry and immobilization of biomolecules. They are also mechanically strong, flexible and easy to modify at the ends and have modifiable electrical conductivity depending on their chirality and diameters. These properties make them a perfect candidate for the third generation of biosensors since they can both work as the medium to transfer the electrochemical signal and immobilization of the probe molecules at the molecule and individual SWNT level with minimum interference from the environment.2-4 During the last two years potential biological applications of CNTs have captured great interest and there have been many investigations relating to the use of CNTs for biological purpose. 5-11
While some examples have been reported for glucose biosensor using CNTs, most of these CNTs based biosensors were built from MWNTs or using physically absorption immobilization.12-15 In this paper we wish to report our preliminary studies for an electrochemical SWNTs biosensor using covalently immobilized glucose oxidase (GOx) as the probe on SWNTs and its utilization for the detection of glucose and H2O2 generated in many biocatalytic processes.
2. Experimental
2.1 Chemicals.
The SWNTs were prepared by a direct current arc-discharge method16 and with about 40-50% purity. 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfo-succinimide sodium salt (NHS), 2-morpholinoethanesulfonic acid monohydrate (MES), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4) and Triton-100 were purchased from Aldrich. Glucose oxidase (GOx, EC 1.1.3.4, Type X-S from Aspergillusniger, 157500 units/g of solid) and β-D-(+) glucose were purchased from Sigma. Hydrogen peroxide (30 wt%) was of analytical grade and used as without any further purification. All solutions were prepared with double distilled water.
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2.2 Apparatus.
Amperometric experiments were performed with a microcomputer-based electrochemical analyzer（LANLIKE, LK98BⅡ）. The working electrode, Ag/AgCl reference electrode，and the platinum counter electrode were inserted into the 20 mL cell through holes in its Teflon cover. A magnetic stirrer provided the convective transport during the amperometric measurement.
Fourier transform Infrared spectroscopy (FT-IR) (BRUKER, Tensor 27), Raman spectroscopy (RENISHAW，inVia), X-ray diffraction (XRD) (RIGAKU, D/MAX-2500), scanning electron microscopy (SEM) (HITACHI, S-3500N), Ultra-visable-near IR absorption spectrum （UV-vis-NIR）（JASCO, V-570）and Fluorescence spectrum（JOBIN YVON, FluoroMax-P） were used to characterize the purified and modified SWNTs.
2.3 Purification of SWNTs
The SWNTs (0.5 g), generated from a modified arcing process,16 was first treated by ultrasonicating in 70 ml of 2.6 M nitric acid for 1 h, followed by refluxing for 45 h.9 It was further purified with a process more like “fractional extraction” consisting of several centrifugation and ultrafiltration cycles using surfactant Triton-X. Four fractional products A, B, C and D were separated from this process. The initial purified SWNTs product from the acid treatment above was sonicated with 0.2 % Triton-X （150 ml） for ~30 mins and then centrifugation was applied. Membrane filtration (0.45 µm) of the upper suspension yielded the first fraction of the product A. The bottom solid was then carried on for the next sonication-centrifugation cycle same as above and product B was collected from the membrane filtration. Fractional product C was similarly separated in the following third cycle. After the third cycle, the final bottom solid by centrifugation was named as product D. In each cycle, the bottom SWNTs residue from centrifugation was sonicated with 150 ml 0.2 % Triton-X thoroughly until a homogeneous suspension was generated and the centrifugation was carried out at 10000 r/min for 100 min.
2.4 Covalent functionalization of SWNTs
Covalent functionalization of SWNTs with GOx was accomplished using similar procedure in the literatures.9,13 Purified SWNTs (1 mg) was first suspended in a solution of 5 ml 0.2 % Triton–100, 100 mM MES (pH=6.0), 100 mM NHS and 100 mM EDC with sonication for 1 h at room temperature and then pH of the mixture was adjusted to 7.4 with 4 M NaOH, followed by addition of Glucose Oxidase (GOX) (25 mg). The reaction mixture was stirred overnight at room temperature, followed by centrifugation to collect the bottom GOx functionalized SWNTs products. It was then washed with double distilled water several times to remove absorbed organic compounds and excess of GOX in the reactions.
2.5 Modification of electrode
The glassy carbon electrode (Φ=4 mm) was polished with 0.05 µm alumina slurries, and then washed ultrasonically with distilled water and ethanol. The purified SWNTs and the enzymes labeled SWNTs were used to modify GC electrode（Φ=4 mm）by casting 10～15 µL of 0.2-0.4 mg/ml suspension of the purified SWNTs or functionalized SWNTs in water on the electrodes.
The measurement was carried out in a phosphate buffer（0.05 M，pH=7.4）and with KCl (0.1 M) as the supporting electrolyte medium at desired working potential. All measurements were performed at room temperature.
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3 Results and disscussion
3.1 Characterization of the purified SWNTs
The purified of SWNTs have been evaluated by XRD、SEM and Raman spectroscopy.
Fig. 1 was the XRD characterization of the four fractional products A, B, C, and D from the above “fractional extraction” purification. Among the four fractional products, the X-ray data shows clearly that the fractional products C and D have more impurities of Ni (peaks at 44o，51o，76 o) and graphite (peak at 26o) and A and B have much less metal and graphite left. 17 SEM images (Fig. 2) shows that fractional B contains almost no impurity and consists of mainly the SWNTs bundles with size of 20-30 nm in diameters overall. The SEM images show that A contains much more amorphous carbon and D contains much graphite impurity. TEM images (not shown) give the similar results.
Figure 1: Powder X-ray diffraction patterns of the purified SWNTs. A, B, C were collected from filtration of the upper suspension after the 1st, 2nd and 3rd sonication-centrifugation cycles. Product D was the final bottom solid after the
3rd cycle.
Figure 2: SEM image of purified SWNTs （fractional B, magnification 20000×）
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Figure 3: Raman (514-nm excitation) spectra of pristine SWNTs and purified SWNTs
Raman spectra (Fig. 3) showed that there was no significant difference between the samples before and after purification (from the fractional B), suggesting that the tubular structure of SWNTs was undisrupted. Typical SWNTs features were observed for the tangential modes (T band) and radial breathing modes (R band) near 1593 and 163 cm-1, respectively. 18 The disappearing of the peak of 147 cm-1 after purification indicated that the tubes with large diameter were more susceptible to acid intercalation during the long time refluxing in nitric acid and this made their Raman resonances suppressed to the laser excitation wavelength energy.19,20 The intensity of the peak around 1350 cm-1 (D-band) increased as expected after purification indicated the conversion of some sp2 C to sp3 C on the nanotube bundles during the process of purification21,22 and furthermore its height increased with increasing sonication-centrifugation cycles from A to D, which may be due to shorter length and higher functionalization degree of SWNTs in the process. The strong peak at 1350 cm-1 and the absence of peak in the range of 100-200 cm-1 from product D indicated that it is mainly graphite, which is consistent with the SEM and X-ray data. From these results, we concluded that the fractional SWNTs product B was the purest and thus was used to for the following works.
3.2 Characterization of the enzymes modified SWNTs
In the FT-IR spectra (not shown), the amide linkage formation was confirmed by the decline of IR peak at 1720 cm-1 corresponding to carboxylic acid and the appearance of the new bands at 1656 cm-1 assigning to amide I band and 1545 cm-1 attributing to amide II band after the immobilization of enzyme on SWNTs. Furthermore, the peaks of 2960 cm-1 and 1400 cm-1 corresponding to the saturate hydrocarbon belong to the enzyme were observed, respectively. 23
Raman spectra of GOx functionalized showed that there is no significant difference between the samples before and after the immobilization of enzyme on SWNTs, suggesting that the tubular structure of SWNTs is undisrupted in this stage as expected.
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Figure 4: UV-Vis-NIR absorption spectra of suspended SWNTs before and after immobilizing GOx. The noise (around
1450nm) was caused by the subtraction of the corresponding aqueous background.
SWNTs before and after the enzyme modification were also evaluated by UV-Vis-NIR absorbance spectroscopy (Fig. 4) 22. It has been reported that three bands M11，S22，S11 were observed in NIR absorption spectrum, which corresponded to the peaks around 740 nm，1000 nm，1800 nm, respectively depending on their diameters. 22,24 Fig. 4 showed that immobilization had no influence on these peaks as expected.
3.3 The electrochemical behavior of H2O2 with SWNTs modified electrode
We first tested the purified SWNTs (from the product B) modified electrode on the detection of hydrogen peroxide, which is a common product liberated from many biocatalystic processes. Figure 5 showed the cyclic voltammetry (cv) curves of different concentrations of hydrogen peroxide on SWNTs modified electrode. A pair of redox peaks was observed around –0.1 V which indicated the redox reaction of carboxylic acid group on the SWNTs. 25 And a much stronger reduction peak of H2O2 was observed at -0.40 V.
Figure 5: Cyclic voltammetry (cv) curves of different hydroperoxide concentration on SWNTs modified electrode. The
curves were taken in a solution of 0.05 M phosphate buffer (pH=7.4) in 0.1 M KCl at a 50 mV/s scan rate.
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The amperometric responses at the SWNTs modified electrode for each addition of 0.4 mM H2O2 were presented in Fig. 6A. Well-defined linear current responses, proportional to H2O2 concentration, were observed. The response of the electrode was also fast in reaching a dynamic equilibrium upon each addition of the sample solution, generating a steady-state current signal within only 2 to 3 s. An excellent linear relationship with correlation coefficient of 0.999 in the range of 0.02 ~ 11.2 mM H2O2 and another coefficient of 0.988 over the range of 11.2~ 26.8 mM H2O2 were obtained (Figure 6B).
Figure 6: Amperometric responses of SWNTs modified GC electrode to successive additions of 0.4 mM H2O2 (A) and the calibration curve (B) at potential of -0.40 V; stirring rate, ~300 rpm. Other conditions were the same as those in
Figure 5.
The current response on the bare GC electrode was also tested for H2O2 detection as reference. Compared with the SWNTs modified electrode, the GC electrode not only has more negative reduction potential (-0.52 V) for H2O2 detection, it was also found that the current response of H2O2 was only 0.08 µA/mM with a linear range of 0.19 ~ 11.2 mM. Combining with the much higher current response (12.2 µA/mM) of SWNTs modified GC electrode, we could conclude that the SWNTs modified electrode showed significant electrocatalytic behavior toward the reduction of H2O2 and this proves that the chemically SWNTs modified electrodes could be used for the electrochemical detection for many enzyme processes with H2O2 generation.
3.4 The electrochemical performance of the Glucose oxidase modified SWNTs
Cyclic voltammetry (cv) curve of glucose on SWNT-GOx modified GC electrode was shown in Fig. 7, in which a pair of redox peaks (~ - 0.1 V) of carboxylic acid groups on the SWNTs were observed same as in Fig. 6. The electrode with GOx bonding on the SWNTs displayed almost symmetrical CV shapes with almost equal reduction and oxidation peaks between -0.40 V and -0.50 V from GOx.26 A good current response with increasing of glucose concentration at –0.36 V and –0.47 V potential was observed in the CV (Fig. 7).
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Figure 7: Cyclic voltammetry (cv) curves of SWNT-GOx modified GC electrode in glucose solution. Other conditions
were the same as those in Figure 5.
Figure 8: Amperometric responses of bare GC electrode (a) and SWNTs-GOx modified GC electrode (b) to successive additions of 4 mM glucose at potential of -0.40 V; stirring rate, ~300 rpm. Other conditions were the same as those in
Figure 6.
The amperometric responses at the GOx modified SWNTs for each successive addition of 4 mM glucose are presented in Figure 8. The reaction occurring at the biosensor is also very fast in reaching a dynamic equilibrium upon each addition of the sample solution, generating a strong current signal within 3 to 5 s. The current response was much higher than that in which immobilized GOx on the SWNTs by physically adsorption. 26 This result showed that the chemically bonded enzyme activity is maintained and the electron transfer on the SWNTs modified electrode with chemical bonding is much better than that by physically adsorption for the electrochemical detection. The linear current response to different glucose concentrations was observed from 0.96 to 12.96 mM glucose (r=0.995) with sensitivity of 1.5 µA/mM. We have noticed that GOx functionalized SWNTs is much more difficult to disperse and form a uniform film on the electrode and this may be the reason for the decaying current and higher noise observed in Fig. 8.
4. Conclusions
In summary, this paper presents our preliminary investigation using chemically bonded enzyme on SWNTs as the probe for electrochemical biosensors. The enzyme GOx functionalized SWNTs was fully characterized and used for the detection for glucose with wide linear range and high sensitivity. Thus, with good conductance, large surface area and high aspect ratio, chemically modified SWNTs directly with
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detecting probes could play a dual role of direct transduction platform and immobilization matrix to build electrochemical biosensors for ultrasensitive and fast chemical and biological detection. To further fully utilize these properties of SWNTs for electrochemical detections, the studies for patterned SWNTs nanoelectrode array biosensors for glucose and other bioactive species are in progress.
Acknowledgments
We gratefully acknowledge the financial support from MOE (20040055020).
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作者简介：陈永胜教授于1997年在加拿大维多利亚大学（University of Victoria）取得博士学位。之后于美国肯特基大学(University of Kentucky)及加州州立大学洛杉矶分校（UCLA）师从国际著名的纳米材料专家Dr. Haddon 及Dr. Wudl从事博士后工作，之后曾任职于加州州立大学圣地亚哥分校（UCSD）。于2003年5月被聘为南开大学特聘教授。主要从事纳米碳管，多功能复合材料及分子器材方面的研究。至2007年，已发表论文40余篇，包括《科学》（Science）一篇，《自然》（Nature）一篇，《美国化学学会会志》（JACS）五篇,< -9- 因篇幅问题不能全部显示，请点此查看更多更全内容