纳米复合材料的制备及其在电化学生物传感器中的应用
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摘要
电化学酶生物传感器因具有灵敏度高、选择性好、易于微型化等优点而具有广泛的应用前景,现已成为当前研究的热点之一。在酶生物传感器的研究和开发中,其活性组分的固定化一直是最为关键和重要的技术。纳米复合材料具有比表面积大、吸附力强、催化活性高和生物相容性好等特性,将其用于生物分子的固载,不仅可以提高生物分子(酶)的吸附量和稳定性,还能很好地保持生物分子的生物活性,从而使传感器的性能得到提高。基于此,本文制备了不同形貌的纳米复合材料,并将这些材料用于生物传感界面的构建。所制备的葡萄糖、胆固醇等生物传感器具有较宽的线性范围、较高的灵敏度、较低的检测下限以及较快的响应速度。具体内容如下：
1.基于普鲁士蓝-金复合膜构建的葡萄糖生物传感器的研究
在本工作中,研制了一种基于普鲁土蓝-纳米金复合物(PB-Au)以及铂纳米簇(Pt-NCs)的葡萄糖生物传感器。作为电子媒介体的PB,通过与氯金酸一起电聚合到玻碳电极表面而形成一层致密的PB-Au复合膜。接着通过电沉积的方式将Pt-NCs固载到PB-Au修饰电极上。最后,将葡萄糖氧化酶(GOD)组装到修饰电极上并修饰一层Nafion膜,防止GOD的泄漏。制得的电流型葡萄糖传感器表现出快速的响应时间,宽的线性范围,当信噪比为3时检出限低至1.01μmol/L。该传感器具有良好的选择性,其原因是由于选择的工作电位较低,与葡萄糖共存的干扰物如抗坏血酸、尿酸、精氨酸等不会产生干扰。
2.基于铂纳米簇-多壁碳纳米管复合物构建的葡萄糖生物传感器的研究
由于具有独特的物理化学性质,铂纳米簇-多壁碳纳米管纳米复合物(PtNCs-MWCNTs)现在已成为极具吸引力的纳米材料。在此工作中,采用一步合成法制得了PtNCs-MWCNTs并以其为固酶基质构建了一种高灵敏的葡萄糖生物传感器。采用循环伏安法(CV),电化学交流阻抗法(EIS)以及原子力显微镜(AFM)表征了传感器的组装过程。在最优条件下,该传感器对葡萄糖表现出良好的催化特性,如线性范围宽(3μmol/L～14.5 mmol/L),检出限低(1.0μmol/L),灵敏度高(12.55μAL/mmol),向应时间短(6s)。更重要的是,此传感器能够用于对人体血.清样品中葡萄糖含量进行分析。该传感器性能优良,是因为PtNCs-MWCNTs具有大的比表面积、良好的导电性、较高的催化活性和良好的生物相容性,从而增大了酶的吸附量且促进电极界面上的电子转移。
3.一步电沉积法制备生物复合膜及其在胆固醇生物传感器中的应用
将多壁碳纳米管(MWCNTs)、金铂合金纳米粒子(Au@PtANPs)和壳聚糖(CS)的优势结合起来,采用一种独特的方法构建了一种新的胆固醇生物传感器。首先采用一步电沉积法将CS-MWCNTs-Au@PtANPs复合膜沉积在玻碳电极表面,再用戊二醛做交联剂将胆固醇氧化酶(ChOx)固载在修饰电极上制得胆固醇生物传感器。从CS-MWCNTs-Au@PtANPs复合膜的扫描电镜图像可以看到在CS基质内分散有大量的MWCNTs和Au@PtANPs。用循环伏安和交流阻抗等方法表征了传感器的组装过程。该传感器的线性范同为0.7-477.7μmol/L,校正曲线的斜率即为传感器的灵敏度,可达到224.5μA L/mmol。
4.基于金铂复合物功能化的氧化锌纳米棒的胆固醇生物传感器的研究
构建了一种基于多壁碳纳米管(MWCNTs)和金铂复合物功能化的氧化锌纳米棒(Pt@Au-ZnONRs)为基质固载胆固醇氧化酶(ChOx)的生物传感器,并考察了该胆固醇传感器的应用潜力。首先,用化学合成法制备了Pt@Au-ZnONRs。然后将其分散液滴涂到MWCNTs修饰的玻碳电极表面,接着通过纳米材料对ChOx的吸附作用以及正电荷ZnO纳米材料与负电荷ChOx之间的静电作用将ChOx固载到电极上。MWCNTs和Pt@Au-ZnONRs为ChOx提供了良好的微环境,从而有利于传感器分析性能的提高。该传感器对胆固醇响应的线性范围为0.1～759.3μmol/L,检测限为0.03μmol/L,灵敏度为26.8μAL/mmol,米氏常数为1.84mmol/L,并具有响应时间短、灵敏度高、稳定性好等优点。
As electrochemical enzyme biosensors have the advantages of high-sensitivity, high-selectivity as well as easy miniaturization, they have gained wide attention and made much more progress. It is reported that the crucial aspect in the fabrication of a biosensor is the immobilization of bio-recognition molecule. Nano-composite materials, exhibiting large specific surface area, strong adsorption capacity, high catalytic activity and good biocompatibility, are favorable for constructing biosensors, which not only retain the biological activities of biomolecules, but also enhance the performance of the biosensors. In this thesis, nano-composite materials with different morphologies were synthesized by the various methods and employed to construct the electrochemical enzyme biosensors. The fabricated biosensors for glucose and cholesterol showed wide linear range, high sensitivity, low detection limit and rapid response. The main works and conclusions are included as follows:
1. Glucose biosensor based on prussian blue-gold nanocomposite films
A glucose biosensor was developed, which was based on prussian blue-gold (PB-Au) nanocomposite films and platinum nanoclusters (Pt-NCs). Prussian blue (PB), as an electron mediator, was electrochemically deposited on the glass carbon electrode (GCE) in the presence of chloroauric acid, forming PB-Au nanocomposite films. Then, Pt-NCs were electrodeposited to construct a bilayer film. At last, glucose oxidase (GOD) was modified on the electrode with the bilayer film and the film of Nafion was used to prevent GOD from leaking off. The resulting amperometric glucose biosensor exhibited a fast response time (within 8 s) and a linear calibration range from 3.0μmol/L to 1.1 mmol/L with a low detection limit of 1.0μmol/L glucose (S/N=3). With the low operating potential, the biosensor showed little interference to the possible interferents, including acetum acid, uric acid and arginine, indicating an excellent selectivity.
2. The construction of glucose biosensor based on platinum nanoclusters-multiwalled carbon nanotubes nanocomposites
One-step synthesis method was proposed to obtain the nanocomposites of platinum nanocluster and multiwalled carbon nanotubes (PtNCs-MWCNTs), which were used as a novel immobilization matrix for the enzyme to fabricate the glucose biosensor. The whole fabrication process of the biosensor was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM) and scanning electron microscope (SEM). Due to the favorable characteristic of PtNCs-MWCNTs nanocomposites, the biosensor exhibited good characteristics, such as wide linear range (3.0μmol/L-12.1 mmol/L), low detection limit (1.0μmol/L), high sensitivity (12.8μA L/mmol), short response time (within 6 s). Finally, it was demonstrated that this biosensor can be used for the assay of glucose in human serum samples. The performance of the resulted biosensor was more prominent than that of most of the reported glucose biosensors.
3. The preparation of the biocomposite film by the method of one-step electrodeposition and its application in the cholesterol biosensor
We reported an ingenious approach for the fabrication of a promising cholesterol biosensor, which integrated the beneficial characteristics of multiwalled-carbon nanotubes (MWCNTs), gold-platinum alloy nanoparticles (Au@PtANPs) and chitosan (CS). Cholesterol oxidase (ChOx) was immobilized via glutaraldehyde as a cross-linker onto CS-MWCNTs-Au@PtANPs nanobiocomposite film deposited onto glassy carbon electrode. Scanning electron microscopy (SEM) images of CS-MWCNTs-Au@PtANPs revealed that MWCNTs and Au@PtANPs were dispersed in CS matrix. Cyclic voltammetry and impedance spectroscopy were used to characterize the assembly process of the biosensor. The proposed biosensor exhibited a linear current response to glucose concentration (0.7～477.7μmol/L) at the potential of 0.5 V. The sensitivity to the change in the concentration of cholesterol as the slope of the calibration curve was 224.5μA L/mmol.
4. Highly-sensitive cholesterol biosensor based on platinum-gold hybrid functionalized ZnO nanorods
A novel scheme for the fabrication of gold/platinum hybrid functionalized ZnO nanorods (Pt-Au@ZnONRs) and multiwalled carbon nanotubes (MWCNTs) modified electrode was presented and its application for cholesterol biosensor was investigated. Firstly, Pt-Au@ZnONRs was prepared by the method of chemical synthesis. Then, the Pt-Au@ZnONRs suspension was dropped on the MWCNTs modified glass carbon electrode, and followed with cholesterol oxidase (ChOx) immobilization by the adsorbing interaction between the nano-material and ChOx as well as the electrostatic interaction between ZnONRs and ChOx molecules. The combination of MWCNTs and Pt-Au@ZnONRs provided a favorable environment for ChOx and resulted in the enhanced analytical response of the biosensor. The resulted biosensor exhibited a linear response to cholesterol in the wide range of 0.1-759.3μmol/L with a low detection limit of 0.03μmol/L and a high sensitivity of 26.8μA L/mmol. The calculated apparent Michaelis constant Km was 1.84 mmol/L, indicating a high affinity between ChOx and cholesterol.
1.基于普鲁士蓝-金复合膜构建的葡萄糖生物传感器的研究
在本工作中,研制了一种基于普鲁土蓝-纳米金复合物(PB-Au)以及铂纳米簇(Pt-NCs)的葡萄糖生物传感器。作为电子媒介体的PB,通过与氯金酸一起电聚合到玻碳电极表面而形成一层致密的PB-Au复合膜。接着通过电沉积的方式将Pt-NCs固载到PB-Au修饰电极上。最后,将葡萄糖氧化酶(GOD)组装到修饰电极上并修饰一层Nafion膜,防止GOD的泄漏。制得的电流型葡萄糖传感器表现出快速的响应时间,宽的线性范围,当信噪比为3时检出限低至1.01μmol/L。该传感器具有良好的选择性,其原因是由于选择的工作电位较低,与葡萄糖共存的干扰物如抗坏血酸、尿酸、精氨酸等不会产生干扰。
2.基于铂纳米簇-多壁碳纳米管复合物构建的葡萄糖生物传感器的研究
由于具有独特的物理化学性质,铂纳米簇-多壁碳纳米管纳米复合物(PtNCs-MWCNTs)现在已成为极具吸引力的纳米材料。在此工作中,采用一步合成法制得了PtNCs-MWCNTs并以其为固酶基质构建了一种高灵敏的葡萄糖生物传感器。采用循环伏安法(CV),电化学交流阻抗法(EIS)以及原子力显微镜(AFM)表征了传感器的组装过程。在最优条件下,该传感器对葡萄糖表现出良好的催化特性,如线性范围宽(3μmol/L～14.5 mmol/L),检出限低(1.0μmol/L),灵敏度高(12.55μAL/mmol),向应时间短(6s)。更重要的是,此传感器能够用于对人体血.清样品中葡萄糖含量进行分析。该传感器性能优良,是因为PtNCs-MWCNTs具有大的比表面积、良好的导电性、较高的催化活性和良好的生物相容性,从而增大了酶的吸附量且促进电极界面上的电子转移。
3.一步电沉积法制备生物复合膜及其在胆固醇生物传感器中的应用
将多壁碳纳米管(MWCNTs)、金铂合金纳米粒子(Au@PtANPs)和壳聚糖(CS)的优势结合起来,采用一种独特的方法构建了一种新的胆固醇生物传感器。首先采用一步电沉积法将CS-MWCNTs-Au@PtANPs复合膜沉积在玻碳电极表面,再用戊二醛做交联剂将胆固醇氧化酶(ChOx)固载在修饰电极上制得胆固醇生物传感器。从CS-MWCNTs-Au@PtANPs复合膜的扫描电镜图像可以看到在CS基质内分散有大量的MWCNTs和Au@PtANPs。用循环伏安和交流阻抗等方法表征了传感器的组装过程。该传感器的线性范同为0.7-477.7μmol/L,校正曲线的斜率即为传感器的灵敏度,可达到224.5μA L/mmol。
4.基于金铂复合物功能化的氧化锌纳米棒的胆固醇生物传感器的研究
构建了一种基于多壁碳纳米管(MWCNTs)和金铂复合物功能化的氧化锌纳米棒(Pt@Au-ZnONRs)为基质固载胆固醇氧化酶(ChOx)的生物传感器,并考察了该胆固醇传感器的应用潜力。首先,用化学合成法制备了Pt@Au-ZnONRs。然后将其分散液滴涂到MWCNTs修饰的玻碳电极表面,接着通过纳米材料对ChOx的吸附作用以及正电荷ZnO纳米材料与负电荷ChOx之间的静电作用将ChOx固载到电极上。MWCNTs和Pt@Au-ZnONRs为ChOx提供了良好的微环境,从而有利于传感器分析性能的提高。该传感器对胆固醇响应的线性范围为0.1～759.3μmol/L,检测限为0.03μmol/L,灵敏度为26.8μAL/mmol,米氏常数为1.84mmol/L,并具有响应时间短、灵敏度高、稳定性好等优点。
As electrochemical enzyme biosensors have the advantages of high-sensitivity, high-selectivity as well as easy miniaturization, they have gained wide attention and made much more progress. It is reported that the crucial aspect in the fabrication of a biosensor is the immobilization of bio-recognition molecule. Nano-composite materials, exhibiting large specific surface area, strong adsorption capacity, high catalytic activity and good biocompatibility, are favorable for constructing biosensors, which not only retain the biological activities of biomolecules, but also enhance the performance of the biosensors. In this thesis, nano-composite materials with different morphologies were synthesized by the various methods and employed to construct the electrochemical enzyme biosensors. The fabricated biosensors for glucose and cholesterol showed wide linear range, high sensitivity, low detection limit and rapid response. The main works and conclusions are included as follows:
1. Glucose biosensor based on prussian blue-gold nanocomposite films
A glucose biosensor was developed, which was based on prussian blue-gold (PB-Au) nanocomposite films and platinum nanoclusters (Pt-NCs). Prussian blue (PB), as an electron mediator, was electrochemically deposited on the glass carbon electrode (GCE) in the presence of chloroauric acid, forming PB-Au nanocomposite films. Then, Pt-NCs were electrodeposited to construct a bilayer film. At last, glucose oxidase (GOD) was modified on the electrode with the bilayer film and the film of Nafion was used to prevent GOD from leaking off. The resulting amperometric glucose biosensor exhibited a fast response time (within 8 s) and a linear calibration range from 3.0μmol/L to 1.1 mmol/L with a low detection limit of 1.0μmol/L glucose (S/N=3). With the low operating potential, the biosensor showed little interference to the possible interferents, including acetum acid, uric acid and arginine, indicating an excellent selectivity.
2. The construction of glucose biosensor based on platinum nanoclusters-multiwalled carbon nanotubes nanocomposites
One-step synthesis method was proposed to obtain the nanocomposites of platinum nanocluster and multiwalled carbon nanotubes (PtNCs-MWCNTs), which were used as a novel immobilization matrix for the enzyme to fabricate the glucose biosensor. The whole fabrication process of the biosensor was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM) and scanning electron microscope (SEM). Due to the favorable characteristic of PtNCs-MWCNTs nanocomposites, the biosensor exhibited good characteristics, such as wide linear range (3.0μmol/L-12.1 mmol/L), low detection limit (1.0μmol/L), high sensitivity (12.8μA L/mmol), short response time (within 6 s). Finally, it was demonstrated that this biosensor can be used for the assay of glucose in human serum samples. The performance of the resulted biosensor was more prominent than that of most of the reported glucose biosensors.
3. The preparation of the biocomposite film by the method of one-step electrodeposition and its application in the cholesterol biosensor
We reported an ingenious approach for the fabrication of a promising cholesterol biosensor, which integrated the beneficial characteristics of multiwalled-carbon nanotubes (MWCNTs), gold-platinum alloy nanoparticles (Au@PtANPs) and chitosan (CS). Cholesterol oxidase (ChOx) was immobilized via glutaraldehyde as a cross-linker onto CS-MWCNTs-Au@PtANPs nanobiocomposite film deposited onto glassy carbon electrode. Scanning electron microscopy (SEM) images of CS-MWCNTs-Au@PtANPs revealed that MWCNTs and Au@PtANPs were dispersed in CS matrix. Cyclic voltammetry and impedance spectroscopy were used to characterize the assembly process of the biosensor. The proposed biosensor exhibited a linear current response to glucose concentration (0.7～477.7μmol/L) at the potential of 0.5 V. The sensitivity to the change in the concentration of cholesterol as the slope of the calibration curve was 224.5μA L/mmol.
4. Highly-sensitive cholesterol biosensor based on platinum-gold hybrid functionalized ZnO nanorods
A novel scheme for the fabrication of gold/platinum hybrid functionalized ZnO nanorods (Pt-Au@ZnONRs) and multiwalled carbon nanotubes (MWCNTs) modified electrode was presented and its application for cholesterol biosensor was investigated. Firstly, Pt-Au@ZnONRs was prepared by the method of chemical synthesis. Then, the Pt-Au@ZnONRs suspension was dropped on the MWCNTs modified glass carbon electrode, and followed with cholesterol oxidase (ChOx) immobilization by the adsorbing interaction between the nano-material and ChOx as well as the electrostatic interaction between ZnONRs and ChOx molecules. The combination of MWCNTs and Pt-Au@ZnONRs provided a favorable environment for ChOx and resulted in the enhanced analytical response of the biosensor. The resulted biosensor exhibited a linear response to cholesterol in the wide range of 0.1-759.3μmol/L with a low detection limit of 0.03μmol/L and a high sensitivity of 26.8μA L/mmol. The calculated apparent Michaelis constant Km was 1.84 mmol/L, indicating a high affinity between ChOx and cholesterol.
引文
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