基于新型纳米复合材料电化学生物传感器的构建及其分析应用
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摘要
生物传感器技术是一种实用的分析技术,具有较强的生物特异性和高灵敏度,装置体积小,成本低。目前,生物传感器在许多领域有很广泛的应用,如环境监测和生物过程控制,食品工业和农业质量控制,以及临床医学的应用。近年来,生物材料,尤其是实用性强的纳米材料的快速发展,以及新的传感技术的出现,给电化学生物传感器的设计和构建带来了新的发展机遇。由于其具有良好的生物相容性,高的催化活性,很强的吸附能力和大的比表面积,这些纳米材料可以大大提高生物传感器的性能。鉴于纳米材料拥有优越的物理和化学性能,本论文制备了不同的纳米材料,将这些纳米材料用于酶的固定化构建了电化学酶传感器。另外,利用金属纳米材料直接作为电氧化活性成份构建了无酶电化学传感器。所构建的生物传感器具有较快的响应速度,较高的灵敏度和较低的检测下限,同时还具有很好的重现性和稳定性,可用于实际样品的检测。本论文主要研究工作如下:
(1)采用水热法合成了大小均一有序的纳米羟基磷灰石(nano-HA),利用nano-HA较强的吸附作用将酪氨酸酶固定到电极界面,构建了一种新颖的酚类传感器。Nano-HA为酪氨酸酶的固定提供了良好的微环境,有利于保持酪氨酸酶的生物活性。酚类化合物通过酪氨酸酶催化产生的醌在-0.25 V(对饱和甘汞电极)直接还原而测定。实验研究了各种因素,如pH、工作电位、温度等对传感器响应电流的影响。在优化的实验条件下,传感器对邻苯二酚响应的线性范围为10 nM~7μM,灵敏度为2.11×103μAmM-1 cm-2,检测下限为5 nM(S/N=3)。传感器对邻苯二酚,苯酚,间甲酚的表观米氏常数分别为3.16, 1.31和3.52μM。同时,该传感器具有良好的稳定性和重现性,可用于实际样品检测(第2章)。
(2)基于纳米氧化镁-壳聚糖(nano-MgO-chit)复合物为基底构建了一种新颖的辣根过氧化酶(HRP)电化学生物传感器。Nano-MgO-chit复合物的形貌通过扫描电镜表征。Nano-MgO和HRP之间的相互作用通过紫外可见光谱进行了表征。该复合材料综合了无机纳米粒子和有机聚合物的优点。固定在纳米复合基底中的HRP对H2O2的还原表现出优良的电催化作用。利用计时电流法对实验变量,如pH,工作电位对传感器响应电流的影响进行了考察。在优化的实验条件下,传感器对H2O2浓度的改变响应非常快速(小于10 s),线性范围为0.1~1300μM,检测下限为0.05μM(S/N=3)。同时,实验结果表明该传感器具有良好的稳定性和重现性(第3章)。
(3)结合碳纳米管(CNTs)优良的导电性能和催化性能以及介孔二氧化硅(SBA-15)大的比表面积和较强的吸附能力,基于CNTs-SBA-15纳米复合膜固定HRP,构建了一种新颖的第三代H2O2传感器。利用紫外可见光谱对固定在复合膜中的酶的生物活性进行了表征。固定在纳米复合基体中的HRP对H2O2有良好的电催化作用。利用计时电流法对实验变量,如溶液pH值和工作电位进行了优化。在最优的实验条件下,传感器对H2O2检测的线性范围为1μm~7 mM,检测下限为0.5μM(S/N=3)。此外,实验结果表明该传感器具有良好的稳定性和重现性(第4章)。
(4)以多孔聚碳酸酯膜为模板结合电化学沉积技术,首次合成高度有序的镍纳米线阵列(NiNWA)。利用扫描电子显微镜和透射电子显微镜对其形貌进行了表征。在碱性介质中,NiNWA电极对葡萄糖表现出很强的电催化作用。基于此,我们构建了一个灵敏的无酶电化学葡萄糖传感器。传感器对葡萄糖检测的线性范围为0.5μM~3 mM,相关系数为0.999。检测下限为0.1μM(S/N=3),灵敏度高达1.043×103μAmM-1 cm-2。同时实验结果表明,该传感器具有良好的重现性,长期稳定性以及很好的选择性(第5章)。
(5)制备了纳米钯-石墨烯(PdNPs-graphene)纳米杂化物并用于构建无酶葡萄糖传感器。PdNPs-graphene纳米杂化物通过原位还原方法合成。首先,将graphene-Nafion组装到电极表面用于化学吸附钯离子,然后用水合肼将钯离子原位还原为PdNPs。在碱性介质中,PdNPs-graphene杂化物修饰电极对葡萄糖的电氧化表现出很高的电催化活性。在最优的实验条件下,所构建的传感器对葡萄糖在10μM~5 mM范围有很好的线性响应,相关系数为0.998。检测下限为1μM(S/N=3)。同时实验结果还表明,该传感器具有很好的重现性,稳定性和良好的选择性(第6章)。
(6)基于原位电聚合罗丹明B(PRhB)传感膜构建了亚硝酸盐传感器。PRhB修饰电极对亚硝酸盐的电化学氧化表现出很好的催化作用。实验研究了各种因素对该传感器性能的影响,如:膜的厚度,溶液的pH值,工作电位。PRhB修饰电极对NO2-响应迅速,在0.5μM~0.7 mM浓度范围之内呈很好的线性关系,检测下限为0.1μM(S/N=3),灵敏度为308.1μAmM-1 cm-2。同时实验结果表明,该传感器具有很好的重现性,稳定性和良好的选择性(第7章)。
Biosensor technology is a powerful alternative to conventional analytical techniques, harnessing the specificity and sensitivity of biological systems in small, low cost devices. Biosensors are being developed for different applications, including environmental and bio-process control, quality control of food, agriculture, military, and, particularly, medical applications. The rapid growth in biomaterials, especially the availability, application and combination of a wide range of nanomaterials with new sensing techniques, has caused a remarkable innovation in the design and construction of electrochemical biosensors. Owing to their biocompatibility, catalytic properties, strong adsorption capacity and high accessible surface area, these nanomaterials can greatly improve the performance of biosensor. Base on the superior physical and chemical properties of nano-materials, many nanomaterials with different morphology were synthesized in this thesis. These nanomaterials were used to fabricate novel biosensors and electrochemically nonenzymatic sensors. The prepared biosensors exhibited fast response, high sensitivity and low detection limit. These biosensors also showed good reproducibility and stability and could be used for the detection of real samples.
(1)A novel tyrosinase biosensor based on hydroxyapatite nanoparticles (nano-HA)-chitosan nanocomposite has been developed for the detection of phenolic compounds. The uniform and size controlled nano-HA was synthesized by hydrothermal method, and its morphological characterization was examined by transmission electron microscope (TEM). Tyrosinase was then immobilized on a nano-HA-chitosan nanocomposite-modified gold electrode. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize the sensing film. The prepared biosensor was applied to determine phenolic compounds by monitoring the reduction signal of the biocatalytically produced quinone species at -0.25 V (vs. saturated calomel electrode). The effects of the pH, temperature and applied potential on the biosensor performance were investigated, and experimental conditions were optimized. The biosensor exhibited a linear response to catechol over a wide concentration range from 10 nM to 7μM, with a high sensitivity of 2.11×103μAmM-1 cm-2, and a limit of detection down to 5 nM (based on S/N=3). The apparent Michaelis-Menten constants of the enzyme electrode were estimated to be 3.16, 1.31 and 3.52μM for catechol, phenol and m-cresol, respectively. Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results(in chapter 2).
(2)A novel horseradish peroxidase (HRP) electrochemical biosensor based on a MgO nanoparticles (nano-MgO)-chitosan (chit) composite matrix was developed. The morpology of nano-MgO-chit nanocomposite was examined by scanning electron microscopy (SEM). The interaction between nano-MgO-chit nanocomposite matrix and enzyme was characterized with UV-vis spectra. This proposed composite material combined the advantages of inorganic nanoparticles and organic polymer chit. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H2O2 in the presence of hydroquinone as a mediator. The effects of the experimental variables such as solution pH and the working potential were investigated using steady-state amperometry. The present biosensor (HRP-modified electrode) had a fast response towards H2O2 (less than 10 s), and excellent linear relationships were obtained in the concentration range of 0.1~1300μM, with a detection limit of 0.05μM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results (in chapter 3).
(3)A new third-generation biosensor for H2O2 assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of carbon nanotubes(CNTs)-mesoporous silicas(SBA-15) modified gold electrode. The biological activity of HRP immobilizing in the composite film was characterized by UV-vis spectra. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H2O2. The effects of the experimental variables such as solution pH and the working potential were investigated using steady-state amperometry. Under the optimal conditions, the resulting biosensor showed a linear range of 1μM to 7 mM and a detection limit of 0.5μM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results (in chapter 4).
(4)Highly ordered Ni nanowire arrays(NiNWAs) were synthesized for the first time using a template-directed electropolymerization strategy with a nanopore polycarbonate (PC) membrane template, and their morphological characterization were examined by scanning electron microscopy (SEM) and transmission electron microscope (TEM). A NiNWAs based electrode exhibited very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium, which has been utilized as the basis of the fabrication of a nonenzymatic biosensor for electrochemical detection of glucose. The biosensor can be applied to the quantification of glucose with a linear range covering from 5.0×10-7 to 7.0×10-3 M, a high sensitivity of 1.043×103μAmM-1 cm-2, and a low detection limit of 1×10-7 M. The experiment results also showed that the sensor exhibited good reproducibility and long-term stability, as well as high selectivity with no interference from other oxidable species (in chapter 5).
(5) A nonenzymatic electrochemical biosensor was developed for the detection of glucose based on an electrode modified with palladium nanoparticles (PdNPs)-functioned graphene (nafion-graphene). The palladium nanoparticle-graphene nanohybrids were synthesized using an in-situ reduction process. Nafion-graphene was first assembled onto an electrode to chemically adsorb Pd2+. And Pd2+ was subsequently reduced by hydrazine hydrate to form PdNPs in-situ. Such a PdNPs-graphene nanohybrids-based electrode shows a very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium. The proposed biosensor can be applied to the quantification of glucose with a wide linear range covering from 10μM to 5 mM (R=0.998) with a low detection limit of 1μM. The experiment results also showed that the sensor exhibited good reproducibility and long-term stability, as well as high selectivity with no interference from other potential competing species (in chapter 6).
(6) The fabrication of an electropolymerized Rhodamine B (PRhB) sensing film based electrochemical sensor and its application for electrochemical detection of nitrite were described. The PRhB film modified GC electrode exhibited good catalytic activity towards the electrochemical oxidation of nitrite. The effects of the experimental variables such as the thickness of the film, solution pH values and the working potential were investigated using steady-state amperometry. The present sensor (PRhB-modified electrode) had a fast response towards NO2- ( less than 10 s), and excellent linear relationships were obtained in the concentration range of 0.5μM~7.0 mM, with a detection limit of 0.1μM (S/N=3). The sensitivity of the sensor was calculated to be 308.1μAmM-1 cm-2. The possible interferences from several common ions were tested. Moreover, the stability and reproducibility of this sensor were evaluated with satisfying results (in chapter 7).
(1)采用水热法合成了大小均一有序的纳米羟基磷灰石(nano-HA),利用nano-HA较强的吸附作用将酪氨酸酶固定到电极界面,构建了一种新颖的酚类传感器。Nano-HA为酪氨酸酶的固定提供了良好的微环境,有利于保持酪氨酸酶的生物活性。酚类化合物通过酪氨酸酶催化产生的醌在-0.25 V(对饱和甘汞电极)直接还原而测定。实验研究了各种因素,如pH、工作电位、温度等对传感器响应电流的影响。在优化的实验条件下,传感器对邻苯二酚响应的线性范围为10 nM~7μM,灵敏度为2.11×103μAmM-1 cm-2,检测下限为5 nM(S/N=3)。传感器对邻苯二酚,苯酚,间甲酚的表观米氏常数分别为3.16, 1.31和3.52μM。同时,该传感器具有良好的稳定性和重现性,可用于实际样品检测(第2章)。
(2)基于纳米氧化镁-壳聚糖(nano-MgO-chit)复合物为基底构建了一种新颖的辣根过氧化酶(HRP)电化学生物传感器。Nano-MgO-chit复合物的形貌通过扫描电镜表征。Nano-MgO和HRP之间的相互作用通过紫外可见光谱进行了表征。该复合材料综合了无机纳米粒子和有机聚合物的优点。固定在纳米复合基底中的HRP对H2O2的还原表现出优良的电催化作用。利用计时电流法对实验变量,如pH,工作电位对传感器响应电流的影响进行了考察。在优化的实验条件下,传感器对H2O2浓度的改变响应非常快速(小于10 s),线性范围为0.1~1300μM,检测下限为0.05μM(S/N=3)。同时,实验结果表明该传感器具有良好的稳定性和重现性(第3章)。
(3)结合碳纳米管(CNTs)优良的导电性能和催化性能以及介孔二氧化硅(SBA-15)大的比表面积和较强的吸附能力,基于CNTs-SBA-15纳米复合膜固定HRP,构建了一种新颖的第三代H2O2传感器。利用紫外可见光谱对固定在复合膜中的酶的生物活性进行了表征。固定在纳米复合基体中的HRP对H2O2有良好的电催化作用。利用计时电流法对实验变量,如溶液pH值和工作电位进行了优化。在最优的实验条件下,传感器对H2O2检测的线性范围为1μm~7 mM,检测下限为0.5μM(S/N=3)。此外,实验结果表明该传感器具有良好的稳定性和重现性(第4章)。
(4)以多孔聚碳酸酯膜为模板结合电化学沉积技术,首次合成高度有序的镍纳米线阵列(NiNWA)。利用扫描电子显微镜和透射电子显微镜对其形貌进行了表征。在碱性介质中,NiNWA电极对葡萄糖表现出很强的电催化作用。基于此,我们构建了一个灵敏的无酶电化学葡萄糖传感器。传感器对葡萄糖检测的线性范围为0.5μM~3 mM,相关系数为0.999。检测下限为0.1μM(S/N=3),灵敏度高达1.043×103μAmM-1 cm-2。同时实验结果表明,该传感器具有良好的重现性,长期稳定性以及很好的选择性(第5章)。
(5)制备了纳米钯-石墨烯(PdNPs-graphene)纳米杂化物并用于构建无酶葡萄糖传感器。PdNPs-graphene纳米杂化物通过原位还原方法合成。首先,将graphene-Nafion组装到电极表面用于化学吸附钯离子,然后用水合肼将钯离子原位还原为PdNPs。在碱性介质中,PdNPs-graphene杂化物修饰电极对葡萄糖的电氧化表现出很高的电催化活性。在最优的实验条件下,所构建的传感器对葡萄糖在10μM~5 mM范围有很好的线性响应,相关系数为0.998。检测下限为1μM(S/N=3)。同时实验结果还表明,该传感器具有很好的重现性,稳定性和良好的选择性(第6章)。
(6)基于原位电聚合罗丹明B(PRhB)传感膜构建了亚硝酸盐传感器。PRhB修饰电极对亚硝酸盐的电化学氧化表现出很好的催化作用。实验研究了各种因素对该传感器性能的影响,如:膜的厚度,溶液的pH值,工作电位。PRhB修饰电极对NO2-响应迅速,在0.5μM~0.7 mM浓度范围之内呈很好的线性关系,检测下限为0.1μM(S/N=3),灵敏度为308.1μAmM-1 cm-2。同时实验结果表明,该传感器具有很好的重现性,稳定性和良好的选择性(第7章)。
Biosensor technology is a powerful alternative to conventional analytical techniques, harnessing the specificity and sensitivity of biological systems in small, low cost devices. Biosensors are being developed for different applications, including environmental and bio-process control, quality control of food, agriculture, military, and, particularly, medical applications. The rapid growth in biomaterials, especially the availability, application and combination of a wide range of nanomaterials with new sensing techniques, has caused a remarkable innovation in the design and construction of electrochemical biosensors. Owing to their biocompatibility, catalytic properties, strong adsorption capacity and high accessible surface area, these nanomaterials can greatly improve the performance of biosensor. Base on the superior physical and chemical properties of nano-materials, many nanomaterials with different morphology were synthesized in this thesis. These nanomaterials were used to fabricate novel biosensors and electrochemically nonenzymatic sensors. The prepared biosensors exhibited fast response, high sensitivity and low detection limit. These biosensors also showed good reproducibility and stability and could be used for the detection of real samples.
(1)A novel tyrosinase biosensor based on hydroxyapatite nanoparticles (nano-HA)-chitosan nanocomposite has been developed for the detection of phenolic compounds. The uniform and size controlled nano-HA was synthesized by hydrothermal method, and its morphological characterization was examined by transmission electron microscope (TEM). Tyrosinase was then immobilized on a nano-HA-chitosan nanocomposite-modified gold electrode. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize the sensing film. The prepared biosensor was applied to determine phenolic compounds by monitoring the reduction signal of the biocatalytically produced quinone species at -0.25 V (vs. saturated calomel electrode). The effects of the pH, temperature and applied potential on the biosensor performance were investigated, and experimental conditions were optimized. The biosensor exhibited a linear response to catechol over a wide concentration range from 10 nM to 7μM, with a high sensitivity of 2.11×103μAmM-1 cm-2, and a limit of detection down to 5 nM (based on S/N=3). The apparent Michaelis-Menten constants of the enzyme electrode were estimated to be 3.16, 1.31 and 3.52μM for catechol, phenol and m-cresol, respectively. Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results(in chapter 2).
(2)A novel horseradish peroxidase (HRP) electrochemical biosensor based on a MgO nanoparticles (nano-MgO)-chitosan (chit) composite matrix was developed. The morpology of nano-MgO-chit nanocomposite was examined by scanning electron microscopy (SEM). The interaction between nano-MgO-chit nanocomposite matrix and enzyme was characterized with UV-vis spectra. This proposed composite material combined the advantages of inorganic nanoparticles and organic polymer chit. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H2O2 in the presence of hydroquinone as a mediator. The effects of the experimental variables such as solution pH and the working potential were investigated using steady-state amperometry. The present biosensor (HRP-modified electrode) had a fast response towards H2O2 (less than 10 s), and excellent linear relationships were obtained in the concentration range of 0.1~1300μM, with a detection limit of 0.05μM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results (in chapter 3).
(3)A new third-generation biosensor for H2O2 assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of carbon nanotubes(CNTs)-mesoporous silicas(SBA-15) modified gold electrode. The biological activity of HRP immobilizing in the composite film was characterized by UV-vis spectra. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H2O2. The effects of the experimental variables such as solution pH and the working potential were investigated using steady-state amperometry. Under the optimal conditions, the resulting biosensor showed a linear range of 1μM to 7 mM and a detection limit of 0.5μM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results (in chapter 4).
(4)Highly ordered Ni nanowire arrays(NiNWAs) were synthesized for the first time using a template-directed electropolymerization strategy with a nanopore polycarbonate (PC) membrane template, and their morphological characterization were examined by scanning electron microscopy (SEM) and transmission electron microscope (TEM). A NiNWAs based electrode exhibited very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium, which has been utilized as the basis of the fabrication of a nonenzymatic biosensor for electrochemical detection of glucose. The biosensor can be applied to the quantification of glucose with a linear range covering from 5.0×10-7 to 7.0×10-3 M, a high sensitivity of 1.043×103μAmM-1 cm-2, and a low detection limit of 1×10-7 M. The experiment results also showed that the sensor exhibited good reproducibility and long-term stability, as well as high selectivity with no interference from other oxidable species (in chapter 5).
(5) A nonenzymatic electrochemical biosensor was developed for the detection of glucose based on an electrode modified with palladium nanoparticles (PdNPs)-functioned graphene (nafion-graphene). The palladium nanoparticle-graphene nanohybrids were synthesized using an in-situ reduction process. Nafion-graphene was first assembled onto an electrode to chemically adsorb Pd2+. And Pd2+ was subsequently reduced by hydrazine hydrate to form PdNPs in-situ. Such a PdNPs-graphene nanohybrids-based electrode shows a very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium. The proposed biosensor can be applied to the quantification of glucose with a wide linear range covering from 10μM to 5 mM (R=0.998) with a low detection limit of 1μM. The experiment results also showed that the sensor exhibited good reproducibility and long-term stability, as well as high selectivity with no interference from other potential competing species (in chapter 6).
(6) The fabrication of an electropolymerized Rhodamine B (PRhB) sensing film based electrochemical sensor and its application for electrochemical detection of nitrite were described. The PRhB film modified GC electrode exhibited good catalytic activity towards the electrochemical oxidation of nitrite. The effects of the experimental variables such as the thickness of the film, solution pH values and the working potential were investigated using steady-state amperometry. The present sensor (PRhB-modified electrode) had a fast response towards NO2- ( less than 10 s), and excellent linear relationships were obtained in the concentration range of 0.5μM~7.0 mM, with a detection limit of 0.1μM (S/N=3). The sensitivity of the sensor was calculated to be 308.1μAmM-1 cm-2. The possible interferences from several common ions were tested. Moreover, the stability and reproducibility of this sensor were evaluated with satisfying results (in chapter 7).
引文
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