【作者】 王波；

【导师】 王敏；

【作者基本信息】 浙江大学 ， 分析化学， 2015， 硕士

【摘要】 电化学生物传感器是一种以生物敏感元件与电化学转换器相连接的装置。基于其灵敏度高、特异性好、检测速度快、设备廉价、操作简单等特点,电化学生物传感器广泛应用于临床诊断、药物与食品分析以及环境监测等领域。无酶生物传感器和细胞传感器是两种重要的电化学生物传感器,本工作在使用生物活性分子如壳聚糖、叶酸分别构建无酶及细胞生物传感器的基础上,进一步将其应用于环境监测和癌细胞检测。第一章,综述了电化学生物传感器的发展史、电化学生物传感器的常用检测技术以及电化学生物传感器的分类。第二章,构建了一种基于壳聚糖与纳米金复合膜修饰金微电极的电化学无酶传感器,将其应用于亚硝酸根离子含量的测定。金电极表面先沉积一层纳米金,随后再沉积一层纳米金胶溶液与壳聚糖的混合凝胶层,从而获得GNP@CS/GNP/Au电极无酶传感器。通过电化学阻抗谱(EIS)表征修饰过程中金电极表面的性质。对修饰后的金电极进行循环伏安法(CV)测试,结果表明电沉积纳米金的存在显著提高了亚硝酸根离子在电极上的响应信号；而GNP@CS复合膜则同时具有促进电子传递和静电吸引亚硝酸根离子的作用。经条件优化后,所构建的GNP@CS/GNP/Au电极对亚硝酸根离子的测定范围为7.94nmol/L-4.74 mmol/L,检测限为4.38μmol/L(S/N=3)。将GNP@CS/GNP/Au电极应用于池水样品中亚硝酸根的测定,回收率在91.6%-104%之间,显示了该传感器具有一定的实际应用潜力。第三章,构建了一种基于L-半胱氨酸与叶酸修饰金微电极表面的电化学细胞传感器。依靠L-半胱氨酸的巯基与金的作用力,将L-半胱氨酸固定于金电极表面,使氨基露于电极表面。通过EDC/NHS的催化作用,将叶酸FA固定至表面带氨基的L-Cys/Au电极,从而构建FA/L-Cys/Au电极。用BSA封闭FA/L-Cys/Au电极表面非特异性吸附后,基于FA与叶酸受体(Folate Receptor, FR)的亲和力,而FR在HeLa细胞表面过度表达,将HeLa细胞接种于BSA/FA/L-Cys/Au电极表面,以阻抗变化信息获知HeLa细胞数目。电化学阻抗谱及循环伏安法的实验结果证明,L-Cys分子总体带正电荷,与负离子存在静电引力,促进电极表面氧化还原反应,起着电子媒介体的作用；固定FA后,FA的空间效应使得Fe(CN)63-与电极之间的电子传递受阻；捕获HeLa后,细胞的存在进一步阻碍了界面电子传递,使Rct大大增加。构建的细胞传感器对HeLa的检测线性范围为1.08×103 cells/mL～1.08×107 cells/mL,检测限低至2.4×102cells/mL(S/N=3),其重现性和抗干扰性良好,具有一定的实际应用前景。更多还原

【Abstract】 Electrochemical biosensors are devices that combine biosensing interface with electrochemical transducer. With the advantages of high sensitivity, fast analysis, good specificity, simple operation and low cost, electrochemical biosensors have been widely used in clinical diagnosis, food and pharmaceutical analysis, environment monitoring and many other fields. There are several kinds of electrochemical biosensors, each of which differs in fabrication method and detection technology. Enzyme-free electrochemical biosensors and cell biosensors are usually produced by layer-by-layer self-assembly technique, which is time-consuming and lack long-term stability. In this work biomolecules such as chitosan, L-cysteine and folic acid are used to construct enzyme-free biosensors and cell biosensors for environmental monitoring and cancer cell detection.In chapter 1, the development history of electrochemical biosensors, classification of electrochemical biosensors and detection techniques of electrochemical biosensors are reviewed.In chapter 2, an enzyme free electrochemical biosensor based on chitosan and nano gold composite film was developed and applied in nitrite analysis. A layer of gold nanoparticles was first electrodeposited onto gold film micro electrode, followed by the deposition of nano gold and chitosan composite layer, to obtain the GNP@CS/GNP/Au enzyme free electrochemical biosensor. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the electrochemical properties of the modified electrode. It was demonstrated that the electrodeposited gold nanoparticles significantly improved the response signal toward nitrite and the GNP@CS composite membrane accelerated electron transfer and improved affinity toward nitrite ions as well. The detection range of the developed GNP@CS/GNP/Au sensor for nitrite ions was 7.94 μmol/L-4.74 mmol/L, with a detection limit of 4.38 μmol/L (S/N=3). The GNP@CS/GNP/Au electrode was used to determinate nitrite ion in pond water with recoveries ranging from 91.6%-104%, which demonstrated its potential in environmental monitoring.In chapter 3, a cell electrochemical biosensor based on L-cysteine and folic acid (FA) was developed and applied in the determination of HeLa cells. L-cysteine was immobilized onto the surface of gold micro electrode via the interaction of thiol and gold. FA was then bound to the L-Cys/Au electrode with the catalysis of EDC/NHS. BSA was used to reduce nonspecific adsorption on the electrode surface. Based on the strong affinity between FA and FR (Folate Receptor) which is over expressed on HeLa cell membrane, the BSA/FA/L-Cys/Au electrode was used for HeLa cell sensing. The results of EIS and CV characterization demonstrated that the positive charge of L-Cys enhanced the redox reaction on the electrode surface while FA hindered the electron transfer process because of its steric effect. After Hela cells were captured by the FA/L-Cys/Au electrode, the film resistance increased further. The detection range of this constructed FA/L-Cys/Au electrode towards HeLa cells was 1.08x103～1.8×107 cells/mL, with a detection limit of 2.4×102 cells/mL (S/N=3).The FA/L-Cys/Au electrode shows a good reproducibility and good anti-interference performance.更多还原