【作者】 刘百祥；

【导师】 邵志清；

【作者基本信息】 华东理工大学 ， 计算机应用技术， 2012， 博士

【摘要】 物联网是新一代信息处理平台,它主要包括信息识别层,终端处理层及传输网络层。其中信息识别层中的传感器技术是物联网架构的重要组成部分。DNA电化学生物传感器是近年迅速发展起来的一种全新的生物传感器,它具有制作简便、使用寿命长、灵敏度高、重现性好、成本低、易于实现微型化等诸多优点,在临床医学检验、遗传工程、药物作用机理、新药筛选、环境监测和食品工程等领域得到广泛的研究与应用。本论文针对DNA电化学生物传感器的设计优化技术展开研究,主要工作包括：靶向单链DNA片段的固定问题；电子媒介体型DNA生物传感器优化过程；基于数据拟合技术的交流阻抗方法的应用；纳米材料在DNA电化学传感器中应用技术优化等。主要创新点如下：(1)研究了在硼掺杂的金刚石电极表面使用电化学方法沉积氧化锆薄膜的新架构,即在室温条件下水溶液中在基体电极表面生成氧化锆多孔状分布的晶体薄膜,薄膜的厚度及氧化锆粒径通过循环电压及沉积时间来改变,循环电压在+O.7至-1.1V之间时,电极表面形成极薄的膜,随着循环电压的变化,在+O.7至-1.6V时,仅仅在10个循环扫描后,电极表面就形成多孔状的氧化锆薄膜,XPS分析显示,电极表面沉积的氧化锆大约增加10倍,为后续传感器的制作打下良好基础。(2)设计了一种简单、方便的电化学DNA生物传感器的制作方法,在硼掺杂的金刚石电极(BDD)表面电化学沉积一层氧化锆薄膜,电极表面的活性区域被惰性的氧化锆分隔成类似微阵列的结构。利用氧化锆的分子结构特点,将5末端修饰磷酸基团的寡核苷酸短链(ssDNA)连接到氧化锆薄膜上。以亚甲基蓝(MB)为氧化还原的电子媒介体,应用循环伏安法和差分脉冲法测试了该电极的性能。由于硼掺杂的金刚石电极背景电流非常小,DNA与氧化锆的连接稳定,使得制备的电化学传感器表现出高的灵敏度、良好的线性、长期的稳定性等。(3)研究了基于数据拟合技术的交流阻抗方法测试非标记型脱氧核糖核酸杂交传感器的机制。先在掺硼金刚石电极表而电化学沉积氧化锆多孔薄膜,再将5’端磷酸基团单链寡聚DNA固定到氧化锆多孔膜修饰的金刚石电极表面,形成杂交识别层,利用交流阻抗技术(EIS)测量出杂交前后工作电极表面电子传递电阻Ret的改变量作为杂交信号。实验得到的非标记型DNA杂交传感器制作方法简便,响应迅速,线性良好。(4)基于DNA/ZrO2/MWCNT/GCE结构设计了高灵敏度的DNA电化学生物传感器。首先将处理过的多壁碳纳米管(MWCNT)适量分散于二甲基甲酰胺中,形成均匀稳定的混合溶液,将其滴涂于玻碳电极(GCE)表面,室温下应用电化学方法将氧化锆沉积至MWCNT/GCE修饰玻碳电极上。碳纳米管大的比表面积和良好的电子传递性能,氧化锆的生物相容性和对DNA极好的吸附能力的协同作用,显著提高了DNA探针的固定量和DNA杂交的检测灵敏度。应用循环伏安法,差分脉冲法和电化学交流阻抗谱分别对传感膜的制备和DNA的固定与杂交进行了测试。线性范围为2.15×10-7至2.15×10-10mol/L,检出限为6.58×10-12mol/L。更多还原

【Abstract】 The Internet of Things is a new information processing platform, including the system of item identification,the terminal processing system and the transmition network. The sensor technology of the system of item identification will be the key factor of the Internet of Things.DNA electrochemical biosensors have been developed rapidly in recent years, as a new kind of biosensors, which has superiority in electrode fabrication, durable years, reproduction quality, higher sensitivity, lower production cost and small size, and convenience for integration and microminiaturization. So they are of significant progress in research and application, such as clinical medical analysis, genetic engineering, mechanism of drug action, new drug selection, environmental monitoring and food engineering.This thesis aims to research on design optimization of DNA electrochemical biosensor, including how to immobilizate the probe DNA onto the working electrode, design optimization of an electrochemical DNA biosensor, application of the data fitting method and impedance measurements,nanotechnology application on electrochemical sensor for performance optimization.The electrochemical deposition of zirconia thin film on boron-doped diamond (BDD) electrode is explored, to be used as a linker to bind biomoleculars on BDD surface for biosensor applications. Two electrodeposition methods are compared, either potential cycling or chronoamperometry in ZrO2Cl2The presence of Zirconia was confirmed by XPS and EDX, and uniform and porous Zirconia film was observed by SEM. Both cyclic voltammetry and electrochemical impedance measurement indicate a partially deactivated BDD electrode, resembling a microelectrode array characteristic with small electrochemically active areas separated by inactive zirconia regions. This demonstrates that uniform and porous zirconia can be electrodeposited on BDD in a well controlled method, suitable for use in biosensor application.Developing an electrochemical DNA biosensor, using a boron-doped diamond (BDD) electrode modified with zirconia(ZrO2), the zirconia thin porous film is fabricated by cyclic voltammatic method in an aqueous electrolyte of ZrOCl2and KCL at room temperature, DNA probes are attached onto the ZrO2/BDD electrode due to the strong binding of the phosphate group of the DNA with the Zirconia film and the excellent biocompatibility of the BDD. DNA immobilization and hybridization are characterized by cyclic voltammetry and differential pulse voltammetry, using methylene blue as indicator. After the hybridization of DNA probe with the complementary DNA, the peak current of MB decrease obviously. the response current are linearly related to the concentration of the target oligonucleotide sequence.Designing a label-free detection system for DNA strands based on data fitting and impedance measurements. A single-stranded5’-PO4mer oligonucleotide (ssDNA) is immobilised via a zirconia porous film on BDD electrodes and serve as probe DNA, The sensor surface clearly distinguished between complementary and non-complementary target ssDNA. The electrode is impedimetrically characterised in the presence of the redox system ferri/ferrocyanide before and after DNA hybridisation. Impedance analysis shows that the charge transfer resistance,Ret, is increasing after DNA duplex formation, The relative change of Ret is used as sensor parameter. The sensor is easy to prepared and respond quickly.A DNA/ZrO2/MWCNT/GCE electrochemical sensor is prepared for performance optimization. The zirconia thin porous film is fabricated by cyclic voltammatic method in an aqueous electrolyte of ZrOCl2and KCl at room temperature.DNA probes are attached onto the ZrO2/MWCNT/GCE electrode due to the strong binding of the phosphate group of the DNA with the Zirconia film and the excellent active surface area of MWCNT, DNA immobilization and hybridization are characterized by cyclic voltammetry and differential pulse voltammetry,using methylene blue as indicatorhe sensor show high sensitivity and good linearity, the peak current of MB is linearly related to the concentration of the target oligonucleotide sequence in the range2.15×107to2.15x10-10mol/L with the detection limit of6.58×10-12mol/L更多还原