基于末端转移酶延伸放大的电化学DNA传感器设计
时间:2017-06-17 17:13 来源:毕业论文 作者:毕业论文 点击:次
| 摘 要生物传感器是一类新型的利用生物特异性识别过程来实现检测的传感器件,一般有两个主要组成部分：生物分子识别元件和信号转换器。生物分子识别元件是具有分子识别能力、并且直接和待测物质接触的生物活性物质。而信号转换器主要基于光学、电化学和声学检测方法等，可以将生物识别事件转换为可检测的信号。 电化学DNA传感器是生物传感器的一个重要分支。由于电化学检测方法本身所具有的灵敏、快速、低成本、低能耗等特点，电化学DNA传感器成为一个非常活跃的研究领域并在近几年中得到了快速发展，在基因相关疾病的诊断、军事反恐和环境监测等方面均具有非常重要的应用。 本论文设计了一种利用末端延伸酶的无模板延伸进行信号放大检测的电化学DNA传感器,实现了无标记、高灵敏的DNA检测。该策略利用巯基与金电极之间的自组装过程进行捕获探针DNA的组装,结合末端转移酶的延伸放大作用，采用电化学检测技术，实现了浓度为1pM目标DNA的检测，并展望了电化学DNA传感器的前景。10332 关键字：电化学DNA传感器；信号放大；末端转移酶 Electrochemical DNA sensor based on the amplification of terminal extension reaction Abstract Biosensor is a variety of sensors based on the specific bio-recognition reaction, which consists of two key units: a bio-recognition layer and a signal transducer. Bio-recognition layer is the bimolecular which can specifically and directly reacted with the target analyte. The signal transducer transduce the bio-recognition into detectable signals such as optical， electronic and acoustic signals. The electrochemical DNA sensor is the most important branch of biosensors. Because electrochemical detectors are inexpensive，portable and power-saving, electrochemical DNA biosensors have been widely recognized to be a highly promising approach to detect clinical，environmental and security relevant nucleic acids especially when time, money or resources are limited Herein, this thesis designed a signal amplified electrochemical DNA sensor for label-free and high sensitive DNA detection, which was based on the terminal extension induced by terminal deoxynucleotidyl transferase (TdT). This DNA sensor employs a capture DNA probe labeled with thiol at its 3’ terminal to be immobilized at gold electrode via gold-thiol chemistry. After the hybridization with target DNA, terminal deoxynucleotidyl transferase (TdT) was used, which can catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3-OH group of an oligonucleotide primer without template. After that, electrochemical methods were employed to realize the detection of target DNA and the detection limit was experimentally found to be 1 pM. Keywords: electrochemical DNA sensors; signal amplification; terminal deoxynucleotidyl transferase (TdT). 目 录 1 绪论 .1 1.1 电化学DNA传感器的原理1 1.2 探针DNA在电极上的组装方法2 1.2.1 不经过修饰的DNA固定2 1.2.2 巯基修饰的DNA（HS-ssDNA）探针的固定..4 1.2.3氨基修饰的DNA（H2N-ssDNA）探针的固定5 1.2.4生物素修饰的DNA（biotin-ssDNA）探针的固定5 1.2.5影响DNA在金电极表面固定的因素..6 1.3 电化学生物传感器的表征8 1.3.1 探针DNA在金电极表面组装的表征8 1.3.2 DNA杂交的检测10 2 电化学DNA传感器的构建.14 2．1仪器与试剂15 2．2 电化学DNA传感器的构建.15 2.2.1 金电极的预处理..15 2.2.2 探针DNA在金电极上的组装16 2.2.3 电化学传感器上进行末端延伸反应..17 (责任编辑：qin) |