【作者】 张伟；

【导师】 丁世家；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2014， 硕士

【摘要】 生物传感器是由生物识别物质（如多肽、核酸、抗体等）、换能器以及信号处理装置构成的分析装置或器件,根据识别体系的不同可分为生物催化型传感器和生物亲和型传感器两类。其中，亲和型电化学生物传感器因其响应速度快、易于使用、廉价以及小型化等优点，受到了研究者广泛的关注。近年来，分子生物技术、纳米材料技术、分析化学技术等不断发展，亲和型电化学生物传感器在各个研究领域得到了快速的发展，能够对多种具有生物活性的物质（细胞、细菌、核酸、无机离子、多肽等）进行快速、准确的检测，在临床检验诊断学中是一个非常活跃的研究前沿。本课题主要研究亲和型电化学生物传感器对多肽和短链DNA的检测新方法，实现其准确、快速和超灵敏的检测，为临床疾病诊疗及预后、病原检测和环境监测等提供有力的工具。研究内容分为两部分：1.基于碳纳米管/壳聚糖修饰玻碳电极的亲和型电化学免疫传感器检测血管紧张素ⅡAng Ⅱ在RAS系统中起着非常重要的作用。同时，在体液平衡、电解质内稳态以及血压的调控中起到一定的作用。Ang Ⅱ结合AT1R形成复合体，在细胞增殖、机体炎症以及癌症等中发挥重要的作用。目前，用于血管紧张素Ⅱ的检测方法有表位免疫测定、放射免疫测定法和高效液相色谱测定法等，这些方法被广泛的应用，但存在着耗时久、操作繁琐、成本高等缺点，同时这些方法需要相对专业的设备和技术人员。因此，发展一种简单、快速且廉价的方法检测血管紧张素Ⅱ，是非常有意义的。本研究基于单壁碳纳米管/壳聚糖复合物修饰电极发展了一种亲和型电化学免疫传感器用于竞争性检测Ang Ⅱ。首先，将血管紧张素Ⅱ共价结合在功能化的电极表面，固定抗原与靶物质血管紧张素Ⅱ竞争性结合其抗体。竞争结合后，ST-AP被捕获到电极表面，用于催化底物α-NP产生信号。采用CV和EIS等电化学方法对电极表面自组装过程进行表征分析。在最优的实验条件下，组装的电化学免疫传感器对Ang Ⅱ产生特异性响应电流信号，在0.05~10μg/mL浓度范围内表现出良好的线性比例关系，其最低检测限（LOD）为0.03μg/mL。该免疫传感器表现出较高的特异性和较好的重复性，并用于血清中Ang Ⅱ的检测分析。本研究首次构建用于检测血管紧张素Ⅱ的免疫传感器，为未来临床诊断中血管紧张素Ⅱ的检测提供了一种潜在的分析方法。2.基于分子灯塔介导的循环链置换和滚环扩增的超灵敏亲和型电化学DNA生物传感器实现对特异序列DNA的超灵敏和高选择性检测，对于临床诊断与治疗、病原菌的检测和环境监测具有重要的作用。基于分子灯塔介导的循环链置换反应、滚环扩增、生物素-亲和素系统等多重级联信号放大策略，一种新颖的超灵敏检测靶DNA的亲和型电化学生物传感策略被发展。在该方法中，靶DNA与固定在金电极表面的分子灯塔进行特异性的结合，触发CISDA，从而产生大量生物素标记的DNA。进而，通过生物素-亲和素系统，引入滚环扩增体系，产生大量可以结合检测探针的重复DNA片段。当加入ST-AP时，发生催化反应，获得超灵敏的电化学DPV信号。该方法表现出较好的选择性和较高的灵敏度，线性范围较宽(1fM~100pM)，其最低检测限（LOD）为0.4fM。该传感策略为临床分子诊断和环境监测提供一个潜在的新技术新方法。更多还原

【Abstract】 Biosensor is a kind of equipment consisting of the bioactive materials,the transducer and the signal analysis device. According to thebiorecognition between the recognition molecules and the target molecules,the biosensor is devided into catalytic biosensor and affinity-basedbiosensor. Owing to its advantages of fast response, easy to use, cheapnessand miniaturization, the affinity electrochemical biosensor is well widelyaccepted. In recent years, with the development of the molecular biology,materials science and analytical chemistry, the electrochemical sensor hasbeen developed rapidly in various areas of research. It can rapidly andaccurately detect a large number of substances, including cells, bacteria,nucleic acids, polypeptide, et al. And the affinity-based biosensor is anactive study in the clinical laboratory diagnostics. In our work, a rapid,simple, and sensitive method for detection of peptides and short DNAsequences based on electrochemical sensor was developed, which wouldbecome a powerful tool for clinical diagnostics, and environmentalmonitoring. The study has two parts as follow:1. A Novel Electrochemical Immunosensor for Angiotensin II Detection based on the Glass Carbon Electrode Modified by CarbonNanotubes/Chitosan FilmAng II is the important peptide of the RAS and plays important roles inthe control of body fluid balance, blood pressure regulation and electrolytehomeostasis. Ang II can bind the type1angiotensin receptor (AT1R), andclosely involve in the regulation of angiogenesis, cell proliferation, cancerand inflammation. So it is a need to detect Ang II for studying the differentactions of Ang II in various systems. Now, a lot of methods are developedfor Ang II detection, for example epitope-immunoassay, high performanceliquid chromatography and radio immunoassay. These assays are widelyused, but they are needing complex and long-time work, requiring high-endinstrument and costliness. Therefore, developing a simple, cheap and fastmethod is a challenge for the direct detection of Ang II.In the assay, we successfully developed a new electrochemicalcompetitive immunosensor for Ang II detection based on the glass carbonelectrode (GCE), which was modified with the single-walled carbonnanotubes/chitosan (SWNTs/CS) nanocomposite film. In the constructionof electrochemical sensor, Ang II was firstly covalently immobilized onSWNTs/CS modified GCE. Then, the mixture solution of target Ang II andBio-Anti-Ang II antibody was dropped on the modified GCE and incubated.After the reaction, ST-AP, which can catalyze the hydrolysis of α-NP in thedetection solution, was captured on the biosensor surface. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) wereused to characterize the every assemble step of the sensor. Under theoptimal assay conditions, the electrochemical biosensor had a specificresponse to the target Ang II. The immunosensor had a wide linear rangefrom0.05to10μg/mL, and the detection limit was0.03μg/mL. Theimmnunosensor presented good specificity and reproducibility, and it wasused to detect Ang II in sample serum. This novel immunosensor methodwas firstly applied to detect Ang II, which might be a potential method forassay of Ang II for clinical diagnosis and treatment in the future.2. Ultrasensitive electrochemical DNA biosensor based onmolecular beacon-mediated circular strand displacement and rollingcircle amplificationHighly sensitive and selective detection of sequence-specific DNAplays essential roles in clinical diagnosis and therapy, pathogen detectionand environmental monitoring. A novel electrochemical biosensing strategywas developed for ultrasensitive target DNA detection using a cascade signalamplification based on molecular beacon (MB) mediated circular isothermalstrand displacement amplification (CISDA), rolling circle amplification(RCA), biotin-strepavidin system and enzymatic amplification. The targetDNA hybridized with the loop portion of MB probe immobilized on the goldelectrode and triggered the CISDA, leading to multiple biotin-tagged DNAduplex. Furthermore, via biotin-streptavidin interaction, the RCA was implemented, producing a lot of long massive tandem-repeat DNAsequences for binding numerous biotinylated detection probes, whichenabled an ultrasensitive electrochemical readout by further employing thestreptavidin-alkaline phosphatase. The proposed biosensor showed veryhigh sensitivity and selectivity with a dynamic linear range from1fM to100pM, and a detect limit as low as0.4fM. The established strategy could havethe potential for applying in clinical molecular diagnostics, pathogendetection and environmental monitoring.更多还原