基于DNA生物分子技术信号放大的电化学免疫传感器的研究

发布时间：2018-03-07 21:52

本文选题：生物传感器　切入点：DNA生物分子技术　出处：《安徽师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：本论文将DNA生物分子技术信号放大策略应用于电化学领域,该技术能够显著地放大传感信号,使构筑的生物传感器的灵敏度提升、检出限降低。在DNA生物分子技术中,杂交链式反应由于过程简单且无需辅助酶的参与受到广大科学工作者的青睐。另外,通过杂交链式反应形成的DNA串联体作为一种理想的载体可以用来标记或负载大量的电化学活性材料(如阿霉素、二茂铁、亚甲基蓝、铜纳米粒子等等),由于携带大量的信号分子,其作为信号探针使电化学响应信号显著增强。本论文主要包括四个方面:第一章:简述生物传感器及其信号放大策略。第二章:制备基于杂交链式反应与DNA模板化的铜纳米粒子相结合的一种超灵敏、免标记的C-反应蛋白电化学免疫传感器。在这项工作中,通过杂交链式反应合成的双链DNA被用作合成铜纳米粒子的模板,而嵌入大量铜纳米粒子的DNA串联体又被用作信号探针来实现检测信号的放大。在最优的实验环境下,差示脉冲伏安技术用于记录该免疫传感器的电化学响应信号,铜纳米粒子的信号强度与C-反应蛋白浓度的对数在1 fg mL-1到100 ng mL-1范围内呈线性关系,其线性相关系数为0.996,检测限为0.33 fg mL-1。第三章:报道了一种基于杂交链式反应信号放大的电化学免疫分析法对甲胎蛋白抗原和前列腺特异性抗原进行同时检测。这项工作中包括了固定在金纳米粒子修饰电极表面的初始抗体、抗原、二抗复合物以及由辅助探针与标记二茂铁、亚甲基蓝的信号探针杂交而成的DNA串联体。在最优的实验环境下,用差示脉冲伏安技术进行信号测定,在-0.35 V和+0.33 V分别出现亚甲基蓝和二茂铁的还原峰,峰电流值与生物标志物浓度的对数在0.5 pg mL-1到50 ng mL-1范围之间呈现良好的线性关系,对于前列腺特异性抗原,其检出限为0.17 pg mL-1,对于甲胎蛋白抗原,其检出限为0.25 pg mL-1。第四章:我们尝试将能够嵌入DNA双螺旋结构中的小分子与杂交链式反应技术相结合构建一种超灵敏的多分析免疫传感器。阿霉素和亚甲基蓝用作信号分子。该免疫分析法通过抗原-抗体特异性识别在初始抗体、目标抗原与合成的二抗复合物之间形成“三明治”型免疫复合物,随后将大量的信号分子阿霉素、亚甲基蓝嵌插到由杂交链式反应形成的双链DNA的沟槽中作为信号探针,并通过DNA杂交反应连接到免疫复合物的表面形成超夹心型免疫结构。在最优的环境下,用方波伏安技术记录阿霉素和亚甲基蓝的电化学响应信号。结果在-0.30 V和-0.70 V分别显示出亚甲基蓝和阿霉素的信号峰,信号峰强度与生物标志物浓度的对数在0.05 pg mL-1到25 ng mL-1范围内呈现优良的线性关系,对于癌胚抗原,其检出限为0.03 pg mL-1,对于甲胎蛋白抗原,其检出限为0.02 pg mL-1。
[Abstract]:In this paper, the signal amplification strategy of DNA biomolecular technology is applied to the electrochemical field. This technique can significantly amplify the sensing signal, enhance the sensitivity of the biosensor and reduce the detection limit. In the DNA biomolecular technology, the sensitivity of the biosensor is improved and the detection limit is reduced. Hybrid chain reaction is favored by many scientists because of its simple process and no coenzyme involvement. In addition, The DNA series formed by hybrid chain reaction can be used as an ideal carrier to label or load a large number of electrochemical active materials (such as adriamycin, ferrocene, methylene blue, adriamycin, ferrocene, methylene blue). Copper nanoparticles and so on, because they carry a lot of signaling molecules, This thesis mainly includes four aspects: chapter 1: brief introduction of biosensor and its signal amplification strategy. Chapter 2: preparation of template based on hybrid chain reaction and DNA. A highly sensitive combination of copper nanoparticles, In this work, double-stranded DNA synthesized by hybrid chain reaction is used as a template for the synthesis of copper nanoparticles. The DNA series embedded in a large number of copper nanoparticles is used as a signal probe to amplify the detection signal. In the optimal experimental environment, differential pulse voltammetry is used to record the electrochemical response signals of the immunosensor. The signal intensity of copper nanoparticles was linearly correlated with the logarithm of C- reactive protein concentration in the range of 1fg mL-1 to 100ng mL-1. The linear correlation coefficient is 0.996 and the detection limit is 0.33 fg mL-1. Chapter 3: an electrochemical immunoassay based on amplification of hybrid chain reaction signal is reported for simultaneous detection of alpha-fetoprotein antigen and prostate specific antigen. This includes the initial antibody immobilized on the surface of the gold nanoparticles modified electrode. Antigens, second antibody complexes and DNA series composed of auxiliary probes and signal probes labeled with ferrocene and methylene blue. In the optimal experimental environment, the signals were determined by differential pulse voltammetry. The reduction peaks of methylene blue and ferrocene appeared at -0.35 V and 0.33 V, respectively. The peak current value showed a good linear relationship with the logarithm of biomarker concentration in the range of 0.5 PG mL-1 to 50 ng mL-1. The detection limit was 0.17 PG mL-1. The detection limit is 0. 25 PG mL-1. 4th: we try to combine small molecules embedded in DNA double helix structure with hybrid chain reaction technique to construct a super-sensitive multi-analytical immunosensor. Adriamycin and methylene blue. As signal molecules. This immunoassay is specifically identified by antigen-antibody in the initial antibody, A "sandwich" type immune complex was formed between the target antigen and the synthesized second antibody complex. A large number of signal molecules, adriamycin and methylene blue, were subsequently inserted into the channel of the double-stranded DNA formed by the hybrid chain reaction as a signal probe. The supersandwich immune structure was formed on the surface of the immune complex by DNA hybridization reaction. The electrochemical response signals of adriamycin and methylene blue were recorded by square wave voltammetry. Results the signal peaks of methylene blue and doxorubicin were observed at -0.30 V and -0.70 V, respectively. The linear relationship between the peak intensity and the logarithm of biomarker concentration was found in the range of 0.05pg mL-1 to 25ng mL-1. The detection limit for carcinoembryonic antigen was 0.03 PG mL -1, and for alpha-fetoprotein antigen 0.02pg mL -1.
【学位授予单位】：安徽师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.1;TP212

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