基于碳纳米材料与电化学发光共振能量转移的免疫传感器检测鼠免疫球蛋白

发布时间：2018-06-11 19:58

本文选题：电化学发光 + 电化学发光免疫传感器　；参考：《西南大学》2015年硕士论文

【摘要】：电化学发光(ECL)分析技术集合了电化学可控性强和发光分析灵敏度高的优点,是一种高的痕量分析检测方法。电化学发光免疫分析法(ECLIA)是电化学发光分析和免疫相结合的产物。这种方法基于抗原抗体之间的特异性免疫反应识别目标物,建立发光物质与目标物之间的定量联系,通过测定发光物质的ECL信号的改变,实现目标检测物的高选择性、高灵敏度检测。电化学发光免疫传感器是生物传感器的一种,其原理是将抗原/抗体固定在电极上,以电化学发光信号强度的变化对抗原/抗体进行测定。电化学发光免疫传感器具有背景低、灵敏度高、线性范围宽、时间/空间可控性好、仪器简单、分析速度快等优点,因此,引起了研究者们广泛兴趣。现已经发展成为生物分析检测领域重要研究手段,可检测不同分子量大小的抗体、抗原和半抗原。与荧光能量转移原理类似,电化学发光物质同样可以作为能量供体光源而发生能量转移。在电化学发光能量转移中(ECL-ET),通过在电极表面施加一定的电位,能量供体光源在电极上发生电化学反应产生电生物质,电生物质之间或电生物质与溶液中某些组分发生化学反应而跃迁到激发态,最后将能量转移给受体而淬灭。钌联吡啶(Ru(bpy)32+)及其衍生物具有发光效率高、可电化学再生、水溶性好、化学性能稳定等诸多优点,是电化学发光领域应用最为广泛的体系之一。本文将Ru(bpy)32+作为能量供体光源应用到ECL-ET体系中,构建了新型一次性电化学发光免疫传感器。具体工作如下：利用蜡烛灰通过简单的方法合成具有高效猝灭效率的无定形碳纳米粒子(ACNPs),研究中发现ACNPs能够淬灭Ru(bpy)32+电化学发光,基于此现象将其用于检测模型蛋白(MIgG)。先将ACNPs标记MIgG,制备ACNP-MIgG纳米复合物,然后与电极表面上Ru(bpy)32+标记的抗原发生免疫反应。借助特异性免疫反应拉近ACNPs与Ru(bpy)32+之间距离,引发高效的ECL-ET过程。当样品中存在游离MIgG时,能够与ACNP-MIgG发生竞争性免疫反应,Ru(bpy)32+标记的抗体所捕获的ACNP-MIgG减少,电化学发光猝灭率降低,进而实现对MIgG定量分析。所得线性范围为0.5-400 ng/mL,检测限为0.35 ng/mL,低于酶联免疫方法的3.2 μg/L,稳定性、特异性和加标回收率均令人满意。一方面,该方法不需要外在激发光源,干扰少,背景低；另一方面,与使用电化学发光物质作为能量受体相比,不会出现发射光谱的重叠。该方法操作简单,成本低廉,具有较高的灵敏度和特异性,适用于床前测试和现场分析,显示出了较大的应用前景。
[Abstract]:Electrochemiluminescence (ECL) analysis technology combines the advantages of high electrochemical controllability and high luminescence analysis sensitivity. It is a high trace analysis method. Electrochemical luminescence immunoassay (ECLIA) is a combination of electrochemiluminescence analysis and immunoassay. This method is based on the specific immune reaction between antigen and antibody to identify the target, establish the quantitative relationship between the luminous substance and the target, and achieve the high selectivity of the target detection by measuring the change of ECL signal of the luminous substance. High sensitivity detection. Electrochemiluminescence immunosensor is a kind of biosensor whose principle is to immobilize antigen / antibody on electrode and measure antigen / antibody with the change of ECL signal intensity. Electrochemiluminescence immunosensor has the advantages of low background, high sensitivity, wide linear range, good time / space controllability, simple instrument and fast analytical speed. It has been developed into an important research tool in the field of biological analysis, which can detect antibodies, antigens and haptens of different molecular weight. Similar to the principle of fluorescence energy transfer, electrochemiluminescence materials can also be used as energy source for energy transfer. In the process of electroluminescent energy transfer, by applying a certain potential on the surface of the electrode, the energy donor light source produces electrochemistry reaction on the electrode to produce electric biomass. The chemical reaction between the electric biomass and some components of the solution leads to the transition to the excited state, and finally the energy is transferred to the receptor and quenched. Ruthenium bipyridyl Rubpyridyl 32) and its derivatives have many advantages, such as high luminescence efficiency, electrochemical regeneration, good water solubility, stable chemical properties and so on. It is one of the most widely used systems in the field of electrochemical luminescence. In this paper, a new type of one-off electrochemical luminescent immunosensor was constructed by using Rubpy32 as a source of energy donor in ECL-ET system. The main work is as follows: the amorphous carbon nanoparticles (ACNPsN) with high quenching efficiency were synthesized by a simple method using candle ash. It was found that ACNPs can quench the electrochemiluminescence (ECL) of Rubpy32. Based on this phenomenon, the ACNPs can be used to detect the model protein. ACNP-MIgG nanocomposites were prepared by labeling ACNPs with MIgG, and then reacted with Rubpy32 labeled antigen on the electrode surface. The distance between ACNPs and RubpyN 32 was narrowed by specific immunoreaction, which induced an efficient ECL-ET process. In the presence of free MIgG in the sample, the ACNP-MIgG could be competitively reacted with ACNP-MIgG. The capture of ACNP-MIgG by the antibody labeled with Rubpyr-32 decreased, and the electrochemiluminescence quenching rate decreased, thus the quantitative analysis of MIgG was realized. The linear range was 0.5-400 ng / mL, and the detection limit was 0.35 ng / mL, which was lower than 3.2 渭 g / L of Elisa. The stability, specificity and recovery were satisfactory. On the one hand, the method does not need external excitation light source, so it has less interference and low background. On the other hand, compared with the use of electroluminescent material as energy receptor, there is no overlap of emission spectra. The method is simple in operation, low in cost, high in sensitivity and specificity. It is suitable for bed test and field analysis, and shows great application prospect.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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