电化学免疫传感器快速检测甲型流感病毒的研究

发布时间：2018-04-20 18:30

本文选题：流感病毒 + H1N1　；参考：《中国人民解放军军事医学科学院》2014年硕士论文

【摘要】：流感病毒长期威胁人类健康，其中甲型流感病毒每年都会出现并不断变异，有时甚至会导致流感疫情大规模爆发流行，而防控的关键环节之一便是通过检测病原体对流感疑似病例的快速筛查和诊断。现有的病毒分离培养、酶联免疫吸附（ELISA）法以及荧光定量PCR等检测方法在疫情检测中发挥了重要作用，但在时效性、灵敏度和操作简便性方面仍存在不足，不能满足病原体现场快速检测的需求。针对上述需求，本论文以A/Beijing/501/2009甲型H1N1流感病毒为检测对象，于一次性丝网印刷碳电极（SPCE）上构建了甲型H1N1流感病毒电化学免疫生物传感器（EIS），探索了流感病毒的电化学免疫传感分析检测的相关机制，建立了甲型H1N1流感病毒电化学免疫生物传感检测技术和方法，主要研究结果如下：（1）对实验研究所需的材料，如流感病毒和相应的单克隆抗体和HRP标记抗体进行了培养和收集，并对这些材料进行了验证。用WHO推荐引物经RT-PCR验证以及流感病毒AB分型检测胶体金试纸条检测，结果表明培养的A/Beijing/501/2009（H1N1）鸡胚尿囊液确属甲型H1N1型流感病毒，血凝实验测定其病毒滴度为128HA Unit，证明所培养的A/Beijing/501/2009（H1N1）鸡胚尿囊液可以用于后续的实验研究。同时还通过ELISA实验考察了所收集到的单克隆抗体A1-24和HRP标记单抗A1-6-HRP分别与甲型H1N1型流感病毒的亲和力，结果表明这两种抗体材料能与甲型H1N1流感病毒形成亲和力较高的免疫夹心复合物，该模式可以用于构建甲型H1N1流感病毒电化学免疫传感器。（2）考察比较了由不同修饰材料和方法修饰电极的电化学特点，发现捕获抗体经物理吸附法固定修饰电极后具有较高的信号/本底值比、操作步骤简单且不需要使用价格昂贵的纳米材料或其他化学修饰材料，成本低廉，故最终选择此方法作为捕获抗体修饰固定电极的方法，并以此构建甲型H1N1流感病毒电化学免疫传感器。通过循环伏安法对该型免疫传感器的电化学行为进行了表征，结果表明该传感器表面的电活性物质主要通过扩散传质方式产生电信号，在传感器上产生的电流响应信号与电极上加入的被测抗原的浓度呈正比关系，适用于对被测抗原进行定量测定。（3）优化了影响传感器检测性能的主要工作条件：检测中捕获抗体H1-24和检测抗体A1-6-HRP的浓度分别采用400μg/ml和80μg/ml，在此浓度条件下检测信号都比较强，均能达到最大信号值的80%以上，能够实现对A(H1N1)流感病毒的灵敏检测。孵育最佳温度均为37℃，在此温度下抗原抗体反应的活性最高，反应能快速达到平衡状态，有利于提高反应效率。抗原及检测抗体的最佳孵育时间分别为45min和30min，在以上孵育时间条件下，检测的灵敏度均较高，经过优化后该免疫传感器的检出时间缩短至2h以内，比传统的ELISA法整体测定时间减少了1~2h，利用这种新型传感器能够实现对A(H1N1)流感病毒的快速检测。（4）建立了电化学免疫传感器测定甲型H1N1流感病毒的标准曲线，以及测定甲型H1N1流感病毒相应的酶联免疫吸附法（ELISA）标准曲线和荧光定量法（qPCR）标准曲线，用于对传感器各项检测性能指标进行考察。针对A(H1N1)流感病毒的检测信号与针对甲型H7N9、H3N2、B型流感病毒、EV71以及腺病毒等阴性对照的信号的统计分析检验结果表明，该传感器的特异性良好。重复性测定结果显示该传感器的RSD10%，回收率为80%~110%，呈现出较高的准确度和精密度。稳定性实验结果显示该传感器储存10天内检测信号几乎不下降，30天后的测定值也能保持在85%以上，具有较好的存储稳定性。EIS法的检测下限可达0.41HA Unit，与ELISA法相当，并能对4~64HA Unit范围内的甲型H1N1流感病毒实现定量测定，，且EIS的总体假阳性率为10.0%，优于ELISA法。所建立的EIS法与qPCR的测定结果的相关系数为0.9517，说明EIS法定量的准确度能达到与qPCR一致的水平。（5）最后应用微型化的Emstat电化学检测设备，基于便携的阵列化电极以甲型H1N1流感病毒电化学免疫生物传感器检测的技术和方法为模型，探索了多种流感病毒的阵列化检测，结果表明该传感器具有推广应用于多种病原体同时检测的前景，且其小型化的检测仪器设备便于现场携带。
[Abstract]:Influenza viruses are threatening human health for a long time. Influenza A virus appears and mutates continuously every year. Sometimes it may even lead to a large-scale outbreak of influenza. One of the key links of prevention and control is rapid screening and diagnosis of suspected influenza cases by detection of pathogens. Existing virus isolation and culture, enzyme linked immunosorbent assay (ELISA) and fluorescence quantitative PCR detection methods have played an important role in the detection of epidemic situation, but there are still shortcomings in the aspects of timeliness, sensitivity and ease of operation, which can not meet the needs of rapid detection of pathogens in the field.
In order to meet the above requirements, the A/Beijing/501/2009 influenza A virus (H1N1) virus was used as the test object to construct an electrochemical immunosensor of influenza A virus (EIS) of influenza A virus (H1N1) on a single screen printed carbon electrode (SPCE). The related mechanism of electrochemical immunoassay for influenza virus was explored, and a influenza A (H1N1) disease was established. The main findings are as follows:
(1) the materials needed for the experimental study, such as influenza virus and the corresponding monoclonal antibody and HRP labeled antibody, were cultured and collected, and the materials were verified. The WHO recommended primers were tested by RT-PCR and the detection of colloidal gold test paper for influenza virus AB typing, and the results showed that the cultured A/Beijing/501/2009 (H1N1) chicken embryo urine was cultured. The bursa solution is a type A influenza A virus (H1N1), and its virus titer is 128HA Unit by hemagglutination test. It is proved that the cultured A/Beijing/501/2009 (H1N1) chicken embryo sac can be used for subsequent experimental study. At the same time, the monoclonal antibody A1-24 and HRP labeled monoclonal antibody A1-6-HRP respectively and a type a H1N1 type flow are also investigated by the ELISA experiment. The affinity of the virus has shown that these two antibody materials can form a high affinity complex with the H1N1 influenza A virus, which can be used to construct an electrochemical immunosensor for influenza A virus (H1N1) virus.
(2) the electrochemical characteristics of modified electrodes modified by different modified materials and methods are compared. It is found that the captured antibody has high signal / base ratio, and the operation procedure is simple and does not need to use expensive nano materials or other chemical modification materials after the modified electrode is fixed by physical adsorption method. Therefore, the cost is low, so this method is finally chosen. The electrochemical immunosensor of a influenza A virus (H1N1) was constructed as a method of capturing a fixed electrode with an antibody modified. The electrochemical behavior of the immunosensor was characterized by cyclic voltammetry. The results showed that the electroactive substance on the surface of the sensor produced electrical signals mainly through the spread mass transfer mode and produced on the sensor. The current response signal is proportional to the concentration of the tested antigen added on the electrode, and is suitable for the quantitative determination of the tested antigen.
(3) the main working conditions that affect the detection performance of the sensor are optimized: the concentration of the capture antibody H1-24 and the detection antibody A1-6-HRP, respectively, are 400 g/ml and 80 mu g/ml respectively. Under this concentration, the detection signals are all strong and can reach more than 80% of the maximum signal value, and can reach the sensitive detection of the A (H1N1) influenza virus. The best temperature of the antigen and antibody reaction at this temperature is the highest, the reaction can quickly reach the equilibrium state and is beneficial to the improvement of the reaction efficiency. The best incubation time of the antigen and detection antibody is 45min and 30min respectively. The sensitivity of the detection is high under the conditions of the incubation time above, and the immune sensor is detected after the optimization. The time is less than 2h, and 1~2h is reduced than the traditional ELISA method. The new sensor can be used to detect the A (H1N1) influenza virus quickly.
(4) the standard curve for the determination of influenza A virus (H1N1) by electrochemical immunosensor, and the standard curve of ELISA and the standard curve of fluorescence quantitative method (qPCR) for the determination of influenza A virus (H1N1), are used to investigate the detection performance of the sensor. The detection signal for the A (H1N1) virus and the detection signal of influenza virus (H1N1) virus are used. The statistical analysis of the negative control signals of influenza A (H7N9), H3N2, B influenza virus, EV71 and adenovirus showed that the specificity of the sensor was good. The reproducibility results showed that the sensor's RSD10%, the recovery rate was 80%~110%, showed high accuracy and precision. The stability test results showed that the sensor was stored in the sensor. Within 10 days, the detection signal was almost no decrease, and the measured value of 30 days later could be kept above 85%. The lower detection limit of.EIS method with better storage stability can reach 0.41HA Unit, which is equivalent to the ELISA method and can be quantified in the 4~64HA Unit range of a H1N1 virus, and the total false positive rate of EIS is 10%, superior to the ELISA method. The correlation coefficient between the established EIS method and the qPCR test result is 0.9517, indicating that the accuracy of EIS method can reach the level consistent with qPCR.
(5) at last, the microminiature Emstat electrochemical detection equipment was used to explore the array detection of a variety of influenza viruses based on the technique and method of portable array electrode based on the detection of electrochemical immunosensors of influenza A virus (H1N1). The results show that the sensor has a wide application to the simultaneous detection of a variety of pathogens. Scene, and its miniaturized testing equipment is easy to carry on the spot.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R446.6;R373

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