基于纳米材料电化学免疫传感器检测禽呼肠孤病毒研究

doi:10.3864/j.issn.0578-1752.2014.05.017

摘要/Abstract

摘要： 【目的】构建一种用于检测禽呼肠孤病毒(ARV)的新型电化学免疫传感器。【方法】将石墨烯(G)和甲壳胺(Chi)分散在乙酸溶液中得到均匀的混合物后，加入HAuCl4溶液于80℃还原成金纳米粒子（AuNPs），得到石墨烯-甲壳胺-金纳米粒子(G-Chi-AuNPs)纳米复合物。将石墨烯(G)和甲壳胺(Chi)分散在乙酸溶液中得到均匀的混合物后，先加入AgNO3溶液于80℃还原成银纳米粒子（AgNPs），再加入禽呼肠孤病毒单克隆抗体(ARV-MAb)，得到石墨烯-甲壳胺-银纳米粒子-禽呼肠孤病毒单克隆抗体（G-Chi-AgNPs-ARV-MAb）纳米复合物。将G-Chi- AuNPs纳米复合物修饰金电极作为传感器平台，固定待检测样品，然后加入G-Chi-AgNPs-ARV-MAb纳米复合物，并对G-Chi-AgNPs-ARV-MAb纳米复合物的孵育时间进行优化，构建了ARV电化学免疫传感器。使用构建的ARV电化学免疫传感器，对禽流感（H5N1、H3N6和H9N2亚型）、新城疫病毒（NDV）、喉气管炎病毒（LTV）、传染性支气管炎病毒（IBV）和传染性法氏囊病毒(IBDV)进行检测，以确定ARV电化学免疫传感器的特异性。使用构建的ARV电化学免疫传感器，对106.5—100.5 TCID50/mL的病毒进行检测，以确定ARV电化学免疫传感器的敏感性试验。使用构建的ARV电化学免疫传感器，对临床样品进行检测，其确定ARV电化学免疫传感器的实用性。【结果】所建立的ARV电化学免疫传感器，G-Chi-AgNPs-ARV-MAb纳米复合物的最佳孵育时间为40 min。当检测的样品为阳性时，ARV与ARV-MAb特异结合，G-Chi-AgNPs-ARV-MAb纳米复合物被固定到电极的表面使其线性伏安曲线出现AgNPs的氧化峰；当检测样品为阴性时，其线性伏安曲线不出现AgNPs的氧化峰。特异性结果表明，所建立的方法仅对ARV的检测为阳性（出现AgNPs的氧化峰），而对非目标禽流感（H5N1、H3N6和H9N2亚型）、NDV、LTV、IBV和 IBDV的检测为阴性（不出现AgNPs的氧化峰）。敏感性结果表明，所构建的ARV电化学免疫传感器，具有很高的敏感性，对ARV检测的敏感性达101.5 TCID50/mL。使用建立的ARV电化学免疫传感器对22份临床样品进行检测，结果与病毒分离方法相符。【结论】所建立的ARV电化学免疫传感器，具有特异性强、敏感度高及快速的特点，为有效检测诊断禽呼肠孤病毒提供了一种新的快速检测诊断技术。

关键词: 石墨烯 , 电化学免疫传感器 , 金纳米粒子 , 银纳米粒子 , 禽呼肠孤病毒 , 禽呼肠孤病毒单克隆抗体

Abstract: 【Objective】A novel assay of nano-materials-based electrochemical immunosensor for the detection of avian reovirus (ARV) was established. 【Method】 Graphene sheet-chitosan homogeneous composite was firstly dispersed in acetic acid solution and then the gold nanoparticles (AuNPs) were in situ synthesized at the composite. A graphene sheet-chitosan-gold nano-particles (G-Chi-AuNPs) modified gold electrode was used as the sensor platform to detect ARV target in samples. Similar method was used to synthesize graphene sheet-chitosan-silver nanoparticles composite (G-Chi-AgNPs), and then avian reovirus monoclonal antibodies(ARV-MAb) was immobilized on the G-Chi-AgNPs composite as the signal amplification materials to construct the immunosensor. This electrochemical immunosensor was used to test avian influenza viruses H5N1, H3N6 and H9N2 subtypes, Newcastle disease virus (NDV), laryngotracheitis virus (LTV), infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV) to ensure the electrochemical immunosensor’s specificity. The electrochemical immunosensor was used to detect 106.5-100.5 TCID50/mL ARV to ensure the electrochemical immunosensor’s sensitivity. Then this electrochemical immunosensor was used to detect the clinical samples to evaluate the practical use of electrochemical immunosensor.【Result】The optimization of the G-Chi-AgNPs-ARV-MAb composite incubation time was studied in detail, and the optimal condition was 40 min. When the sample is tested positive, ARV and ARV-MAb specific combination, G-Chi-AgNPs-ARV-MAb composite are fixed onto the electrode surface, so it’s linear sweep voltammetry curve shows an AgNPs oxidation peak. When the sample is tested negative, it’s linear sweep voltammetry curve dose not show an AgNPs oxidation peak. The developed immunosensor is specific to ARV and does not react with non-target avian viruses when a test sample consists of avian influenza viruses H5N1, H3N6 and H9N2 subtypes, Newcastle disease virus (NDV), laryngotracheitis virus (LTV), infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV).The electrochemical immunosensor detected the ARV target at a titer higher than 101.5 TCID50/mL. Twelve specimens of ARV were identified from the 22 clinical samples by electrochemical immunosensor assays developed, which was consistant with that of virus isolation. 【Conclusion】 The novel nano-materials-based electrochemical immunosensor for ARV is highly specific and sensitive, and has great potential for efficient diagnosis and monitoring of ARV in affected poultry.

Key words: graphene sheet , electrochemical immunosensor , gold nano-particles , Ag nano-particles , avian reovirus , avian reovirus monoclonal antibodies

黄娇玲, 谢芝勋, 谢丽基, 滕丽琼, 黄莉, 刘加波, 庞耀珊, 邓显文, 谢志勤, 范晴, 罗思思. 基于纳米材料电化学免疫传感器检测禽呼肠孤病毒研究[J]. 中国农业科学, 2014, 47(5): 1006-1012.

HUANG Jiao-Ling, XIE Zhi-Xun, XIE Li-Ji, TENG Li-Qiong, HUANG Li, LIU Jia-Bo, PANG Yao-Shan, DENG Xian-Wen, XIE Zhi-Qin, FAN Qing, LUO Si-Si. Nano-Materials-Based Electrochemical Immunosensor for the Detection of Avian Reovirus[J]. Scientia Agricultura Sinica, 2014, 47(5): 1006-1012.

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