基于导电气凝胶信号放大电化学免疫传感器的构建及ALVs检测的研究

The wide spread of Avian leukosis virus (ALVs) has not only caused serious economic loss to poultry farming, but also posed severe threat to human’s food safety. Thus, the specific, sensitive and real-time detection of ALVs is of great significance to prevent and control Avian leukosis.x In this project, we focus on the development of novel electrochemistry immunosensor based on nanofibrillated cellulose-graphene conductive aerogel for ALVs determination with high selectivity and sensitivity. Firstly, the conductive aerogel is synthetized via in situ reduction method by means of the reducing hydroxyl of cellulose and the deoxygenation of graphite oxide in strong alkaline solutions. Due to the high porosity, high specific surface area and the abundant active groups (-OH), cellulose-graphene aerogel can be used as carrier material of biomolecules and electroactive molecules. Secondly, novel electrochemical immunosensor is fabricated using cellulose-graphene aerogel as carrier of antibodies, enzyme or electroactive probes. Lastly, ALVs are detected with high specificity and sensitivity under complex biological environment combining the specificity of immune-reaction and the signal amplification of aerogel. The proposed research will not only expand the application of conductive aerogel in electroanalysis, but also develop novel methods and techniques for various viruses detection in complicated biological environment with high selectivity and sensitivity, which will provide new ideas for the early diagnosis of Avian leukosis, population purification of poultry, and human’s food safety.

禽白血病病毒（ALVs）的广泛传播给禽类养殖业和人类食品安全造成了严重危害，实现ALVs特异、灵敏、实时的检测对防控ALVs的流行具有重要意义。针对目前检测方法存在的昂贵、费时、操作繁琐、灵敏度低等缺点，本项目提出制备新型纳米纤维素-石墨烯导电气凝胶，构建信号放大型电化学免疫传感器对ALVs进行灵敏检测的研究计划。首先通过调控制备条件，利用纤维素多羟基的还原性和氧化石墨烯强碱性条件下自身脱氧的特性，采用一步式原位还原法制备纳米纤维素-石墨烯导电气凝胶，利用气凝胶多孔结构、大的比表面积和多活性基团的优势，负载更多的抗体、酶或电活性探针，从而放大检测信号，再结合免疫反应的高特异性，实现复杂生物样品中ALVs特异、灵敏的检测。本研究的实施，可以拓展气凝胶在电化学领域的应用，还将为禽白血病的提前诊断、禽类的种群净化和人类的食品安全提供准确有效的检测手段和技术支持。

对禽白血病病毒进行灵敏检测是有效防控禽白血病爆发、减少养殖业经济损失、降低食品安全隐患的有效措施。针对传统检测方法昂贵、费时、操作繁琐、灵敏度低等不足，本项目提出，结合电化学的灵敏性和免疫反应的特异性，构建基于纳米复合材料的电化学免疫传感器，对J亚群禽白血病病毒(ALV-J)进行特异性灵敏检测的研究计划。本研究首先利用纤维素、木质素等生物质原料制备了纳米纤维素-石墨烯气凝胶、纤维素-纳米金复合物、纳米纤维素/聚酰胺-胺/钌复合物、掺N多孔碳及中空MnO2、金属有机框架材料ZIF-8等纳米复合材料，以这些纳米材料为电极修饰材料、信号探针或抗体载体，构建了多种“夹心型”电化学免疫传感器，对ALV-J进行了检测。以上制备的纳米材料具有表面积大、孔隙率高、导电性强、生物相容性好等优点，用以构建传感器时起到了促进电子传递、保持生物分子生物活性、提高信号探针负载量等作用，因此可以提高检测灵敏度。ALV-J病毒滴度在10^1.80-10^4.30 TCID50/mL范围内，传感器的电化学检测信号和病毒滴度成正比，最低检测限低至95 TCID50/mL。实际样品检测中，ALV-J在SPF鸡血清中的加标回收率为95.5-107.15%。本项目构建的电化学免疫传感器灵敏度高、选择性好，还具有良好的重现性和稳定性。以上研究结果可以为拓展电化学传感器在病毒检测、疾病诊断领域的应用提供理论依据和技术支持。