农药残留快速检测适配体传感器构建中的关键问题

At present, there are many methods of rapid detection for pesticide residues. Particularly, the immunoassay methods, such as ELISA and immunosensor, are based on the antibodies. However, the period of antibody screening and preparation is time-consuming, and its reuse is limited, which restricts the development of immunoassay methods. Considering the problems of the rapid detection of pesticide based on immunoassay in this project, we will put forward the thought that the aptamers are used for replacing the antibodies to study the new aptasensors for rapid detection of pesticide residues. After studying the spatial conformation between aptamers and pesticides under an electrochemical system, we will identify the recognition mechanism between them,and then design the structure of aptamers suitable for the electrochemical system. Meanwhile the reaction kinetics process between aptamers and pesticides will be studied as well. A reaction kinetics model will be established and an electrochemical reaction mechanism will be found, thus a target recognition ability between them will be improved. By studying the relationships between the loading capacity and the pesticides recognition capacity which lies on the sensing interface, we will acquire the immobilization methods of aptamers with its controllable surface density and orientation,by which the reproducibility and stability of the aptamer biosensors will be improved.It is extpected that the studies will provide a theoretic guide and technical support to establish a rapid method for the detection of pesticide residues with high sensitivity, high through-put and selectivity.

目前农药残留快速检测方法如酶联免疫试剂盒、免疫传感器等分析方法均使用抗体进行检测。抗体筛选和制备周期长，重复使用受限，制约了免疫分析方法发展。本项目针对农药快速检测免疫分析方法存在的问题，拟以适配体代替抗体，研究适配体传感器农药残留快速检测新方法。拟研究电化学体系适配体与农药作用前后的空间构象，明确电化学体系适配体和农药识别机理，设计适合电化学体系的农药适配体结构。研究电化学体系适配体和农药相互作用的动力学过程，建立农药与适配体作用的动力学模型，明确电化学体系适配体和农药电化学反应机理，改善适配体靶向识别能力。研究电化学体系传感界面适配体负载量与农药识别能力关系，获得表面密度与取向可控的适配体固定方法，提高适配体传感器重现性与稳定性。以上研究为建立高灵敏、高通量、高选择性农药残留快速检测技术提供理论指导和技术支撑。

农业生产中农药的广泛应用甚至不规范使用，对农产品安全和人体健康构成严重威胁。寻找一种现场、快速、准确的农药残留检测技术是保障农产品安全的有效途径。农药是小分子半抗原物质，难以制备相应的抗体，并且抗体存在重复使用性差、重现性不能满足实际样品检测要求等问题。与目标分析物高特异性反应结合的适配体为农药快速检测提供了新思路，将高灵敏的传感器技术与适配体结合实现了一种检测农药残留的新方法。围绕上述问题，本项目通过分子斑点杂交实验对农药适配体的特异性进行测定，并根据需要和特异性设计了适合电化学体系的农药适配体结构。比较适配体序列5’端和3’端修饰氨基、巯基以及二茂铁等基团对适配体与农药作用的空间构象、适配体识别能力、适配体稳定性的影响，选择了最佳效果的二茂铁修饰方法，并对设计成发卡结构的适配体进行二茂铁标记，通过电子供体二茂铁电化学阻抗谱测量，建立模拟等效电路，探讨了适配体与农药电化学反应机理。分子斑点杂交实验测定农药适配体的特异性较高。建立了适配体与农药作用的Butler-Volmer动力学模型，明确了传感界面适配体负载量与农药识别能力的关系，改善了适配体在电化学体系下对农药的靶向识别能力。通过对滴加适配体浓度和体积的优化，对适配体负载量进行控制优化，研究了电化学体系传感界面适配体负载量与农药识别能力关系，获得了表面密度与取向可控的适配体固定方法，提高了适配体传感器重现性与稳定性。并通过生物模板法在适配体上制备CdS和PbS量子点，达到CdS和PbS量子点分别标记不同适配体的目的，并根据Cd2+和Pb2+在不同的电位下可以同时产生电流的原理制备了可以同时检测多种目标物的适配体传感器。以上研究为建立高灵敏、高通量、高选择性农药残留快速检测技术提供理论依据和技术支撑。