纳米电致化学发光免疫传感新体系及肝癌相关同工酶的检测新方法研究

Electrochemiluminescent (ECL) biosensing possesses various advantages, such as high sensitivity, convenient operation and easy control. Thus nevol methods for detection of serum isoenzymes using ECL technique is of good clinical prospect. This project will focus on hepatocellular carcinoma related isoenzymes to design and construct novel nano-ECL immunosensing systems, develop convenient, low-cost and highly sensitive immunoassay methods and evaluate the clinic applications of these methods in detection of isoenzymes. Green nano-ECL probe with higher ECL efficiency and lower ECL emission potential could be obtained by controlling of the chemical compositions, micro morphology, size and surface structure of the nanomaterials. Based on the study of nano-ECL mechanisms, it could be more clearly of the relationships between the ECL properties and the nanostructures, with the instruction of which novel efficient coreactants could further be developed. Using isoenzymes of gamma-glutamyl transpeptidase and alkaline phosphatase as model analytes, the nano-ECL matierals could be used to label the immunoreagents to prepare stable ECL biosensing probes. With the help of further designed signal amplification strategies, rapid and ultra-sensitive detection methods with high sensitivity for the isoenzymes could be constructed. The clinical performance of those novel immunosensors will be preliminarily evaluated by testing of a large number of clinical specimens (human sera). The expected results of this project will greatly extend the research domain of ECL systems, advanced the clinic application of nano-ECL technology, and provide novel approaches for clinical diagosis and the detection of disease biomarkers.

电致化学发光（ECL）生物传感具有灵敏、操作简便等优势，发展血清同工酶的ECL检测新方法具有很好的临床应用前景。本课题拟以肝癌相关血清同工酶为对象，设计并构建纳米ECL免疫传感新体系，建立便捷、价廉、高灵敏的同工酶检测新方法，并对其临床应用价值进行评估。通过调控纳米材料的化学组成、尺寸、形貌和表面结构，降低其激发电位，获得具有高发光效率的新型绿色ECL探针，探讨其ECL机制及其构效关系，提出温和高效的ECL共反应剂，并以谷氨酰转肽酶、碱性磷酸酶为模型同工酶系，将获得的ECL探针用于免疫试剂的标记，制备稳定的ECL免疫示踪探针，设计信号放大新策略，建立稳定、灵敏的同工酶免疫检测新方法。进一步将提出的新方法用于临床标本的检测，初步评价其临床应用前景。本项目将拓宽ECL的研究范围，有望推进纳米ECL技术的临床应用，为临床诊断与疾病标志物检测提供新途径。

项目系统的研究了CdTe量子点（QDs）以及TiO2-CdTe QDs复合物、介孔SiO2-CdTe QDs复合物等纳米结构的电致化学发光（ECL）机制和行为，构建了生物相容性材料增强的QDs ECL体系，通过ECL发光机制的研究提高了纳米ECL体系的发光效率，并基于纳米ECL技术提出了适用于生物分析及临床检测的传感平台。具体研究内容和结果包括：.1.利用两种不同外壳CdTe QDs材料的微观表面性质，获得较大的ECL发光电位差（～360 mV），并依此性质提出了基于电位分辨的QDs-ECL传感平台。依靠溶解氧作为唯一的共反应剂，实现了单方向循环伏安扫描过程中的双组分同时检测，对血清AFP-L3%检测具有较高的灵敏度、准确度和精确度，为AFP-L3％临床检测及原发性肝癌的实验室诊断提供新方法和新思路。.2.基于QDs-ECL技术，通过功能化QDs特异性生物识别体系的特征，设计了分子识别介导的ECL策略，为ECL细胞诊断技术奠定基础。.3. 研究TiO2纳米晶体（NPs）的ECL行为，尤其是发光峰电位与检测溶液pH之间的依赖效应，提出基于TiO2纳米粒子ECL峰电位示踪的pH传感体系，为相关生物分析平台的搭建提供了新的角度。.4.引入纳米TiO2、介孔SiO2等低生物毒性材料，与传统CdTe QDs进行复合，可大大提高单纯CdTe QDs ECL体系的发光效率，降低含Cd和Te成分QDs的用量。以金属离子对ECL信号的猝灭效应为基础，研究了蛋白与金属离子的结合效率以及 Cu-蛋白结合物对ECL体系的猝灭效率。