"Click"化学法标记抗体技术在免疫传感分析中的应用及其对Raf激酶抑制蛋白的定量检测研究

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding protein family, which is a widely expressed protein in a variety of different species. It plays a pivotal modulatory role in EGFR signaling pathway, and recently, RKIP has become a novel prognostic marker in several cancers. However, there still exist some "bottlenecks" in the accurate, rapid and quantitative detection technique to RKIP. Traditional analysis methods (such as immunohistochemical method, the Western blot, etc) only investigated qualitative or relative quantification in tissue or cell level, which operate tediously and will be difficult to ensure reproducibility. The development of electrochemical immunoassay technology provides a new way and means for it. The project aims to prepare a new electrochemical immunosensor based on a good conductivity, excellent compatibility, multi-coordinated bionic nano-sensing interface for the establishment of a new method of fast, ultrasensitive electrochemical detection of RKIP protein. In this project, for the first time, we employ "click" chemistry method to co-label antibody on the surface of the hollow gold nanoshells and the electron mediator, obtaining a multifunction signal antibody with biological and electrochemical redox activity. The proposed biosensor can significantly improve the sensitivity of the immunosensor through layer-by-layer amplifying electrochemical signal to achieve the purpose of the trace detection of RKIP protein. To sum up, such strategy explicates a method to detect the RKIP protein from a completely new view, which will provide a new ideas, new targets and new technology platform for the prevention and treatment of tumors, as well as the development of anticancer drugs.

Raf激酶抑制蛋白（RKIP）在EGFR信号传导通路的调控中起关键作用，现已成为一个新的肿瘤诊断标志物。但对其准确快速定量检测技术还存在"瓶颈"。传统分析方法（如免疫组化法、Western blot等）仅仅考察其在组织或细胞水平上的定性或相对定量，其操作繁琐、重现性难以保障。电化学免疫传感技术的发展为其提供新的途径和手段。本项目旨在构筑导电性好、兼容性佳、多元协调的纳米传感界面，制备新型的电化学免疫传感器及建立快速、超灵敏的检测RKIP蛋白新方法。首次采用 "click"化学法将含有双酶的中空纳米金壳和媒介体共标记抗体技术，获得兼具生物功能和电化学氧化还原活性的信号抗体，形成层层催化放大电化学响应信号的模式，显著提高传感器的灵敏度，达到对RKIP蛋白分子微量及痕量检测的目的。这种策略从全新的角度检测RKIP蛋白分子，为肿瘤的防治和治疗以及对抗肿瘤药物的研发提供了新思路、新靶点和新技术平台。

本项目构建了多种导电性好、兼容性佳、多元协调的生物仿生纳米传感界面，利用电化学传感器的方法对Raf激酶抑制蛋白（RKIP）等为模板的多种免疫蛋白进行了定量研究。通过苝四甲酸、石墨烯、碳纳米管、离子液体、壳聚糖等物质为固载基质，引入了电子媒介体为探针，多种复合纳米材料来增强基质的导电性，并采用“click”化学法、生物素-亲和素法、直接吸附法、EDC/NHS法等不同方法来固定生物蛋白或用来标记二抗，获得了同时具有生物功能和电化学活性的信号抗体，起到了层层催化放大电化学响应信号的作用，用于对葡萄糖氧化酶、RKIP、netrin 1等蛋白的检测。试验结果表明“click”化学法在传感器的稳定性和灵敏度方面有较好的优势，将这种方法用于传感器的研制过程，对多种生物蛋白进行定量分析，进而评估生物蛋白水平与疾病之间的关系，为肿瘤的防治和治疗、以及对抗肿瘤药物的研发提供了新思路、新靶点和新技术平台。目前已发表论文12余篇，其中SCI论文11篇，待发表论文3篇，培养了3名硕士研究生。