基于多功能磁探针和生物发光信号放大系统的免疫分析方法研究

We propose an ultrasensitive immunoassay to provide a new strategy for detection of procalcitonin (PCT) in human serum samples. In this approach, multifunctional magnetic probes and bioluminescent signal amplification strategy are emploited to develop the immunoassay with ultrasensitivity. The main contents and innovations of the proposal are: 1）The antibody(Ab) and alkaline phosphatase (ALP) are conjugated to the surface of magnetic beads (MBs) layer by layer using click chemistry. This multi-functional magnetic probe (“Ab-MBs-ALP” conjugate) not only can enrich target from samples by immunomagnetic enrichment, but also can be used for enzymatic signal amplification. Therefore, immunomagnetic enrichment and enzymatic signal amplification can be completed in one step in this immunoassay. 2) The “luciferin -luciferase -ATP” bioluminescence reaction is used as signal amplification and readout system in this method, thus a novel immunoassay based on immunomagnetic enrichment and double enzyme (alkaline phosphatase and luciferase) cascade signal amplification is developed. This immunoassay combines effective immunomagnetic enrichment mode, click chemistry reaction and efficient signal amplification strategy (both enzymatic cascades and nanomaterials for signal amplification) to achieve high sensitivity and speediness, which provides a new strategy for the development of a simple and ultrasensitive immunoassay. In addition, this immunoassay can be further applied for the early diagnosis of infectious diseases, the ultrasensitive and rapid detection of trace toxic substance in food and environment, which is of great importance in fields like disease control and prevention, environment monitoring and food safety.

本项目立足于构建一种高灵敏、快速的免疫分析方法，并应用于人血清样品中降钙素原的检测。主要研究内容为：1）通过click可控反应将抗体和碱性磷酸酶组装在纳米磁珠的不同表层，制备一种同时具有免疫识别和酶促信号放大的多功能磁探针，实现免疫磁富集和信号放大一步完成；2）将“荧光素-荧光素酶-ATP”生物发光反应作为该方法的信号读出放大系统，进而构建一种基于免疫磁富集和双酶（碱性磷酸酶和荧光素酶）级联信号放大的免疫分析方法。该方法将有效的免疫磁富集模式、click可控反应和高效的信号放大策略（酶促级联放大和纳米材料信号放大）结合起来，实现了高灵敏和快速检测的统一，对发展一种简便的高灵敏免疫分析方法提供了一条新思路。此外，这种新型的免疫分析方法可应用于重大传染病的早期诊断和食品环境中痕量有毒物质的高灵敏、快速检测，对疾病防控和保障环境和食品安全具有重要的意义。

本项目系统地研究了“荧光素-荧光素酶-ATP”生物发光反应作为免疫反应信号读出系统的优势和可行性，并成功将便携式的ATP检测仪作为信号收集系统，结合免疫磁分离技术，构建了一种分析速度快，灵敏度高的生物发光免疫传感器，成功应用于血清中细菌感染生物标志物（降钙素原）的快速分析。该方法的整个检测时间为 1 h，灵敏度可以达到飞摩尔。该方法实现了高灵敏和快速检测的统一，这对合理使用抗生素具有重要的意义。该项目对发展一种简便的高灵敏免疫分析方法提供了一条新思路，对疾病防控和保障环境和食品安全具有重要的意义。. 同时，将点击反应的可控组装的优势和微流控芯片多指标同时分析的优势结合起来，构建了一种可以同时检测降钙素原、白介素和C-反应蛋白的免疫分析方法，该方法不仅灵敏度高，而且可以实现线性范围的可调(pg/mL-μg/mL)，这部分内容属于原项目的继续和拓展。同时分析多个生物标志物对于提高诊断率，降低分析成本具有重要的意义。