表面等离子体共振成像之高通量免标记识别与筛选分析方法

In this project, surface plasmon resonance imaging (SPRi) will further be explored, on the base of our previous studies, to establish label-free, high throughput recognition analytical and screening methods. These new methods are designed to be able to work with simulated physiological conditions, aiming at the in situ and real time or dynamic monitoring the multi-molecular recognition events. The targeted recognitions will be directed to some key biomolecules such as proteins, nucleic acids, carbohydrates and their conjugates, and some cells. The interested recognition events to be studied are the interaction(s) of the targeted biomolecules happening in some mimic multi-molecular networks. The project is planned to start from the modification of presently existed chip preparation techniques and SPRi devices that have been established in this laboratory. They need to adapt to the requirements of high throughput screening and/or analysis by increase of the density of the recognition spots on each chip and by upgrade of the batch throughput of the present SPRi devices, including the equipped imaging workstation. A standard method will then be established by feedback adjustment of the primarily established approach using protein, nucleic acid and carbohydrate standards and/or purified cells as testing samples. This standard method will then be extended to the screening and/or analysis of multi biomarkers used for cancer diagnosis and to the high throughput measurement of omic samples. This technical route map for conducting the research of this project is in theory logic and in practice executable. It can thus be expected that several label-free biological SPRi methods of high throughput will be available for clinic diagnosis and omic investigations. They should be able to accelerate the development of SPRi toward the requirements of life science, clinic medicine, drug chemistry, and other complex-sample-bothersome fields by offering new methods. This is hence a significant project worth of pushing forward as early as possible.

拟对我们在SPRi上的多年研究进行提升，发展出新型的高通量免标记识别与筛选分析新方法，拟模仿生物体系中的网络多分子识别过程，实现在生理条件下原位静态或实时动态观测生物分子识别现象，揭示其与生物功能间的关系，为癌症等重大疾病诊断或组学研究等提供新的方法选项。已制定了从仪器改造建立雏形方法，到以标准生物样品为对象构建标准方法，再到以癌症标志物、组学样品分析为应用出口发展目标方法的可操作研究方案和可行的实施路线。预计能建立可同时静态识别分析5000个样点或动态分析20个样点识别反应的实用方法并发表25篇研究论文、申请5件发明专利。本研究如能取得成功，将会为组学、医学、药学等面向复杂样品分析的学科领域提供新的方法学选择，并推动SPRi自身研究向更具挑战性的前沿领域发展，因此是一项具有重要科学和实用价值的研究项目，期待能获得支持。

在生理条件下，免标记、高通量研究分子识别事件既是生命科学研究的梦想，也是分析化学面临的严峻挑战并期待突破。本项目基于我们的基础，发展表面等离子体共振成像（SPRi）法，研究涉及高效芯片研制、仪器改造、方法发展和相关平台建设等众多内容。研究始于标准样品，终于实际样品检验，不断遭遇困扰，其严峻者有不灵敏、非特异性吸附和点阵间交叉干扰等，经专题研究已获突破，主要进展有：改造和新搭建了高通量/高分辨SPRi实验仪器与装置3类5种；升级图像工作站1个；建立高通量SPRi芯片制备方法2类9种（常规和长程SPRi芯片2类及6种共价修饰法、2种可寻址点样法、1种边界自约束点样法）；建立方法发展平台1个，可在生理条件下对5000个微点进行识别筛查分析，能原位、动态观测>20个微点的识别反应过程，可测定反应动力学与热力学参数；建立了从小分子到细胞范围内的原位静态或动态识别过程分析法；建立了抑制非特异性吸附和点阵交叉干扰的新方法；设计建立了3类9种级数信号放大新方法（保真高负载桥接探针、折射率调控、纳米颗粒增强、聚多巴胺原位还原金属沉积增强、循环识别增敏、多级聚合放大、样品触发循环反应放大等）；建立了混合样品和混合探针点阵的高通量分析方法，发展了与恶性肿瘤相关的多种标志物的生理条件识别分析法。所建方法已用于人血样品中microRNA-15a、癌胚抗原等恶性肿瘤标志物的高灵敏分析和抗癌药物-蛋白互作动态研究等，突破了SPRi难以检测小分子的限制，实现了μmol/L级单糖、亚fmol/L级microRNA的测定。该项目还形成了SPRi方法发展研究队伍，培养毕业7名博士研究生；发表期刊研究论文25篇、英文书2章；申请中国发明专利11件，其中授权8件，项目前申请执行期间授权9件。本项目攻克了许多障碍，可能会推动SPRi自身研究向更具挑战性的前沿领域发展。