基于氧化石墨烯和核酸末端保护的蛋白质传感新方法研究

It is highly important to establish easy operation, rapid, accurate and ultra-sensitive methods for biological molecules detection in early diagnosis of major diseases. These detection methods have vital significance to prevent of disease outbreaks and protect the safety of people's lives. In this project, we explore graphene oxide based fluorescent biosensors for protein detection via terminal protection of small molecule-linked DNA, where probe DNA was covalently attached to graphene oxide and nuclease-aided DNA recycling method was employed, to solve the serious problem of many methods which have low sensitivity in complex matrix. The advantage of this biosensor is reducing nonspecific probe displacement and ultra-high sensitive, which holds considerable potential for practical samples analysis. On the basis of these excellent performances, the terminal protection mechanism and the graphene oxide-assisted strategies for protein assay is expected to hold considerable potential for molecular diagnostics, genomic research, and drug development.

生物分子检测方法以其操作简便、快速准确和超高灵敏度等优势在重大疾病的预防和早期诊断，保障人民群众生命安全等方面发挥了重要作用。本研究针对复杂基质中蛋白质检测灵敏度低，实际应用困难的严重问题，以新型纳米材料氧化石墨烯为平台，采用共价偶联法和核酸信号放大法，结合核酸末端保护策略，构建新型生物传感器用于蛋白质检测研究。该传感器不仅可消除非特异性蛋白的竞争吸附，而且可实现目标蛋白超高灵敏检测，预计在分子诊断、基因组学研究以及药物开发等方面具有较大的应用潜力。

本项目针对复杂基质中蛋白质检测灵敏度低，实际应用困难的问题，以新型纳米材料氧化石墨烯为平台，采用共价偶联法和核酸信号放大法，结合核酸末端保护策略，构建新型生物传感器用于蛋白质检测研究。具有单原子层石墨平面结构的氧化石墨烯表面存在环氧基、羟基以及羧基等多种含氧功能基团，我们采用共价偶联的方式将末端带有氨基的寡核苷酸分子偶联在氧化石墨烯的表面。采用共价偶联法所制备的探针具有较好的稳定性和抗干扰能力，将其用于蛋白质的检测，可有效避免复杂基质的干扰问题，使得该方法可以直接用于血清中蛋白质的检测。接着，我们构建了基于核酸外切酶Ⅲ的酶切信号放大技术，经过反复“构象转换-切割”循环，在溶液中产生大量信号探针DNA，用于蛋白质的检测，可有效提高检测的灵敏度。本项目所构建的蛋白质检测方法具有灵敏度高、选择性好、抗干扰能力强的特点，实现了复杂基质中靶蛋白痕量检测的需求。