基于单微球信号富集机制的单细胞水平蛋白激酶及基因标志物分析

In this project, we wish to propose a new sensing strategy by using a single microbead as the reaction vessel for signal amplification and fluorescence enrichment. Based on this new principle, we aim to develop a highly sensitive detection platform for various genetic markers and protein kinases at single-cell and single-molecule level. On one hand, towards different genetic markers, rationally designed nucleic acid amplification systems will be integrated and performed on the surface of a single microbead. As a result, the nonspecific amplification background will be effectively suppressed on such a microamplification zone, while the fluorescence signal produced by the target will be highly concentrated on the single microbead, which will lead to significantly enhanced detection sensitivity. Therefore, the single microbead-based sensing platform will enable the accurate detection of various genetic markers such as gene mutation, DNA methylation, mRNA as well as microRNA at the single-cell and single-molecule level. On the other hand, by systematically screening new rare earth-based phospho-recognizing materials, we aim to prepare phospho-recognizing microbeads which exhibit high binding specificity towards phosphopeptides. By employing a single phospho-recognizing microbead as the carrier for specific enrichment of protein kinase-catalyzed phosphopeptides, highly sensitive detection of protein kinase activity at the single-cell level will be achieved. Therefore, the single microbead-based detection strategy in this project may provide a powerful platform for highly sensitive detection of gene mutation, DNA methylation, mRNA, microRNA as well as protein kinase activity at the single-cell level, which is of great significance for further understanding the pathogenesis of diseases such as cancers at the single-cell level.

本项目创新性提出以单个微球为信号放大及荧光富集载体的传感新策略，开展单细胞、单分子水平上多种基因标志物及蛋白激酶活性的超高灵敏度分析。一方面，针对不同基因标志物，在单个微球表面合理设计相应的核酸信号扩增体系，在有效抑制非特异性扩增信号的同时，所有检测信号将高度集中于单一微球的表面，使分析检测基因突变、DNA甲基化、mRNA、microRNA等不同基因标志物的灵敏度达到单细胞乃至单分子水平。另一方面，筛选性能优异的新型稀土基磷酸化多肽亲和识别材料，并制备表面负载该亲和材料的复合微球材料，构建以单个亲和微球为磷酸化多肽底物富集载体的单细胞水平蛋白激酶活性检测方法。本项目研究将系统建立普遍适用于单细胞中基因突变、DNA甲基化、mRNA、microRNA以及蛋白激酶等多种疾病标志物的超高灵敏度检测平台，对于在单细胞水平深入了解疾病发生机制及疾病早期诊断与预防具有重要意义。

本项目主要针对癌症相关核酸（microRNA、基因突变等）、蛋白及酶类标志物的高灵敏度、高特异性分析开展了系统的研究。取得的主要研究成果如下：（1）创新提出并拓展了以单个微球为信号放大及富集载体的新分析方法，以简便的操作步骤实现了microRNA、蛋白激酶等多种重要生物标志物的单细胞、单分子分析。并以单个微球为载体，通过多通道荧光成像分析，实现了多种核酸、蛋白标志物的同时灵敏测定；（2）基于流式细胞仪对微球表面光学信号优异的多信息读取能力，结合微球表界面信号扩增新策略，创新发展了简便高效的流式微球检测技术平台，在有效消除了复杂基质干扰的同时，提高了检测结果的准确性和可靠性。相关成果在Angew. Chem. Int. Ed., Chem. Sci.、Anal. Chem.、Chem. Commun.等期刊发表SCI论文21篇（通讯作者），授权中国发明专利3项，为复杂临床样本中痕量生物标志物的高特异性准确分析提供了高灵敏度、高特异性的检测手段。