基于信号放大的高灵敏检测循环microRNAs化学发光生物传感新方法研究

在我们多年来对化学发光分析及生物传感研究的基础上，借鉴前人有关信号放大和miRNA检测的研究成果，本项目拟以miRNA的生物属性为基础，充分利用分子生物学和集群多重标记信号放大技术，揭示信号放大规律，探索简单、高灵敏检测循环miRNA的化学发光生物传感新方法。将多种化学发光信号分子（如HRP、碱性磷酸酶、荧光素）用于标记探针，系统研究化学发光体系（如1,2-二氧杂环丁烷-碱性磷酸酶）及化学发光共振能量转移体系（如Luminol-H2O2-HRP-荧光素）检测循环miRNA的分析特性；探索提高化学发光检测循环miRNA灵敏度的信号放大策略，从而建立测定miRNA新方法。此新方法既具有信号放大技术在生物传感方面的优势，又具有化学发光在检测方面的优势。本项目给设计高灵敏的miRNA生物传感方法提供一种新的思路，为miRNA基础研究和应用等提供新的检测方法和技术，同时也推动化学发光研究向纵深发展。

Circulating microRNAs are useful biomarkers for cellular events or early diseases diagnosis. Because of the short size and low abundance of microRNAs, it is challenging to develop fast, inexpensive, and simple biosensors to detect them. In this proposal, we aim to find a chemiluminescence (CL) biosensing for simple and sensitive detection of circulating microRNAs. In order to improve the sensitivity of the analytical method, one signal amplification strategy by combination of multi-labels and nuclease-based amplification will be employed. Many CL signal molecules (HRP, alkaline phosphatase (ALP), G-quadruplex-hemin DNAzyme, and fluorescein) will be used to label the DNA probe. The CL systems (such as luminol-H2O2, and ALP-AMPPD) and CL resonance energy transfer systems (such as luminol-H2O2-HRP-fluorescein) will be explored for detection of circulating microRNAs. The new analytical method will have both the advantages of signal amplification and the advantages of CL detection. The features make the proposed assay particularly useful in clinical biochemistry laboratory settings. Therefore, the proposal not only will find a new analytical method of circulating microRNAs, but also will open a new research area for chemiluminescence analysis.