基于DNA聚合酶和nicking酶放大信号检测肺癌miRNA的生物传感器研究

MicroRNAs (miRNAs) are a group of short endogenous noncoding RNAs, which is mainly functioned as post-transcriptional regulation and play an important roles in a wide range of biological processes. Recently, miRNAs have been emerged as new biomarkers for early diagnosis of a variety of diseases, including cancer. However, their unique characteristics such as small sizes, low expression levels in cells, and high sequence homology among family members have made a great challenge for quantitative analysis. On the basis of construction of enzyme-based sensitive biosensor and the study of the lung carcinoma biological characteristics effects with the presence of miRNA, we develop a simple and sensitive miRNA biosensor using DNAzyme and hairpin DNA probe in this study, which based on the cooperative amplification of nicking endonuclease and polymerase. The strand displacement amplification, which will result in the generation of a number of triggers, has been initiated after binding the target miRNA to the DNA template probe. These triggers could unfold the hairpin probes and generate catalytic G-quadruplex DNAzyme sequence. The amplification signal can be obtained by being assisted by nicking endonuclease and polymerase in the mentioned two steps. The proposed biosensor shows great potential application in quantification of other miRNA and become a universal biosensing platform. This biosensor provides a new thought of detection of miRNAs and offers an economic, simple and convenient potential tool for early clinical diagnosis.

miRNA是一类内源性具有调控功能的非编码小RNA，主要在基因转录后水平起调控作用，被认为是癌症等重大疾病早期预警、早期诊断的新型标志物而广受关注。但因分子片段小、在细胞中表达水平偏低、同源性高等，导致miRNA特异性定量检测成为亟待解决的难题。项目组在基于工具酶构建高灵敏生物传感器及miRNA对肺癌生物学特性影响研究的基础上，提出基于DNA聚合酶和nicking酶的信号放大作用，利用发夹型核酸探针研究高灵敏检测miRNA的生物传感器并应用于临床检测。目标miRNA能诱发以模板链为模板的聚合反应并释放出能结合发夹型核酸探针的触发链，继而释放出DNA酶序列，这两步反应均能在DNA聚合酶和nicking酶共同作用下实现信号循环放大。本研究如顺利完成，则可拓宽到其他疾病中miRNA的检测，有望成为一种通用的生物传感技术平台，为癌症等重大疾病早期预警、早期诊断提供经济、便捷、实用的新技术、新方法。

项目组基于目标miRNA能诱发以模板链为模板的聚合反应并释放出能结合发夹型核酸探针的触发链，继而释放出DNA酶序列，而这两步反应均能在DNA聚合酶和nicking酶共同作用下实现信号循环放大的原理，设计了一种基于DNA聚合酶和nicking酶的信号放大作用及发夹型核酸探针的miRNA生物传感器。我们对发夹型探针的结构、浓度，模板链的浓度等条件进行了优化，实验结果表明，miRNA-205的浓度在0.1-1000 pM浓度范围内呈良好的线性关系，该方法对miRNA-205的检测限为0.1 pM。在此基础上，利用本基金的资助，我们还进行了以下相关研究：（1）构建了一种基于血红素/石墨烯纳米复合材料的人工酶，运用该人工酶，建立了一种用于ATP检测的适配体电化学生物传感方法；（2）运用该人工酶，还构建了一种用于白血病检测的生物传感器；（3）针对白血病的早期检测，发展了一种基于适配体标记的荧光硅纳米颗粒检测白血病细胞的高灵敏生物传感方法；（4）发现了还原型氧化石墨烯能够催化银沉积反应，并基于此构建了一种检测甲基化酶的生物传感方法。.在本项目的支持下，共发表SCI学术论文2篇，申请国家专利2项，均已获得授权。