基于酶剪切量子点荧光放大技术的双元miRNA定量检测

Quantitative Detection of Dual miRNAs Based on Enzyme-sheared Quantum Dot Fluorescence Amplification

将量子点荧光特性与双链特异性核酸酶的DNA剪切特性相结合,提出一种高灵敏度、高特异性的双元miRNA定量检测方案.首先,将量子点和四氧化三铁磁性纳米粒子分别与捕获DNA链接形成捕获探针,再与待测miRNA互补配对形成异源双链杂合结构,随后双链特异性核酸酶对杂合结构中的捕获DNA进行特异性剪切,实现量子点和待测miRNA从捕获探针分离,且分离的待测miRNA与捕获探针上未配对的DNA开始新一轮杂交和再剪切.经过上述循环过程,量子点从捕获探针大量释放,荧光信号不断增强,实现肿瘤标志物miRNA的高灵敏检测.实验结果表明,基于酶剪切量子点荧光放大技术,在1fmol/L至100pmol/L的浓度范围内,同时实现了肿瘤标志物miRNA-141及循环miRNA内参miRNA-1228的特异性定量检测,其检出限分别达到0.69fmol/L和0.21fmol/L.与实时荧光定量多聚核苷酸链式反应方法相比,该方案获得了相同的检测结果,且具有更高灵敏度.

In order to realize the highly specific and sensitive detection for miRNA,a multiple miRNA quantitative detection protocol was designed by combined the fluorescence characteristics of quantum dots with the DNA shearing properties of double-strand specific nuclease.First,the two ends of the capture DNA are separately linked with the quantum dots and the ferroferric oxide magnetic nanoparticles to form a capture probe,and then the capture DNA and the detected miRNA will form a heteroduplex DNAmiRNA hybrid structure as they are complementary gene sequences.And then,the DNA is specifically sheared by double-strand specific nuclease to separate the quantum dots and the detected miRNA from the capture probe,and the isolated miRNA and the unpaired capture probe start a new round of hybridization and specific shear of the double-strand specific nuclease.Through the above cyclic process,quantum dots are continuously released from the capture probe,resulting in the continuous enhancement of the detected fluorescent signal,thereby achieving highly sensitive detection of tumor marker miRNA.The experimental results show that the specific quantitative detection of miRNA-141 and miRNA-1228 is successfully achieved by double-strand specific nuclease-assisted circular amplification of quantum dot fluorescence signals in the concentration range of 1 fmol/L to 100 pmol/L,and the detection limits reached 0.69 fmol/L and 0.21 fmol/L,respectively.In addition,compared with the real-time fluorescence quantitative polynucleotide chain reaction method,the proposed protocol obtains consistent detection results with a higher sensitivity.