Electrochemical determination of microRNAs based on isothermal strand-displacement polymerase reaction coupled with multienzyme functionalized magnetic micro-carriers

Abstract

MicroRNAs (miRNAs) show great potential for disease diagnostics due to their specific molecular profiles. Detection of miRNAs remains challenging and often requires sophisticated platforms. Here we report a multienzyme-functionalized magnetic microcarriers-assisted isothermal strand-displacement polymerase reaction (ISDPR) for quantitative detection of miRNAs. Magnetic micro-carriers (MMCs) were functionalized with molecular beacons to enable miRNAs recognition and magnetic separation. The target miRNAs triggered a phi29-mediated ISDPR, which can produce biotin-modified sequences on the MMCs. Streptavidin–alkaline phosphatase was then conjugated to the MMC surface through biotin–streptavidin interactions. In the presence of 2-phospho-L-ascorbic acid, miRNAs were quantitatively determined on a screen-printed carbon electrode from the anodic current of the enzymatic product. We show that this method enables detection of miRNAs as low as 9fM and allows the discrimination of one base mismatched sequence. The proposed method was also successfully applied to analyze miRNAs in clinical tumor samples. This paper reports a new strategy for miRNAs analysis with high sensitivity, simplicity, and low cost. It would be particularly useful for rapid point-of-care testing of miRNAs in clinical laboratory.