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 9 fM 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.