title: 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.
authors: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensor, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province 510515, PR China.
authors: Wen
authors: W
authors: Ma
authors: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensor, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province 510515, PR China.
authors: Bo
authors: B
authors: Situ
authors: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensor, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province 510515, PR China.
authors: Weifeng
authors: W
authors: Lv
authors: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensor, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province 510515, PR China.
authors: Bo
authors: B
authors: Li
authors: Department of Laboratory Medicine, The Fifth Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong Province 510900, PR China.
authors: Xiaomao
authors: X
authors: Yin
authors: School of Engineering and Materials Science, Queen Mary University of London, London E1 4NS, UK.
authors: Pankaj
authors: P
authors: Vadgama
authors: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensor, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province 510515, PR China. Electronic address: nfyyzhenglei@smu.edu.cn.
authors: Lei
authors: L
authors: Zheng
authors: School of Engineering and Materials Science, Queen Mary University of London, London E1 4NS, UK. Electronic address: wen.wang@qmul.ac.uk.
authors: Wen
authors: W
authors: Wang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D001710:Biotin / Q000737:chemistry; D001940:Breast / Q000473:pathology; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics*; D055664:Electrochemical Techniques / Q000379:methods*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D059346:Magnets / Q000737:chemistry; D035683:MicroRNAs / Q000032:analysis*; D053758:Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D001710:Biotin; D019809:Streptavidin
keywords: Electrochemical sensing assay; Magnetic microcarriers; MicroRNA; Signal amplification