title: An immobilization-free electrochemical impedance biosensor based on duplex-specific nuclease assisted target recycling for amplified detection of microRNA.
abstract: An immobilization-free electrochemical impedance biosensor for microRNA detection was developed in this work, which was based on both the duplex-specific nuclease assisted target recycling (DSNATR) and capture probes (Cps) enriched from the solution to electrode surface via magnetic beads (MBs). In the absence of miR-21, Cps cannot be hydrolyzed due to the low activity of duplex-specific nuclease (DSN) against ssDNA. Therefore, the intact Cps could be attached to the surface of magnetic glass carbon electrode (MGCE), resulting in a compact negatively charged layer as well as a large charge-transfer resistance. While in the presence of miR-21, it hybridized with Cp to form a DNA-RNA heteroduplex. Due to the considerable cleavage preference for DNA in DNA-RNA hybrids, DSN hydrolyzed the target-binding part of the Cp while liberating the intact miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. In this way, a single miR-21 was able to trigger the permanent hydrolysis of multiple Cps. Finally, all Cps were digested. Thus, the negatively charged layer could not be formed, resulting in a small charge-transfer resistance. By employing the above strategy, the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with a detection limit of 60aM. Meanwhile, the method showed little cross-hybridization among the closely related miRNA family members even at the single-base-mismatched level. Successful attempts were made in applying the approach to detect miR-21 in human serum samples of breast cancer patients.
authors.affiliation: Department of Chemical Biology, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian Province 350002, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Pharmaceutical Analysis, The higher educational key laboratory for Nano Biomedical Technology of Fujian Province, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350108, PR China.
authors.forename: Dong-Zhi
authors.initials: DZ
authors.lastname: Wu
authors.affiliation: Department of Pharmaceutical Analysis, The higher educational key laboratory for Nano Biomedical Technology of Fujian Province, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350108, PR China.
authors.forename: Shu-Xian
authors.initials: SX
authors.lastname: Cai
authors.affiliation: Department of Pharmaceutical Analysis, The higher educational key laboratory for Nano Biomedical Technology of Fujian Province, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350108, PR China.
authors.forename: Mei
authors.initials: M
authors.lastname: Chen
authors.affiliation: Department of Pharmaceutical Analysis, The higher educational key laboratory for Nano Biomedical Technology of Fujian Province, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350108, PR China.
authors.forename: Yao-Kun
authors.initials: YK
authors.lastname: Xia
authors.affiliation: Department of Pharmaceutical Analysis, The higher educational key laboratory for Nano Biomedical Technology of Fujian Province, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350108, PR China.
authors.forename: Fang
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Pharmaceutical Analysis, The higher educational key laboratory for Nano Biomedical Technology of Fujian Province, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350108, PR China. Electronic address: cjh_huaxue@126.com.
authors.forename: Jing-Hua
authors.initials: JH
authors.lastname: Chen
mesh_terms: D015374:Biosensing Techniques; D001943:Breast Neoplasms / Q000097:blood*; D004277:DNA, Single-Stranded / Q000737:chemistry; D017097:Electric Impedance; D055664:Electrochemical Techniques; D005260:Female; D006801:Humans; D035683:MicroRNAs / Q000097:blood / Q000302:isolation & purification*
chemical_list: D004277:DNA, Single-Stranded; C524940:MIRN21 microRNA, human; D035683:MicroRNAs
keywords: Duplex-specific nuclease assisted target recycling; Electrochemical impedance; Immobilization-free; MicroRNA detection