Label-free detection of microRNA: two-stage signal enhancement with hairpin assisted cascade isothermal amplification an
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ORIGINAL PAPER
Label-free detection of microRNA: two-stage signal enhancement with hairpin assisted cascade isothermal amplification and light-up DNA-silver nanoclusters Mei Li 1,2,3 & Xiong Xu 3 & ZhongGao Zhou 3 & GuoHai Xu 3 & YongRong Xie 3 & QingYou Cai 4 Received: 24 August 2019 / Accepted: 22 December 2019 # Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract A method is described for the determination of microRNAs via two-stage signal enhancement. This is attained by combining hairpin (HP) assisted cascade isothermal amplification with light-up DNA-Ag nanoclusters. A rationally designed dual-functional HP is used, and microRNA-21 is chosen as a model analyte. At the first stage, upon the hybridization of the microRNA-21 with HP, microRNA recycling via polymerase-displacement reaction and a circulative nicking-replication process are achieved. This generates numerous G-abundant overhang DNA sequences. In the second stage, the above-released G-abundant overhang DNA sequences hybridize with the dark green Ag NCs, and this results in the appearance of bright red fluorescence. Thanks to the two signal enhancement processes, a linear dependence between the fluorescence intensity at 616 nm and the concentration of microRNA-21 is obtained in the range from 1 pM to 20 pM with a detection limit of 0.7 pM. The strategy clearly discriminates between perfectlymatched and mismatched targets. The method was applied to the determination of microRNA-21 in a spiked serum sample. Keywords Fluorescence detection . MicroRNA-21 . DNA polymerase . NEase . Hairpin assisted target recycling . Light-up Ag NCs . Strand displacement amplification . Serum analysis
Introduction MicroRNA is a single-stranded endogenous non-coding small RNA about 19–23 nucleotides in length. It can regulate gene Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00604-019-4094-1) contains supplementary material, which is available to authorized users. * YongRong Xie [email protected] * QingYou Cai [email protected] 1
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, Jiangsu, China
2
School of Medicine,Chemistry and Chemical Engineering, Taizhou University, Taizhou 225300, Jiangsu, China
3
Key Laboratory of Jiangxi University for Functional Material Chemistry, College of Chemistry & Chemical Engineering, Gannan Normal University, Ganzhou 341000, Jiangxi, China
4
College of Mathematics and Computer Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi, China
expression at post-transcriptional level in biology [1, 2]. MicroRNA plays a pivotal regulatory role in growth and development [3], cellular proliferation [4], differentiation [5] and apoptosis [6], as well as immune system [7]. Besides, it is also involved in regulating the pathological processes of various diseases [8]. For example, microRNA-126, microRNA-143, and microRNA-145 have been identified to be remarkably down-regulated in mos
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