Label-free and sensitive microRNA detection method based on the locked nucleic acid assisted fishing amplification strategy

Abstract

Herein, a label free and sensitive miRNA detection method with enhanced practical applicability was developed based on the locked nucleic acid (LNA) assisted repeated fishing amplification strategy. The working mechanism of the proposed method is as follows: 1) a DNA probe (i.e, L-DNA) with LNA bases is immobilized onto the surface of a gold foil. The L-DNA hybridizes with the 3' terminus of the first strands of complementary deoxyribonucleic acid (cDNA) of the target miRNA in the test samples; 2) The protruding 5' terminus of the cDNA serves as a 'fishhook' to repeatedly fish the products of a hybridization chain reaction (HCR) out from a 'reaction tube'; 3) The HCR products can be unloaded from the gold foil into a 'product tube' through temperature-controlled dehybridization; 4) The concentration of the target miRNA is determined based on the fluorescence intensity generated by the addition of SYBR-Green I (SG) into the 'product tube'. The proposed platform was applied to the detection of miRNA-122 in cell lysate samples and obtained quantitative results with accuracy comparable to the quantitative reverse transcription PCR method (qRT-PCR). It is worth pointing out that the proposed platform achieved a limit of detection value of 2.9 fM for miRNA-122 by a simple but effective LNA-assisted repeated fishing amplification strategy instead of complicated enzyme-based amplification techniques. It is reasonable to expect that the proposed method provides a competitive alternative for designing practically applicable, cost-effective and label-free miRNA detection methods.

Keywords: Hybridization chain reaction; Locked nucleic acid; MicroRNAs; Repeated fishing amplification strategy; Temperature-controlled dehybridization.