Multicomponent nucleic acid enzymes as signal amplification strategy for the detection of microRNA based on fluorescence resonance energy transfer

TL;DR

A new method using nucleic acid enzymes and fluorescence transfer detects microRNA in milk, which could help diagnose bovine mastitis.

Contribution

A novel amplification strategy using multicomponent nucleic acid enzymes and FRET for highly sensitive miRNA detection.

Findings

The method achieved a detection limit of 2.3 fM for miR146a, a highly sensitive result.

The technique can distinguish miR146a from similar miRNAs with single base mismatches.

Successful detection of miR146a was demonstrated in raw milk samples.

Abstract

A novel and simple methodology is introduced that allows accurate and highly sensitive detection of microRNAs (miRNAs), taking advantage of an amplification strategy based on multicomponent nucleic acid enzymes (MNAzymes), combined with a fluorescence resonance energy transfer (FRET) phenomenon. For this purpose, a fluorescent dye (FAM) has been selected as an energy donor, while gold nanoparticles (AuNPs) are employed as energy acceptors, located close to each other through hybridisation with the substrate. The research object was miR146a, which is a biomarker whose overexpression in milk is associated with inflammation in bovine mammary glands caused by bovine mastitis. The presence of a genetic target activates the MNAzyme cleavage capability, splitting the substrate into two parts. Hence, the presence of the target increases the distance between donor and acceptor, recovering the quenched fluorescence. Experimental parameters have been optimised, achieving a limit of detection (LOD) of only 2.3 fM (highly competitive as compared to other similar approaches) and a wide linear response range between 15.9 fM and 10 nM. In addition, the proposed methodology allows discriminating miR146a from other similar miRNAs differing in a single base mismatch. Detection of miR146a has been successfully carried out in spiked raw milk samples.

The online version contains supplementary material available at 10.1007/s00604-025-07002-6.

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