Highly sensitive FRET-based miRNA assay using target-catalyzed hydrogel formation

Abstract

Enzyme-free target-catalyzed signal amplification which can overcome the limitations of the existing technologies has been recently attracting attention with the goal of rapid and early diagnosis of disease-derived target nucleic acid. In particular, studies have been reported applying this to the amplification of a quantum dot (QD)-based FRET signal. However, most of these are toxic cadmium QDs, which have a significant limitation that they cannot be applied to clinical diagnosis. In this study, the multivalent, non-toxic FRET donor was prepared using a streptavidin-biotin coupling, and the hydrogel network was formed by applying an enzyme-free target-catalyzed DNA nanostructure amplification method. It showed 3 nM of limit of detection (LOD) for miR-133A-1, a biomarker of acute coronary syndrome. In addition, to solve the false positive problem, which is essential for highly sensitive detection, the blocker DNA strategy using DNA base complementary binding and the strategy of removing overhang of amplified DNA nanostructure were used. As a result, the FRET signal that can be distinguished from a target negative sample was obtained. This is a proof-of-concept study using non-toxic FRET donor for miRNA detection based on enzyme-free amplification of DNA nanostructure. The approaches discussed in this study are expected to help develop a non-toxic target nucleic acid detection system that replaces cadmium QD.