Enzyme-free and protein-assisted dual-amplified fluorescence anisotropy for sensitive miRNA detection in tumor cells.

We here report a double amplification strategy to construct a fluorescence anisotropy sensor for microRNA analysis in practical biological samples. In this strategy, one target can trigger cyclic catalyzed hairpin assembly (CHA), with streptavidin incorporated as an amplifier of molar mass to enhance the signal intensity. The proposed strategy has a good linearity in the range of 5 pM - 0.5 nM with a detection limit down to 2.3 pM. More importantly, by using fluorescence anisotropy as the signal output, the strategy can be used directly for detection of miRNA in practical samples without any tedious sample pretreatment, holding the practical value in real biological systems.

2'-O-Methyl molecular beacon: a promising molecular tool that permits elimination of sticky-end pairing and improvement of detection sensitivity.

An innovative 2'-O-methyl molecular beacon (MB) has been designed and prepared with improved thermal stability and unique nuclease resistance. The employment of 2'-O-methyl MBs helps efficiently suppress the background signal, while DNase I is responsible for the signal amplification and elimination of sticky-end pairing. The coupled use of 2'-O-methyl MBs and DNase I makes it possible to develop an enzyme-aided strategy for amplified detection of DNA targets in a sensitive and specific fashion. The analysis requires only mix-and-measure steps that can be accomplished within half an hour. The detection sensitivity is theoretically determined as 27.4 pM, which is nearly 200-fold better than that of the classic MB-based assay. This proposed sensing system also shows desired selectivity. All these features are of great importance for the design and application of MBs in biological, chemical, and biomedical fields.