MicroRNAs
(miRNAs) are found in extremely low concentrations in
cells, so highly sensitive quantitation is a great challenge. Herein,
a simple dual-amplification strategy involving target-activated catalytic
hairpin assembly (CHA) coupled with multiple fluorophores concentrated
on one X-shaped DNA is reported. In this strategy, four hairpin probes
(H1, H2, H3, and H4) are modified with FAM and BHQ1 at both sticky
ends, while a circulating hairpin probe (H0) is used to activate CHA
circuits once it binds to complementary sequences in the target miR-21
(T). The powerful dual-amplification cascades in Förster resonance
energy transfer (FRET)-based nonenzymatic nucleic acid circuits are
triggered by T–H0-activated formation of the X-shaped DNA nanostructure,
freeing T–H0 for the next CHA reaction cycle. CHA circuits
increase the fluorescence due to the wide distance between FAM and
BHQ1 in the formed X-shaped DNA nanostructure, resulting in signal
amplification and highly sensitive detection of miR-21, with a limit
of detection (LOD, 3σ) of 0.025 nM, which is 25.6 or 57.6 times
lower than that obtained through a single-amplification strategy without
multiple fluorophores on one X-shaped DNA or CHA circuit. Furthermore,
this cascade reaction was completed in 45 min, effectively avoiding
target degradation. This new enzyme-free signal amplification strategy
holds promising potential for sensitively detecting different DNA
or RNA sequences by simply adapting the fragment of the H0 sequence
complementary to the target.
Nonalcoholic
fatty liver disease (NAFLD) can progress gradually
to liver failure, early warning of which is critical for improving
the cure rate of NAFLD. In situ imaging and monitoring of overexpressed
miR-21 is an advanced strategy for NAFLD diagnosis. However, this
strategy usually suffers from the high background imaging in living
cells owing to the complexity of the biological system. To overcome
this problem, herein, we have developed a one-donor–two-acceptor
nanoprobe by assembling gold nanoparticles (AuNPs) coupled with BHQ2
(AuBHQ) and quantum dots (QDs) through DNA hybridization for imaging
of miR-21 in living cells. The fluorescence of QDs was quenched up
to 82.8% simultaneously by the AuNPs and the BHQ2 via nanometal surface
energy transfer and fluorescence resonance energy transfer, reducing
the background signals for target imaging. This low background fluorescent
nanoprobe was successfully applied for imaging the target miR-21 in
nonalcoholic fatty liver cells by catalyzing the disassembly of QDs
with the AuBHQ and the fluorescence recovery of QDs. In addition,
the sensitivity of this nanoprobe has also been enhanced toward detecting
miR-21 in the range of 2.0–15.0 nM with the detection limit
(LOD, 3σ) of 0.22 nM, which was 13.5 times lower than that without
BHQ2. The proposed approach provides a new way for early warning,
treatments, and prognosis of NAFLD.
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