Nanoscale assembly line composed of dual DNA-machines enabling sensitive microRNA detection using upconversion nanoparticles probes.

DNA machines are smart artificial devices that perform well-organized DNA hybridization reactions or nanoscale mechanical movements. Herein, a nanoscale assembly line composing of dual DNA machines is meticulously designed by coupling a catalytic hairpin assembly (CHA)-based machine with a 3D DNA walker machine. Equipped with upconversion nanoparticles (UCNPs) as signal tags, the dual DNA machines-based assembly line (DDMAL) can efficiently amplify the fluorescent signal of target recognition event, enabling sensitive detection of microRNA (miRNA). In detail, once activated by target miRNA-21, the CHA machine is initiated to constantly produce a single-stranded DNA (named binding DNA) via the strand displacement reaction. The binding DNA as a trigger factor can initiate the DNA walker machine by linking a walking strand DNA with an anchor strand DNA immobilized on the surface of magnetic beads (MBs). The movement of walking strand on the surface of MBs is then driven by Mn-dependent DNAzyme formed through the hybridization of walking strand with a UCNPs-linked substrate strand. The DNAzyme-catalyzed cleavage of substrate strand is accompanied by the release of numerous UCNPs from MBs. By measuring the fluorescent signal of released UCNPs after the magnetic separation, target miRNA-21 can be detected by the DDMAL system in a linear range from 1.0 fM to 10 nM, with a limit of detection (LOD) of 0.62 fM (3σ). Moreover, the practicability of DDMAL system was demonstrated by using it to evaluate the expression levels of miRNA-21 in cell lines and assay miRNA-21 in human serum.