Despite the progress on the analysis of miRNA either in vitro or in vivo, intracellular imaging of lowly expressed microRNA remains a challenge. Here we develop a novel dual-enzyme-propelled DNA walking nanomachine, which is tailored to accomplish this mission. The nanomachine is constructed with nanoparticles-loaded DNA tracks, on which the targeted miRNA working as a single-foot DNA walker can move autonomously under the catalysis of two cooperative enzymes. Cleavage of the DNA tracks like a "burnt-bridge" mechanism is thereafter triggered, resulting in an amplified fluorescent signal. After the comprehensive study and optimization of the DNA nanomachine, miR-892b, a significantly down-regulated miRNA in breast cancer cells, is selected as a model target. Sensitivity detection in vitro is achieved with a superior detection limit of 4 pM. While being delivered into cells, the DNA nanomachine is available for the imaging of the lowly expressed microRNA, which is totally missing using the conventional fluorescence in situ hybridization (FISH) method. Up-regulation or down-regulation of the miRNA by exogenous regulatory factors can be also well evaluated. This DNA nanomachine provides a competitive approach for the analysis of miRNA, and has the potential to be extended to some other biomolecules.

Nanozymes have received great attention owing to the advantages of easy preparation and low cost. Unlike natural enzymes that readily adapt to physiological environments, artificial nanozymes are apt to passivate in complex clinical samples (e.g., serum), which may damage the catalytic capability and consequently limit the application in biomedical analysis. To conquer this problem, in this study, we fabricated novel nanozyme@DNA hydrogel architecture by incorporating nanozymes into a pure DNA hydrogel. Gold nanoparticles (AuNPs) were adopted as a model nanozyme. Results indicate that AuNPs incorporated in the DNA hydrogel retain their catalytic capability in serum as they are protected by the hydrogel, whereas AuNPs alone totally lose the catalytic capability in serum. The detection of hydrogen peroxide and glucose in serum based on the catalysis of the AuNPs@DNA hydrogel was achieved. The detection limit of each reaches 1.7 and 38 μM, respectively, which is equal to the value obtained using natural enzymes. Besides the mechanisms, some other advantages, such as recyclability and availability, have also been explored. This nanozyme@DNA hydrogel architecture may have a great potential for the utilization of nanozymes as well as the application of nanozymes for biomedical analysis in complex physiological samples.