DNAzyme shows great promise in designing a highly sensitive and specific sensing platform; however, the low cellular uptake efficiency, instability, and especially the insufficient cofactor supply inhibit the intracellular molecule sensor applications. Herein, we demonstrate a novel type of DNAzyme-based self-driven intracellular sensor for microRNA (miRNA) detection in living cells. The sensor consists of a metal–organic framework [zeolite imidazole framework (ZIF-8)] core loaded with a shell consisting of a rationally designed DNAzyme, where the substrate strand is modified with FAM and BHQ-1 nearby both the sides of the restriction site, respectively, while the enzyme strand consists of two separate strands with a complementary fragment to the substrate strand and the targeting miRNA, respectively. The ZIF-8 nanoparticles enable the efficient delivery of DNAzyme into the cell and protect the DNAzyme from degradation. The pH-responsive ZIF-8 degradation is accompanied with the release of the DNAzyme and Zn2+ cofactors, and the intracellular target miRNAs recognize and activate the DNAzyme driven by the Zn2+ cofactors to cleave the substrate strand, resulting in the release of the FAM-labeled shorter product strand and increased fluorescence for miRNA detection. The self-driven approach can be generally applied to various miRNAs’ detection through DNAzyme engineering.

Since their first discovery in 1994, DNAzymes have been extensively applied in biosensing and therapy that act as recognition elements and signal generators with the outstanding properties of good stability, simple synthesis, and high sensitivity. One subset, RNA‐cleaving DNAzymes, is widely employed for diverse applications, including as reporters capable of transmitting detectable signals. In this review, the recent advances of RNA‐cleaving DNAzyme‐based amplification strategies in scaled‐up biosensing are focused, the application in diagnosis and disease treatment are also discussed. Two major types of RNA‐cleaving DNAzyme‐based amplification strategies are highlighted, namely direct response amplification strategies and combinational response amplification strategies. The direct response amplification strategies refer to those based on novel designed single‐stranded DNAzyme, and the combinational response amplification strategies mainly include two‐part assembled DNAzyme, cascade reactions, CHA/HCR/RCA, DNA walker, CRISPR‐Cas12a and aptamer. Finally, the current status of DNAzymes, the challenges, and the prospects of DNAzyme‐based biosensors are presented.