AND-gated DNAzyme circuits with hybridization chain reaction amplification for high-fidelity profiling of dual miRNA biomarkers

DNA logic-gate technology has significantly advanced cancer diagnostics by enhancing detection specificity through programmable molecular recognition. However, conventional implementations face critical limitations including inadequate sensitivity from dependence on nucleic acid strand displacement reactions without intrinsic amplification, alongside operational complexity imposed by enzymatic dependencies and diverse reagent requirements. To overcome these constraints, we developed an enzyme-free Logic-gated Cascade with Multi-DNAzyme Activation (LCMDA). This system employs an AND-gated architecture where target miRNAs such as miRNA-141 and miRNA-375 convert into specific DNAzymes. These DNAzymes then cleave substrates to release initiators for hybridization chain reaction, whose products self-assemble into catalytically active multi-component deoxyribozymes that drive secondary signal amplification. The platform delivers three pivotal advantages through its enzyme-free AND-gated architecture. First, cascaded signal amplification enables ultrahigh sensitivity, achieving detection limits of 4.11 fM for miRNA-141 and 7.97 fM for miRNA-375. Second, complete elimination of proteolytic enzymes reduces costs while enhancing operational stability. Third, room-temperature operation facilitates point-of-care deployment without compromising performance. Validated in prostate cancer biomarker analysis, LCMDA achieves single-base mismatch discrimination and demonstrates near-quantitative recovery rates between 102.23 % and 95.04 % in human serum samples. Its modular architecture permits straightforward adaptation to diverse miRNA targets through simple reconfiguration of binding domains, establishing a versatile tool for precision oncology research and clinical diagnostics.