The Trend of Nonenzymatic Nucleic Acid Amplification: Strategies and Diagnostic Application

Nonenzymatic nucleic acid amplification reactions, especially nonenzymatic DNA amplification reactions (NDARs), are thermodynamically driven processes that operate without enzymes, relying on toehold-mediated strand displacement (TMSD) and branch migration. With their sensitive and efficient signal amplification capabilities, NDARs have become essential tools for biomarker detection and intracellular imaging. They encompass four primary amplification methods: catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), DNAzyme-based amplification, and entropy-driven circuits (EDC). Based on amplification mechanisms, NDARs can be categorized into three types: stimuli-responsive NDARs, which employ single amplification strategies triggered by specific stimuli like pH, light, or biomolecules; cascade NDARs, which integrate two or more amplification reactions for stepwise signal enhancement; and autocatalytic NDARs, which achieve exponential amplification through self-sustained cycling. These advanced designs progressively improve amplification efficiency, enhance sensitivity, and minimize background noise, enabling precise detection of proteins, viruses, and nucleic acids as well as applications in cancer cell imaging and therapy. Compared with classical NDARs, these approaches significantly reduce signal leakage, offering broader applicability in diagnostics, imaging, and therapeutic contexts. This review summarizes recent advancements, addresses existing challenges, and explores future directions, providing insights into the development and applications of NDARs.