Integration of click ligation chain reaction with split DNAzyme for enzyme-free cascade amplification and ultrasensitive nucleic acid detection

Nucleic acid-templated click ligation offers a powerful enzyme-free amplification route for nucleic acid detection with high specificity and simplicity. However, achieving rapid, low-cost, portable, and ultrasensitive detection remains challenging. Herein, we develop an enzyme-free cascade amplification system, nucleic acid-templated split DNAzyme click ligation chain reaction (NA-SpDzyme-CLCR), by rationally integrating Cu₂O-catalyzed nucleic acid-templated click ligation chain reaction with a split Mg^{2 +} -dependent DNAzyme for ultrasensitive nucleic acid detection. In this system, target-triggered ligation products undergo covalent reassembly to form catalytically active DNAzymes, which exhibit robust recycling cleavage with high single turnover ( k _obs.s = 0.94 ± 0.07 min⁻¹) and multiple turnover ( k _obs.m = 0.37 ± 0.02 min⁻¹) numbers, enabling efficient secondary signal amplification. As a proof of concept, we established fluorescence and lateral flow strip assays using the NA-SpDzyme-CLCR system, achieving detection limits of 0.3 aM (fluorescence) and 2 aM (visual), while exhibiting excellent specificity in discriminating single-base mismatches and reliable performance in complex biological matrices. Furthermore, these assays enable versatile detection of the target of interest by customizing the probe recognition sequences. Benefiting from its enzyme-free, high sensitivity, versatility, and compatibility, the NA-SpDzyme-CLCR provides a robust platform for low-abundance biomarker detection and holds great promise for point-of-care diagnostics.