Inception-level signal amplification: Cascaded DNAzyme-Cas9 nickase achieves sub-nanomolar kanamycin tracking

Background Kanamycin's persistent contamination in agricultural products poses significant human health risks due to its nephrotoxicity and bioaccumulation via the food chain. Existing methods for on-site kanamycin monitoring lack sufficient sensitivity and portability, limiting their utility in field settings. The urgent need for rapid detection technologies remains unmet. This work addresses the critical gap in developing a field-deployable platform for ultrasensitive kanamycin residue screening. Results We engineered a fluorescence biosensor integrating DNAzyme-assisted Cas9 nickase-based amplification reaction (Cas9nAR). Kanamycin binding induces aptamer conformational changes, triggering Cas9nAR-driven cascade amplification that continuously generates DNAzymes. These cleave reporter probes to enable quantitative detection. The system achieved a broad linear range (1 nM – 5 μM) with an ultralow detection limit (0.3 nM), surpassing conventional methods. It exhibited high specificity against interfering antibiotics and delivered consistent recoveries (97% to 103%) in spiked water and milk samples. Following pretreatment, analysis can be completed within 120 min, validating its operational simplicity and robustness for complex matrices. Significance and novelty This work reports the first CRISPR-Cas9 nickase/DNAzyme cascade amplification platform for small-molecule detection, establishing a new paradigm that integrates programmable nucleic acid amplification with catalytic signal turnover. By using Cas9nAR-generated ssDNA as an in situ template for autonomous DNAzyme assembly, the biosensor achieves ultrasensitive, and homogeneous detection of kanamycin-addressing a critical gap in field-deployable antibiotic residue monitoring. The modular design offers a generalizable strategy for translating non-nucleic acid recognition events into amplified fluorescent outputs, with broad implications for point-of-need diagnostics in food safety and environmental analysis.