Two-pot ready-to-use reagents achieved quadruple-signal amplification for the ultra-sensitive biosensing of Salmonella Typhimurium in foods.

The achievement of both ultra-sensitivity and operational simplicity remains a major challenge in the development of nanozymatic biosensors for foodborne pathogen detection. Here, we report a two-pot, ready-to-use nanozymatic colorimetric biosensor that integrates a cascade-based quadruple signal amplification strategy for the rapid detection of Salmonella Typhimurium (S.T.) in foods. The biosensor deploys rolling circle amplification (RCA) to generate repetitive aptamer motifs and hybridization sites, which are densely anchored onto generation 6.5 poly(amidoamine) dendrimers (G6.5) and hybridized with gold nanoparticles (AuNPs) to form RCA-G6.5-AuNP nanozymes. This architecture enables the first dual amplification via multivalent target recognition and high-density AuNP loading. Upon recognition of S.T. via magnetic nanoparticle-assisted capture, the nanozymes catalyze glucose oxidation, generating HO and gluconic acid which synergistically decompose MnO nanosheets and inhibit 3,3',5,5'-tetramethylbenzidine (TMB) oxidation. This constitutes the second dual amplification through a catalytic cascade. Collectively, the quadruple amplification yields a 21-fold enhancement in detection sensitivity and a 3569-fold reduction in the detection limit (5 CFU/mL) compared to horseradish peroxidase-based systems. The entire assay is completed within 50 min using a two-pot reaction workflow, eliminating the need for enzymatic labelling, multi-step conjugation, or complex pretreatment and thus dramatically enhancing operational simplicity and reducing contamination risk. The biosensor exhibits a broad dynamic range (10-10 CFU/mL), high specificity, and robust performance in real food samples, with recovery rates of 93.3%-107.3% and RSD < 9.85% in milk and beef. This work offers a sensitive and practical biosensing platform, bridging high-performance detection with real-world usability for food safety monitoring.