A Novel Steady-state Chemiluminescent Aptasensor using Rolling Circle Amplification Products DNA Hydrogel for Rapid and Ultra-sensitive Detection of Salmonella Typhimurium in Chicken

Salmonella typhimurium ( S.typhimurium ) is one of the common foodborne pathogens, posing a serious global food safety risks. It is thus important to develop a rapid and sensitive detection method for S.typhimurium . In this study, a steady-state chemiluminescent DNA hydrogel aptasensor was constructed for ultrasensitive detection of S.typhimurium in chicken. The sensor exploited the dense network of rolling circle amplification products (RCPs) DNA hydrogel to restrict luminol diffusion, thereby generating a stable chemiluminescent signal. A dual rolling circle amplification (DRCA) strategy was employed to accelerate hydrogel formation within 33 min, which increased RCP yield and strengthened intermolecular interactions. In the presence of S.typhimurium , the aptamers specifically binds to the bacteria, leading to the dissociation of the aptamer–primer complex and the subsequent release of primers that initiate two rolling circle amplification (RCA) reactions on partially complementary circular templates. When mixing the two parallel RCA products, the long ssDNA strands instantaneously assembled into hydrogels through complementary base pairing and physical entanglement, subsequently incorporating luminol for quantitative analysis. The chemiluminescence intensity exhibited a concentration-dependent enhancement with S.typhimurium , displaying a well-defined linear response from 10 to 7.6 × 10⁸ CFU/mL under optimal conditions, with a limit of detection of 8 CFU/mL. The assay also exhibited reliable specificity and stability in chicken samples, supporting its potential application in food safety monitoring.