Precision screening of DNAzymes and aptamers, and an ultrasensitive isothermal integrated biosensor for Cronobacter...

Cronobacter sakazakii (C. sakazakii), an opportunistic pathogenic bacterium resistant to adverse environments, can induce meningitis and sepsis in infants with high mortality, which necessitates ultra-sensitive detection. In this study, the core binding region was derived by tailoring existing C. sakazakii aptamer, and novel aptamer screening libraries were designed based on this core sequences with high affinity and specificity. Additionally, we performed the first screening of C. sakazakii DNAzyme and explored its binding sites with the aid of molecular docking. Based on the aptamer, DNAzyme, G-quadruplex, and RCA reaction, an ultra-sensitive visual C. sakazakii sensor was developed. This sensor achieves C. sakazakii enrichment via aptamer-magnetic bead conjunction and converts bacterial count to nucleic acid count signals through DNAzyme. Rolling circle amplification (RCA) generates massive G-quadruplexes for exponential signal amplification; the complex formed by G-quadruplex and hemin catalyzes TMB chromogenesis to complete the final visual signal output. Under optimized conditions, the sensor exhibits a linear relationship in the range of 9 × 10⁰ ∼ 9 × 10³ CFU/mL, with a linear equation of y = 0.2282LgC + 0.1957 (R²=0.9992) and a limit of detection (LOD) of 2 CFU/mL. In summary, this study developed an ultra-sensitive sensor based on multiple functional nucleic acids for C. sakazakii detection.