Sandwich capture ultrasensitive sensor based on biohybrid interface for the detection of Cronobacter sakazakii

A simple, rapid and ultrasensitive visual sensing method for the detection of Cronobacter sakazakii (C. sakazakii) based on a biohybrid interface was established. During the entire sensing process, quadruple-cascade amplification showed its superior sensing performance. First, the prepared immunomagnetic beads (IMB) were used to isolate and enrich specific targets from the food matrix. After adding the fusion aptamer, the aptamer sequence specifically recognized the target and formed the immune sandwich structure of antibody-target-fusion aptamer. In addition, the fusion aptamer also included the template sequence of exponential amplification reaction (EXPAR), which contained the antisense sequence of the G-rich sequence. Therefore, a large number of G-rich sequences can be generated after EXPAR can be triggered in the presence of Bst. DNA polymerase, nicking endonuclease, cDNA, and dNTP. They were self-assembled into G-quadruplex structures and then combined with hemin to form G4/hemin DNAzyme, resulting in visible coloration and measuring absorbance at 450 nm for quantitative detection. The assay showed a limit of detection (LOD) of 2 CFU/mL in pure culture and 12 CFU/g in milk powder in optimal conditions. This method provides a promising strategy for rapid and point-of-care testing (POCT) since it does not require DNA extraction, medium culturing, and expensive instrumentation. KEY POINTS: •Single-cell level detection of C. sakazakii with ultrasensitive and rapidness •The fusion aptamer integrated recognition and amplification •Sensing analysis of C. sakazakii based on cascade amplification of biohybrid interface.