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In this study, an aptamer-functionalized magnetic bead-based fluorescence sensor for the detection of Cronobacter sakazakii in milk powder using hybridization chain reaction (HCR) amplification was constructed. First, the sequence HP, combining a trigger sequence and an aptamer sequence which complement each other to form a stable secondary structure, and the hairpin sequences H1 and H2 were cleverly designed. Then, aptamer-functionalized magnetic beads were prepared by pentanediol reaction and avidin-biotin reaction. C. sakazakii was incubated with the aptamer magnetic beads. The aptamer sequence in HP recognized the target, causing conformational change of HP to expose its trigger sequence. The chain assembly of H1 and H2 was triggered by HCR to produce long double-stranded DNA, and the fluorescent dye SYBR Green I (SG) bound to the long double strands of HCR products by intercalation and slot binding. Finally, graphene oxide (GO) was added to adsorb free H1, H2 and SG on its surface via π-π stacking, so the fluorescence signal was quenched. However, the HCR products could not be adsorbed on the surface of GO, so SG binding to the HCR product emitted a strong fluorescence signal dependent on the target concentration, thus allowing quantitative detection of C. sakazakii. The detection limit of this method was 2 CFU/mL for pure culture and 8 CFU/g for milk powder. It gave results for milk powder samples in good agreement with those obtained by the traditional microbial culture method. This method has the advantages of no requirement for DNA extraction, fast operation, high stability, specificity and sensitivity, so it provides a potential method for the on-site rapid detection of C. sakazakii. |
