Integrating Bacteriophage onto the Magnetic Nanozyme for Effective Enrichment and Colorimetric Detection of Cronobacter sakazakii in Powdered Infant Formula

<i>Cronobacter sakazakii</i> is a dangerous pathogen easily found in powdered infant formula (PIF), causing severe infections and even death in infants. Herein, a bacteriophage-immobilized magnetic nanozyme (Fe₃O₄@EspYZU13) was prepared for <i>C. sakazakii</i> detection. Bacteriophage EspYZU13 isolated and identified by our group exhibits specific lytic capacity. Fe₃O₄@EspYZU13 possesses remarkable enrichment capacity towards <i>C. sakazakii</i>, efficiently enriching different concentrations of <i>C. sakazakii</i> from a mixed bacterial solution. Furthermore, Fe₃O₄@EspYZU13 shows peroxidase-like activity, which can catalyze the 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reaction in the presence of H₂O₂. Upon introduction of <i>C. sakazakii</i>, it can be specifically captured by Fe₃O₄@EspYZU13, inhibiting its peroxidase-like activity. Based on it, <i>C. sakazakii</i> ranging from 3.2 × 10¹ to 3.2 × 10⁷ CFU mL^-1 can be determined, offering a detection limit (LOD) of 26 CFU mL^-1. Moreover, this reaction system keeps high specificity towards <i>C. sakazakii</i>, which can resist interferences of other possible coexisting bacteria. <i>C. sakazakii</i> in the artificially contaminated PIF can be detected, offering good recoveries (96.76% to 103.13%). These results indicate that our proposed reaction system demonstrates its practical application potential for efficient enrichment of <i>C. sakazakii</i> from complex samples and accurate determination of <i>C. sakazakii</i> in PIF.