A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

Abstract

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H2O2) and H2O2-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H2O2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H2O2, as well as the incubation time between H2O2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 102 CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 103 CFU/mL to 1.18 × 106 CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control.