A Fluorescent Aptasensor Coupled with Nanobead-Based Immunomagnetic Separation for Simultaneous Detection of Four Foodborne Pathogenic Bacteria

Abstract

<abstract> <b><sc>Abstract.</sc></b> There is a growing need for rapid detection of multiple foodborne pathogens. The objective of this study was to develop an aptasensor for rapid, sensitive, specific, quantitative, and simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus, Listeria monocytogenes, and Salmonella Typhimurium in food using magnetic nanobeads (MNBs) for separation and quantum dots (QDs) as fluorescence reporters. Streptavidin-coated 25 nm MNBs, conjugated with four corresponding biotin-labeled antibodies, respectively, were used to simultaneously capture and magnetically separate four bacterial pathogens from the food matrix in 45 min. Streptavidin-coated QDs with emission wavelengths of 528, 572, 621, and 668 nm, conjugated with four corresponding biotin-labeled aptamers, were used to label the separated MNB-cell complexes. The fluorescence intensities of all reporting QDs in the MNB-cell-QD complexes were measured simultaneously with a portable spectrometer for quantitation of four different types of bacterial cells. SEM and confocal microscopy were used for characterization of the binding between nanobeads, QDs, and bacterial cells, and a simulation model was used to analyze the magnetic separation. Results showed that the capture efficiencies of antibodies with 25 nm MNBs were 90.4%, 87.5%, 92.0%, and 92.0% for E. coli O157:H7, S. aureus, L. monocytogenes, and S. Typhimurium, respectively. The limits of detection for E. coli O157:H7, S. aureus, L. monocytogenes, and S. Typhimurium were 80, 100, 47, and 160 CFU mL^-1, respectively, in pure culture and 320, 350, 110, and 750 CFU mL^-1, respectively, in ground beef. The developed aptasensor was capable of simultaneously detecting four bacteria within 2.5 h in a broad range of 10¹ to 10⁴ CFU mL^-1, showing great potential for multiplex detection of other foodborne pathogens.