A Fluorescent Aptasensor Coupled with Nanobeads-based Immunomagnetic Separation for Simultaneous Detection of Four Foodborne Pathogens

Abstract

<abstract> <bold>Abstract.</bold> The objective of this study was to develop an aptasensor for rapid, sensitive, specific, quantitative, and simultaneous detection of multiple foodborne pathogens using magnetic nanobeads for separation and quantum dots (QDs) as fluorescence reporters. Salmonella Typhimurium, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus were used as four model pathogens. Streptavidin-coated 25 nm magnetic nanobeads (MNBs), conjugated with four corresponding biotin-labeled antibodies, respectively, were used to simultaneously capture and magnetically isolate four bacterial pathogens in 45 min. The magnetic separation was simulated using Comsol Multiphysics software. 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 MNBs-cell complexes simultaneously. The fluorescence intensities of the reporting QDs on the MNBs-cell-QDs complexes were measured with a portable spectrometer for quantitation of bacterial cells. SEM and confocal microscopy were used for characterization of the binding between nanobeads, QDs, and bacterial cells. Results showed the capture efficiencies of antibodies to E. coli, S. aureus, L. monocytogens, and S. Typhyimurium were 97, 95, 98, and 97%, while those of aptamers were 3, 54, 81, and 4%, respectively. The magnetic separation time of the MNBs-cell complex was calculated to be 44 min based on simulation results. The limit of detections for E. coli, S. aureus, L. monocytogens, and S. Typhyimurium were found to be 4, 6, 20, and 8 CFU/mL, respectively. The developed aptasensor was capable of simultaneously detecting four bacteria within 2.5 h in a broad range, having great potential in multiplex detection. Ongoing research focuses on the development of a prototype for detection of multiple foodborne pathogens in foods.