Development of a novel FRET-based biosensor for detection of foodborne pathogens

Salmonella or Listeria contamination in food products results in not only foodborne outbreaks, but also a large economical burden for the food industry due to product recalls. Traditional testing methods for pathogens are relatively costly and time-consuming. Therefore, rapid, accurate, and real-time methods for detecting pathogens in food processing facilities are urgently needed.;The objective of this research was to develop and demonstrate the feasibility of a fluorescence resonance energy transfer (FRET) biosensor technique to detect Salmonella and Listeria in homogenized pork and to implement the technique into an on-line instrumental pathogen detection system.;Salmonella or Listeria antibodies were labeled with FRET donor fluorophores (Alexa Fluor 546) while protein A or G was labeled with the acceptor fluorophores (Alexa Fluor 594). The labeled antibody-protein A or G complex was formed via incubation of the labeled antibody with protein A or G. The labeled antibody-protein A or G complex was tested in phosphate buffered solution and specific and non-specific antigens were added to the solution. Changes in fluorescence were monitored by a spectrofluorometer. For the in-solution tests, the optimal acceptor/donor fluorophore (A/D) ratios were 0.2 and 1.0 for Listeria and Salmonella, respectively, and both Listeria and Salmonella antigen detection limits were 2.0mug/ml. These results indicated the potential of the FRET technique to detect pathogens.;To improve sensitivity, the antibody/protein complexes were immobilized onto the surface of silica optical fibers. The polymeric cladding was removed from the silica core along the distal end of the 600 micron core fiber, and then the labeled antibodies-protein G complexes were immobilized on the decladded and tapered core to form the probe sensing region via a Silanization method. Different packing densities were examined and it was determined that a packing density of 0.033 mg/ml produced the highest energy transfer. The fiber optic sensors were interfaced to a Jasco spectrofluorometer. The fiber tips with the immobilized antibody-Protein G complex were placed in a PBS solution containing Salmonella Typhimurium (0 to 106 cells/ml) or L. monocytogenes (0 to 106 cells/ml) for 5 min at each concentration. Then the sensor response was recorded. A limit of detection of 102 and 103 cells/ml were obtained for S. Typhimurium and L. monocytogenes, respectively. Additionally, the fiber optic sensors were interfaced to the custom-built benchtop fluorometer. The tips of the fibers were placed in homogenized pork sample solutions spiked with S. Typhimurium and L. monocytogenes. A limit of detection of the sensor exposed to S. Typhimurium and L. monocytogenes was 10 5 and 106 CFU/g respectively. The results of this work demonstrated the feasibility of a FRET-based evanescent wave biosensor for the detection of foodborne pathogens. In addition, the results suggest that this on-line instrumental detection system will provide food processors and producers the ability to detect food safety problems, thus reducing the incidents of food pathogen outbreaks and recapturing lost dollars due to inadequate food safety.