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The detection of pathogens in our food supply is of high importance to ensure the safety of consumers nationwide. Lacking are technologies that provide rapid and sensitive analysis of surfaces in food processing plants, which can be performed by non-trained personnel and are inexpensive. The proposed research focuses on the development of biosensors for this application. The main objective is the integration of biosensor and nanofiber technologies previously developed by Drs. Baeumner and Frey to design a highly sensitive biosensor in the format of a swab and the detection of the model analyte E. coli O157. Tasks to accomplish this include: <ol type="a">
<li> Electrospinning polylactic acid nanofibers presenting biotin on their surface and characterization of the biologically available biotin on the surface; <li> Immobilization of anti E. coli O157 antibodies on the fibers; <li> Investigating the uptake of bacteria from plastic surfaces into the fiber; <li> Detection of bacteria present using dye-encapsulating liposomes tagged with a second antibody specific for E. coli O157; <li> Optimization of the detection. </ol>In the end, it is expected that a novel biosensor for food pathogens is obtained that allows the analysis of surfaces such as table tops, packages, fruits and vegetables.
NON-TECHNICAL SUMMARY: The detection of pathogenic organisms in our food supply is of high importance to ensure the safety of consumers nationwide. However, we are lacking technologies that provide rapid and sensitive analysis of surfaces in food processing plants, which can also be carried out by non-trained personnel on a routine basis and are inexpensive. The proposed research focuses on the development of biosensors for this application. The integration of novel nanofiber and highly sensitive biosensor technologies will enable the development of a sensing swab in the format of a Q-tip. It will be simple to use, inexpensive, very rapid, and ideal for detecting pathogens present on surfaces such as table tops, conveyer belts, pipes, fruits and vegetables.
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APPROACH: Biosensors for the detection of pathogens have been developed previously in the format of a dip stick. Dye-encapsulating liposomes and conventional nitrocellulose membrane materials were used. Limits of detection in the fmol range of RNA and 1000 cells/sample were obtained. Electrospun nanofibers provide an enormous surface to volume ratio, enable the presentation of biotin on the surface, and thus a fast and efficient possibility for antibody immobilization via streptavidin bridging. Thus, combining these two technologies, lower limits of detection will be achievable that are crucial for the desired application field. Nanofibers will be prepared using electrospinning of polylactic acid (PLA) with biotin in the spinning dope. This process has been developed previously by Prof. Frey. Non-woven mats of the nanofiber will be cut into strips in the Baeumner lab and anti- E. coli O157 antibodies will be immobilized. Liposomes will be synthesized entrapping sulforhodamine B. The liposomes will be tagged with a second antibody directed against the pathogen. Subsequently, the strips will be used in a dip stick assay determining limits of detection for E. coli O157. Blocking reagents such as gelatin, bovine serum albumin etc. needed to avoid non-specific binding of liposomes to the PLA fibers will be determined. Finally, swabs in the format of a Q-tip will be prepared of the PLA, antibodies immobilized and surfaces seeded with E. coli O157 will be analyzed. The need for moisture, effect of surface contaminants and a limit of detection in the swab format will be determined.