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The goal of this project is to use the Salmonella typhimurium and E. coli O157:H7 as a model for Raman spectroscopic detection in foods. The major uniqueness of the proposed detection scheme lie in the fact that Raman detection, fluorescence immunoassay, and topographic imaging can be performed sequentially in a short period of time, resulting in rapidly providing multi-faceted information about the bacteria. The ultimate goal of our research is to devise a detection scheme at the level of single bacterium.
Non-Technical Summary: High sensitivity of rapid detection methods for food inductry and gobernmental regulatory agencies is not available. The purpose of the study is to increase the sensitivity for rapid detection of foodborne pathogens using Raman/fluorescence biosensing. <P> Approach: Near-field scanning optical microscopy (NSOM) is a scanning probe microscopy (SPM) technique which utilizes near-field optics to enhance spatial resolution in optical imaging and spectroscopy. By using near-field optics, spatial resolutions beyond the diffraction limit can be achieved. In the proposed work, we will use the cantelivered optical fiber coated with silver nanoparticle (or islands) as an apertured-NSOM probe. The silver nanoparticles and islands will be formed via chemical method and evaporation, respectively. For apertureless NSOM work, we will use Ag metallic tip and Ag-nanoparticle attached cantilevered nanopipette (manufactured by Nanonics). The bacteria will be bound onto a substrate through the antibodies. The surface of the bacterium will be covered with fluorescein-conjugated antibody for fluorescence immunoassay (imaging). Raman identification of the specific bacterium will be confirmed when the fluorescent light from the fluorescein-conjugated antibody is detected. The sensor platform will be prepared from microscope cover glass (0.17 mm thickness) by cutting it into 5 mm squares. These squares will be ultrasonically cleaned in deionized (DI) water and then in 95% ethanol. The cleaned glass will be coated with gold at 140 nm thickness using the sputtering machine. The 100 micro l of 1B4 monoclonal antibody at 5 micro g/ml in phosphate buffered saline (PBS) will be immobilized onto the gold coated sensor platform for 1 hr at room temperature, and then the unbound antibodies will be removed by washing with PBS three times. The uncoated areas of the sensor platform will be blocked with 1% bovine serum albumin (BSA) for 30 min, followed by washing with PBS three times. The prepared biosensors will be dried by a stream of argon and then used immediately or stored for up to 60 days at -20oC. Upon use, the biosensor will be placed in 100 micro l of properly diluted bacterial suspension at room temperature for 1 hr, and then washed with DI water four times. The anti-Salmonella antibody (1B4) and polyclonal antibody (S48) have been developed by the Food Microbiology Laboratory at Auburn University. The specificity of the 1B4 and S48 has been tested with 12 different bacterial strains by indirect ELISA. The 1B4 shows a high specificity that only reacts with Salmonella enterica Typhimurium and S. paratyphi; the S48 reacts with S. enterica Typhimurium, Paratyphi, Enteritidis, Mission, and Montevideo. However, the S48 has cross-reactivity with Staphylococcus aureus and E. coli O157:H7, but the reactivity is very low. The capture of S. enterica Typhimurium by the 1B4 immobilized gold surface has also been tested, and the capture efficiency is very high. The detection level can be as low as 100 cfu/ml. Due to the high specificity, 1B4 will be immobilized onto the biosensor platform to capture the target bacterium (Salmonella enterica Typhimurium) in this study, subsequently the fluorescein conjugated S48 will be applied to bind the captured bacteria for detection.