Rapid Detection of Foodborne Pathogenic Bacteria by Surface Enhanced Raman Spectroscopy using AG Nanorod Array Substrates

NON-TECHNICAL SUMMARY: The potential risk for deliberate contamination of the environment, food and agricultural products has recently increased due to the global war on terrorism, making biosensing an important issue for several federal agencies. The current trend is to decentralize large stationary laboratory facilities such that tests can be performed anywhere and under field conditions. Consequently, the development of portable, rapid and sensitive biosensors with on-the-spot interpretation of results is gaining momentum. From a food safety point of view, real-time microbial detection and source identification are becoming increasingly important due to the growing consumer concerns over foodborne disease outbreaks and economic loss from the outbreaks. The conventional culture method recommended by the USDA for detection and identification of foodborne pathogens usually requires three general steps: enrichment, colony isolation, and confirmation. Other methods including polymerase chain reaction (PCR), antibody-based systems and mass spectrometry have been developed as a diagnostic tool to detect pathogens; however, these approaches have fundamental restrictions that limit the use outside of a laboratory. False negative/ false positive identification with PCR method1, the multi-steps, chemical reagents required for procedures for immunoassay, and the expensive, non-portable mass spectrometry, make these methods neither fast nor robust enough for field detection. The research directions for improvement of analytical methods obviously falls on 1) the reduction or elimination of the sample preparation procedure, 2) continual and routine analysis of large numbers of samples with minimum reagent usage and cost, 3) ease to operate under most conditions, and 4) short data accumulation time. An alternative approach that satisfies most of the above requirements is spectroscopic techniques that are specific, noninvasive, nondestructive, and can be rapidly performed. Through the results of this proposal, we expect to achieve the detection of foodborne bacteria on food quickly and accurately with nanorod-based SERS techniques.

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APPROACH: General methods as follows will be performed throughout the entire experiment to accomplish objectives of the proposal. (1). Fabrication of SERS Substrates: The SERS active substrate used to obtain spectra will be silver nanorod arrays fabricated by OAD technique using a custom-designed electron beam/sputtering evaporation (E-beam) system (Torr International, New Windsor, NY). (2). Bacterial Sample Preparation: The following bacteria will be used in the analyses: Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus and Staphylococcus epidermidis. Bacterial cells will be grown in trypticase soy broth (TSB, Difco, Detroit, MI) over night at 37 degree C with 240 rpm shaking. Following incubation, the cultures will be washed three times with sterilized deionized (DI) water before re-suspending in DI water. Desired dilutions will be made in sterilized DI water. Two different types of mixed cell cultures will be prepared by mixing equal amount of E. coli O157:H7 (108 CFU/mL) and Staphylococcus aureus (108 CFU/mL); also equal amount of E. coli O157:H7 (108 CFU/mL) and Salmonella typhimurium 1925-1(108 CFU/mL). (3). SERS Measurements: SERS spectra will be acquired using a HRC-10HT Raman analyzer system (Enware Optronics Inc. Irvine, CA) consisting a diode laser, spectrometer, integrated Raman probe head for both excitation and collection, and separate delivery and collection fibers. The excitation source will be a frequency stabilized, narrow linewidth near IR diode laser with a wavelength of 785 nm. SERS spectra will be collected from multiple spots across the substrate and from multiple substrates. Generally, these solution spot should be 2 mm in diameter. If the concentrations of a culture solution containing ~108 CFU/ml, then there will be roughly 500 cells on the laser spot. (4). Data Analysis: Enwave Raman analyzer software (Enware Optronics Inc. Irvine, CA) will be used for instrument control and data collection. Principle component analysis (PCA) will be carried out by Unscrambler version 9.7 (Camo, AS, Norway). Prior to PCA analysis, each SERS spectrum will be smoothed using the Savitsky-Golay method with first derivative, a second order polynomial and nine-point smoothing. Spectra will be normalized with respect to it most intense peak. The methods used will develop a SERS technique to quickly and accurately detect the bacteria agents on food and to further validate the SERS detection technique with real samples in terms of background interference testings and model and real food systems.