The overall goal of this project is to develop rapid detection techniques for pathogens, toxins and bioactive substances using nanotechnology. Particular emphasis will be given to develop a nanorod-based biosensor by using Surface Enhanced Raman Spectroscopy to detect pathogenic bacteria. Focuses will also be given to determine a method for toxins and bioactive substances that may cause foodborne illness or benefit human health. <P> Specific objectives are to: <OL> <LI> Develop nanorod based substrates using OAD for rapid detection of bacteria using E. coli O157:H7 as a model<LI> Distinguish different strains of bacteria by using the nanorord substrate-based probe and a fiber Raman spectroscopy<LI> Apply the sensor to detect pathogens, toxins and bioactive substances in various foods system. </ol> Outputs include: <OL> <LI> Develop a portable sensor with rapid, reproducible and reliable results for detection of pathogens, toxins and bioactive substance<LI> Publish results in scientific journal and present at national or international conferences<LI> Prototypes will be technologically trasferred to the industry.
NON-TECHNICAL SUMMARY: Escherichia coli O157:H7 was first recognized as a cause of illness in 1982 during an outbreak of severe bloody diarrhea and is one of hundreds of serotypes of Escherichia coli. Although most strains are harmless and live in the intestines of healthy humans and animals, strains of E. coli O157:H7 produce powerful toxins and can cause severe illness. In United States, an estimated 73,000 cases of infection, 2100 hospitalizations and 61 deaths occur each year. Infection often leads to bloody diarrhea, and occasionally to kidney failure. Most cases of E. coli O157:H7 infections have been associated with eating undercooked, contaminated ground beef. Person-to-person contact in families and child care centers are also important modes of transmission. Infection can also occur after drinking raw milk and after swimming in or drinking sewage-contaminated water. The conventional culture method recommended by the USDA for detection and identification of E. coli O157:H7 in meats requires three general steps: enrichment, colony isolation, and confirmation. Although it needs at least 4-5 days to confirm an isolate as E. coli O157:H7, conventional culture is the most sensitive detection methodology available, the standard from which other methods are evaluated. Conventional culture method apparently is time-consuming and laborious; therefore it is unsuitable for commercial and industrial applications. There are numerous commercial assays for E. coli O157:H7, many of these assays target the unique antigens, and therefore they are immunoassays and dependent on antibody-antigen recognition and interactions. Almost all of them proceed from the initial step of enrichment and have shortened the time of analysis considerably compared to conventional culture isolation and identification methods. In recent year, application of nanotechnology in detection devices has been advancing. Nanoparticle has ability to amplify the optical signals through an effect known as surface-enhanced Raman scattering (SERS). We plan to develop a sensor to detect pathogen using E. coli O157:H7 as an example. In addition, we also plan to use the sensor developed to detect toxins that may be produced by bacteria or other sources. The technology will also be applied to detect bioactive substances which may affect public health. We are going to develop a rapid technique for pathogen, toxin and bioactive substance detection using nanotechnology. Particular emphasis will be given to develop a nanorod-based biosensor by Surface Enhanced Raman Spectroscopy to detect foodborne pathogenic bacteria. Focuses will also be given to determine a method for detection of toxins that cause foodborne illness or bioactive substances that may affect public health. This research project will increase the efficiencies of the pathogens, toxins and bioactive substances detection process. It will result in rapid real-time detection so that will be able adapted to monitor the critical control point of the HACCP plan. It may also use for quick detection for antibioterrorism scheme.<P>APPROACH: (A) The scientific methodology includes (1) Fabrication of Silver Nanorod Arrayby the oblique angle deposition (OAD) method using a custom-designed electron-beam/sputtering evaporation system. The source material for evaporation is Ag pellets obtained from Alfa Aesar. Throughout the deposition, the overall thickness of the metal deposited is monitored by a quartz crystal microbalance positioned at normal incidence to the vapor source. 500 nm Ag film first deposited onto the glass slides. The vapor incident angle was tuned to 86 degree, and deposited 2000 nm silver nanorod. (2) SERS spectra are acquired using a HRC-10HT Raman analyzer system (Enware Optronics Inc. Irvine, CA). Fiber-optic coupled interfaced to a CCD-equipped spectrograph (6/cm resolution). A fiber-optic interfaced 785 nm near-IR diode laser is used to provide the incoming radiation. The incident laser light is focused to a 1 mm spot size on the substrate at normal incidence. All spectra are acquired with a 10s integration. (3) SERS Molecular Probe used in this study is trans-1, 2-bis (4-pyridyl) ethene (BPE, Aldrich) and BPE solutions are prepared by sequential dilution of HPLC grade methanol. 2 microliter of BPE solution is applied onto silver nanorod substrate and allow it to dry before the measurement of spectra. (4) E. coli O157:H7 (ATCC 43888) used as target bacteria. Bacterial populations are determined by the conventional surface plating-count method. Other pathogenic bacteria such as Salmonella spp. and Staphylococcus aureus in different strains will be included in the projects. (5) Detection of E. coli O157:H7 in Food System including meat, milk and apple juice. 25 grams of ground beef purchased from retail markets are placed in a sterile stomach bag and 225 ml sterile 0.1 percent peptone water are added before shaking for 3 min in a Stomacher. Uninoculated apple juice, milk and ground beef are used as controls and the presence of indigenous E. coli O157:H7 are examined. Spiking is done with the addition of culture E. coli O157:H7 solution of pre-determined counts. Comparison of uninoculated and spiked samples will be made to evaluate the practicality of using SERS to detect pathogen in a complex food system. The methodology developed will also be applied to other pathogenic bacteria. (6) Surface-enhanced Raman Spectroscopy provides a higher local optical field, due to redistribution and concentration of electromagnetic energy. Coupled with the incoming electric field from the incident radiation, this generates a larger spectroscopic signal for an analyte caught in both fields. (B) The effect results of the project will be evaluated by using criteria at planned time frame. Deliverables at each milstone dates will be used for evaluation in the progress reaport annually. The publications of each year's goal will be used to evaluate the impact on the intended audiences.