<p>The objective of this program is to develop, demonstrate and field test an inexpensive, accurate, easy to use biosensor for the detection of Salmonella contamination of fresh globe fruits (tomatoes, cantaloupes and watermelons). This biosensor can be used in the field to identify critical hazard points, i.e. when and where bacterial contamination enters the system. With this biosensor technology, the grower will be able to monitor Salmonella contamination on globe fruits:</p>
<p>1) as a function of location in the field to determine site specific hazards, e.g., Whether contamination is due to runoff from adjacent fields, proximity to stagnant water/ponds, farm building sewer systems or compost piles;</p>
<p>2)as a function of activities conducted during the growing operation, e.g., Did contamination result after application of organic fertilizers, pesticides, irrigation with a pond source, heavy rain resulting in flooding;</p>
<p>3)during harvesting, e.g., Did contamination occur as a result of workers, contaminated wash water or harvesting equipment that was improperly cleaned; and</p>
<p>4) during transportation, e.g., Were the trucks properly cleaned or previously used to carry other contaminated fruits/vegetables, Did cross contamination occur due to mixing with contaminated produce from other farms.</p>
<p>Once the above sources of contamination have been identified, the farmer can adopt an integrated growth, harvest and transportation management program that minimizes potential hazards of Salmonella contamination. The technical deliverables that will be accomplished during the project are:</p>
<p>1) Demonstrate AU-free resonators;</p>
<p>2) Demonstrate SAM phage binding;</p>
<p>3) Demonstrate prototype in-field biosensor measurements;</p>
<p>4) Complete laboratory biosensor</p>
<p>5) Complete in-field measurement device;</p>
<p>6) Complete laboratory PCR evaluations;</p>
<p>7) Perform in-field evaluations of biosensors;</p>
<p>8) Perform in-field PCR comparisons with biosensors; and</p>
<p>9) Complete Agro-economic evaluations.</p>
<p>NON-TECHNICAL SUMMARY:<br/> The objective of this program is to develop, demonstrate and field test an inexpensive, accurate, easy-to-use biosensor for the detection of Salmonella contamination of fresh globe fruits (tomatoes, cantaloupes and watermelons). Globe fruits will be picked in the field using an appropriate sampling methodology and placed in sterilized, resealable plastic bags. The biosensors will be placed directly on the surface of the globe fruit, the bag sealed and the biosensors interrogated wirelessly to determine whether the surface of the fruit is contaminated with Salmonella. Data from these sensors will be used to spatially and temporally track Salmonella contaminations. In this manner potential sources of contamination such as run-off from heavy rains, compost piles, contaminated pond water, or applications such as organic fertilizers can be identified
and incorporated into an integrated growth, harvest and transportation management plan to ensure a safe food product. The biosensors will be constructed using wireless, microelectronically fabricated magnetoelastic resonators as the transducer. Highly robust and specific, genetically-engineered phage will be immobilized onto the resonators as the bio-molecular recognition element. The biosensors will be smaller than a dust particle and cost less than 1/1000 of a cent per sensor. More than 50 individual ME biosensors may be placed on a single globe fruit and simultaneously interrogated to detect Salmonella contamination. By spatially and temporally monitoring Salmonella contamination, critical hazard sources can be identified and best farming, harvesting and transportation practices can be developed to improve the food safety of fresh fruit and vegetable crops.
<br/>The following is a list of methods that will be used to perform the research: 1). Method of Magnetoelastic Resonator Fabrication. The ME resonators will be fabricated from an amorphous binary alloy of iron-boron. The following fabrication process will be used. First, a layer of photoresist is applied to the surface of the wafer by spin coating. This photoresist is then UV exposed using a positive mask comprised of evenly spaced rectangles. The wafer is then developed. Next, the magnetoelastic film is formed by co-depositing iron (DC) and boron (RF) simultaneously using the dual-cathode method. Once deposition is complete, lift-off of the resonators is accomplished by rinsing the wafer in acetone. 2). Measurement of Resonant Frequency. A solenoid coil is used to measure responses of the ME biosensor. The biosensor is placed in the center of the solenoid
coil. A time-varying current applied to the solenoid generates a time-varying magnetic field. This time-varying magnetic field causes the sensors to oscillate. The resonant frequency of the sensor's oscillations is measured using a HP8751A network analyzer. 3). Confirmation of Salmonella Concentration by Plate Counts. The concentrations of the serial Salmonella Typhimurium dilutions in water are confirmed by plate counts. A 0.1 mL Salmonella Typhimurium suspension of each dilution is spread on Luria-Bertani agar plates with two plates for each dilution. All plates are incubated at 37 degrees C for 24 hours. The colonies on each plate are counted and the number of bacteria per milliliter is calculated. 4). Scanning Electron Microscope (SEM) Observations. SEM provides a visual verification of Salmonella cells bound to the sensor surface. Osmium tetroxide is commonly used as a stain of
biological samples for electron microscopy. The diffusion of Os (heavy metal) into the cell membrane provides a better contrast to the images in an electron microscope. The SEM micrographs are taken at different regions on the sensor surface. The number of bacterial cells attached to the sensor surface is counted manually for each of the photographs taken. 5). Detection of Salmonella on Globe Fruit Surfaces by Polymerase Chain Reaction.The procedure used by Thompson, et al will be used to minimize inhibitors and run the qPCR procedure. The three steps in the procedure are 1) hybridization of the target DNA with a biotin-labeled probe; 2) binding of the hybrid to streptavidin coated magnetic beads; and 3) PCR amplification. The target DNA in the wash water will be captured magnetically by capture hybridization. M-280 Streptavidin coated beads (Dynal) are added to 1X hybridization solution
(Sigma) before resuspension in distilled water. The amplification procedure will be performed in an ABE 7000 Sequence Detection System (Applied Biosystems). The target DNA is the InvA gene in Salmonella. The thermocycle profile is 50 degrees C for 2 minutes, 95 degrees C for 10 minutes, then 40 cycles of 95 degrees C for 15 seconds and 60 degrees C for 1 minute.
PROGRESS: 2011/09 TO 2012/08OUTPUTS: Drs. Bryan A. Chin (Materials Engineering), James Barbaree (Biological Sciences), and Tung- Shi Huang (Poultry Sciences) of Auburn University's Colleges of Engineering, Science and Mathematics and Agriculture are involved in this project to develop and demonstrate new technologies and methodologies for the rapid detection of foodborne pathogens (spores and bacteria) on globe fruits. The globe fruits being studied are tomatoes, cantaloupes and watermelons. The research focuses on the detection of Salmonella and Bacillus anthraces spores as demonstration pathogens representing bacteria and spore species. In the first year of the research, the scientists conducted experiments in laboratories and gathered samples from farms and local retail outlets (grocery stores and farmer outlets) located in Alabama. The experiments and studies
were conducted on soil, irrigation water, plants, tomatoes, cantaloupes, watermelon and spinach leaves in an effort to research and develop detection technologies for use in the field to rapidly detect foodborne contamination. Members of the research team met with other food scientists and engineers, growers, state food inspectors, state and national agricultural specialists, corporate farm managers and corporate food processing engineers and scientists individually, in training seminars, and at the February, 2012 state-wide conference of the Alabama Farmers Association, at the SPIE annual conference (April, 2012) in Baltimore, MD on biosensors, and at the ECS annual conference (May 2012) in Seattle, WA on agricultural sensors to disseminate the findings of their research. Research results were also presented in University wide seminars and undergraduate/graduate classes of the College
of Engineering, College of Science and Mathematics, and College of Agriculture. Undergraduate, masters, doctorate of philosophy and post-graduate students participated in the research. Outreach to K-12 classes occurred during faculty visits to individual classes and special event days at Auburn University for high school students, such as E-day (Engineering visitation day), Regional Science Fair, and Science Olympiad Competitions. A web based teaching module targeted for eighth to ninth graders was developed on food safety and is available on our website for download by any teacher. This teaching module consists of prepared lessons for a week of class and was developed using a nearby class of 20 students from the Lagrange, GA school system. Dissemination of the Knowledge: The participants of this project attended three (3) different international/national conferences and published 3
refereed journal publications to disseminate the scientific and engineering knowledge that was developed. During the first year of this project, faculty participating in the project awarded 1 Ph.D. degree to a professional educated in a multidisciplinary environment including the disciplines of food science, chemistry, microbiology and engineering. The research team also worked with the Alabama agricultural extension service, ALFA, and local farms and food industries to obtain feedback from end users on food safety/security concerns.
<br/>PARTICIPANTS: The following individuals worked on this project and their role is described below. Dr. Bryan Chin, the PD, worked on the development of in-situ magnetoelastic sensors, the measurement system, data analysis and evaluation software. Dr. Chin coordinated the overall research and activities of the project. Dr. James M. Barbaree, one of the co-PDs,
worked on the development of the biomolecular recognition elements required to capture and bind the target pathogens. Dr. Tung-Shi Huang, who was the third co-PD, worked on the food science related to pathogen detection and served as the extension contact for the project.
<br/>TARGET AUDIENCES: This project is designed to develop new technologies that improve the safety of the food that we eat. Our research project concentrates on the development of economical, rapid and accurate methods of food sampling, sample preparation, detection of food pathogens, and rapid tracking to determine the source of the contamination. The technology will hopefully enable direct detection of foodborne pathogens on globe fruits in the agricultural field, reducing or eliminating the tedious and time consuming sample preparation and pathogen enrichment steps now required by current technologies. Our new
magnetoelastic biosensor technology may have additional applicability in the medical field. The biosensor technologies may be used in clinical diagnosis to identify the specific pathogen causing the foodborne illness while the patient is in the doctor's office. This will lead to the prescription of the right drugs and hence reduce the time and severity of the illness. This research will improve food safety, reduce suffering, enable better treatment of illnesses by the physician, as well as advance biological research and improve our fundamental understanding of science. Based on the above, the project has the following target audiences. The primary audiences are the food industry, food inspectors, food scientists, researchers, educators, federal/state/local governments. Students are being trained to become future employees of the food industry and government regulators. The researchers
of this team use the classroom, laboratory, and outreach activities to disseminate the information, technologies, and knowledge gained/acquired in this project. The team also participated/conducted workshops to disseminate the research achievements to its final customers, such as food inspectors and food industry scientists.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.</p>