Source: AUBURN UNIVERSITY submitted to
MAGNETOELASTIC BIOSENSORS FOR DETECTION OF PATHOGENS ON GLOBE FRUITS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0225754
Grant No.
2011-51181-30642
Project No.
ALA061-4-11013
Proposal No.
2011-01378
Multistate No.
(N/A)
Program Code
SCRI
Project Start Date
Sep 1, 2011
Project End Date
Aug 31, 2016
Grant Year
2011
Project Director
Chin, B.
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
College of Engineering
Non Technical Summary
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.
Animal Health Component
(N/A)
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7111420202010%
7111460110010%
7111499202010%
7116220301010%
7117210202020%
7121420202010%
7121499110010%
7126220301010%
7127210202010%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources;

Subject Of Investigation
7210 - Remote sensing equipment and technology; 1499 - Vegetables, general/other; 6220 - Marketing, processing, and supply firms other than cooperatives; 1420 - Melons; 1460 - Tomato;

Field Of Science
2020 - Engineering; 1100 - Bacteriology; 3010 - Economics;
Goals / Objectives
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: 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; 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; 3)during harvesting, e.g., Did contamination occur as a result of workers, contaminated wash water or harvesting equipment that was improperly cleaned; and 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. 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: 1) Demonstrate AU-free resonators; 2) Demonstrate SAM phage binding; 3) Demonstrate prototype in-field biosensor measurements; 4) Complete laboratory biosensor characterization; 5) Complete in-field measurement device; 6) Complete laboratory PCR evaluations; 7) Perform in-field evaluations of biosensors; 8) Perform in-field PCR comparisons with biosensors; and 9) Complete Agro-economic evaluations.
Project Methods
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 09/01/11 to 08/31/16

Outputs
Target Audience:The target audiences were researchers and scientists developing and seeking new technologies for the detection ofpathogens on fresh globe fruits, companies interested in commercialization and selling of pathogen detection technologies,agricultural producers of fresh globe fruits and K-12 teachers/classes/students interested in food safety. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students and post-doctorates received chemical and biological safety trainings annually, through which they strengthened their knowledge of safety management in laboratories. Microelectronic fabrication of biosensors and surface-scanning detectors, biological sample preparation, PCR experiments, and management of chemical and biological wastes were appropriately conducted. Every member of the research team was given opportunities to attend and present their work at domestic and international conferences to develop their presentation and networking skills. One post-doctorate received the best poster presentation award ($500 prize) at the 228th ECS meeting held in Phoenix, Arizona. In addition, two graduate students received the 2nd ($250 award) and 3rd place awards ($150) for their presentations. During the entire timeframe of the project, 10refereed journal publications were published in biosensor and applied physics journals of high impact factors. In addition, 10conference papers were accepted in electrochemical and optical journals. Faculty participating in the project awarded 4 Ph.D. degrees and 1 master's degree to graduate students educated in a multidisciplinary environment including the disciplines of food science, chemistry, microbiology and materials engineering. How have the results been disseminated to communities of interest?The members of the research team disseminated their knowledge, technologies, and findings through classroom, laboratory, and outreach activities. The team members also presented the results at domestic and international conferences, where they met with other engineers, food scientists, state food inspectors, and agricultural specialists at the SPIE annual conferences on biosensors and food safety, and at the ECS biannual conferences on agricultural biosensors. The research team hosted several companies (BASF, Immulogix, and Zoetis) and demonstrated technologies and applicability of ME biosensors to detecting pathogens in the field. The 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. 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. The surface-scanning biosensing system has been featured in several news media including ScienceDaily, Food Safety News, Global Biodefense, and Homeland Security News Wire. The ME biosensor method combined with the surface-scanning system has been selected as one of the five top methods in the first FDA's food safety challenge that was held in July 2015 (http://www.foodsafetychallenge.com/). The technology was awarded a $20,000 prize (See a video of our testing at http://eng.auburn.edu/food-safety). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACTOF THIS PROJECT Foodborne illness outbreaks due to contaminated fresh produce are on a dramatic rise to the individual consumer. Current bacterial detection methods require the collection of the samples followed by the analysis of the samples in the lab, which is an expensive and time consuming process. More importantly, because of cost, only a limited number of samples can be taken and analyzed from what may be a very large field.In this project, the research team led by Auburn University developed a magnetoelastic (ME) biosensor technique that can directly detect and quantify foodborne pathogens on the surfaces of globe fruits and vegetables in the agriculture field. The ME biosensors cost less than a cent per piece and are designed for use by minimally trained personnel. ACCOMPLISHMENTS The research conducted under this project led to the following outcomes: 1. Demonstration of SAM phage binding A chemical adsorption method was evaluated as a potential way to enhance the surface coverage and immobilization stability of phage. The surface functionalization of biosensors was performed through the construction of a carboxyl-terminated self-assembled monolayer (SAM), which forms a strong peptide bond with the N-terminus of phage. The results, obtained by atomic force microscopy, showed that SAM phage binding yielded as high a surface phage coverage as the conventionally used physical adsorption method with an enhanced immobilization stability. 2. Engineered Phage That Binds to All Salmonellae C4-22 phage, binding to allpathogenic Salmonellae, was produced and found to havevery low cross-reactivity toEscherichia coliandShigella, and no cross-reactivity toStaphylococcus aureusandListeria monocytogenes. 3. Laboratory Biosensor Characterization As away to enhance the detection capabilities, submillimeter-sized ME biosensors made of Fe79B21were microelectronically fabricated and evaluated. The sensor size ranged from 500 μm to 100 μm in length. These submillimeter-sized biosensors have much higher sensitivity than conventionally used 1-mm long biosensors, approximately by a factor of up to 3,000. Down to 11 bacterial spores were successfully detected with 100×25×4 μm biosensors. 4. Model for Probability of Detection Calculations The effect of food surface roughness on the ability of the ME biosensor to detectSalmonellabacteria was studied. In order to detectSalmonella, the biosensor must come into direct contact with the bacteria.The probability of detection as a function of sensor size and quantity (number) wasstudied. By using the model, the required number of biosensors to obtain a desired limit of detection can be determined. 5. Laboratory PCR Evaluation A PCR primer set for Salmonella Typhimurium was selected and tested with 74 bacterial strains (29-Salmonella and 45 non-Salmonella strains) for selectivity. This primer set was used in the in-field PCR testing. 6. In-Field Measurement Device 6.1. Surface-Scanning Detection System Surface-scanning coil detectors were designed, constructed, and evaluated. The primary objective of this work was to demonstrate the feasibility of this new detection system to perform real-time,in-situdetection of bacterial pathogens on fresh produce. Microelectronically fabricated planar coils were used and found to have stand-off distances on the order of millimeters, which allows one to scan food surfaces with high roughness and curvatures. The measurement setup consists of the surface-scanning detector, magnet plates for the supply of a bais magnetic field, a 3-axis translation stage, and an environmental chamber. 6.2. Pulsed Wave Excitation System To develop a portable measurement system for in-field pathogen detection, a pulsed wave excitation system was designed and constructed. By using a specially designed coil in combination with low-noise cascade amplifiers, the current system is capable of measuring ME biosensors as small as 200 um in length. In this system, a square pulse current is applied to an excitation coil to excite the ME biosensors, and a pick-up coil is used to detect the corresponding magnetic flux change, which is converted into an output electrical signal. The output signal is amplified by a signal amplification circuit, and the output waveform is shown on an oscilloscope. Based on the acquired damped oscillating signal, the frequency change due to the mass change on the surface of the ME biosensor can be calculated. 6.3. Development of Software for Real-Time Data Acquisition and Analysis Software that performs real-time acquisition and analysis of sensor frequency data was developed. It allows: (1) storing data on an external computer at a desired sampling interval; (2) calculating the resonant frequency shifts of multiple biosensors in real time and (3) displaying the results as a function of time. 7. Demonstration of prototype in-field biosensor measurement To demonstrate the capability of simultaneous measurement of multiple sensors, two sensors (one measurement sensor and one control sensor) were tested side-by-side on aSalmonella-spiked watermelon. The resonant frequency shifts of these sensors were measured simultaneously as a function of time. As expected, the resonant frequency of the measurement sensor was found to decrease largely over time, whereas that of the control sensor showed a very small change. This capability of simultaneous multiple sensor measurement is of great importance because it allows: (1) real-time determination of pathogen contamination by comparing measurement and control sensors that are tested under nearly identical conditions and (2) detection of multiple pathogen types if biosensors with different phages targeting different pathogens are used. 8. In-Field Biosensor Evaluation Various fresh produce and food processing surfaces (tomatoes, grapes, strawberries, blueberries, polyethylene and polyethylene terephthalate) were tested to demonstrate the applicability of the ME biosensor method. The speed of detection was from 2 to 10 minutes, largely depending on surface pathogen concentration and the roughness of the tested surface. The specificity of detection was 2 in 106background bacteria. The limit of detection was found to be in a range of 102and 104CFU/mm2. 9. In-Field PCR Comparisons with Biosensors To demonstrate the feasibility of the ME biosensor method, it was compared with a real-time, quantitative PCR (qPCR) method by detectingS. Typhimurium on tomato, spinach, and cantaloupe surfaces. The LODs for the ME biosensor and qPCR methods were found to be (1) 3 log cfu and 2 log cfu/tomato; (2) 1.94 log cfu and 1.37 log cfu/spinach, and (3) 2.47 log cfu and 1.35 log cfu/cantaloupe, respectively. These studies demonstrated that the ME biosensor method was rapid and robust, as well as competitive in LOD with qPCR forS. Typhimurium detection on fresh produce. 10. Development of an Automated Sensor Measurement System A new method of rapid pathogen detection by automated indexing plate measurement has been demonstrated. Multiple biosensors can be placed on an indexing plate consisting of an array of rectangular cavities approximately 10% larger in size than the ME biosensors. A vibrating table was used to vibrate the ME biosensors into motion until each cavity of the plate was occupied by a single ME biosensor. The biosensors were then measured by sequentially positioning the sensors under a surface scanning detector for measurement of the resonance frequency of the biosensors using an automated translation system. In this manner, a single sensor can be measured in less than 2 seconds with 1,000 sensors being measured in less than 30 minutes.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: J. Hu, Y. Chai, S. Horikawa, H. C. Wikle, F. Wang, S. Du, B. A. Chin and J. Hu, "The blocking reagent optimization for the magnetoelastic biosensor," Proc. SPIE 9488, Sensing for Agriculture and Food Quality and Safety VII, 94880W.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: J. Hu, Y. Chai, S. Horikawa, B. A. Chin and J. Hu, "Blocking Non-Specific Binding for Phage-Based Magnetoelastic Biosensors," Biosensors Journal 4, 130 (2015).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: I. Chen, J. Hu, F. Wang, S. Horikawa, J. M. Barbaree and B. A. Chin, "Alternative soaking media for the FDA procedure in the detection of salmonella from tomatoes and spinach leaf using phage magnetoelastic biosensors," Proc. SPIE 9864, Sensing for Agriculture and Food Quality and Safety VIII, 986412.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: S. Horikawa, I. Chen, S. Du, Y. Liu, H. C. Wikle, S. Suh, J. M. Barbaree and B. A. Chin, "Method for detection of a few pathogenic bacteria and determination of live versus dead cells," Proc. SPIE 9864, Sensing for Agriculture and Food Quality and Safety VIII, 98640H.
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: S. Horikawa, Y. Chai, I. Chen, H. C. Wikle, S. R. Best, S. Li, J. M. Barbaree and B. A. Chin, "Direct detection of bacteria on fresh fruits and vegetables," submitted to Biosensors and Bioelectronics.


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:The target audience were researchers and scientists developing and seeking new technologies for the detection of pathogens on fresh globe fruits, companies interested in commercialization and selling of pathogen detection technologies, agricultural producers of fresh globe fruits and K-12 teachers/classes/students interested in food safety. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the fourth year of the project, new graduate students and post-doctorates received chemical and biological safety training. This training strengthened their knowledge of safety management in laboratories. Microelectronic fabrication of biosensors and surface-scanning detectors, biological sample preparation, and management of chemical and biological wastes were appropriately conducted. In addition, every member of the research team was given opportunities to attend domestic/international conferences and present their work to develop presentation and networking skills. During this reporting period, 4conference papers were accepted in electrochemical and optical journals. One post-doctorate received the best poster presentation award ($500 prize) at the 228th ECS meeting held in Phoenix, Arizona. In addition, two graduate students received the 2nd ($250 award) and 3rd place awards ($150) for their presentations. How have the results been disseminated to communities of interest?The members of the research team disseminated their knowledge, technologies, and findings through classroom, laboratory, and outreach activities. The team members also presented the results at domestic and international conferences, where they met with other engineers, food scientists, state food inspectors, and agricultural specialists at the SPIE conference (May,2015) in Baltimore, MD on biosensors and food safety, and at the ECS conference (May 2015) in Chicago, IL on agricultural biosensors. In addition, the ME biosensor method combined with the surface-scanning system has been selected as one of the five top methods in the first FDA's food safety challenge that was held in July (http://www.foodsafetychallenge.com/). The technology was awarded a $20,000 prize (See a video of our testing at http://eng.auburn.edu/food-safety) and has been featured in several reporting media such as: http://www.ecsblog.org/preventing-foodborne-illness-with-novel-biosensors/. What do you plan to do during the next reporting period to accomplish the goals?Design and construction of a prototype of a hand-held wand device for in-field measurement of multiple ME biosensors will be investigated. The prototype will consist of a planar surface-scanning coil detector, portable network analyzer, portable DC bias field supply, and laptop computer. Alternatively, another prototype that is based on the transient response method (i.e., the pulse method without the need of a DC bias-field supply) will be investigated. These prototypes will be first evaluated in our labs and then used in the first field tests of ME biosensors. The first field tests are planned for tomato crops grown on Auburn University test plots. Specific tomatoes will be picked by location, photographed and tested for Salmonella with ME biosensors throughout the growing season at periodic intervals.

Impacts
What was accomplished under these goals? Five major advances in knowledge were made during the fourth year of this project: 1. Improvement in surface blocking for the ME biosensor Surface blocking is a common technique for preventing unwanted substances from attaching to biosensor surfaces and eliminating possible false detection. Previously, bovine serum albumin (BSA) was used as the surface blocker that fills the gaps among filamentous phage particles coated on the ME biosensor. To further improve the biosensor performance, different surface blockers (SuperBlock and Blocker BLOTTO, purchased from Thermo Fisher Scientific, Inc.) were tested and compared to BSA. Measurement and control sensors treated with each surface blocker were prepared and exposed to Salmonella Typhimurium on tomatoes (1×106 CFU/mm2) for 30 min at 85% relative humidity. The biosensors were then collected and rinsed with sterile distilled water. Finally, a crystal violet dye was used to stain the Salmonella cells bound on the biosensors for cell counting by optical microscopy. As anticipated, a large cell count was found on bare biosensors without a surface blocker due to non-specific binding of Salmonella cells. The number of non-specifically bound cells was then reduced to a large extent by surface blocking. However, at the same time, the cell counts on the measurement sensors were also found to be decreased, resulting from the undesired covering of a portion of phage binding sites by the surface blockers. In order to select the best surface blocker that gives the maximal statistical difference between the measurement and control sensors, a one-tailed, unpaired Student's t-test was conducted. The SuperBlock was found to be the best surface blocker with the highest confidence level of difference (99.5%). The number of non-specifically bound Salmonella was reduced by 300% compared to BSA. 2. Development of software for real-time data acquisition and analysis Software that performs real-time acquisition and analysis of sensor frequency data was developed. It allows: (1) storing data on an external computer at a desired sampling interval; (2) calculating the resonant frequency shifts of multiple biosensors in real time and (3) displaying the results as a function of time. With the use of this software, the speed of detection and the rate of sensor response as a function of Salmonella concentration can be determined easily. 3. Simultaneous measurement of multiple ME biosensors To demonstrate the capability of multiple sensor measurement, two sensors (one measurement sensor and one control sensor) were tested side-by-side on a Salmonella-spiked watermelon. The resonant frequency shifts of these sensors were measured simultaneously as a function of time using the software described above. As expected, the resonant frequency of the measurement sensor was found to decrease largely over time, whereas that of the control sensor showed a very small change. To further investigate the method, pairs of measurement and control sensors were prepared and tested simultaneously at different concentrations of Salmonella. Similar to the previous result, there were large differences in the responses of measurement and control sensors at high Salmonella concentrations (> 7.4 × 103 cfu/mm2), indicating that large amounts of the bacteria were attached to the measurement sensors due to the phage-based specific binding. These binding results were also confirmed by scanning electron microscopy. This capability of simultaneous multiple sensor measurement is of great importance because it allows: (1) real-time determination of pathogen contamination by comparing measurement and control sensors that are tested under nearly identical conditions and (2) detection of multiple pathogen types if biosensors with different phages targeting different pathogens are used. 4. Sensor measurement on various fresh produce and food processing surfaces In addition to globe fruits, various fresh produce and food processing surfaces (tomatoes, grapes, strawberries, blueberries, polyethylene and polyethylene terephthalate) were tested to demonstrate the applicability of the ME biosensor method. The speed of detection was from 2 to 10 minutes, largely depending on surface pathogen concentration and the roughness of the tested surface. The specificity of detection was 2 in 106 background bacteria. The limit of detection was found to be in a range of 102 and 104 CFU/mm2. 5. Development of a 3D biomolecular filter and an automated sensor measurement system A new method of rapid detection of pathogens by 3D biomolecular filtering and automated indexing plate measurement has been demonstrated. In this process, a liquid containing pathogens is passed through a 3D biomolecular filter composed of planar arrays of ME biosensors held by a magnetic field. This filter captures specific target pathogens (in our demonstration case, Salmonella) in the liquid as it passes over the filter. Capture of the pathogens is by specific biomolecular recognition, not size exclusion as with traditional filters. After the entire liquid has passed over the filter, the magnetic field is shut off and the individual biosensors collected and dried. The biosensors are then placed on an indexing plate consisting of an array of rectangular cavities approximately 10% larger in size than the ME biosensors. A vibrating table is used to vibrate the ME biosensors into motion until each cavity of the plate is occupied by a single ME biosensor. The biosensors are then measured by sequentially positioning the sensors under a surface scanning detection coil for measurement of the final resonance frequency of the biosensors using an automated translation system. In this manner, a single sensor can be measured in less than 2 seconds with 1000 sensors being measured in less than 30 minutes.

Publications

  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2015 Citation: S. Horikawa, Y. Chai, H. C. Wikle, S. Suh, J. M. Barbaree and B. A. Chin, Direct detection of Salmonella on fresh produce, ECS Transactions.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2015 Citation: Y. Liu, J. Hu, S. Horikawa, S. Du, Y. Chai, F. Wang and B. A. Chin, Rapid and sensitive detection of Salmonella Typhimurium on plastic food processing plates by using wireless biosensors, ECS Transactions.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2015 Citation: S. Horikawa, Y. Chai, H. C. Wikle, J. M. Barbaree and B. A. Chin, Direct Detection of Bacteria on Fresh Produce, Biodevices, the 9th International Joint Conference on Biomedical Engineering Systems and Technologies.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Y. Chai, S. Horikawa, J. Hu, I. Chen, J. Hu, J. M. Barbaree and B. A. Chin, In-situ detection of multiple pathogenic bacteria on food surfaces, Proc. SPIE 9488, Sensing for Agriculture and Food Quality and Safety VII, 2015, 948805.


Progress 09/01/13 to 08/31/14

Outputs
Target Audience: The target audience for this year's efforts were researchers and scientists developing new technologies for the detection of pathogens on fresh globe fruits, companies interested in commercialization and selling of pathogen detection technologies, food safety/quality scientists (ARS and AI Food Inspection Service), agricultural producers of fresh globe fruits, and K-12 teachers/classes/students interested in food safety. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? In the third year of the project, post-doctorates and graduate students received chemical and biological safety trainings, through which they strengthened their knowledge of safety management in laboratories. Microelectronic fabrication of biosensors, biological sample preparation, and management of chemical and biological wastes were appropriately conducted. In addition, every member of the research team was given opportunities to attend domestic and international conferences and present their work to develop presentation and networking skills. During this reporting period, 3 refereed journal publications were published in microbiology and applied physics journals of high impact factors. Faculty participating in the project awarded 1 Ph.D. degree to a graduate student educated in materials engineering. How have the results been disseminated to communities of interest? The members of the research team disseminated their knowledge, technologies, and findings through classroom, laboratory, and outreach activities. The team members also presented the results at domestic and international conferences, where they met with other engineers, food scientists, state food inspectors, and agricultural specialists at the SPIE conference (May, 2014) in Baltimore, MD on biosensors and food safety, and at the ECS conference (May 2014) in Orlando, FL on agricultural biosensors. In addition, the surface-scanning biosensing system has been featured in several news media including ScienceDaily, Food Safety News, Global Biodefense, and Homeland Security News Wire. What do you plan to do during the next reporting period to accomplish the goals? Design and construction of a prototype of a hand-held interrogator for in-field measurement of multiple ME biosensors will be completed. The prototype will consist of a planar surface-scanning coil detector, portable network analyzer, portable DC bias-field supply, and laptop computer. Alternatively, another prototype that is based on the transient response method (i.e., the pulse method without the need of a DC bias-field supply) will be developed. These prototypes will be first evaluated in our labs and then used in the first field tests of ME biosensors. The first field tests are planned for tomato crops grown on Auburn University test plots. Specific tomatoes will be picked by location, photographed and tested for Salmonella with ME biosensors throughout the growing season at periodic intervals. Tests will be conducted using the optimal size and number of biosensors determined in the second year of the project.

Impacts
What was accomplished under these goals? Four major advances in knowledge were made during the third year of this project: 1. Development of an engineered phage that binds all Salmonella enterica serovars Phage display technology allows us to synthesize engineered phages for selective capture of the pathogens of interest. In this work, the filamentous M13 bacteriophage was used to acquire an engineered phage specific for all Salmonella enterica serovars. The approach was to add an affinity-selected oligopeptide to the pIII region of the M13 phage. A phage pIII library was used to perform biopanning in this oligopeptide selection where common LPS Salmonellae antigens were used as binding targets. The selected peptides were then sequenced and tested for specificity using ELISA procedures. Finally, the engineered phage with the highest binding affinity was immobilized on a magnetoelastic (ME) sensor platform to complete the fabrication of an ME biosensor. The phage-immobilized ME biosensor provides us the ability to detect all Salmonella enterica serovars in real time by measuring the changes in sensor’s resonance frequency induced by the mass attached (i.e., Salmonella cells attached). Our data with an ELISA procedure verify the phage probe’s high affinity for Salmonellae, very low cross-reactivity to Escherichia coli and Shigella, and no cross-reactivity to Staphylococcus aureus and Listeria monocytogenes. The phage-immobilized biosensor was also found to possess thirty times higher capture ability than the control sensor (without phage). 2. Construction and evaluation of surface-scanning coil detectors Following the second year’s investigation into the design of surface-scanning coil detectors, a number of coil detectors were constructed and evaluated. The objectives of this work are to demonstrate the feasibility of our proposed detection system and to perform real-time, in-situ detection of bacterial pathogens on fresh produce. The two-fold roles of the coil detectors are: (1) supply of a time-varying magnetic field to place the ME biosensor into mechanical resonance (i.e., excitation); and (2) detection of the magnetic flux change caused by the resonating biosensor. A network analyzer operating in the S11 reflection mode was used to measure the biosensor’s resonant frequency changes as a function of time. In addition, an equivalent electric circuit model of the biosensor system (consisting of an ME biosensor, coil detector and the network analyzer) was developed. The signal amplitude as a function of detection distance was measured and compared to the results calculated by the model. The coil design was then re-evaluated. At last, the final coil detector was fabricated and tested with ME biosensors. 3. Study of simultaneous measurement of multiple biosensors Simultaneous measurement of multiple biosensors was accomplished with a newly designed surface-scanning coil detector. This coil detector provides a longer detection distance and larger detection space. Multiple ME biosensors of different length were employed to demonstrate the feasibility of measurement. Due to the differences in sensor length, these biosensors theoretically possess different characteristic resonant frequencies. As expected, multiple distinguishable frequency peaks were successfully detected. Previous research has shown that the use of multiple biosensors is necessary since bacterial cells tend to be non-uniformly distributed over food surfaces. Hence, the capability of measuring multiple biosensors simultaneously holds promise for greatly enhancing the chance of physical contact of biosensors to pathogens and thus detection. 4. Design, construction, and evaluation of a pulsed wave excitation system In order to develop a portable measurement system for in-field pathogen detection, a pulsed wave excitation system was designed and constructed. By using a specially designed coil in combination with low-noise cascade amplifiers, the current system is capable of measuring ME biosensors as small as 200 um in length. In this system, a square pulse current is applied to an excitation coil to excite the ME biosensors, and a pick-up coil is used to detect the corresponding magnetic flux change, which is converted into an output electrical signal. The output signal is amplified by a signal amplification circuit, and the output waveform is shown on an oscilloscope. Based on the acquired damped oscillating signal, the frequency change due to the mass change on the surface of the ME biosensor can be calculated. The impact of signal amplification on the resonant frequency, amplitude, and Q-factor of the resonant frequency peak has been studied.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Y. Chai, S. Horikawa, H. C. Wikle, Z. Wang and B. A. Chin, Surface-scanning coil detectors for magnetoelastic biosensors: A comparison of planar-spiral and solenoid coils, Applied Physics Letters, Vol. 103(17), 173510 (2013).
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Y. Chai, H. C. Wikle, Z. Wang, S. Horikawa, S. Best. Z. Cheng, D. F. Dyer and B.A. Chin, Design of a surface-scanning coil detector for direct bacteria detection on food surfaces using a magnetoelastic biosensor, Journal of Applied Physics, Vol. 114, 104504 (2013).
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: I-H. Chen, K. Vaglenov, Y. Chai, B.A. Chin, J.M. Barbaree, Use of LPS extracts to validate phage oligopeptide that binds all Salmonella enterica Serovars, Advances in Microbiology, Vol. 4(9), 11 (2014).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: S. Horikawa, Y. Chai, R. Zhao, H.C. Wikle, B.A. Chin, Effects of food surface topography on phage-based magnetoelastic biosensor detection, SPIE Sensing Technology and Applications 910802 (2014).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Y. Chai, S. Horikawa, A. Simonian, D. Dyer, B.A. Chin. Wireless magnetoelastic biosensors for the detection of Salmonella on fresh produce. Sensing Technology (ICST), 2013 Seventh International Conference 174-177 (2013).
  • Type: Theses/Dissertations Status: Accepted Year Published: 2014 Citation: Real-Time, in-situ Detection of Pathogenic Bacteria on Food Surfaces Using a Surface-Scanning Coil Detector and Phage-Based Magnetoelastic Biosensors, Yating Chai, Auburn University, 2014.


Progress 09/01/12 to 08/31/13

Outputs
Target Audience: The target audience for this year's efforts were researchers and scientists developing new technologies for the detection of pathogens on fresh globe fruits, companies interested in commercialization and selling of pathogen detection technologies, food safety/quality scientists (ARS and AI Food Inspection Service), agricultural producers of fresh globe fruits, and K-12 teachers/classes/students interested in food safety. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? In the second year of the project, post-doctorates and graduate students received additional chemical and biological safety trainings, through which they strengthened their knowledge of safety management in laboratories. Microelectronic fabrication of biosensors, biological sample preparation, PCR experiments, and management of chemical and biological wastes were appropriately conducted. In addition, every member of the research team was given opportunities to attend and present their work at domestic and international conferences to develop presentation and networking skills. During this reporting period, 3 refereed journal publications were published in biosensor and applied physics journals of high impact factors. Faculty participating in the project awarded 1 Ph.D. degree and 1 master’s degree to graduate students educated in materials engineering. How have the results been disseminated to communities of interest? The members of the research team disseminated their knowledge, technologies, and findings through classroom, laboratory, and outreach activities. The team members also presented the results at domestic and international conferences, where they met with other engineers, food scientists, state food inspectors, and agricultural specialists at the SPIE conference (April, 2013) in Baltimore, MD on biosensors and food safety, and at the ECS conference (May 2013) in San Diego, CA on agricultural biosensors. In addition, the team hosted several companies (BASF, Immulogix, and Zoetis) and demonstrated technologies and applicability of ME biosensors to detecting pathogens in the field. What do you plan to do during the next reporting period to accomplish the goals? Design and construction of a prototype of a hand-held interrogator for in-field measurement of multiple ME biosensors will be initiated. The prototype will consist of a planar surface-scanning coil detector, portable network analyzer, portable DC bias-field supply, and laptop computer. Alternatively, another prototype that is based on the transient response method (i.e., the pulse method without the need of a DC bias-field supply) will be developed. These prototypes will be first evaluated in our labs and then used in the first field tests of ME biosensors. The first field tests are planed for tomato crops grown on Auburn University test plots. Specific tomatoes will be picked by location, photographed and tested for Salmonella with ME biosensors throughout the growing season at periodic intervals. Tests will be conducted using the optimal size and number of biosensors determined in the second year of the project.

Impacts
What was accomplished under these goals? Changes in Knowledge: Five major advances in knowledge were accomplished during the second year of this project. 1. Biomolecular-recognition study The objective of this study is to produce an engineered phage that binds to all Salmonellae, excluding S. Typhi. To facilitate the selection of such a phage, instead of testing millions of molecular combinations for binding to the cell surfaces of over 2,500 Salmonella enterica serovars, a new approach that extracts common antigens within representatives of O antigen Groups B, C, and D (A is S. Typhi) was investigated. To isolate the common antigens, lipopolysaccharide (LPS) was extracted from seventeen foodborne Salmonella serovars representing the three groups B, C, and D. In order to efficiently extract the LPS, a modified phenol-chloroform-petroleum ether (PCP) extraction method was used. The modified PCP LPS extraction method enabled us to test Salmonella enterica serotypes antigens to produce peptides that bind with the cell surface of all representative Salmonella (O-antigen groups B, C, and D) without testing numerous whole cells. The phage C4-22 was found to be a strong candidate for use with rapid biosensor testing platforms for Salmonella detection in foods. As a proof-of-concept experiment, S. Typhimurium solutions at three concentrations of 5 × 107, 1 × 108, and 5 × 108 cfu/ml were exposed to C4-22 phage-coated sensors and BSA only sensors. S. Typhimurium cells detected on the sensors were washed with TBS/0.5% Tween three times and eluted with 0.1M Glycine (pH 2.2) to break phage-Salmonella binding. The percent of Salmonella binding to the phage-coated sensor was determined using a standard aerobic plate count method. The C4-22 phage-coated sensors demonstrated 30 times higher Salmonella binding capacity than the control sensors at a Salmonella loading concentration of 5 × 108 cfu/ml. 2. Study of the probability of detection Following the first year's investigation into the effect of food surface topography on the ability of biosensors to detect Salmonella bacteria, the probability of detection as a function of sensor size and quantity (number) was studied. Smaller biosensors have theoretically higher sensitivity; however, they have smaller coverage on a food surface, potentially leading to false-negative detection. Hence, a simple statistical model to calculate the probability of detection was constructed. In this model, the detection probability can be expressed as a function of the size, quantity, and mass-sensitivity of the biosensor, and it is also correlated with the concentration and surface distribution of S. Typhimurium. By using the model, the required number of biosensors to obtain a desired limit of detection can be determined. 3. Laboratory biosensor characterization As a potential way to enhance the detection capabilities of the system, smaller ME biosensors made of Fe79B21 were microelectronically fabricated and evaluated. The sensor size ranged from 500 μm to 100 μm in length. These micron-scale biosensors have much higher sensitivity than 1 mm-long biosensors that were initially used, approximately by a factor of up to 3,000. For these biosensors, corrosion resistance was first tested as a measure of environmental stability. A 50 nm-thick layer of gold was found to well protect the sensors from corrosion in liquids. In addition, the dependence of the sensitivity on the position of attached masses was investigated by locally placing a small amount of model masses (Bacillus anthracis spores) on the sensor surface. An improvement in sensitivity was observed as the mass position got closer to the end of the sensor's longest dimension. In this way, down to 11 spores were successfully detected with 100 μm × 25 μm × 4 μm biosensors. Numerical simulations using the ANSYS and CalculiX software packages were also conducted and compared with the experimental results, showing a good agreement between these results. 4. Laboratory PCR evaluation To demonstrate the feasibility of the ME biosensor method, it was compared with a real-time, quantitative PCR (Q-PCR) method by detecting S. Typhimurium on tomato surfaces. In this work, standard curves, correlations, and limits of detection (LODs) were first determined for tomato surfaces inoculated with various concentrations of S. Typhimurium (1 to 8 log cfu/tomato). The LODs for the ME biosensor and Q-PCR methods were found to be 3 log cfu/tomato and 2 log cfu/tomato, respectively. In addition, both methods were tested to detect S. Typhimurium directly grown on tomato surfaces. Drops of a Salmonella suspension were inoculated on 65 tomato surfaces and incubated at 37°C and 100% RH for 24 h. After incubation, S. Typhimurium was positively detected by both methods, and the quantified concentrations were nearly the same, 6.35 ± 2.03 and 6.34 ± 0.17 log cfu/tomato for the ME biosensor and Q-PCR methods, respectively. These quantified concentrations were greater by one order of magnitude than that determined by a BGS-plate count method, 5.33 ± 0.21 log cfu/tomato. Scanning electron microscopy was used to confirm the growth of S. Typhimurium on the tomato surfaces and its specific binding to the phage-coated ME biosensors. This study demonstrated that the ME biosensor method was rapid and robust, as well as competitive in LOD with Q-PCR for S. Typhimurium detection on fresh produce. 5. Design, construction, and evaluation of surface-scanning coil detectors To be able to field-test the ME biosensors, surface-scanning coil detectors were designed, constructed, and evaluated. The primary objective of this work is to demonstrate the feasibility of this new detection system to perform real-time, in-situ detection of bacterial pathogens on fresh produce. Previously, the resonant frequency of the ME biosensor had to be read by inserting it in the center of a cylindrical electromagnetic coil, which hinders user-friendliness as well as increases testing time. Hence, there is a need for a new detection system that enables measurement of the biosensor’s characteristic resonant frequency directly on food surfaces as a function of time. The two-fold roles of the new coil detector are: (1) supply of a time-varying excitation magnetic field to place the biosensor into mechanical resonance; and (2) detection of the resultant magnetic flux change caused by the resonating biosensor. A network analyzer operating in the S11 reflection mode was then used to determine the biosensor’s resonant frequency. Microelectronically fabricated planar coils were used and found to have standoff distances on the order of millimeters, which allows one to scan food surfaces with high roughness and curvatures. To demonstrate the detection capabilities of this new methodology, multiple biosensors (length × width × thickness: 1 mm × 0.2 mm × 30 μm) were directly placed on a tomato surface contaminated with a known concentration of S. Typhimurium. The coil detector was then brought close to the sensors for frequency measurement over time. The coil detector was able to monitor frequency increases of the measurement biosensors temporally.

Publications

  • Type: Theses/Dissertations Status: Accepted Year Published: 2013 Citation: Shin Horikawa, Low-cost, rapid, sensitive detection of pathogenic bacteria using phage-based magnetoelastic biosensors, PhD dissertation, Auburn University, 2013.
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Mi-Kyung Park, Kanchana A. Weerakoon, Jun-Hyun Oh, Bryan A. Chin, "The analytical comparison of phage-based magnetoelastic biosensor with TaqMan-based quantitative PCR method to detect Salmonella Typhimurium on cantaloupes," Food Control 33, 330-336 (2013).
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Yating Chai, Shin Horikawa, Suiqiong Li, Howard C. Wikle, and Bryan A. Chin, A surface-scanning coil detector for real-time, in-situ detection of bacteria on fresh food surfaces, Biosensors and Bioelectronics, 50, 311 (2013).
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Mi-Kyung Park, Suiqiong Li, Bryan A. Chin, "Detection of Salmonella typhimurium Grown Directly on Tomato Surface Using Phage-Based Magnetoelasti," Food Bioprocess Technol, 6, 682-689 (2013). Biosensors
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Mi-Kyung Park, Jang Won Park, Howard C. Wikle III, Bryan A. Chin, "Evaluation of phage-based magnetoelastic biosensors for direct detection of Salmonella Typhimurium on spinach leaves," Sensors and Actuators B, 176 1134-1140 (2013).


Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: 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. 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. 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. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Changes in Knowledge: The research conducted under this project led to three major advances in knowledge during the first year of this project. 1). The objective of the biomolecular recognition research that is being performed is to broaden the binding affinity to include other serovars of Salmonella that are pathogenic to humans. Using our landscape phage library, we have selected M13 filamentous phage with peptides specific for Salmonella enterica species. These peptides have been inserted in the PIII minor capsid protein. In the early biopanning process, possible related bacteria such as Escherichia and Enterobacter spp. were depleted to avoid cross-reactions with the final phage proteins. Since S. enterica has numerous serotypes, proteins that bind the common O antigen structures for phases I and II plus the flagella proteins were sought and generated via phage display techniques. Additionally, common amino acid sequences (motifs) were identified in the binding proteins to substantiate the selection of phages/proteins to be immobilized onto the sensor platform to complete the package. 2). The effect of food surface roughness on the ability of the magnetoelastic biosensors to detect Salmonella bacteria was studied during the first year. In order to detect the target pathogenic bacteria (Salmonella), the biosensor must come into direct contact with the bacteria. Rough food surfaces may allow the target bacteria to avoid contact with the biosensor, thereby avoiding detection. Tomatoes have a very smooth surface, while cantaloupe and spinach leaves have very rough surfaces. Spinach leaves were investigated because the surface topographies can easily be measured because of their small size, versus cantaloupe. Also leaves have stomas that serve as ideal areas providing moisture and nutrients necessary for survival. Spinach leaf roughness and curvatures of both adaxial (top) and abaxial (underside) surfaces were measured using the laser confocal microscope. Solutions of different concentrations of Salmonella in water were then distributed uniformly over leaf surfaces and the leaves incubated for 24 hours, prior to examination under SEM. As the concentrations of Salmonella in water decreased below 10000 CFU/ml, the distribution of Salmonella on the leaf became highly nonuniform with most of the bacteria migrating to deep valleys and stomas on the leaf surface. Detection experiments were conducted with different size sensors. As anticipated, smaller biosensors improve the ability to detect small concentrations of nonuniformly distributed bacteria on foods with rough surfaces. 3). Construction of a new measurement system for use in the field was initiated. The new system will use an array of permanent magnets to generate the uniform magnetic field in which the magnetoelastic biosensors are measured. The permanent magnets will replace the electromagnets that are currently used. This will eliminate the need for electrical power to run the electromagnets. One postdoc, 2 Ph.D., 1 M.S. and several undergraduates were supported and trained by this project.

Publications

  • Suiqiong Li, Shin Horikawa, Mi-kyung Park, Yating Chai, Vitaly J. Vodyanoy and Bryan A. Chin, "Amorphous Metallic Glass Biosensors," Intermetallics 30 (2012) pg 80-85.
  • Shin Horikawa and Bryan A. Chin, "Harvesting nanometer displacements for biosensor applications," Proceedings of the International Symposium on Plasticity, Puerto Valarta, Mexico, In-Press, January 2012, 5 pages.
  • Shin Horikawa, Deepa Bedi, Suiqiong Li, Wen Shen, Shichu Huang, I-Hsuan Chen, Yating Chai, Maria L. Auad, Michael J. Bozack, James M. Barbaree, Valery A. Petrenko and Bryan A. Chin, "Effects of surface functionalization on the surface phage coverage and the subsequent performance of phage-immobilized magnetoelastic biosensors," Biosensors & Bioelectronics, Vol. 26(5), pp. 2361-2367, 2011. doi:10.1016/j.bios.2010.10.012