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Increasing the Safety and Shelf Life of Idaho Commodities

Branen, A. Larry
University of Idaho
Start date
End date

Objective 1: Target Capture Optimization:
a) Use BNT system to specifically recognize and discriminate live organisms of Salmonella serovar Typhimurium and Escherichia coli O157 and staphylococcal enterotoxin B (SEB) in a single sample, b) Use BNT System to detect above organisms in food and environmental samples, c) Examine the application of the BNT system for detection of other organisms and toxins of interest in bioterrorism and food and environmental safety, d)Adapt a micro-TAS system to the target capture system to food and environmental samples;

Objective 2: Signal Generation Optimization:
a)Optimize the time and temperature conditions for signal generation, b)Develop and adapt a micro-TAS system module for the signal generation step of the BNT system;

Objective 3: Signal Detection Optimization:
a)Optimization of the reusable gold electrochemical arrays for detection of nanosignals, b) Optimization of disposable screen printed gold electrodes for detection of nanosignals, c)Development of an inexpensive VLSI Potentiostat for amperometric measurements and adaptation to a micro-TAS system;

Objective 4: Application of new technologies to the BNT System:
a)Evaluation of silica based nanowires for use in development of DNA-antibody conjugates, b) Use of silica based nanowires for use in the changing the electrical/flow characteristics of target capture in the micro-TAS system


Objective 1: Target Recognition and Signal Generation:
a) Continue to develop aptamers against Staphylococcus aureus surface markers, b) Optimize generation of electrical, optical and electrochemical signal generation;

Objective 2: Signal Detection Platforms:
a) Fabricate electronic platforms which include 250 nm microscale electronics and 10 nm nanoFET, nano scale electronics, b)Investigate optical imaging technology composed of bioluminescence and NASA imaging techniques

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NON-TECHNICAL SUMMARY: Maintaining the expected quality and safety of food and agricultural products could be challenged by chemical and biological contamination. Of particular concern is the threat of food poisoning. To protect the safety of the food supply and to assure continued high quality of agricultural commodities, it is essential to have methods available for predicting the potential contamination of foods and other agricultural commodities. The purpose of this project is to develop bioelectronic detectors that can quickly detect the presence of microbial pathogens in food projects.

APPROACH: The Bio-nanotransduction (BNT) system will be optimized for the recognition and discrimination of live organisms and toxins in pure cultures and in food products using a multi-analyte approach. Magnetic beads functionalized with commercially available polyclonal or monoclonal specific antibodies will be used to capture the organisms and toxins. Signal generation will be accomplished using a biological recognition element/ DNA template conjugate (BRED) and in vitro transcription to produce the RNA nanosignals. Nanowires will be evaluated for their ability to replace the BRED in some assays. An enzyme linked oligonucleotide assay (ELONA) will be used in combination with a gold electrochemical array as the detection system using an electrochemical analyzer to measure current. Target capture, signal generation, and detection of the BNT assay will be optimized for sensitivity, specificity and reliability using live organisms of Salmonella serovar Typhimurium and Escherichia coli O157 and SEB. Once optimized, the system will be evaluated for ability to detect Listeria, Campylobacter, capsular antigen F1 of Yersenia pestis, Aspergillus flavus mold spores and aflatoxin. The development of sensors and complete micro-Total Analysis Systems (micro-TAS) will be evaluated including the addition of an integrated electrode and potentiostat. Both reusable and disposable electrodes will be integrated into the system. New capture techniques will be developed through the use of SELEX to generate aptamers showing a high affinity that for recognition and capture of Staphylococcus aureus. At least 90 clones will be sequenced in order to find aptamer families and the aptamers will be characterized through structural analysis and binding affinity assays. New signal generation methods will be developed through the use of bioluminescence detection using an enzyme linked reaction. Luciferases which have much lower non-specific noise background will be studied. The light produced by the reaction of luciferases with their substrate will be detected by both CCD image sensor and luminometer. Detection surface materials, sample concentrations, reaction temperature and times will be optimized fro the detection. A micro FET device has been designed and verified using standard CAMBR chip design CAD tools used in other NASA designs and will be fabricated, gold plated, packaged, and wire bonded. Fundamental testing will be used to characterize the device and testing will model the effects of charge deposits on the gold electrodes. The ability of the electrical circuits to resolve the small signals will be evaluated Biochemical evaluation will include ISFET behavior; insulation from buffer with SAMs; ability to detect tethered charge; and electrochemical testing of electrode array. The nano FET device represents fundamental research as the nano structure level. Nano wires will be created from various compounds, placed on gold contacts and wired in a research test structure. Modeling at the basic physics level will be used to characterize modulation of electrical properties of the nano FET devices.

PROGRESS: 2007/09 TO 2008/08
OUTPUTS: Detection systems were established for detection of organisms and toxins in food (meat and milk) and environmental samples (water and wastewater). Parameters and conditions for the most efficient capture of target analytes using immunomagnetic particles were determined and applied to these products. A sensitive detection method was developed for detection of the captured targets based upon using a disposable magnetized screen-printed carbon electrode (SPCEs) strip. The estimated lowest detection of Staphylococcal enterotoxin B (SEB) in milk was 3 pg/ml. Using the system for detection of Escherichia coli O157:H7 in ground beef showed a sensitivity of <25 cfu/ ml (250 cfu/ gram with no culturing). Two silica nanospring mat electronic biosensor devices were fabricated and were shown to be useful in sequence specific detection of DNA. Methods were also developed for the synthesis of fluorescent silica nanoparticles which can be used to enhance detection methods. Locked Nucleic Acids (LNA) are being evaluated as a novel nonmaterial for biosensor applications. Specific sequences were designed and synthesized and hybridization efficiency experiments are underway with the newly synthesized LNA. Two types of nano-FET devices have been designed and fabricated. Both devices have more than one nanowire between source and drain. Device characterizations were performed by using electrochemical impedance spectrum and conventional electronic parametric means (Keithly). Field effect transistor behavior of the nanowires was observed in both types of devices. The new devices demonstrated stability in the electrical field and operated over long test time periods. Further improvement of the yield of nano-FET devices is needed. Staphylococcal enterotoxin B and C were used as the targets in the detection model using nano-FET devices . Bio-conjugation of antibody and protein was achieved by using a novel and highly efficient approach. Five PNA capture molecules were designed and custom synthesized. Hybridization of PNA and signal molecules was achieved at room temperature with no mis-match. Further investigation is required in the sensitivity of detection. Microelectronic logical readout chip has been designed and fabricated. There were 1500 logical readout units on a single die. Surface modification of microelectronics will be performed in Cornell Nano-Facility for characterization of device functions.
PARTICIPANTS: Individuals: Application of biosensors to food products and development of biosensor system: Larry Branen: Principal Investigator, general oversight for project; Josh Branen: Co-Principal Investigator and Research Scientist and lead for biosensor applications; Seth Gibbon: Lab technician; Martha Hass: Lab Technician. Electrical engineering and molecular biology expertise and development of nano transistors and bioluminescence detection methods: Wusi Maki: Co-Principal Investigator, lead on molecular techniques; Gary Maki: Co-Principal Investigator, lead on microelectronics; N.N. Mishra, Res.Sci./Nano-FET development; B. Filanoski, Sci. Aide/Bioconjugation; S. Ristogi, Post-Doc/PNA Synthesis. Partner Organizations, Collaborators and contacts: Dave Newcombe, Rhena Cooper, Dave McIlroy, Eric Aston, Patrick Hrdlicka, Jim Nagler, Cornell NanoScale Facility (CNF), USDA North Central Project on Biosensors and Nanotechnology. Training or professional development Internships and practicum opportunities are being used to provide hands-on training in the area of diagnostic technologies and technology development for North Idaho College and University of Idaho undergraduate students.
TARGET AUDIENCES: Target audiences: Large and small scale food producers, Idaho small businesses and technology-based companies, farmers/ on-site researchers. Efforts: Internship and practicum opportunities to provide hands-on training in the area of diagnostic technologies and technology development for North Idaho College, Eastern Washington University and University of Idaho undergraduate students. Assistance to local biosensor companies. Numerous talks given locally and regionally regarding the work.

IMPACT: 2007/09 TO 2008/08
The early, fast and accurate detection of biological pathogens is an important element for ensuring the safety and shelf life of agricultural commodities. Each year, foodborne bacterial pathogens account for over 3.5 million illnesses and $6.9 billion in costs in the United States. In addition the potential for contamination of water supplies is significant and a growing problem around the world. Current technologies for the detection of microbial pathogens in food or water are slow in producing a positive diagnosis, can be costly (PCR) and require the use of skilled professionals. The technology advanced in this work will produce sensitive, easy to use and low-cost detection results. Currently many food products must be held in storage an additional time to allow accurate testing for pathogens. With the proposed electronic detection, this time delay can be greatly reduced assuring greater freshness and safety to the consumer and lower cost to the producer. Being able to quickly determine a change in water quality in streams, rivers and lakes is also essential. The ability to detect multiple organisms in the multiplex system will allow simultaneous screening for several organisms and toxins at one time. The detector could also be used to follow potential food or water contamination and poisoning outbreaks. Through the use of a microprocessor on-board the detector, information could be directly sent and collected via the internet to the USDA or other federal and state agencies. Such knowledge in a near real time fashion would be extremely valuable in response to emergency situations. The program can also lead to significant economic development through the development of industries that manufacture and utilize the electronic biosensors developed through this research. The biological materials and the hardware already developed in this project will have significant impact in preserving the safety of food and water systems.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Natural Toxins
Bacterial Pathogens
Food Defense and Integrity