Increasing the Safety and Shelf Life of Agricultural Commodities

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.

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APPROACH: Develop a model prototype biosensor capable of analyzing for select food poisoning organisms, ie., enterotoxin-producing staphylococci in pure culture. Subsequent goals will be to authenticate the use of the biosensor and develop methods for use of the biosensors for detection of organisms within or on the surface of food and other agricultural commodities, water, air and food processing equipment.

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PROGRESS: 2004/09 TO 2007/08<BR>
OUTPUTS: The primary goal of this project is to develop bioelectronic detectors that can quickly detect the presence of microbial pathogens in food products. A biosensor system has been designed consisting of three steps: target capture, signal generation, and detection. We have determined that each step is distinct from the next and can be optimized in a parallel fashion to the other steps. In the target capture step, magnetic beads that have been functionalized with antibodies, DNA and DNA/RNA aptamers were used to capture and concentrate proteins, DNA and whole cell microorganisms in both model and food systems. Simultaneous capture of all of these components through the use of specific capture materials was achieved and cross reactions were minimized. Whole cells of Escherichia coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes and toxins from Staphylococcus aureus were detected simultaneously. A new signal generation and amplification system (bio-nanotransduction) has been designed and tested which is predicated on the use of a nano-sized biomolecule (RNA) as a general biological signal for biological recognition events. By transducing the specific biomolecular interaction event into a nano-sized generic biological signal (RNA), the detection platform is uncoupled from the specific biological recognition event. As a universal signal molecule, the RNA can be detected directly on a charge detection device or indirectly using an electrochemical or optical device through a labeled secondary signal element. Two electronic systems, enzyme based amperometry and biological charge detection on a field effect transistor, were compared with enzyme based fluorescence detection. The system can be used for multiplex detection of several proteins and microorganisms simultaneously using a newly designed electrochemical test device using reusable or disposable electrodes. The system can be monitored through a hand-held potentiometer. A nanogold-ferrocene signal structure and several other electrochemically active molecules have been evaluated for use to report biological interaction events electrochemically. The RNA nano-signal can also be detected using a field effect transistor based on detection of the intrinsic charge of the RNA nano-signal. A relationship with the Cornell NanoScale Facilty (CNF) has been established and two sets of nano transistors were fabricated by CAMBR scientists at CNF. The first set of devices produced nano wire devices with diameters ranging from 70 nm to 500 nm. The second set of devices with feature sizes of 30 nm plus or minus 5 nm were formed using e-beam lithography. Electrical tests using Electrochemical Impedance Spectroscopic methods have shown that the nano transistors are functional. Biomolecular testing is underway. Peroxylate chemiluminescence and bioluminescence detection methods for the development of a CMOS imaging chip based pathogen detector were also studied. A new optical element to increase spatial resolution for the development of high throughput assay on an imaging chip has been invented. One patent application resulted from the work entitled " Gene Expression Luminescent Biosensors". <BR> PARTICIPANTS: Larry Branen, PI; Josh Branen, Research Scientist; Martha Hass, Technician; Seth Gibbon, Technician; Erin Douthit, Sr. Technician- Application of biosensors to food products and development of biosensor system and bionanotransduction signal generation system. Expertise in food science, molecular biology, chemistry and microbiology. Greg Bohach, Co-PI- Expertise on microbiology. Gary Maki, Co-PI and Wusi Maki, Co-PI- Electical engineering and molecular biology expertise and development of nano transistors and bioluminescence detection methods. Collaborators: Cornell NanoScale Facilty (CNF) USDA North Central Project on Biosensors and Nanotechnology Training provided to graduate students at University of Idaho in Molecular Biology and Electrical Engineering; and undergraduate students from local community college. <BR> TARGET AUDIENCES: Food and agricultural industry and related governmental agencies are primary targets. Consulting has been provided and written and oral reports distributed to these groups and to a national research group in USDA.
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IMPACT: 2004/09 TO 2007/08<BR>
The early, fast and accurate detection of biological pathogens is an important element for ensuring the safety and shelf life of agricultural commodities. Staphylococcal food poisoning is the most common type of food-borne illness in the US and in a typical year, affects 1.5 million Americans, causing $1.2 billion in economic losses. Current technologies for the detection of microbial pathogens are slow in producing a positive diagnosis, can be costly if PCR techniques are employed and require the use of skilled professionals. The technology advanced in this work will produce near real time 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. 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 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 bioelectronic devices developed through this research.

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PROGRESS: 2006/01/01 TO 2006/12/31<BR>
The primary goal of this project is to develop bioelectronic detectors that can quickly detect the presence of microbial pathogens in food products. In a previous project, IDA00302-SG, a biosensor system was designed consisting of three steps: target capture, signal generation, and detection. We have determined that each step is distinct from the next and can be optimized in a parallel fashion to the other steps. In the target capture step, magnetic beads that have been functionalized with antibodies, DNA and DNA/RNA aptamers were used to capture and concentrate proteins, DNA and whole cell microorganisms in both model and food systems. Simultaneous capture of all of these components through the use of specific capture materials was achieved and cross reactions were minimized. Whole cells of Escherichia coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes and toxins from Staphylococcus aureus were detected simultaneously. A new signal generation and amplification system (bio-nanotransduction) has been designed and tested which is predicated on the use of a nano-sized biomolecule (RNA) as a general biological signal for biological recognition events. By transducing the specific biomolecular interaction event into a nano-sized generic biological signal (RNA), the detection platform is uncoupled from the specific biological recognition event. As a universal signal molecule, the RNA can be detected directly on a charge detection device or indirectly using an electrochemical or optical device through a labeled secondary signal element. In the signal detection step, two electronic systems developed in this grant, enzyme based amperometry and biological charge detection on a field effect transistor, are being compared with enzyme based fluorescence detection. The system can be used for multiplex detection of several proteins and microorganisms simultaneously using a newly designed electrochemical test device using reusable or disposable electrodes. The system can be monitored through a hand-held potentiometer. A nanogold-ferrocene signal structure and several other electrochemically active molecules have been evaluated for use to report biological interaction events electrochemically. The RNA nano-signal can also be detected using a field effect transistor based on detection of the intrinsic charge of the RNA nano-signal. A relationship with the Cornell NanoScale Facilty (CNF) has been established and two sets of nano transistors were fabricated by CAMBR scientists at CNF. The first set of devices produced nano wire devices with diameters ranging from 70 nm to 500 nm. The second set of devices with feature sizes of 30 nm plus or minus 5 nm were formed using e-beam lithography. Electrical tests using Electrochemical Impedance Spectroscopic methods thus far has shown the nano transistors functional. Biomolecular testing is underway. Peroxylate chemiluminescence and bioluminescence detection methods for the development of a CMOS imaging chip based pathogen detector are also being studied. A new optical element to increase spatial resolution for the development of high throughput assay on an imaging chip has been invented.
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IMPACT: 2006/01/01 TO 2006/12/31<BR>
The early, fast and accurate detection of biological pathogens is an important element for ensuring the safety and shelf life of agricultural commodities. Staphylococcal food poisoning is the most common type of food-borne illness in the US and in a typical year, affects 1.5 million Americans, causing $1.2 billion in economic losses. Current technologies for the detection of microbial pathogens are slow in producing a positive diagnosis, can be costly if PCR techniques are employed and require the use of skilled professionals. The technology advanced in this work will produce near real time 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. 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 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 bioelectronic devices developed through this research.
<BR> <BR>

PROGRESS: 2005/01/01 TO 2005/12/31<BR>
The primary goal of this project is to develop bioelectronic detectors that can quickly detect the presence of microbial pathogens in foods and food products. In the related project, IDA00302-SG, a biosensor system has been designed consisting of three steps: target capture, signal generation and detection. We have determined that each step is distinct from the next and can be optimized in a parallel fashion to the other steps. Two target capturing methods, two signal generation and amplification systems, and two detection devices have been developed. In the target capture step, magnetic beads that have been functionalized with antibodies, DNA and DNA/RNA aptamers were used to capture and concentrate proteins, DNA and whole cell microorganisms in both model and food systems. Simultaneous capture of all of these components through the use of specific capture materials was achieved and cross reactions were minimized. Whole cells of Escherichia coli and Listeria monocytogenes and toxins from Staphylococcus aureus were detected simultaneously. A target capturing microfluidic device design has been tested with computer simulations. In the signal generation step, a new signal generation and amplification system (bio-nanotransduction) has been designed and tested. Bio-nanotransduction is predicated on the use of a nano-sized biomolecule (RNA) as a general biological signal for biological recognition events. By transducing the specific biomolecular interaction event into a nano-sized generic biological signal (RNA), the detection platform is uncoupled from the specific biological recognition event. As a universal signal molecule, the RNA can be detected directly on a charge detection device or indirectly using an electrochemical or optical device through a labeled secondary signal element. A nanogold-ferrocene signal structure was also developed to report biological interaction events electrochemically. In the signal detection step, two electronic devices have been designed and tested. Through the use of bio-nanotransduction, the detection platform can be optimized to detect the RNA nano-signal. The system can be used for multiplex detection of several proteins and microorganisms simultaneously using a newly designed electrochemical test device using reusable or disposable electrodes. The system can be monitored through a hand-held potentiometer. The RNA nano-signal can also be detected using a field effect transistor based on detection of the intrinsic charge of the RNA nano-signal. A carbon nanotube field effect transistor based device has been designed which combines nanotechnology, microelectronics and a microfluidic system to achieve an advanced Lab-on-chip biosensor. This project will further the development of this biosensor system and apply it to the analysis of microorganisms in several food products. A provisional patent application has been filed on the use of the nanogold-ferrocene signal structures.
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IMPACT: 2005/01/01 TO 2005/12/31<BR>
The early, fast and accurate detection of biological pathogens is an important element for ensuring the safety and shelf life of agricultural commodities. Staphylococcal food poisoning is the most common type of food-borne illness in the US and in a typical year, affects 1.5 million Americans, causing $1.2 billion in economic losses. Current technologies for the detection of microbial pathogens are slow in producing a positive diagnosis, can be costly if PCR techniques are employed and require the use of skilled professionals. The technology advanced in this work will produce near real time 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. 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 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 bioelectronic devices developed through this research.
<BR> <BR> PROGRESS: 2004/01/01 TO 2004/12/31<BR>
The primary goal of this project is to develop bioelectronic detectors that can quickly detect the presence of microbial pathogens in foods and food products. In the related project, IDA00302-SG, a biosensor system has been designed consisting of a capture and amplification system and analysis via conventional fluorescent techniques and transistor based electronic detection. This project will further the development of this biosensor system and apply it to the analysis of microorganisms in several food products. The system will be optimized using Staphylococcus aureus as the test organism. The initial goal will be to optimize the conditions for production of efficient capture probes through the use of chemical conjugation procedures. Subsequently these probes will be used to optimize the signal generation systems for S. aureus DNA, whole cells, and enterotoxin and to further the development of the electronic detection system. Ultimately the biosensor system will be authenticated for use in detection of organisms in food products and methods will be developed for use of the biosensor system for detection of organisms within or on the surface of food and other agricultural commodities, water, air, and food processing equipment.
<BR> <BR>
IMPACT: 2004/01/01 TO 2004/12/31<BR>
The early, fast and accurate detection of biological pathogens is an important element for ensuring the safety and shelf life of agricultural commodities. Staphylococcal food poisoning is the most common type of food-borne illness in the US and in a typical year, affects 1.5 million Americans, causing $1.2 billion in economic losses. Current technologies for the detection of microbial pathogens are slow in producing a positive diagnosis, can be costly if PCR techniques are employed and require the use of skilled professionals. The technology advanced in this work will produce near real time 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. The detector could also be used to follow potential food 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 bioelectronic devices developed through this research.