The overarching goal of this project is to develop detection systems that integrate efficient sample handling with rapid, low-cost, ultra-sensitive detection of microbial pathogens and toxins in agricultural, food and environmental sources and samples. <P>
The early, fast and accurate detection of biological pathogens is an important element for ensuring the safety and shelf life of agricultural commodities resulting from both accidental and intentional food contamination. <P>This work will play a major role in preventing bioterrorism in our food supply. Previously, we have developed a biosensor system capable of detecting multiple organisms and/or toxins allowing simultaneous screening for several agents. <P>We propose to further the development of these electronic, electrochemical, and optical biosensor systems through the development and application of new nanotechnology and microelectronics toward several agricultural, food, environmental and health systems. <P>Our goal is addressed by four subprojects: Development of a real-time food sampling sensor utilizing silica nanosprings; biofunctionalization of nanomaterials for pathogen detection; nanomaterial-based methods development for gene regulation in preharvest meat muscle and fat; nanoelectronic sensor fabrication for Listeriolysin O detection.<P> The four projects are unique, yet complementary, and cover a broad range of food pathogen detection methodologies. The first project objective is to model the flow characteristics of our nanospring-based sensor platform and to characterize the performance of biofunctionalized nanosprings therein for detecting antibodies, DNAs and locked nucleic acids (LNAs). Parallel objectives are the development of a dry reagent test strip as an optical biosensor (also antibody, DNA and LNA sensing), the application of surface-enhanced Raman spectroscopy in pathogen detection in an integrated immunomagnetic assay, and the fabrication and testing of a nano field-effect device (nanoFED) for the capacitive detection of Listerolysin O via antibody immobilization. Complementary objectives are directed at shelf life extension and prevention of meat product contamination via lipid oxidation that leads to rancidity; the synthesis and testing of biofunctional gold nanoparticles for gene knockdown will be evaluated for effectiveness at targeting muscle cells and the lipid oxidation connected with meat quality deterioration. These approaches use antisense oligonucleotides and muscle-targeting peptides in gene regulation to understand mechanisms for improving shelf life of meat through pre-harvest treatments that would reduce post-harvest fatty acid oxidation. The same targeting protocols can be implemented in sensor platforms toward muscle cell interrogations for quality control. Each of these objectives are expected to be successfully addressed over a 12-month funded period. <P>The expected outputs will be at least three different sensor platform demonstrations with the above targets successfully tested for detection limits and the commensurate improvements in understanding the effectiveness of the targeting protocols, which will lead to further sensor optimization and new targets.
NON-TECHNICAL SUMMARY: The threat of food poisoning and other agricultural related contamination is a particular concern and the challenge of maintaining expected product quality and safety for humans and animals is great. Over 40 different food-borne microbial pathogens cause an estimated 30 million cases of human illness each year costing >$12 billion annually, and the potential for bioterrorism through the food supply is a clear and present threat. To sustain food safety and to assure the continued high quality of agricultural commodities, it is essential that we have methods to detect, predict and/or prevent contamination of foods and other agricultural commodities, which includes preservation and shelf-life strategies. Rapid detection of bacterial pathogens and toxins is critical for prevention. Current detection methods cannot meet the requirements for rapid, ultra-sensitive and accurate diagnostics. Electrical biosensors have a number of characteristics (low cost prodcution, robust function, improved performance, easy handling) that will contribute to point-of-care/field-based technologies for food quality analysis. We have previously developed electrical and optical biosensor systems for rapid and low-level detection of micro-organisms and toxins using low-cost materials and small equipment. The biosensors are based on the use of various nanomaterials (e.g., fluorescent and magnetic nanoparticles, modified nanowires and nanosprings) to isolate and concentrate targets from food products and to integrate these materials and detection schemes into sensor platforms. The future of electrical biodetection will be based on this successful integration strategy, resulting in rapid and sensitive bionanosensors as highly-compact devices unrivaled by standard materials and techniques. This project is designed to take our four prototypes into further development for improving consistency, performance, sample handling and expanding to other targets for proof of versatility in application. Four distinct aims will support our overarching project goal of producing sensor platforms that integrate efficient sample handling with rapid, low-cost, ultra-sensitive detection of microbial pathogens and toxins in agricultural, food and environmental sources and samples: Development of a real-time food sampling sensor utilizing silica nanosprings; biofunctionalization of nanomaterials for pathogen detection; nanomaterial-based methods development for gene regulation in preharvest meat muscle and fat; nanoelectronic sensor fabrication for Listeriolysin O detection. The four projects are unique, yet complementary, and cover a broad range of food pathogen detection methodologies. The research teams bring a breath of experience in electronic detection, nanomaterials development and characterization, and biological sciences. The integrated teams will establish new protocols for pathogen detection that dovetail with advanced food safety nanotechnology in sensing.
APPROACH: The method of assembly and testing of the first, nanospring-based sensor will be through a two-electrode capacitive design that also is used in a flow-through microreactor detection scheme. The sensor platform has already been designed and its equivalent circuit model is understood, so the flow-through operation will first be fine-tuned and tested to determine range of feasible function. The nanosprings will be modified with antibodies, DNAs and LNAs, in parallel, for selective targeting of paired moieties and characterized for sensor response via impedance spectroscopy analysis. The second sensor platform (dry reagent test strip) for optical detection is designed to conduct lateral flow of the fluorescent nanoparticles with biorecognition elements to transport the colored targets only. Lowest detection limits will be determined as well as off-target or noncomplementary detections for false positives. The third detection methodology of using Raman-enhancing nanoparticles with biorecognition elements will be evaluated by determining the effective amplification of Raman spectra from the target molecule. These materials may be dispersed in a sample, retrieved and concentrated for testing using magnetic separation or integrated into a fixed sensor device. Methods for the fourth detection approach include fabrication with continued optimization of existing and modified nano field-effect devices (nanoFEDs), electrical behavior characterization through I-V sweep analysis, demonstration of selective capacitive-type alternating current measurements on the nanoFED with a concentration study on response to Listerolysin O (LLO) via antibody caption and a layer-by-layer LLO deposition study to understand and model equivalent circuit. All of these efforts will provide substantial increases in knowledge of biodetection methodologies and result in useful sensor materials and devices as outputs, which will be evaluated against other known sensor types for performance, ease of use, and relative cost and complexity. Final evaluations will be proved through external review and publication of results and/or adaptation of sensor technologies into patents, inventions, and technology transfer to industry.