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The primary goal of this project is to develop integrated electronic and nano-electronic devices and systems for rapid, low-cost, ultra-sensitive detection of bacterial pathogens and toxins in food samples. Rapid detection of bacterial pathogens and toxins in the food supply at or near the source of contamination is critical for preventing contaminated food distribution. The proposed project will support our goal through the completion of three objectives: development of an integrated nano-FET for ultra-sensitive, multiplex detection of bacterial toxins; development of a compact, Enzyme-linked Immunomagnetic Electrochemistry (ELIME) biosensor for detection of Staphylococcal enterotoxin B (SEB) in milk; and development and characterization of food pathogen detection assays based on utilizing silica nanosprings and locked nucleic acids (LNA). Based on our previous research in the development of nano-FET biosensors, we will develop an integrated nano-electronic device for ultra-sensitive detection of bacterial toxins in food samples. The overall system concept consists of the following elements: a) target recognition, b) nano-FET device surface modification and characterization, c) detection of the presence of the target molecules on the sensing surface of nano-FET, and d) microelectronics to amplify nano-FET signals to produce digital signals. A rapid, on-site biosensor will be developed using the integration of ELIME assay protocol with an extremely low-cost detection and sample handling components. Components and assay parameters will be modified and evaluated to determine fast, simple and sensitive detection of SEB in milk. Finally, we will develop methods to incorporate two novel nanomaterials (silica nanosprings and LNA) into diagnostic assays for food pathogens. Each material will be characterized for diagnostic capabilities in isolation and investigation of the combined properties of the materials will be initiated.
NON-TECHNICAL SUMMARY: Maintaining the expected quality and safety of food products is often challenged due to potential chemical and biological contamination. Of particular concern is the threat of food poisoning. Over 40 different foodborne microbial pathogens cause an estimated 30 million cases of human illness each year costing >$12 billion annually. A new threat to the food supply is the potential for bioterrorism. Intentional contamination of livestock, crops and foodstuffs is possible and deliberate attacks can imitate natural or common outbreaks of disease, and may be difficult to identify quickly. To protect the safety of the food supply and to assure the continued high quality of agricultural commodities, it is essential that we have methods available to predict the potential contamination of foods and other agricultural commodities and to increase the preservation and shelf-life of these commodities. Rapid detection of bacterial pathogens and toxins in the food supply is critical for preventing contaminated food distribution. Current detection methods used in the tests cannot meet the requirement for rapid, ultra-sensitive and accurate diagnostics. Electrical biosensors have a number of characteristics (low cost, robust, and easy to handle components) that will help open the door to point-of-care/field-based sensor technologies required for on-site food quality analysis. We have previously developed an electrical biosensor system for the rapid and sensitive detection of micororganisms and toxins using low-cost materials and equipment. The biosensor system is based on the use of magnetic particles to rapidly isolate and concentrate targets from food products and integration of these materials into a magnetic electronic sensor system. This project is designed to further tune assay parameters, sensor materials and sample handling. The future of electrical biosensor development is based on the integration of nanomaterials with sensor designs. This can result in sensors with rapid and sensitive detection in highly compact devices not achievable with standard materials. Recently, nanowire Field Effect Transistor (nano-FET) based electronic devices have shown promising results in the detection of bio-molecules with the attractive features of being label-free and ultra-sensitive. Based on our previous research in the development of nano-FET biosensors for the detection of Staphylococcus aureus enterotoxin B, we propose to develop integrated nano-electronic devices for ultra-sensitive detection of bacterial toxins in food samples. The above system is envisioned to reside on a single silicon die and embody an on-chip intelligent system that is easy for an end user to deploy for real time detection and can also be cost effective when produced in large quantities. The development and characterization of novel nanomaterials in the scope of specific applications will allow further advances to nano-based diagnostics (e.g., electrical nanobiosensors) and two novel materials,nanosprings and locked nucleic acids, will be included in the study.<P>
APPROACH: We will complete three objectives in this project that will lead to improved electrical biosensors for food pathogens and toxins. <P>Objective 1: Development of integrated nano-FET for ultra-sensitive, multiplex detection of bacterial toxins. Task 1: Improve the quality and yield of nano-electronic devices. Task 2: Demonstrate toxin detection models on nano-FET devices and characterize detection sensitivity and specificity. Task 3: Design and fabricate microelectronics for future integrated detection system. <P>Objective 2: Development of compact, Enzyme-linked Immunomagnetic Electrochemistry(ELIME) biosensor for detection of Staphylococcal enterotoxin B in milk. Task 1: Design and construct active carbon epoxy electrodes and magnet-carbon epoxy working electrode and integrate into three electrode device. Task 2: Measure the collection and detection of enzyme-labeled magnetic particles. Task 3: Optimize assay procedures for rapid detection of SEB in milk. <P>Objective 3: Development and characterization of food pathogen detection assays utilizing silica nanosprings and LNA. Task 1: Immunodetection of SEB on silica nanospring mats (SN) and gold (Au) nanoparticle decorated silica nanospring mats (AuNpSN). Task 2: Detection of Salmonella species-specific DNA sequence using LNA. Task 3: Integration of AuNPSN and LNA by bio-funtionalizing AuNPSN using thiolated LNA probes.