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<p>Goals: The overall objective of the research effort will be to demonstrate the feasibility of replacing antibody based immunoassays with extremely stable synthetic glycoligands as target probes for the real-time detection of E. coli and its toxins. The project will demonstrate the detection using a rapid detection electronic platform. Objectives: </p>
<p>1. Synthesize tailored glycans for E. coli O157:H7, Shiga-like toxins Stx-1 and Stx-2. </p>
<p>2. Integrate the capture ligands onto sensor platform and test for specific detection of target pathogens individually. </p>
<p>3. Develop a methodology for rapid isolation of pathogens from complex food matrices. </p>
<p>4. Demonstrate the assay capabilities in relevant food matrices such as ground meat, hamburgers, dairy etc. </p>
<p>5. Determine the figures of merit such as specificity, limit of detection (LOD), detection time, false-positive and false-negative probabilities. </p>
<p>Expected Outputs: A major advancement is expected in real-time portable sensors as an alternate to currently employed delayed, laborious, and costly detection methods for positive identification of food-borne pathogens. A technologically and practically simple test is envisioned so that this novel system of detection can be quickly implemented in a food handling/processing setting. The label-free technology, real-time response, high sensitivity and increased stability, combined into one sensor platform, is expected to revolutionize the way foodborne pathogens are detected. Additionally, the sensor components are "generic" so that ligands against other pathogens, toxins can be incorporated into the system, opening up a wide array of public health commercial potential for the sensor system.</p>
<p>NON-TECHNICAL SUMMARY:<br/> This project involves the development of a label-free detection technique for real-time and high-sensitivity detection of foodborne pathogens in meat processing and packaging facilities. It will be based on the development of highly specific and temporally-stable capture ligands for target pathogen detection. The detection technique will be based on measuring extremely small changes in the physical environment of a surface due to the binding of target pathogens. The capture ligands are extremely stable and make the assay suitable for field operations. The system to be developed will perform sample collection and be compatible with stand-alone operation in a wireless network setting. This approach will provide an alternate to the gold-standard antibodybased assays without compromising the binding affinities for target pathogens. Currently, there
is no label-free sensor technology to detect foodborne pathogens and toxins in in a real-time. The technology to be developed will incorporate three distinct key aspects: (1) a label-free capture probe for target pathogens, (2) a real-time detection platform, and (3) testing in relevant food matrices. The work proposed here will offer great benefits to foodborne pathogen detection, disease prevention and combating disease outbreak through contaminated food. It will result in a real-time technology that will replace traditional laboratory based assays for contamination analysis, which take several hours to identify pathogens. It will also result in a pathogen detection assay technology having the unique ability to rapidly detect biological pathogens without the need for labeling the captured targets, and offer the ability to also determine the concentration of the pathogens while
simultaneously confirming their presence. It will possess the reliable and portable detection capability needed to assess the quality and safety of meat and food products, and also be widely applicable in sectors such as water monitoring, agriculture, medical diagnostics, etc. The key to the proposed system is the reliability and small-size that will allow for implementing a network of sensors across a large food processing plant and at the same time can be used by small farms and meat packaging plants. The development and field testing of stand-alone systems to satisfy the necessary performance characteristics, such as accuracy, sensitivity and reliability, will be a tremendous step towards the successful commercial large-scale manufacturing of biosensors for foodborne disease detection and prevention.
<p>APPROACH:<br/> A majority of the current techniques in foodborne detection have huge variations (1 h to 8 h) in their total analysis time, which is the total time from the beginning of sample collection to a signal response. The proposed system with a microsecond response time, is expected to be several orders of magnitude faster. The technology based on label-free detection and highly stable reagents which do not require freezing, will greatly reduce the cost of consumables and make it economically viable for both small-scale and large commercial meat processing facilities. From a device point of view, automatic sample collection compatible with meat processing operations, easy readout and low power consumption will provide the necessary requirements and performance capabilities to replace currently existing systems for food-borne pathogen detection. Synthetic
glycoligands specific to E. coli and its toxins will be designed and developed followed by conjugation of these ligands to the sensor platform using standard conjugation chemistry. The sensor will be designed with control tests built into the platform for analyzing the cross reactivity. The sensor will be exposed to "spiked" real-world food samples such as ground meats, dairy etc. to test the detection limts and capabilities. These detection limits will be compared to traditional antibody-based immunoassays that will be performed on the same samples.