The overall goal of the project is to develop improved sensors, biosensors and methodologies to measure the quality and safety of food products during production, processing, storage, or retail sale. <P>
Specific project objectives are to: <OL> <LI> Improve the understanding of receptor-ligand binding forces through measurement of single-molecule interactions <LI> Improve methods of immobilizing sensing molecules, i.e. enzymes or antibodies, to improve stability, activity, and/or specificity to the target analyte or antigen <LI> Develop novel sensing strategies for food applications.
NON-TECHNICAL SUMMARY: The entire food industry from producers to consumers are increasingly concerned about the quality, safety, traceability, and origin of products and ingredients. To improve quality and safety, sensors are needed to rapidly measure quality and safety related parameters throughout the production chain. This project examines various antibody or enzyme-based sensing methods to evaluate their effectiveness in detecting safety and quality of selected food products. <P>
APPROACH: Procedures to meet the objectives include biosensing technologies based on antibody-antigen recognition or enzyme activity. The main limitations of many of the current methods are lack of sensitivity, specificity, speed of response, sensor lifetime, and reusability. All of these factors can be related to the micro-environment and function of the sensing molecules. <P>Obj 1:For measuring the force interactions between biomolecules, technologies such as a Scanning Force Microscope (SFM) will be used. Unbinding forces will be measured between biomolecules (receptor-ligand pairs) of interest for sensing food safety or quality. For example, antibodies specific for Listeria monocytogenes will be immobilized to cantilever tips and brought into contact with the bacteria. After the antibody binds to an antigen (epitope) on the surface of the bacteria, the cantilever will be retracted and force versus distance will be recorded. These unbinding force experiments can be repeated on the same cell surface with the same antibodies from 10 to 100 times to obtain a statistical distribution of forces. This distribution of forces can then be analyzed to estimate the force for single-molecule interaction. <P>Obj 2:Immobilization of the sensing molecule(s) is key to retaining the activity of the enzyme or antibody while keeping it in close proximity to the transducer, e.g. electrode or fiber optic detector. Various immobilization methods for sensing molecules will be investigated to: maintain the activity of the sensing element, immobilize an optimum number of sensing molecules, and subsequently improve sensor lifetime. Most antibody-based sensors for detection of pathogenic bacteria in foods use surface immobilization methods to hold the antibody in a proper orientation for detection of the specific antigen. For all immobilization methods, the effects of environmental conditions on the bioactivity of the sensing molecules will be evaluated. For enzyme-based sensing methods, the enzyme can be immobilized on a surface using methods similar to those used for antibodies, or the enzymes can be trapped in a porous matrix. Again, the effect of the immobilization strategy on the activity of the enzyme under various environmental conditions will be evaluated. <P>Obj 3:Procedures for detecting contamination of ready-to-eat (RTE) meat will focus on the development of fiber-optic fluorescent immunosensors and bioelectrical impedance of mammalian cells exposed to pathogenic microorganisms. The study of immobilization methods and binding forces from objectives 1 & 2 will aid in the development of improved sensors. These sensing strategies normally will require extraction of the bacteria from food samples and selective culture to increase their numbers for detection. The antibody-antigen binding force should be related to the capture efficiency of the antibodies for their target antigen. We will attempt to demonstrate that antibodies with higher binding forces have improved immunocapture efficiency by comparing their performance in various food systems.