We will meet the need for a fundamentally new method of detection for biotoxin agents. Our strategy is based on using the cell surface receptor targeted by biological toxins or pathogens as the core of the sensor. Receptor-cell membrane mimics will be generated using self-assembly techniques where monolayers and bilayers are bound directly to an optical transducer surface. In addition, advanced transduction techniques that mimic the ability of natural systems to amplify signals will be developed. Our ultimate target is sensor arrays based on multiple receptor-cell membrane mimics that can serve as a """"""""smart"""""""" sensing system capable of rapid identification and quantification of a wide range of biological toxins. Many toxins and pathogens enter cells through a general mechanism that begins with their binding of receptors on the animal cell surface. Most of the primary receptors have been identified for biological agent toxins, many of them have been identified for bacterial pathogens, some of them have been identified for viral pathogens. While cholera toxin was chosen for initial studies because of its experimental accessibility, ganglioside binding is a common feature of bacterial toxins. For example, the toxins from tetanus, botulinum, perfringens, and shiga and the toxin ricin all bind gangliosides before entering the cell. Thus, everything we learn about cholera toxin will be directly applicable to developing sensors for other important toxins such as botulinum and ricin. We have established two systems for detecting cholera toxin binding to its glycolipid receptor; one based on fluorescence detection by flow cytometry, and the other based on mass detection by surface plasmon resonance. In both cases the receptor is embedded in a biomimetic surface and toxin binding to the surface is detected. In flow cytometry (FC), the surface is a microsphere and the toxin is labeled with a fluorescent probe, whereas for surface plasmon resonance (SPR), a gold-coated planar surface is used and toxin binding is detected as a change in refractive index at the surface. These two systems represent our first generation of toxin sensors and will provide platforms with which we will optimize molecular recognition, the biomimetic substrate, and detection of binding. Systematic investigation of the receptor and toxin concentration dependence of these kinetics is in progress and will be interpreted in terms of a rigorous kinetic model to determine the individual rate constants (binding affinities) for the mixed, mono,-and multivalent binding events we envision. We will investigate the effect of receptor immobilization and of various biomimetic substrates on this interaction. These results will be used to guide the design of sensor surfaces with the desired sensitivity, specificity, reversibility, and stability.
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