This project develops a ultra-miniaturized microarray platform for the detection of bacterial exotoxins. Bacterial exotoxins are the primary virulence factors for many human diseases. As they are easily carried through food and water borne pathways, they pose a significant threat to public health, especially in underdeveloped countries where food and water sources are not secure. Their use as an instrument of bioterrorism cannot be overlooked. The first line of defense against these pathogens is a rapid detection, but this has become more difficult over the past several years as the number of pathogens has proliferated.
The microarray platform used in this project specifically addresses the needs for detection systems which are rapid and can identify a toxin from among hundreds of possibilities. The microarray displays an assembly of capture molecules, each of which selectively binds to a different toxin. The capture molecules used by the array are the natural membrane receptors which the exotoxins use to target and attach to cells before infecting them. As such, these receptors bind the toxins with high selectivity and affinity. The receptors are displayed in the array by first sequestering them in supported phospholipids bilayers which are formed around polystyrene particles of the order of ten microns in size. Batches of lipobeads are prepared separately, with each batch displaying a single receptor whose identity is encoded by a spectral barcode composed of the emission spectrum of luminescent nanocrystals (quantum dots or Qds)encapsulated in the bead. Lipobeads, each with a different receptor and identifying barcode, are then arrayed on a surface by attaching them to microwells pre-patterned on the surface. This research will develop a fluorescence detection system for identifying the binding of a toxin to a particular capture molecule in the array without having tolabel the toxin or perform subsequent assays on the bound toxin. The label free system is based on measuring the changes in the resonance energy transfer between donor and acceptor fluorescence pairs pre-labeled in the bilayer. This as is assaying will allow a much shorter screening time compared to present approaches which usually require further analytical steps. The miniaturized design also allows for the display of receptors at different concentrations in the bilayers which will allows the binding affinity to be measured. This additional information allows toxins to be identified with greater accuracy, particularly in the case where two bind to the same receptor.
Intellectual Merit:
The ultra-miniaturized, label-free microarray for toxin detection developed in this proposal allows for the rapid screening of analyte samples for the detection of a particular toxin from a potential list of hundreds. The design can be adopted to the general display of membrane receptors for understanding binding interactions in drug discovery, and the lipobeads used as array elements can alo be used in flow cytometry.
Broader Impact:
The research will support two graduate students and undergraduates, each of which will be educated in interfacial and biological sciences. Their research will also be in collaboration with the Soft Material IGERT Program in the Department of Chemical Engineering at City College whose focus it is to educate students in the various disciplines of soft matter physics.
