Membrane proteins are one of the most important classes of molecules, representing over half of drug targets, and also one of the most poorly understood classes. This award is to leverage new technologies in nanofabrication, nano-optics, microfluidics and protein engineering to develop a nanopore membrane biosensor, which has the potential to transform membrane protein research. This real-time imaging instrument will exploit periodic nanopores patterned in a thin gold film to measure the binding kinetics and affinities between membrane-bound receptors and ligands - a task that is highly desirable but is rarely achieved with existing affinity biosensors such as BIAcoreTM. A thin gold film perforated with periodic nanopores will concurrently act as a mechanical scaffold to support cell membranes as well as a label-free surface plasmon resonance (SPR) sensor. Binding of ligands to immobilized membrane receptors will sharply modulate the light transmission through metallic nanopores, which is recorded as a real-time "movie" with high resolution. The system will be integrated with microfluidics to reduce sample consumption and enable multiplexing. While this instrument will be applicable to analyze potentially any membrane protein in a near-native lipid bilayer environment, initial studies will be performed with the key recognition events controlling cellular immunity. The ability to perform ligand screening for membrane receptors at high throughput and high resolution would present a breakthrough in studying these molecules and the fundamental life processes they enable. The integration of supported and free-standing lipid membranes with nanopore SPR sensors will provide an entirely novel approach for dynamic probing of membrane protein interactions, rather than adding incremental improvements to existing SPR instruments. To encourage adoption of the technology by biological groups, a two-day short course at the University of Minnesota (www.nano.umn.edu/biomems09/) will be expanded to include nanopore array fabrication, while the software and fabrication protocols will be freely available on the research website. To excite underrepresented groups about STEM research, SPR and protein-protein binding experiments will be incorporated into a training plan spanning middle-school through graduate students, placing special emphasis on undergraduates.
