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.
The goal of this project is to develop a new biological instrument based on gold nanopores for optical sensing of membrane protein interactions. Intellectual merit: Surface plasmon resonance (SPR) sensors utilizing gold films have been the stardard tool for studying protein-ligand interactions to measure their affinity and binding kinetics. Commercially available SPR sensors, however, suffer from several drawbacks, because it is difficult to combine conventional SPR sensors with lipid bilayer membranes and membrane proteins incorporated therein. Membrane proteins play key roles in a variety of biological processes, yet they are difficult to integrate with solid-state sensors because the presence of the underlying surface often perturbes their functions or even denature them. This project aims to develop SPR sensors using nanoporous gold films to naturally incorporate transmembrane proteins inside free-standing lipid membranes inside such pores. To address this technological challenge, the PI's team first developed a high-throughput nanofabrication technique to produce uniform and high-quality gold nanopore films with low cost. Subsequently, the substrate was incorporated into a fully functional instrument, and such effort required a series of instrument development efforts including surface modification protocols, data acquisition and analysis, as well as optical design and buildup. All of these goals have been accomplished, and the details on instrument development have been documented in more than 10 journal articles published. Proof-of-concept experiments demonstrated that membrane proteins can be incorporated into nanopore-spanning lipid membranes and used for real-time SPR biosensing. This is a unique capability that is very difficult to perform using other types of SPR instruments. Broader Impact: The chip fabrication protocol as well as instrumentation techniques developed in this project are relatively easy to duplicate, and the PI expects that more researchers will gradually adopt this technology. Additinally, to facilitate the dissemination of the knowledge and techniques developed herein, the PI's lab members annually held hands-on workshop and invited people from industry and academic, as well as local college to learn about microfluidic chip fabrication, the use of the nanopore sensors, and to perform protein binding measurements. The PI's graduate course on biological instrumentation consists of lab sessions to demonstrate the nanopore chip fabrication and SPR biosensing of proteins. The PI's team members also annually visited the Science Museum of Minnesota during their NanoDays events in April for interaction with school kids and their parents, and performed optics demo experiments.
