David Walt develops new array technologies and uses them to collect large amounts of data that can be used to understand fundamental aspects of genetics and cell biology. His HHMI project integrated this experience into undergraduate research, teaching, and outreach efforts and will make scientific research accessible to students who would not normally have the opportunity.
Enzyme Mechanisms and High-Sensitivity Disease Diagnosis Using Single-Molecule Arrays
Single-molecule studies provide unique information about heterogeneous molecular behaviors that are hidden using bulk methods. We enclose single enzyme molecules with substrate in an array of 50,000 individually addressable microchambers etched into a glass optical fiber bundle. With fluorescence microscopy, we monitor the substrate turnover of thousands of individual enzyme molecules simultaneously. The large number of single enzyme molecules provides excellent statistics for analyzing the activity distributions between different molecules. From these differences, we can learn about the reaction mechanisms of the enzymes. If we add an inhibitor to the enzyme/substrate solution in the microchamber array, we are able to observe inhibition kinetics on a single-molecule basis. In addition to these fundamental studies, we have also developed high-sensitivity detection methods, which allow us to count single DNA and protein molecules in the microchambers. This approach is a thousand-fold more sensitive than other analytical methods, such as ELISAs. We are attempting to use the unprecedented sensitivity of this method for early diagnosis of diseases, such as breast cancer and infectious disease, from blood samples. Both projects will allow the student to work on the cutting edge of single-molecule detection. The project will include preparing fiber-optic microarrays, loading enzymes and other molecules into the microchambers, working with fluorescence microscopy, and analyzing substrate turnover of individual molecules.
Single-Cell Protein Analysis
The underpinnings of this project are a series of technological innovations based on using the ends of optical fiber bundles as very small, femtoliter-sized reaction wells containing single cells whose properties can be individually interrogated using fluorescent probes. Varying fiber diameters allows us to optimize the size of the microwells to diminish occupancy by multiple cells. We are investigating the ability to perform single-molecule protein analysis to examine biological phenomena at the single-cell level. We are developing methods to capture and lyse cells and analyze their content for low levels of proteins using a single-molecule counting scheme. Individual cell protein concentration information is presently unattainable despite tremendous advances in the ability to perform single-cell sequencing. By coupling protein concentrations with sequencing or genotyping information, a more comprehensive understanding of cell-to-cell variation will be possible. We will apply these protocols to the solution of biological problems in mammalian tumor cells where genetic heterogeneity is already known to be biologically important: Cell-to-cell variation can lead to different clinical outcomes. If successful, this project will provide a powerful new technology broadly applicable to multiple areas of biological research. Examples include the ability to study variations within tumor cell populations, selection rates for particular cell phenotypes during tumor progression, responses to chemotherapeutic regimens at the cellular level, and characterizing the spread of viral infections through the organism.