Title: "ENHANCING THE TEMPORAL AND SPATIAL RESOLUTION OF SOLID-STATE NANOPORE SENSORS"
Prof. Amit Meller (*Advisor, BU, BME & Physics)
Prof. Mark Grinstaff (BU, BME & Chemistry)
Prof. Michael Smith (*Chair, BU, BME)
Prof. Kenneth Rothschild (BU, Physics)
Prof. Björn Reinhard (BU, Chemistry)
Nanopore-based single-molecule sensors have grown substantially more versatile since the initial detection of single-stranded nucleic acids with alpha-hemolysin. Genetic and chemical modifications of biological nanopores, fabrication of synthetic nanopores in solid-state materials, and the development of hybrid biological/solid-state nanopores have collectively enabled detection of analytes ranging in size from small single molecules and nucleic acids to large protein complexes. Cheap and fast nanopore-based next-generation sequencing promises long read lengths and the detection of epi-genetically modified bases, both of which are weaknesses of current sequencing-by-synthesis techniques. Yet in order to further develop nanopores as useful tools for basic research as well as commercial applications, temporal and spatial limitations must be addressed. Free electrophoretic threading of nucleic acids through a nanopore occurs on the order of 10-100 ns/nucleotide with a typical separation of 0.34 nm between adjacent nucleotides. This translocation velocity allows for discrimination based on large features such as molecule length, but is too fast to resolve smaller features such as individual nucleotides. The first aim of this research is to enhance the temporal resolution of nanopores through modification of the nanopore surface to both better understand and tune electrostatic and electro-osmotic effects on translocation time. To this end, we designed and fabricated pH-sensitive chemically coated nanopores that show the capability to retard the translocation of DNA molecules. A practical nanopore sensing device relies on taking measurements from many pores in parallel to provide sufficient robustness (through redundancy) and throughput. Optical detection facilitates such parallelism, but requires coupling between an analyte feature and a fluorescence source. The second aim is to enhance nanopore spatial resolution via optical detection of chemically activated fluorescence signals associated with single nanopores under total internal reflectance (TIR) illumination. We performed numerical simulations of the concentration field of donor molecules near a nanopore and showed that nanopores are theoretically capable of discriminating between features located near a pore surface separated by ~1 nm or less. The third and final aim is to use fluorescence signals to detect unlabeled DNA translocation through spatially addressed single pores and arrays of pores. With this aim we experimentally validate our theoretical predictions and demonstrate a novel highly parallel near-field chemo-optical detection scheme.