Enhancing the temporal and spatial resolution of solid-state nanopore single-molecule sensors

Abstract

Since the first report of single-molecule detection using the biological nanopore alpha-hemolysin in 1996, nanopores have grown substantially more versatile. The genetic and chemical modification of biological nanopores and the fabrication of synthetic nanopores in solid-state membranes have enabled detection of analytes ranging in size from single nucleotides to large protein complexes. Among the most promising applications of nanopores is single-molecule sequencing, which has the potential to become a routine part of medical care, is compatible with long read lengths, and can detect epigenetically modified bases. Yet in order to further develop nanopores as useful tools for basic research as well as commercial applications, their temporal and spatial limitations must be addressed. Free electrophoretic threading of nucleic acids through a nanopore allows for discrimination based on large features (e.g., molecular length), but is too fast to resolve smaller features (e.g., single nucleotide identity). The first aim of this research is to enhance the temporal resolution of nanopores by tuning their electrostatic interaction with translocating molecules via chemical modification of the nanopore surface. To this end, we designed and fabricated pH-sensitive chemically coated nanopores to slow 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 parallel throughput, 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 reflection (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 separated by ~ 1 nm or less, a distance that far exceeds the resolution offered by TIR illumination. Finally, we use fluorescence signals to detect unlabeled DNA translocation through spatially addressed nanopores. With this aim we experimentally validate our theoretical predictions and demonstrate a novel highly parallel near-field chemo-optical detection scheme.