Highly Sensitive, Mechanically Stable Nanopore Sensors for DNA Analysis.

Understanding the biophysics governing single-molecule transport through solid-state nanopores is of fundamental importance in working toward the goal of DNA detection and genome sequencing using nanopore-based sensors. Even with significant advances in semiconductor fabrication technologies, the state-of-the-art in nanopore technology still falls well short of mimicking the elegance and functionality found in biology. Kasianowicz et al. pioneered the first in vitro studies of biomolecule transport through single nanopore channels by translocating individual ssDNA and ssRNA molecules through a-hemolysin protein pores inserted into a lipid bilayer membrane. More recently, focus has shifted to the solid-state domain with numerous groups studying biomolecule transport through solid-state nanopores. Solid-state nanopores exhibit superior chemical, thermal, and mechanical stability over their biological counterparts, and can be fabricated using conventional semiconductor processes, thereby facilitating mass fabrication and size tunability. They are typically formed in thin Si3N4 or SiO2 membranes using a combination of decompositional ion/ electron-beam-based sputtering and surface-tension-driven shrinking processes. Other techniques for creating individual nanopores include the track-etch method for the formation of conical nanopores in polycarbonate membranes. The translocation of negatively charged DNA molecules through these nanometer-sized solid-state pores is conventionally performed using two-terminal electrophoresis, resulting in characteristic blockades in the measured ionic current. This technique has been used to study various physical phenomena at the single-molecule level, including unzipping kinetics of hairpin DNA, detection of single-nucleotide polymorphisms, stretching transitions in dsDNA, biomolecule folding, discrimination of long DNA molecules based on length, and nanopore-based DNA force spectroscopy. Though this technology shows much promise, major hurdles still remain. Fabrication challenges (stress-induced membrane deformation and mechanical failure in SiO2 structures), [5] limited nanopore lifetime, electrical noise, and a lack of chemical specificity limit the feasibility of this technology in high-end applications such as DNA sequencing. Thus, there is a need for highly sensitive, mechanically robust nanopore sensors with welldefined surface-charge properties for the detection of specific biological molecules (ssDNA, dsDNA, mRNA). This paper reports on the development and characterization of a new solid-state nanopore sensor for the detection of single DNA molecules. The Al2O3 structures reported here exhibit enhanced mechanical properties (increased hardness, decreased stress) and improved electrical performance (low noise, high signal-to-noise ratio) over their SiO2 and Si3N4 counterparts. The fabrication process described results in high device yield and a ten-fold reduction in process time/complexity relative to techniques demonstrated in SiO2. [5] High-temperature process steps, wet-etch steps, and electron-beam lithography (EBL) were eliminated, allowing for possible integration with metal electrodes and optical probes. Al2O3 nanopore sensors fabricated using this process have all the advantages of existing SiO2 and Si3N4 architectures (size control with sub-nanometer precision, controlled contraction/expansion, chemical modification with biomolecules) but also exhibit superior noise performance and increased lifetime over their solid-state counterparts. Interestingly, the formation of nanopores in Al2O3 membranes resulted in the localized crystallization and facetted grain growth of hexagonal g -Al2O3 nanocrystallites in the vicinity of the pore, attributed to nanoscale thermal annealing and electron-beam assisted diffusion. This phenomenon has not been reported in Si3N4 and SiO2 topologies, and could potentially enhance the mechanical hardness and localized structure of the nanopore. Bulk membrane properties (crystallinity, composition, and thickness) were studied using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and Auger electron spectroscopy (AES). A 3D symmetric double-cone structure for the nanopore was extracted from conductance