Properties of the nanopore membrane as a solid support for ion channel recordings and application towards identifying single nucleotide modifications

The nanopore membrane is a single conical-shaped pore in a solid glass or fused quartz membrane at the end of a capillary; it can be used to support a planar lipid bilayer for ion channel recordings with a reconstituted biological nanopore. The work presented here explores the nature of the nanopore membrane and its influence on the suspended bilayer. The nanopore membrane is then used for ion channel recordings with the protein ion channel ?-hemolysin (?-HL) to detect single oxidative damage sites within a DNA sequence. Chemical modifications to the surface of the glass nanopore membrane with hydrophobic silanes (trimethylchlorosilane, n-butyldimethylchlorosilane, and n-octadecyldimethylchlorosilane) are explored to understand their influence on the pore wettability and the bilayer structure (seal resistance, voltage stability, and lifetime). Further, fused quartz was used to fabricate fused quartz nanopore membranes (QNMs) and these were compared with the traditional soda lime glass membranes as bilayer supports. The leakage current across the membrane was compared for fused quartz and soda lime glass capillaries. The structure of the suspended bilayer is investigated as a function of applied pressure across the orifice of a QNM using fluorescence microscopy. Ion channel reconstitution within lipid bilayers suspended across nanopore membranes is a pressure-dependent process; a positive pressure must be applied to the inside of the nanopore relative to the exterior for protein channel insertion to occur. Lastly, the nanopore membrane was used to perform ion channel recordings to detect the presence of a single oxidative damage site within a DNA sequence. The kinetics of the DNA duplex unzipping process within the ?-HL nanopore were monitored to determine the presence of a single DNA lesion, 8-oxo-,8-ihydroguanine (OG). The presence of OG influences the duplex stability which is reflected in the unzipping event duration. Additionally, the detection of a single oxidative damage site is examined using DNA immobilization experiments to determine the presence of the damage site based on the ion channel current. A single OG site within a DNA strand is adducted with a larger molecule and held within the ?-HL protein ion channel. The resultant current blockage level and noise level are shown to be unique to the adducted molecule.