Doctor of Philosophy

dissertationThe 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

Decreasing the Limits of Detection and Analysis Time of Ion Current Rectification Biosensing Measurements via a Mechanically Applied Pressure Differential

Improving on the analytical capabilities of a measurement is a fundamental challenge with all assays, particularly decreasing the limit of detection while maintaining a practical associated analysis time. Of late, ion current rectification (ICR) biosensing measurements have received a great deal of attention as an analyte-specific, label-free assay. In ICR biosensing, a nanopore coated with an analyte specific binding molecule (e.g., an antibody, an aptamer, etc.) is used to detect a target analyte based on the ability of the target analyte to alter the ICR response of the nanopore upon it binding to the aperture interior. This binding changes the local surface charge and/or size of the nanopore aperture, thus altering its ICR response in a time dependent manner. Here, we report the ability to enhance the transport of a target analyte molecule to and through the aperture of an antibody modified glass nanopore membrane (AMGNM) with the application of a mechanically applied pressure differential. We demonstrate that there is an optimal pressure that balances the flux of the target analyte through the AMGNM aperture with its ability to be bound and detected. Applying the optimal pressure differential allows for picomolar concentrations of the cleaved form of synaptosomal-associated protein 25 (cSNAP-25) to be detected within the same analysis time as micromolar concentrations detected without the use of the pressure differential. The methodology presented here significantly expands the utility of ICR biosensing measurements for detecting low-abundance biomolecules by lowering the limit of detection and reducing the associated analysis time

Antigen Detection via the Rate of Ion Current Rectification Change of the Antibody-Modified Glass Nanopore Membrane

Ion current rectification (ICR), defined as an increase in ion conduction at a given polarity and a decrease in ion conduction for the same voltage at the opposite polarity, i.e., a deviation from a linear ohmic response, occurs in conical shaped pores due to the voltage dependent solution conductivity within the aperture. The degree to which the ionic current rectifies is a function of the size and surface charge of the nanopore, with smaller and more highly charged pores exhibiting greater degrees of rectification. The ICR phenomenon has previously been exploited for biosensing applications, where the level of ICR for a nanopore functionalized with an analyte-specific binding molecule (e.g., an antibody, biotin, etc.) changes upon binding its target analyte (e.g., an antigen, streptavidin, etc.) due to a resulting change in the size and/or charge of the aperture. While this type of detection measurement is typically qualitative, for the first time, we demonstrate that the rate at which the nanopore ICR response changes is dependent on the concentration of the target analyte introduced. Utilizing a glass nanopore membrane (GNM) internally coated with a monoclonal antibody specific to the cleaved form of synaptosomal-associated protein 25 (cSNAP-25), creating the antibody-modified glass nanopore membrane (AMGNM), we demonstrate a correlation between the rate of ICR change and the concentration of introduced cSNAP-25, over a range of 500 nM–100 μM. The methodology presented here significantly expands the applications of nanopore ICR biosensing measurements and demonstrates that these measurements can be quantitative in nature