Nanopore biosensors are a relatively new tool for single-molecule detection, whose inception was inspired by molecular transport through transmembrane pores in nature and the operating principle of the Coulter Counter, so-called resistive pulse sensing. In recent years, nanopores have been integrated with alternative detection modes, such as fluorescence spectroscopy, with the goal of increasing structural resolution and analytical throughput. The integration of fluorescence spectroscopy is particularly useful as fluorescent labels can be used to identify different regions of a molecule; discriminate molecules in heterogeneous solutions and probe molecular properties such as distance.
This thesis describes the development and application of a unique low-noise nanopore platform, composed of a predominately pyrex substrate and silicon nitride membrane, for synchronized optical and electrical detection of biomolecules. The use of a pyrex substrate was pursued as commonly used Si substrate based nanopore sensors exhibit high ionic current noise with and without laser illumination. This limits their applicability to high-laser-power, high- bandwidth electronic measurements, which in-turn restricts the range of molecules that can be studied and the structural resolution provided by resistive pulse sensing. Proof-of-principle experiments are presented that show a pyrex substrate greatly reduces ionic current noise arising from both platform capacitance and laser illumination. Furthermore, using a confocal microscope and a pyrex based platform with a partially metallic nanopore, thereby acting as a zero mode waveguide, we demonstrate synchronized optical and electrical of dsDNA.
The high translocation velocity of biomedically relevant molecules such as proteins and nucleic acids means there is a continual drive for low-noise high-bandwidth measurements within the nanopore community. The use of these low-noise platforms for synchronized measurements increases the sensitivity of resistive pulse sensing and therefore the range of molecules that can be studied and potential applications of the sensor.