Development of a Solid-State Nanopore Platform Toward High-Throughput, Ultra-Sensitive, and Robust Single-Molecule Analysis of Nucleic Acids and Proteins

Abstract

Solid-state nanopore sensors consist of a nanoscopic hole in a thin free-standing dielectric film (a membrane) bridging independent reservoirs of salt solution to interrogate the analytes present therein. Nanopores have demonstrated promising results in applications spanning DNA sequencing, biomarker detection, information storage and energy harvesting. Recently, researchers in the field have aimed to interrogate proteins with enough precision to extract their sequences. Despite their versatility, solid-state nanopores have advanced slowly experimentally due to obstacles and knowledge gaps that need to be explored. A few challenges are addressed in this thesis, attempting to provide insight for future steps in this direction, namely: 1) extracting surface charge density is key to studying the effect of membrane material properties on nanopore transport, fabrication conditions and subsequent chemical functionalization, 2) unwanted adsorption between the biomolecules and the sensor’s surface during the electrophoretic capture and translocation process hinders sensing performance and results in clog events, 3) small proteins or DNA nanostructures can be challenging to fully resolve, leading to missed or attenuated molecular event signatures, and 4) reducing membrane thickness to enhance the signal-to-noise ratio and the spatial resolution leads to faster sensor degradation over time for SiN. For the first objective, I present an experimental characterization platform capable of measuring the surface charge density of silicon nitride as a membrane material. The platform allows high-pressure streaming current measurements in the picoampere range, providing enough resolution to extract the zeta potential and surface charge density of new materials. Next, I explore an alternative anti-fouling protocol for the possibility of increasing sensing time while interrogating long and flexible single-stranded DNA (ssDNA). This analyte clogs the sensor in less than 15 seconds, while after surface modification, we collect relevant data for ⁓45–60 minutes before the first clog event. This improved protocol preserves noise performance comparable to pristine nanopores, even after surface modification. Furthermore, I evaluate the surface properties of these modified nanopores to confirm the successful binding of the polymer on the surface through zeta potential measurements. Moreover, I develop a trapping procedure that takes advantage of the surface characterization platform to reduce the translocation velocity of complex analytes, thus investigating pressure-voltage actuation for analytes of known velocity while determining the impact of this mode of translocation on dwell time. This actuation approach provides an up to 6× reduction in translocation velocity for DNA nanostructures and proteins respectively, facilitating the extraction of high-fidelity event signatures. Finally, I perform an exploration of nanopore membrane microfabrication processes and materials to improve signal amplitude while reducing noise limitations. Here, I present an alternative deposition approach for SiN thin films with a lower thermal budget, namely ion beam deposition (IBD) compared to the traditional Low-Pressure Chemical Vapor Deposition (LPCVD). Independently, I explore the properties of a sub-5 nm annealed Hafnium oxide (HfO2) membrane deposited by Atomic Layer Deposition (ALD), supported by an LPCVD SiN, in terms of film stability and sensing performance. Future work involves combining the lower thermal budget SiN deposition as a mechanical support for a sub-3 nm ALD HfO2 membrane. This approach will offer an ultra-thin solid-state nanopore architecture with reduced degradation, enhanced signal-to-noise ratio, and bypass the annealing step due to the temperature mismatch between depositions. Ultimately, my goal is to pave the way for more efficient and sensitive nanopore-based technologies, contributing to the fields of genomics, proteomics, and beyond.