The capability to detect single molecules using nanopore sensors has rapidly gained prominence in developing next generation biosensors. Solid state nanopores have shown great potential in this field due to their unique features, including a robust structure, tunable size, and versatility, making them an attractive area of research. This thesis explores the electrokinetic effects for enhanced single molecule detection through nanopore sensing system. Specifically, we focus on investigating the electrophoretic force and electroosmotic flow to control the trajectory of biomolecules including DNA and proteins through the conical glass nanopores. The research study is divided into four specific aims, each targeting a specific aspect of nanopore sensing. The first chapter explains the theory of noise specifically high-frequency noise component in nanopore sensing systems. Then a straightforward approach is proposed to provide signal enhancement and improve the electrophoretic capture of DNA at the system level including the liquid-air interface. The second chapter seeks to explore diffusion limited capture mechanism of DNA under volumetric flow to propose a novel DNA quantification approach that eliminates external calibration requirement. The third chapter focuses on reducing the hydrodynamic forces to achieve high throughput and high-resolution DNA detection at low salt condition. Additionally, multi-pass DNA measurements is investigated under several factors including surface charge, permittivity, and voltage both experimentally and computationally. The last chapter explores DNA amplicons of sars-cov-2 viral genome in +10 electrolyte settings including symmetric and asymmetric conditions to quantify specificity of dCas9 binding to wild type and delta variant sequences using nanopore sensing system. This approach can provide an alternative to full genome DNA sequencing for highly sensitive and specific viral variant analysis with no antibodies or detection reagents requirement. Overall, this thesis provides new insights into the single molecular analysis of biological processes and reveals the potential of electrokinetic-based sensing for a broad range of applications, including biomolecular detection, disease diagnosis, and drug discovery. Furthermore, the ability to control and manipulate the transport of charged biomolecules using electrokinetic phenomena opens up new avenues for the design of advanced sensing platforms, with potential applications in areas such as point-of-care testing, and lab-on-a-chip devices.

Modern diagnostics strive to be accurate, fast, and inexpensive and aim to properly identify the presence of a disease, infection, or illness. Early diagnosis is key to earlier therapeutic intervention, which can improve prognosis and reduce mortality. The challenge with many diseases is that detectability of the disease scales with disease progression. Modern diagnostic techniques exhibit a specific threshold to be surpassed for accurate, reliable tests to be performed. Often, symptoms do not appear until disease progression has reached a significant stage. Since single molecule sensors, e.g., nanopores, can sense biomolecules at extremely low concentrations, they have the potential to become clinically relevant in many of today’s medical settings. The nanopore-detected current measurements can be used to identify disease biomarkers, determine the presence of pathogens, and monitor the efficacy of drugs. Specifically, solid-state nanopores offer several advantages over traditional diagnostic tools such as speed, accuracy, and cost-effectiveness. As a result, they have become an increasingly popular tool in the diagnostic field and are expected to play a critical role in the development of personalized medicine. In the first chapter, we investigate the origins of current enhancing events under low ionic strength conditions and propose an alternative theory for the observation of conductive events. Utilizing electroosmotic flow, we show that a flux imbalance in favor of cations allows for detection of DNA and protein to be divergent. In the second chapter, we expand upon low ionic strength conditions to high and asymmetric electrolyte conditions to examine differences in signal-to-noise ratio, molecule dwell time, and configuration. In asymmetric salt, we show the ability of detecting differences in DNA configuration as well as protein alone and bound to DNA complexes, superior to that of the gold standard sensing conditions. Following that, several denaturing agents are integrated into the nanopore system, serving to linearize and provide a uniform negative charge to both whole proteins and peptides of varying lengths. With this, a novel electrolyte sensing condition is introduced that allows for peptide length to be distinguishable by changes in the current amplitude upon molecule translocation via electrophoretic forces. Lastly, we critically analyze peptide fragments generated by protease digestion in whole blood using a portable device. The utilization of nanopores in this study goes beyond serving as only a sensing device; rather, it represents a breakthrough in the field of diagnostics, as it opens up the possibility of monitoring protease activity levels within the body.