Solid-State Nanopore Sensors for Infectious Disease Detection Based on Ion Current Rectifying Nanopipettes

This thesis explores the use of ion current rectification, a phenomenon observed in conical-shaped nanopipettes that arises from overlapping electrical double layers, as a diagnostic tool for infectious disease detection. By functionalizing the internal walls of nanopipettes with analyte-specific probes, detectable modulation of ion-rectification upon analyte binding can be exploited for sensing applications. Laser-assisted fabrication using a Sutter P-2000 pipette puller provided an inexpensive and simple method for producing conical-shaped quartz nanopipettes with radii ranging from 6 to 310 nm. It was demonstrated that numerous factors, including pore size, electrolyte concentration, and pH, play a crucial role in determining the transport properties of these nanopipettes. We selected an optimal pore size of 110 nm and an electrolyte concentration of 10 mM KCl to conduct ion rectification-based sensing, conditions that exhibited reproducible rectification and were compatible with further biomolecule immobilization. A versatile surface modification strategy for modifying the internal walls of nanopipettes was developed, which involved coating the quartz surface with an amine-terminated silane layer, then reacting it with 3-maleimidopropionic acid n-hydroxysuccinimide ester to form a thiol-reactive maleimide group. A thiolated aptamer selective for the S1 domain of SARS-CoV-2 was employed to detect the spike protein, achieving a detection limit of 0.05 pg ml-1. The discrimination between positive and negative patient samples from nasal swabs was also readily achieved. DNA-functionalized nanopipettes were also employed to selectively detect complementary DNA sequences associated with infectious diseases, with DNA hybridization events modulating ICR. It was demonstrated that the probe surface density on the nanopipette walls could be varied by controlling the probe DNA concentration utilized during functionalization and subsequently used to tune the dynamic range and sensitivity of the ion-rectifying sensors, with a detection limit as low as 350 fM achieved. Utilizing a probe DNA sequence complementary to the mecA gene, which encodes antibiotic resistance in Methicillin-resistant Staphylococcus aureus, we integrated this novel sensing platform with a nucleic acid amplification reaction to achieve sensitive and selective identification of this pathogen in clinical isolates. Overall, the work shown in this thesis has demonstrated the remarkable promise and viability of ion-rectifying nanopipettes for infectious disease detection in clinical applications.