Transducing Signals and Pre-Concentrating Molecules for Enhanced Solid-State Nanopore Biosensing
Creators: name: Roelen, Zachary
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2024
Subjects: Single-molecule
Biosensors
Nanopores

description: Single-molecule biosensors offer distinct advantages over their ensemble-averaged counterparts by being able to extract information related to rare targets and specific molecular configurations within a sample. In particular, solid-state nanopores embody a promising single-molecule technique that is based on detecting target molecules by the amount of ionic current they block as they pass through a nanoscale aperture across a thin membrane. In this thesis, I present extensions of the basic nanopore system aimed at addressing some of its main limitations at present, namely: 1) the low rates at which nanopores capture molecules from a bulk volume, which restricts their ability to work with dilute (≲ nM) samples, and 2) the difficulty in using nanopores to distinguish small or closely related molecules by their direct current blockage signatures alone. I begin by describing the design and construction of a nanopore-based instrument that integrates an optical detection channel in parallel with ionic current sensing. A particular emphasis was placed on minimizing the electrical noise contributions of the added optical equipment on the original ionic current channel. Measuring the optical signals of translocating molecules together with their current blockages can improve the discrimination of two fluorescently labelled targets (or two configurations of a single target) that normally produce similar ionic current signatures. I next investigate the combination of nanopore sensing with target pre-concentration, specifically, by embedding a nanopore membrane within a fluidic cell that features an insulator-based dielectrophoretic (iDEP) trap. Applying large (≳ 100 V) AC voltages across the iDEP channels of the cells resulted in the accumulation of polarizable targets (dsDNA, polystyrene beads) at the locations of the membranes, thus pointing toward a convenient method for the detection of ultra-dilute target samples in future nanopore devices. Finally, I introduce improved protocols for the synthesis and nanopore signal analysis of dsDNA-based molecular carriers. In a molecular carrier scheme, in order to enhance the target specificity of the system, target molecules are not sensed directly by a nanopore but instead interact specifically with secondary molecules (“carriers”) to recognizably alter the carrier translocation signals. Here, I present proof-of-principle analyses of DNA carrier experiments that highlight the multiplexing capabilities of our carrier design, which are based on separating targets by their interactions with carriers of different lengths. Developments of the nanopore sensing platform such as those presented in this work, which leverage the intrinsic versatility of solid-state nanopores to be integrated within complex devices and to detect a wide range of target molecules, will play an important role in continuing to increase the precision of single-molecule measurements into the future and to expand their breadth of potential applications.