Broadly speaking, the work herein discussed encompasses the development of biomolecular assays for biomarker detection. Specific to the assays in this thesis is the design of reaction schemes that consider the unique requirements of one class of single-molecule sensors in particular: solid-state nanopores formed using a novel fabrication and conditioning technique discovered during this research at the University of Ottawa. We present three unique assays for the detection of different biomolecular targets. The first uses a class of DNA origami structures termed nanoswitches to translate the presence of a short segment of single-stranded DNA Zika virus biomarker to a large configurational change in a double-stranded DNA scaffold. The signal amplification inherent in this topological change allowed us to a achieve a high degree of specificity for detecting a small nucleic acid target by requiring two separate binding events. Furthermore, through careful design of the configurational change, the number of topological states that a solid-state nanopore can sense is limited, providing unambiguous signals in ionic current recordings. Quantification of the Zika gene was performed by sensing the relative amounts of nanoswitches in looped and linear configurations from only hundreds of individual molecules. We then explored the sensitivity of solid-state nanopores for detecting small molecular features along short DNA scaffolds. Leveraging the ability of our nanopores to detect the presence of these protrusions, we present results in which ATP, a molecule significantly too small to be directly detected by the nanopore sensor, initiated an aptamer-based DNA displacement reaction to form a protrusion along scaffolds, producing measurable changes in ionic current signatures in nanopore recordings. Finally, we present an assay in which a microRNA, a biomarker linked to various cancers, was detected through the conjugation of two probes, each of which contained a binding site to different segments of the microRNA. In addition to examining different probe set structures for optimal performance, our two-probe design aimed to improve specificity over conventional single-probe-based assays which only require one recognition step, while still providing unambiguous signals due to the greater-than-doubling in molecular complex size upon conjugation. Furthermore, the use of two individual small probes, rather than one large nanoswitch, increased the resolution with which we could differentiate microRNA concentrations. The assay enabled the quantification of six concentrations of microRNA spanning a single order of magnitude, in only several hundred events, and allowed us to take advantage of the reduced cost, material and labour, as well as increased nanopore capture rates, associated with small assembled molecules.

Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible. Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.

Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible. Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.

Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible. Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.