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DNA Labels for Improved Detection and Capture with Solid-State NanoporesKarau, Philipp 16 May 2018 (has links)
Nanopores have emerged as a simple but effective tool to investigate the behavior of polymers in solution. They have shown great potential to simplify expensive and time consuming procedures like DNA sequencing, protein detection, and disease biomarker detection. With the development of in situ fabrication of solid-state nanopores by controlled breakdown (CBD) of a dielectric material, nanomanufacturing of nanopore-based technologies became feasible. However, there are still a lot of challenges to overcome for these applications to become reality. One of the major problems with solid-state nanopores is the rapid passage time of analytes going through the pore, complicating detection and reliable identification of molecules. In this thesis molecular structures are proposed that increase passage times due to increased interactions between analyte and pore wall, and at the same time increase signal amplitude due to increased blockage of the pore. These structures are short, branched DNA molecules that were assembled with built-in modifications and matching sequences to assume their structure. Nanopore experiments reveal that these structurally defined DNA produce higher detection rates than their linear DNA counterparts, making them better candidates for labels in single-molecule sensing experiments.
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DNA Nanostructures for Nanopore-based Digital AssaysHe, Liqun 08 November 2022 (has links)
Solid-state nanopores are a versatile class single-molecule sensors to electrically characterize a range of biological molecules. Nanopores operate on the simple premise that when a voltage is applied across a pore immersed in a salt solution, the passage of a biomolecule results in a transient blockage in the ionic current that provide information about the translocating molecule. This thesis presents studies employing various DNA nanostructures with solid-state nanopore electrical readout for the development of high sensitivity digital single-molecule assays to detect low-abundance biomarkers. Toward this ultimate goal, work presented in this thesis use nanopores to probe DNA nanostructures, their assembly, mechanical properties, and monitor their dynamics with time and temperature. DNA nanostructures are self-assembled via specific base pairing of DNA, their programmability make them particularly useful for applications including drug delivery, molecular computation and biosensing. Here, I first show results of translocation profiles and discuss folding characteristics, mobility, and molecular configuration during passage for different DNA nanostructures such as the short star-shaped DNA nanostructures and large helix-bundle DNA origami structures under various experimental conditions in an effort to understand the passage characteristics through nanopores of these structures before using them in biological assays. I conclude by presenting a magnetic bead-based immunoassay scheme using a digital solid-state nanopore readout. Nanopore has the ability to count molecules one at a time, this allows accurate and precise determination of the concentration of a biomarker in solution. Coupled with the use of specific choice of DNA nanostructures, as proxy labels for proteins of interest, I establish that nanopores sensors can reliably quantify the concentration of a protein biomarker from complex biofluids and overcome the traditional challenges associated with nanopore-based protein sensing, such as specificity, sensitivity, and consistency. I demonstrate the quantification of thyroid stimulating hormone (TSH) with a high degree of precision down to the femtomolar range by using a nanoparticle-based signal amplification strategy. The proposed assay scheme is generalizable to a framework for the detection and quantification of a wide range of target proteins, and given that its performance can further be improved with the use of parallelization, preconcentration, or miniaturization, it opens up exciting opportunities for the development of ultra-sensitive digital assay in a format that is compatible for point-of-care.
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