Rapid Single Molecule FRET Biosensing Assay for Nucleic Acid Detection
Khanna, Kunal
2021
Abstract
Cancer is a complex, multi-faceted, and varied disease that comes with a substantial economic and health burden. Due to this immense burden and its increasing prevalence, diagnostic tools capable of detecting cancer and aiding treatment, especially at an early stage, have grown into a major area of interest for researchers and clinicians alike. There has especially been a growth in interest in liquid biopsies, which are assays that utilize biomarkers found in typical body fluids like blood and urine in order to diagnose cancer in the same way tissue biopsies normally do, but without the need for invasive procedures. Although cancer biomarkers come in many different forms, two in particular that have received the attention of researchers for liquid biopsies are circulating tumor DNAs and microRNAs due to their stability in body fluids and differential expression in healthy individuals versus cancer patients. Several technologies have arisen and established themselves as gold standards for detecting and analyzing nucleic acid biomarkers, in particular, Polymerase Chain Reaction (PCR) and Next Generation Sequencing (NGS). While these techniques possess excellent sensitivity and throughput, respectively, they have drawbacks that include false negatives from amplification bias, primer mismatching causing false positives for short sequences like those of miRNAs, and laborious preparation for both techniques and extensive analysis for NGS. Our group recently developed a kinetic fingerprinting-based detection method that counts single target molecules directly from sample with ultra-high specificity. This amplification-free approach, termed single-molecule recognition through equilibrium Poisson sampling (SiMREPS), immobilizes potential target molecules on a microscope slide surface and fingerprints them via binding and dissociation patterns of a freely diffusing fluorescent probe. This approach, however, had an upper limit on analysis speed as a higher probe concentration is necessary to accelerate binding speed and the associated background worsens the signal-to-noise ratio. This dissertation focuses on the development of a kinetics-based biosensor design, termed iSiMREPS, which incorporates intramolecular Förster Resonance Energy Transfer (FRET) to rapidly identify nucleic acid targets through conformational changes in the biosensor. Chapter 2 highlights the biosensor design including the three component strands that comprise it called the anchor, capture and query strands and the development of a standardized design capable of rapidly alternating between two conformations to generate a target-identifying FRET signal pattern. In this chapter, I detail the optimizations necessary for the biosensor to perform effectively including capture probe modifications, optimization of the query probe and a competing sequence on the anchor for conformational flexibility and desirable kinetic behavior, and the introduction of the denaturant formamide to provide faster signal generation. Chapter 3 focuses on developing a full assay with the biosensor and details my optimizations to the imaging and slide preparation protocol, maximizing the effectiveness of formamide, improving sensitivity through toehold mediated strand displacement of target-unbound biosensors, and establishing limits of detection for a microRNA target as well a DNA target performed alongside a collaborator. Chapter 4 addresses multi-target detection and shows my development and refinement of biosensors for additional targets, the establishment of design principles and guidelines for generalized target detection, and the development of a multi-target well setup for use in panel assays. Combined, this thesis has developed a proof-of-concept and case for using a biosensor-based assay for liquid biopsy detection, eventually in a spatially addressable microarray format.Deep Blue DOI
Subjects
Single molecule fluorescence microscopy smFRET Kinetic Fingerprinting Biosensor Nucleic Acids Rapid biomarker detection
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