Browsing by Author "Drezek, Rebekah A"

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    EXPLORATION OF NUCLEIC ACID-BASED PLATFORMS FOR MICROBIAL IDENTIFICATION
    (2020-08-20) Bugga, Pallavi; Drezek, Rebekah A
    The rapid and accurate identification of microbes is critical for a variety of industries, notably healthcare, bioterrorism/defense, food and agriculture, and environmental testing. Nucleic acid-based identification platforms, in particular, have introduced marked improvements in the overall specificity and sensitivity of pathogen detection. While tremendous technical progress has been made in addressing the specific demands of these various sectors, there still exists a significant unmet need for a rapid and universal microbial identification platform in the clinic. Using a set of universal, target-agnostic probes, microbial species can be readily distinguished from one another based upon the observed variability in the total number of unique hybridization events between each probe and each target genome. In this way, both the identity of the microbe and its infectious load can be determined. To that end, this work first establishes the efficacy of a specific universal-probe that builds off of existing toehold-probe technologies. Given the overly narrow thermodynamic constraints of single-mismatch protectors in traditional toehold-probes, and the inherent noisiness of standard molecular probes, we herein introduce “sloppy” or mismatch-tolerant universal toehold-probes, and validate their efficacy by demonstrating successful detection and characterization of viral subpopulations or quasi-species in patient-derived viral DNA. This work also investigates several novel schemes that utilize a set of target-agnostic universal toehold-probes to rapidly and accurately identify bacterial species with high sensitivity. These include probe-capture, endonuclease cleavage, size-exclusion chromatography, and fluorescence in situ hybridization.
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    A New Framework for Rapid, Scalable Bacterial Diagnostics with Microfluidics and Compressed Sensing
    (2022-08-02) Kota, Pavan Kumar; Drezek, Rebekah A; Baraniuk, Richard G
    Critically ill patients with suspected invasive bacterial infections can be treated effectively once the responsible pathogens are characterized. Rapid and sensitive diagnostic tests rely on a specific sensor for each target pathogen, a paradigm that cannot practically cover the dozens to hundreds of plausible pathogens. As a result, sample culture is a prerequisite for downstream testing but can take days. Clinicians are forced to use broad-spectrum antibiotics in the interim which are ineffective for some patients and contribute to rising drug resistance. To address these challenges, I present a new approach with compressed sensing (CS) and microfluidics. CS focuses on the efficient recovery of sparse signals. Patient samples from normally sterile sites are sparse because among many pathogens to consider, at most a few are causing any given infection. Microfluidic partitioning technologies split a sample into thousands of compartments, capturing bacterial biomarkers across the partitions according to a Poisson distribution. Leveraging this statistical prior can yield scalable systems that break previous theoretical barriers in CS, enabling the detection of more analytes with arbitrarily few sensors. First, I cover the theory and our newly developed Sparse Poisson Recovery (SPoRe) algorithm. I present SPoRe’s superior performance over baseline CS algorithms, including its high sensitivity and tolerance of measurement noise. Second, I generalize the theoretical results to apply to many common types of biosensors and present the first in vitro realization of SPoRe with droplet digital PCR. We use five DNA probes to barcode the 16S rRNA gene to quantify nine pathogen genera within hours. Given two fluorescent channels, we measure portions of the barcodes in four groups of droplets, pool the data, and infer bacterial quantities with SPoRe. I highlight how the underlying principles of this demonstration enable sensor-constrained systems to scalably cover large panels of analytes, an advance that could unlock new biosensing solutions for multiple industries.
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    Quantifying changes in the optical spectra of plasmonic nanoparticles following interaction with biological cells: Towards optimized design in biomedical applications
    (2015-04-22) Chen, Allen L; Drezek, Rebekah A; Raphael, Robert; Link, Stephan
    As plasmonic nanoparticle (NP)-based diagnostic and therapeutic technologies—such as Plasmon Resonance Energy Transfer (PRET)-based intracellular analysis or NIR photothermal cancer therapy—continue to be developed with the goal of eventual clinical translation, clearer understanding of the interactions between NPs and biological environments is critical. In biological media, proteins adsorb to NPs, leading to physicochemical changes which affect the cellular uptake of NPs, and NPs can further agglomerate within intracellular vesicles. Since the plasmonic properties of NPs are highly dependent on their geometry and local environment, these physicochemical changes can also change the optical spectra of NPs in cellular environments. Understanding how NPs’ optical properties change in biological environments is especially important as medical nanotechnologies move toward increased multiplexing capabilities, and shifted optical spectra may result in unintended outcomes. In this thesis, we develop a darkfield hyperspectral (HS) imaging approach for systematically studying and quantifying how the NP optical spectra change in a cellular environment in order to inform biomedical NP design. We begin by establishing methods for measuring and analyzing spectra of plasmonic NPs in a cellular environment, showing that 100 nm gold NPs (AuNPs) experience up to a 79 nm spectral shift and substantial spectral broadening after exposure to Sk-Br-3 breast adenocarcinoma cells for 24 h. Then, we apply this HS imaging approach to characterize how NP design factors and biological environment factors impact the extent of spectral changes experienced by NPs within cellular environments. We find that NP functionalization with poly(ethylene glycol) (PEG) can reduce spectral shifting and decrease spectral broadening exhibited by NPs upon cellular uptake, and we show the impact of serum concentration on the magnitude of spectral shift exhibited by NPs. Finally, in order to more specifically focus characterization on the optical response from cell-internalized NPs and improve understanding of intracellular delivery of NPs, methods are needed to differentiate membrane-adsorbed NPs from cell-internalized NPs. Using confocal imaging, we investigate the efficacy of emerging approaches for differentiating cellular internalization from cell membrane adsorption of NPs to enable further advances in understanding of nano-bio interactions. Together, this thesis demonstrates the development of a HS imaging and analysis approach for quantitatively characterizing changes in the optical properties of NPs within cellular contexts, and the application of this approach towards developing quantitative relationships to enable plasmonic NPs to be engineered to exhibit desired optical properties in biomedical environments for maximal efficacy and safety.
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    Structured Illumination in a Fiber-Optic Microendoscope to Image Nuclear Morphometry in Columnar Epithelium
    (2015-03-23) Keahey, Pelham; Richards-Kortum, Rebecca Rae; Tkaczyk, Tomasz S; Natelson, Douglas; Drezek, Rebekah A
    Fiber-optic microendoscopes have shown promise to image the changes in nuclear morphometry that accompany the development of precancerous lesions in tissue with squamous epithelium such as in the oral mucosa and cervix. However, fiber-optic microendoscopy image contrast is limited by out-of-focus light generated by scattering within tissue. The scattering coefficient of tissues with columnar epithelium can be greater than that of squamous epithelium resulting in decreased image quality. To address this challenge, I present a small and portable microendoscope system capable of performing optical sectioning using structured illumination (SI) in real-time. Several optical phantoms were developed and used to quantify the sectioning capabilities of the system. Columnar epithelium from cervical tissue specimens was then imaged ex vivo, and I demonstrate that the addition of SI achieves higher image contrast, enabling visualization of nuclear morphology.
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    The Development of a Low-Resource Appropriate Diagnostic for the Detection of Malaria DNA
    (2015-03-11) Cordray, Michael Scott; Richards-Kortum, Rebecca Rae; Drezek, Rebekah A; Hafner, Jason H; Storthz, Karen
    Despite recent progress, malaria remains one of the most serious global health threats, especially since it disproportionately affects low-resource areas. One of the keys to management of the disease is access to effective diagnostics. Nucleic acid tests (NATs) are the most sensitive and specific type of diagnostic for malaria, but require too much infrastructure, training, and cost to be widespread in many malaria affected regions. The aim of this thesis was to develop a NAT for malaria that can detect 50 copies/µL or less of target DNA in blood and then package that assay into an easy to use and self-contained system. An assay was investigated to detect small amounts of target DNA using gold nanoparticle (AuNP) aggregation. A method was developed to quantify the results using spectroscopy and found a limit of detection (LOD) of 150 amoles of target DNA and a linear dynamic range that spans 150 amoles – 15fmoles. The conditions of the assay were optimized and a novel method of measuring the assay results was developed, eliminating the need for heating and reducing the time to result to 10 minutes (from 2 hours). These changes also dropped the LOD to 50 amoles (approximately 107 copies/µL) and increased the linear dynamic range of the assay to 50 amoles - 500 fmoles. Since the LOD of the AuNP assay was not low enough to detect clinically relevant amounts of target on its own, I investigated recombinase polymerase amplification (RPA) and a lateral flow dipstick assay to detect the product. Blood was found to inhibit the RPA reaction and protocols were developed to minimize this inhibition. A LOD of 15 copies/µL of target DNA spiked into blood was found. Finally, a paper and plastic platform was developed to carry out the RPA reaction and lateral flow detection of the amplified products. A novel set of RPA primers were designed which target a sequence found in all of the species of malaria which infect humans. Testing these primers in the paper and plastic device I found an LOD of 5 copies/µL in aqueous solution, and 200 copies/µL in blood.