THESIS
2022
1 online resource (xiv, 102 pages) : illustrations (some color)
Abstract
Infectious diseases cause morbidity and mortality worldwide, with the COVID-19
pandemic, caused by a coronavirus, underscoring this fact. Rapidly mutating and proliferating
infectious agents perpetuate the threat of future pandemics, making pathogen monitoring
imperative; however, a lack of scalable and convenient multiplexed diagnostic technologies
makes this challenging. To address this deficit, new methods and technologies for nucleic acid
diagnostics were investigated in this work. Centrifugal microfluidic chips were developed which
aliquoted liquid to multiple reaction chambers with 97.5% accuracy. These chips were
fabricated using variotherm desktop injection molding, a method innovated in this project
capable of faithfully molding 100-micron wide channels with a coefficient of va...[
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Infectious diseases cause morbidity and mortality worldwide, with the COVID-19
pandemic, caused by a coronavirus, underscoring this fact. Rapidly mutating and proliferating
infectious agents perpetuate the threat of future pandemics, making pathogen monitoring
imperative; however, a lack of scalable and convenient multiplexed diagnostic technologies
makes this challenging. To address this deficit, new methods and technologies for nucleic acid
diagnostics were investigated in this work. Centrifugal microfluidic chips were developed which
aliquoted liquid to multiple reaction chambers with 97.5% accuracy. These chips were
fabricated using variotherm desktop injection molding, a method innovated in this project
capable of faithfully molding 100-micron wide channels with a coefficient of variation averaging
only 3.6%. Microfluidic designs prototyped using this method are guaranteed scalable, since
they can be mass-manufactured with minimal alteration via industrial injection molding. Loop-mediated
isothermal amplification (LAMP) and reverse transcription LAMP (RT-LAMP) were
used for detecting DNA and RNA fragments, respectively. A colorimetric method for SARS-CoV-2 detection was initially developed utilizing triarylmethane dyes for assay readouts and
had a limit of detection of 170 RNA copies per reaction. Next, halochromic indicators were
used to generate readouts in minimally buffered RT-LAMP reactions, followed by the novel
usage of fluorescent pH sensors comprising poly(2-hydroxyethyl methacrylate) conjugated
with fluorescein isothiocyanate. The acidification caused by positive diagnostic LAMP
reactions was converted into a fluorescent signal by the optical pH sensors. Sensor precursor
solution was drop-cast into the centrifugal microfluidics’ reaction chambers and using a
custom-built imaging rig, allele-specific LAMP (AS-LAMP) and RT-LAMP assays could be
performed for multiplexed discrimination of human CYP2C19 alleles and respiratory virus
RNA, respectively. Assays were completed within an hour and sensitivities of only 50 RNA
copies per reaction were obtained for SARS-CoV-2. This technology should therefore help
public health officials, medical practitioners, and even home users to diagnose genetic
predispositions and disorders and monitor infectious agents.
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