Simplifying assay chemistry via complex sample preparation integration for point-of-care diagnostics
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Although therapeutics are undeniably important in the fight against any disease, a crucial step in making timely and effective therapeutic choices is diagnostics. Early treatments often lead to better patient outcomes, however early treatments are only possible with accessible diagnostics. Additionally, in public health emergencies, tracking the spread of disease via diagnostic tools is crucial to slowly and stopping it. Currently, standardized diagnostic testing is typically limited to central laboratories, requiring expensive, bulky instrumentation, trained technicians to perform these diagnostic tests, and the means to collect samples from patients, deliver the samples to the central laboratory, then return the results to the patient. In resource-limited settings (e.g., rural clinics), near-patient settings (e.g., at home), and in time-sensitive settings (e.g., emergency care), where accessibility and speed to a diagnosis are critical, the current central laboratory approach to diagnostic testing slows or even prevents an informed diagnosis and thus proper treatment. Subsequently, a large sector of the diagnostic field has focused away from a central laboratory approach and instead toward a near-patient paradigm, where samples can be testing immediately at the site of the patient such that results are rapidly available, allowing for faster treatment response. The diagnostic community refers to this idea of near-patient testing as point-of-care.
Although point-of-care diagnostics have seen some commercial progress, the field is still struggling to develop tests for complex sample mediums. For complex samples, it is crucial that any sample preparation steps are integrated into the overall device to be truly considered point-of-care. Specifically, a diagnostic that can produce an easy-to-interpret result with little effort exerted by the user once the sample has been input is considered to be sample-to-answer. This dissertation highlights scientific advancements in the field of sample-to-answer diagnostics through optimized approaches that integrate higher order alkanes acting as pseudo-valves with point-of-care assay techniques. These higher order alkanes are solid at ambient temperatures and liquify when warmed and these alkanes can act as either as (1) a breachable barrier to allow for hands-free, controlled reagent mixing as the alkane melts or (2) as a permeable barrier that remains in place to separate assay regions, while magnetic beads can be pulled through. The behavior can be controlled by modifying the specific geometry in which the assay is confined.
Developments involving thermally responsive alkane partitions enable complex sample processing and assay integration with intervention-free operation in low-cost, easily manufacturable cartridges. The work presented in this dissertation aims to address the need for sample-to-answer diagnostics, especially for complex samples, using these alkane partitions in three sequential avenues. This dissertation first details a point-of-care method to detect proteins, specifically histones, in whole blood, which can be a biomarker for severe internal trauma. This work demonstrates a hands-free technique for temperature-controlled reagent mixing as well as automated blood separation to allow for quantitative fluorescent measurements from whole blood samples. Although this work contributes to the field of sample-to-answer diagnostics, by integrating blood sample preparation, there was a need to generalize the diagnostic device to detect not just histones, but a broader category of protein biomarkers, like antibodies. Next, this dissertation assesses a sample-to-answer immunoassay, designed to quantify antibodies from whole blood which is useful to not only help track community spread of a disease, but also aid in validating vaccines and determining immunity. Although this diagnostic device was optimized to be as sensitive as its gold standard, central laboratory equivalent, in order to branch into nucleic acid biomarker detection, there was a need to include an exponential amplification step. Thus, finally, this dissertation looks at a sample-to-answer approach to monitor viral RNA genome in an additional complex sample, wastewater, in order to monitor the spread of disease community-wide. These sample-to-answer advancements to the field of point-of-care diagnostics enable low-cost, user-friendly solutions that increase overall accessibility to healthcare.