Development, Validation, and Deployment of a Portable, Automated Fluorescence Microarray Scanner for Point of Care Diagnostics

Abstract

The accurate detection and quantification of disease biomarkers are pivotal for early diagnosis, monitoring disease progression, and guiding treatment decisions. Immunoassays have significantly advanced clinical diagnostics, offering sensitive and specific measurements of protein biomarkers and antibody responses. However, the gold standard immunoassay, enzyme-linked immunosorbent assay (ELISA), is typically confined to centralized clinical laboratories. This restriction arises from its dependence on skilled personnel, extensive manual intervention, complex reagent handling, and multiple washing steps, thereby limiting widespread accessibility and practical implementation, especially in resource-limited environments.In response, point-of-care (POC) diagnostics have emerged as a promising alternative, enabling diagnostic testing outside traditional hospital settings and holding substantial potential to democratize healthcare. These technologies could profoundly impact clinical outcomes by enabling rapid and early detection of infectious diseases, timely diagnosis of cancers in remote or underserved areas, and convenient monitoring of chronic conditions. The lateral flow assay (LFA) currently represents the most widely adopted POC immunoassay technology, celebrated for its simplicity, affordability, and rapid turnaround time. Despite their advantages, LFAs predominantly yield binary (qualitative) results and exhibit limitations in multiplexing, hindering their effectiveness in scenarios requiring precise quantification of multiple biomarkers simultaneously. Consequently, there remains a critical need for simpler, automated, and multiplex-capable POC diagnostic platforms that combine the ease-of-use and accessibility of LFAs with the quantitative accuracy and reliability of laboratory-based assays. Such innovations are crucial to bridge the existing gap and expand high-quality diagnostics into broader, diverse healthcare settings. To bridge this gap in clinical diagnostics, this dissertation introduces the D4Scope, a portable and automated platform designed to scan, analyze, and readout fluorescent microarray immunoassays, facilitating accurate quantification of biomarkers from small-volume, complex biological samples within seconds with minimal user intervention. Chapter 1 introduces the D4 assay as a POC test capable of in-field, quantitative diagnosis, emphasizing microfluidic automation to reduce user error and comparing various fluorescence scanners (including the original “Leo”) used for Ebolavirus detection. Chapter 2 focuses on validating the second-generation D4Scope (“Einstein”) for COVID-19 and Talaromycosis diagnosis, detailing the specific challenges encountered and the solutions devised to address them. Chapter 3 outlines the development and deployments of the D4Scope (version 3.0), detailing its optomechanical and software components, and highlights its successful application for detecting Ebolavirus, Talaromycosis, biowarfare agents, and in therapeutic drug monitoring. Chapter 4 discusses ongoing research into the design of a smartphone-based detector for an alternative scattering-based D4 assay. Chapter 5 outlines the design of a point-of-care lactate assay and accompanying colorimetric detector unrelated to the D4. Collectively, this dissertation provides robust evidence supporting the D4Scope's performance, versatility, and scalability. It establishes a foundation for its widespread adoption as a highly effective diagnostic platform capable of addressing critical global health challenges by enabling rapid, accurate, and user-friendly disease detection in diverse healthcare settings.