A “Zero-Background” Multiplexed, Point-Of-Care Testing Platform for Disease Diagnosis, Management, and Surveillance

Bioanalytical techniques such as immunoassays are ubiquitous in clinical and basic research laboratories and have transformed how we diagnose patients, monitor health, and study disease. Immunoassays typically use capture reagents, such as antibodies or antigens, to detect and quantify a biomarker of interest from a clinical specimen based on highly sensitive and specific binding interactions. While laboratory-based assays, such as enzyme-linked immunosorbent assay (ELISA), are the workhorses of clinical laboratories, they have several shortcomings that limit their overall utility, especially in low resource settings. Of note, ELISA requires multiple timed incubation steps, trained personnel, expensive equipment, and suffers from long times to result. To democratize access to clinical-grade tests, researchers have sought out different methods for point-of-care (POC) testing that are easy to perform and maintain high sensitivity and specificity. This dissertation describes the use of a “zero-background” polymer coating—poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)—as a substrate for highly sensitive and specific POC diagnostic tests. The POEGMA coating eliminates nearly all non-specific protein adsorption and cellular adhesion, thus leading to high signal-to-noise ratios, even from complex biological samples, such as whole blood. In addition, the POEGMA brush contains all biomolecules necessary to complete an assay after addition of a liquid sample, thus allowing assays to be conducted in a single step. Further, the POEGMA coating stabilizes biomolecules on the surface, which allows tests to be stored at ambient conditions without refrigeration. Assays are read out using a fluorescence detector which quantifies the concentration for a given biomarker of interest. By inkjet printing capture biomolecules at discrete spatial addresses on the POEGMA-slides, multiplexing can be accomplished using a single fluorophore which greatly reduces the complexity and costs for assay readout. This dissertation focuses on adapting and applying this platform to several clinically relevant applications. First, we developed a test for molecular and cellular credentialing of breast cancer tissue at the POC (Chapter 2). With the onset of the coronavirus 2019 (COVID-19) pandemic, we adapted the platform to detect several different relevant biomarkers for COVID-19, including total antibody concentration against several viral proteins (Chapter 3), neutralizing antibodies (Chapter 4), and viral proteins (Chapter 5). All the tests developed for COVID-19 use multiplexed sensing strategies and can be conducted with minimal/no user intervention or clinical infrastructure. Taken together, these studies highlight the great potential for bioanalytical assays built upon POEGMA-coated substrates to be used for clinical applications in disease diagnosis, surveillance, and management.