This dissertation focuses on developing assays with potential applications in point-of-care (POC) diagnostics.

The first chapter provides an overview of POC diagnostics, HIV, and recent advancements in POC diagnostics for the HIV-1 p24 antigen.

The second chapter describes developing assays to detect Azidothymidine (AZT), a key drug in Highly Active Anti-Retroviral Therapy (HAART). While adherence to HAART is important for HIV treatment success, it is often compromised by side effects, interactions, and forgetfulness. Additionally, detecting small molecules, such as AZT, often requires advanced tools like Mass Spectroscopy or HPLC. We developed a novel "add, mix, and measure" assay that detects AZT within minutes in simulated urine using three fluorophore-releasing probes. This rapid and simple POC test is a valuable tool for clinicians, especially in resource-limited settings, to monitor AZT levels effectively.

Chapters three and four focus on the development of highly sensitive multilayer sandwich immunoassays to detect HIV-related biomarkers, Galectin-9 (Gal-9) and HIV-1 p24 antigen, using bioorthogonal chemistry and dye-encapsulated fluorescent silica nanoparticles. This approach achieved significant signal enhancement through a layer-by-layer assembly, with two platforms explored: a magnetic bead-based system and a microwell plate-based system.

For Gal-9 detection, we performed extensive studies for optimizations, including adjusting fluorescent silica nanoparticle concentrations, selecting optimal antibody pairs, and fine-tuning the synthesis conditions for Gal-9 Ab2-TCO (temperature, reagent equivalents, and pH). This resulted in a detection limit (LOD) of 100 pg/mL and a broad linear range (100 pg/mL − 10 μg/mL), fully meeting the requirements for HIV viral load monitoring.

For p24 detection, the magnetic bead-based assay achieved a femtomolar LOD of 17 fg/mL with a broad linear range (17 fg/mL−10 ng/mL), while the microwell plate-based assay simplified the procedure and further improved sensitivity, achieving a LOD of 8 fg/mL with a broad linear range (8 fg/mL−10 ng/mL), making it more suitable for clinical applications than traditional ELISAs. The introduction of site-specific labeled antibodies using an enzyme-catalyzed method preserves antibody functionality while enabling precise modifications, demonstrating the potential to further enhance the assay's performance. These developments present a promising approach for early HIV detection and viral load monitoring, particularly in resource-limited areas.