Development of Point-of-Care Assays to Monitor Biologically Relevant Analytes

This thesis focuses on the development of assays that could potentially be in Point-of-Care (POC) diagnostics. This research targets four analytes: Tacrolimus, interleukin-2 (IL-2), and Dengue Virus biomarkers nonstructural protein 1 (NS1) and interferon gamma-induced protein 10 (IP10). The first chapter introduces the requirements for POC diagnostics and reviews notable advancements in this broad field. The second chapter discusses the challenges of detecting small-molecule therapeutics without the need for complex equipment, such as mass spectrometry or HPLC. A novel "add, mix, and measure" assay was developed for the rapid detection of Tacrolimus, a crucial immunosuppressive drug. Utilizing mesoporous silica nanoparticles (MSNs) as a sensing platform, this assay improves the speed and accessibility of testing, offering a simplified method for real- time monitoring in clinical settings. The transition from traditional MSNs to hollow mesoporous silica nanoparticles (HMSNs) resulted in a significant improvement in detection sensitivity, reducing detection limits from 90 nM to 5.2 nM. The third chapter focuses on the development of an amplification method to enhance the sensitivity of POC assays. This technique was applied to monitor interleukin-2 (IL-2), a pro- inflammatory cytokine associated with autoimmune diseases. By using a layer-by-layer signal amplification strategy, which combines fluorescent dye encapsulated silica nanoparticles and bio- orthogonal click chemistry that exhibits high specificity, the assay demonstrated a limit of detection (LOD) of 1.1 pg/mL in PBS, and a broad linear range that is 7 orders of magnitude from 1 pg/mL to 10 ng/mL. In comparison, commercial assays have a LOD and linear range of 9.1 pg/mL and 18.8-1200.0 pg/mL, respectively. In human serum, the LOD was 65.5 pg/mL, and the linear range of detection was 100 pg/mL to 10 ng/mL, respectively. This broad range and low LOD set the stage for translation into POC diagnostics for frequent home or resource limited testing. In the fourth chapter, we describe the application of this layer-by layer sandwich assay using fluorescent dye-encapsulated nanoparticles and biorthogonal chemistries, to quantify Dengue Virus biomarkers, including the viral biomarker NS1 and host biomarker IP-10, aiming to create a robust and sensitive method for early-stage detection. In human serum, the assay achieved a LOD 65.8 pg/mL (~7.6 nM) for IP-10, ranging from 10 pg/mL to 100 ng/mL; and 351.0 pg/mL (8.6 nM) for NS1, ranging from 100 pg/mL to 10 µg/mL. We further monitored host biomarkers in dengue virus-infected AG129 mice using a Milliplex Mouse Cytokine/Chemokine Magnetic Bead Panel. IP-10 levels ranged from 150 to 300 pg/mL (mean 188.0 pg/mL) in infected mice and 50 to 100 pg/mL (mean 69.4 pg/mL) in controls. These results aligned with our multilayered fluorescent assay, demonstrating its potential for sensitive dengue biomarker detection. Each chapter highlights the key advancements and discusses the potential for translating these technologies into practical POC solutions for resource-limited settings. We also include initial efforts to translate these findings to a point-of-care prototype.