Quantification of Antithrombin III via Flow Velocity Profiling in Microfluidic Paper-Based Devices

Abstract

Antithrombin III (ATIII) is a protein which plays a critical role within the regulation of the coagulation cascade. When the concentration of ATIII is found to be insufficient, thrombotic conditions that are caused by hypercoagulability increase in its likelihood to occur. This can result in life-threatening events, such as myocardial infarction or stroke. Methods that are currently available in order to detect ATIII levels lack the ability to be portable and prompt. These inefficiencies result in the inability to effectively serve as a point-of-care (POC) test in urgent and critical settings. This thesis presents a novel method for detecting levels of ATIII through the use of a particle-based assay which is driven through a model involving capillary flow. The platform uses a microfluidic paper-based device (µPAD) in order to measure flow velocity profiling through the quantification of capillary flow alterations which are caused by the immunoagglutination of the antigen, ATIII and anti-ATIII antibody conjugated microparticles. A smartphone was used to take video recordings of the tested samples which traveled through the channels of the µPAD and in addition, cloud-based Google Colab was used to analyze the videos in order to automatically provide raw data tracking the fluid front of the sample as it moves down the channel. ATIII spiked concentrations of 0 ng/mL, 0.5 ng/mL, 1 ng/mL, 3 ng/mL, 6 ng/mL and 12 ng/mL were tested in PBS, 0.5% plasma and 0.1% plasma. In addition, 0 ng/mL, 0.5ng/mL and 1 ng/mL spiked concentrations were tested in 0.001% plasma. All the samples and their flow profiles were determined. Our definition of flow profile represented the flow velocity slope. The average flow velocity slope was found which allowed for a correlation to be seen between the degree of immunoagglutination of ATIII and anti-ATIII antibody conjugated microparticle as it caused alterations in interfacial tension and viscosity for each of the concentration tested. Samples that had a higher concentration of ATIII led to a higher profile of flow velocity slope. The gold standard immunoassay for ATIII quantification is the enzyme linked immunosorbent assay (ELISA) which provided us with proper quantification of endogenous levels of ATIII in 0.001% plasma and this allowed for steps in the optimization of the assay. This assay does not require intensive training but rather, is affordable, portable, and also capable of rapidly detecting ATIII levels which make it appealing to use within the clinical setting. The work done in this thesis has proven logarithmic linear trends of increasing flow velocity in relation to increasing concentrations of ATIII, providing insight towards developing a foundation in future POC tests targeting ATIII and potentially other biomarkers.