Advancement of an agarose bead-based immunoassay platform towards point-of-care applications
McDevitt, John T
Doctor of Philosophy
Efficient and affordable technologies for early detection of disease are of high priority in modern practice of medicine in order to effectively manage diseases and improve outcomes. The programmable bio-nano-chip (p-BNC) platform is an in-development miniaturized micro-device aimed for broad-scale clinical practice at the point-of-care which utilizes agarose beads as a solid-support surface for immunoassay testing in a lab-on-a-chip format. This dissertation work describes research directed towards advancing the p-BNC platform. It begins by introducing a new custom-designed flow-through biochip which holds the agarose beads in an increased pressure-driven flow environment, allowing for an enhancement in analyte capture inside the three-dimensional fibrous network of agarose. This part of the thesis addresses a major concern of how to further lower limits of detection for quantitative measurements of samples composed of low concentrations of biomarkers while using inexpensive, disposable biochips. The thesis next presents novel experimental work exploring the use of agarose beads with an extended range of porosity to maximize binding capacity for three biomarkers of varying molecular weights and sizes. This work demonstrates the importance of pores in the agarose solid support structure for increasing analyte binding capacity and sensitivity, as well as showed that agarose beads with one pore size are unsuitable for all biomarker sizes. Next, a novel extension of the p-BNC system for measuring the binding kinetics of antibodies is demonstrated which reveals the importance of binding strength for optimizing immunoassay sensitivity and dynamic range. Lastly, long-term stability of antibody-functionalized agarose beads stored in the p-BNC system is examined. Here, it is demonstrated for the first time that, by stabilizing with polyhydoxyl compounds, agarose beads can be stored in wet form in the p-BNC device at room temperature for up to 2 months while maintaining their structural integrity and biological function.