Browsing by Author "Kulla, Eliona"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Advancement of an agarose bead-based immunoassay platform towards point-of-care applications
    (2015-12-04) Kulla, Eliona; McDevitt, John T
    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.
  • Thumbnail Image
    Item
    Enhancement of performance in porous bead-based microchip sensors: effects of chip geometry on bio-agent capture
    (Royal Society of Chemistry, 2015) Kulla, Eliona; Chou, Jie; Simmons, Glennon; Wong, Jorge; McRae, Michael P.; Patel, Rushi; Floriano, Pierre N.; Christodoulides, Nicolaos; Leach, Robin J.; Thompson, Ian M.; McDevitt, John T.
    Measuring low concentrations of clinically-important biomarkers using porous bead-based lab-on-a-chip (LOC) platforms is critical for the successful implementation of point-of-care (POC) devices. One way to meet this objective is to optimize the geometry of the bead holder, referred to here as a micro-container. In this work, two geometric micro-containers were explored, the inverted pyramid frustum (PF) and the inverted clipped pyramid frustum (CPF). Finite element models of this bead array assay system were developed to optimize the micro-container and bead geometries for increased pressure, to increase analyte capture in porous bead-based fluorescence immunoassays. Custom micro-milled micro-container structures containing an inverted CPF geometry resulted in a 28% reduction in flow-through regions from traditional anisotropically-etched pyramidal geometry derived from Si-111 termination layers. This novel "reduced flow-through" design resulted in a 33% increase in analyte penetration into the bead and twofold increase in fluorescence signal intensity as demonstrated with C-Reactive Protein (CRP) antigen, an important biomarker of inflammation. A consequent twofold decrease in the limit of detection (LOD) and the limit of quantification (LOQ) of a proof-of-concept assay for the free isoform of Prostate-Specific Antigen (free PSA), an important biomarker for prostate cancer detection, is also presented. Furthermore, a 53% decrease in the bead diameter is shown to result in a 160% increase in pressure and 2.5-fold increase in signal, as estimated by COMSOL models and confirmed experimentally by epi-fluorescence microscopy. Such optimizations of the bead micro-container and bead geometries have the potential to significantly reduce the LODs and reagent costs for spatially programmed bead-based assay systems of this type.
  • Thumbnail Image
    Item
    High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study
    (Elsevier, 2014) Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Dominguez-Medina, Sergio; Kulla, Eliona; Kang, Marci; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.
    The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of ?-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.