Date of Completion
6-10-2016
Embargo Period
6-8-2026
Major Advisor
Prof. James F. Rusling
Associate Advisor
Prof. Challa V. Kumar
Associate Advisor
Prof. Mark W. Peczuh
Field of Study
Chemistry
Degree
Doctor of Philosophy
Open Access
Campus Access
Abstract
This thesis is focused on development of Surface Plasmon Resonance imaging (SPRi) and electrochemical immunoarrays for sensitive and accurate detection of biomarker proteins using offline capture of analytes onto the antibody coated magnetic beads. First, it elucidates kinetic characterization of super-paramagnetic particles decorated with over 100,000 antibodies binding to protein antigens attached to flat surfaces. Surface plasmon resonance (SPR) was used to show that these antibody-derivatized MPs (MP-Ab2) provide essentially irreversible binding and 100-fold increased association rates compared to free antibodies. These findings were consistent with highly efficient capture of protein antigens in solution by the MP-Ab2, and explain in part the utility of these beads for ultrasensitive protein detection into the fM and aM range.
SPR imaging microarrays were developed for detection of epitope specific immunoglobuling E (IgE) antibodies relevant to the diagnosis of severe nut allergies. Microarrays feature 16-25 gold sensor spots equipped with peptide and carbohydrate epitopes from major peanut allergen glycoprotein Arachis hypogaea 2 (Ara-h2). A monoclonal anti-IgE antibody was included as positive control. IgEs were pre-captured onto magnetic beads loaded with polyclonal anti- IgE antibodies to enhance sensitivity and minimize non-specific binding for serum proteins. As low as 0.1 attomole of IgE antibodies were detected from serum samples in 45 min assay time.
Enzyme linked electrochemical immunoassays were developed on 8 screen-printed carbon electrode arrays for detection of prostate cancer biomarker proteins. These proteins are Insulin growth factor-1 (IGF-1), pigment epithelium derived factor (PEDF), vascular endothelium growth factor-D (VEGF-D), and prostate specific antigen (PSA). Serum levels of all these proteins are elevated during different stages of prostate cancer malignancy. Massively enzyme labeled magnetic beads carrying secondary antibodies were used to capture analyte proteins from serum samples. The beads were then magnetically separated, washed and transferred to an electrochemical device housing an 8 nano- structured electrode array modified with capture antibodies. Amperometric signal generated after injection of reagent solution containing 0.1 mM hydrogen peroxide and 1 mM hydroquinone mediator was directly proportional to the amount of captured antigen. Using this strategy, specific and ultrasensitive immunoassays are developed with detection limits in lower femtomolar ranges.
Recommended Citation
Joshi, Amit, "Surface Plasmon Resonance and Electrochemical Microarrays for Multiplexed Protein Detection" (2016). Doctoral Dissertations. 1145.
https://digitalcommons.lib.uconn.edu/dissertations/1145