Date of Award

5-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Chemistry

Advisor

Creager, Stephen E

Committee Member

Guiseppi-Elie , Anthony

Committee Member

Chumanov , George

Committee Member

Arya , Dev Priya

Committee Member

Christensen , Kenneth A

Abstract

Applications such as point-of-care medical diagnosis and in-the-field biological warfare agent detection require portable bioassays which are less laborious yet cost effective. Conventional sandwich bioassays see limited use for such applications as it involves time consuming steps of reagent treatment and washings, and are restricted to use inside laboratories. We explore new platforms for sandwich bioassays required by such applications. The platform we have developed is based on electrochemical methods of detection. Flow-through three-dimensional electrodes suitable for use in electrochemical sandwich bioassays were fabricated. A composite of superporous agarose and reticulated vitreous carbon (SPA-RVC) material was used for the fabrication of these electrodes. These SPA-RVC electrodes provide confined spaces to trap the redox molecules within the electrode making it possible for coulometric detection. The flow-through design allows for ease in treatment of multiple solutions and washing steps. The electrode materials were characterized for their physical features like pore-size distribution, total free volume, densities, and structural characteristics. The characteristics of the SPA-RVC electrode to perform electrochemical measurements were also studied. Avidin affinity molecules were covalently bound onto the superporous agarose (SPA) matrix to create binding sites to immobilize biotinylated capture probes. The loading of these avidin moieties inside the SPA-RVC electrodes was determined. SPA-RVC electrodes were used for the determination of kinetic parameters of immobilized avidin alkaline phosphatase labels required for carrying out electrochemical bioassays. The electrodes were tested for their feasibility in carrying out sandwich assays. Biotinylated bovine serum albumin was detected in a sandwich assay scheme using these electrodes. DNA hybridization detection with the proposed scheme posed problem such as high non-specific and undesirable specific binding of the biomaterials used. Alternative bioassay schemes for the improvement of the DNA hybridization assays are discussed.

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