Doctoral Dissertations

Date of Award

12-1991

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

Michael J. Sepaniak

Committee Members

E. L. Wehry, J. Q. Chambers, P. Bienkowski

Abstract

By combining the analytical specificity and selectivity of immunological reagents with fiber optic transmission of spectral signals, the area of fluoroimmunoassay-based fiber optic chemical sensors has grown considerably in the last several years. Building on past work within this research group, a new sensor (the microscale regenerable biosensor, MRB) has been developed which is capable of performing remote in situ direct fluorimmunoassay protocols. The sensor employs a unique delivery system consisting of six capillary columns arranged in a symmetrical fashion about a 400 μm core diameter optical fiber. The columns can be utilized to perform any number of reagent delivery steps including a regeneration step. Regeneration is accomplished by completely replacing the solid reagent phase. All solid reagent phases are made by immobilizing the desired bioaffinity reagent (usually antibodies to a given antigen) on silica beads and referred to as immunobeads. Initial evaluation of the first design MRB (MRBl) was conducted using rabbit-immunoglobulin G (rab-IgG) and a fluorescently labeled (with fluorescein isothiocyanate, FITC) antibody to rab-IgG (anti-rab- IgG-FITC), to mimic a direct fluoroimmunoassay (FIA). Results of the initial evaluation of the MRB presented herein indicate that the MRB can be utilized for performing direct FIAs. The MRBl is subsequently used to perform a direct FIA for benzo(a)pyrene tetrol (BPT), a metabolic product of the carcinogenic compound, benzo(a)pyrene. A dose-response curve for BPT results in an absolute limit of detection of 65 picomoles in a 10 μL sample volume. To address certain difficulties with the MRBl (most notably inadequate signal retention), a second generation MRB was fabricated utilizing a molding concept. By using resin injection molds, the two components of the MRB2 (sensing tip and chamber) can be fabricated more reproducibly and with greater flexibility of design and materials than was possible with the MRBl. Modification of the sensing chamber is discussed. Results of a direct FIA for FITC utilizing anti-FITC immunobeads indicates that signal retention for the MRB2 is higher than that seen for the MRBl. A dose-response curve for the determination of FITC is constructed, and an absolute limit of detection is calculated to be 94 femtomoles in a 10 μL sample volume.

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