Doctoral Dissertations

Winter 1989

Dissertation

Chemistry

Degree Name

Doctor of Philosophy

Rudolf Seitz

Abstract

An optical glucose sensor has been developed using competitive binding in conjunction with energy transfer. Sensor response is based on competition between glucose and dextran for a limited number of binding sites on the protein concanavalin A (conA). The system is optically monitored using fluorescent donor-acceptor dye pairs labeled to concanavalin A and dextran. When the dyes are sufficiently close, on the order of 50 A, energy is transferred from the donor emission band to the overlapping excitation band of the acceptor. This nonradiative, singlet-singlet transfer of energy enhances the acceptor emission at the expense of donor emission.

In absence of glucose, the conA and dextran are bound together and energy transfer takes place. Upon addition of glucose, the dextran is displaced, and energy transfer is disrupted. The ratio of the two emission intensities can be related to glucose concentration. Donor-acceptor systems investigated included energy transfer from fluorescein (FITC) to three different types of rhodamine, TRITC, XRITC, and Texas Red, and both coumarin and fluorescamine donating energy to FITC. The system that gave the largest change in intensity involved FITC labeled conA as the donor and TRITC labeled dextran as the donor.

Fluorescence was measured with both conventional fluorescence instrumentation and a computer controlled, laser excited spectrometer. The laser instrument was developed specifically for the optical glucose sensor, but was designed to support a wide range of fiber optic sensors. Instrument components include a nitrogen pumped dye laser, fiber optic beam splitters, photomultiplier tubes fitted with interference filters for wavelength selection, and boxcar averagers.

Instrument development included calibration of the dye laser, evaluation of different fiber optic beam splitter arrangements, reduction of stray light, and evaluation of the boxcar averagers. The spectrometer was interfaced to an Apple IIc computer which was programmed to collect the data, perform baseline corrections, ratio the two channels, and trigger the laser to initiate the next data point.

The instrument's detection level, using Rhodamine 6G standards, is 1.0 $\times$ 10$\sp{-9}$molar and is limited by stray light. Precision of the instrument is approximately 3% and is limited by drift of the boxcar averagers.

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