Date of Award

Spring 2005

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

Michael J. McShane

Abstract

Minimally and non-invasive glucose sensing techniques are instrumental in improving the quality of life of diabetics, point-of-care testing, critical care monitoring, development of new anti-diabetic drugs or therapeutic compounds, and the study of basic physiology. The overall goal of this project was to build a fluorescent glucose sensor by incorporating relevant sensing chemistry within microcapsules. The specific aims for this research were to study various processes used for the fabrication of micro-containers, the use of these for encapsulation purposes, and the application of the encapsulated system towards glucose sensing. The following is a brief description highlighting the key areas of the research.

Different materials such as polystyrene sulfonate (PSS), polyallylamine hydrochloride (PAH), polydiallyldimethyl ammonium chloride (PDDA), chitosan (CHT), and polyvinyl sufate (PVS) were used to construct the capsule walls. Fluorescence recovery after photobleaching (FRAP) was used to study the effect of capsule wall architecture on the permeability of capsules to FITC labeled GOx (FITC-GOx). Long-term stability and activity studies proved that {PVS/CHT} 2-{PSS/PAH}2 capsules were the most stable and showed minimal leaching (79% of enzyme retained) and the activity of the encapsulated enzyme did not change significantly over four weeks. Hence, they were selected for demonstration of glucose sensing. Fluorescence sensors for glucose using pH-sensitive Pyrene 8-hydroxy-1,4,6,-trisulfonylchloride (HPTS), which facilitates ratiometric monitoring of pH changes, was used to label GOx (HPTS-GOx), and encapsulated in these capsules. Changes in glucose concentration were monitored as a function of the pH changes during glucose consumption by GOx, and the sensors showed a linear response to sequential additions of glucose in the physiological region of interest (0mM–30mM).

In summary, the ideas, techniques and discussion of the results presented in this dissertation are a significant contribution to the science of enzyme encapsulation. The preliminary data demonstrating glucose sensing using a labeled pH-sensitive dye is an important contribution to this application. Some of the novel techniques presented here serve as a foundation for future application of these to other areas. The experimental techniques and methods of characterization described in this dissertation can serve as a comprehensive guide to researchers involved in similar study.

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