Microcapsule biosensors based on competitive binding and fluorescence resonance energy transfer assays
Date of Award
Doctor of Philosophy (PhD)
Micro and Nanoscale Systems
Fluorescent sensing systems offer the potential for minimally invasive monitoring with implantable devices, but they require carrier technologies that provide suitable immobilization, accessibility, and biocompatibility while maintaining adequate response characteristics. Towards the development of this goal, a general design of a biosensor with the capability of detecting different metabolites was investigated. The approach is based on the encapsulation of a competitive binding assay in microcapsules and monitoring the changes in fluorescence resonance energy transfer (FRET) in the presence of analyte. To experimentally demonstrate this type of sensing system, glucose was chosen as the model target analyte. The design, fabrication, and characterization of several embodiments of a non-consuming fluorescence affinity glucose sensor are described in this dissertation. The novel feature of this system used through out the work is the employment of microcapsules for entrapping the sensing assay, which allows for the free movement of sensing elements while maintaining their constant concentrations with continuously-varying analyte concentration.
Initially, a FRET based glucose sensor was demonstrated by encapsulating multilayers of Concanavalin A (Con A)/dextran in microcapsules. Even though microcapsules comprised of Con A/dextran complexes showed reasonable glucose sensitivity, there are some significant obstacles to practical use of this system due to toxicity, aggregation, and irreversible binding. Therefore, to overcome the limitations of Con A, an improved FRET assay was developed by replacing Con A with apo-glucose oxidase (apo-GOx). Apo-GOx is highly specific toward β-D-glucose, reduces the concern over aggregation as it can only bind to one glucose molecule (whereas, Con A binds to four glucose molecules), and also could be more biocompatible than Con A by recombinant production.
The first attempt at the apo-GOx/dextran assay encapsulated in microcapsules used a blue-light-excited FRET pair (FITC/TRITC). The assay elements were encapsulated in microcapsules using photosensitive polymers (poly(styrene sulfonate) and diazoresin) in the shell structure. The results of glucose sensitivity experiments showed a controllable and reversible sensor response with sensitivity in the range of 2--6%/mM over the range of 0--40 mM glucose. In spite of the advantages of this system, it is not ideal for in vivo studies, as the short-wavelength dyes will be difficult to interrogate transdermally due to high tissue scattering. Additionally, diazoresin contains formaldehyde groups that could prove to be toxic. (Abstract shortened by UMI.)
Chinnayelka, Swetha, "" (2005). Dissertation. 584.