Oxidoreductase immobilization in reprecipitated polyaniline nanostructures for optical biosensing applications.

摘要

To maintain good health, patients with diabetes mellitus must keep careful control over their blood glucose levels. For many, however, this requires painful and inconvenient fingerstick tests several times each day, a regimen that can still miss periods of time when blood sugar levels are dangerously high or low. This thesis describes an effort to develop a less invasive, continuous glucose monitoring system using the optical properties of polyaniline, a biocompatible conjugated polymer. This proof-of-concept study demonstrates the detection of glucose levels in vitro using a novel reagentless biosensing platform consisting of a nanostructured thin-film of polyaniline containing immobilized glucose oxidase, an oxidoreductase enzyme with high specificity for glucose. Enzyme entrapment is accomplished via a facile reprecipitation method, in which the polymer is held at the edge of solubility in an aqueous-organic binary suspension. By adjusting the ionic strength of the suspension, non-covalent intermolecular crosslinking of the chains can be induced, leading to rapid gelation. This transition, which is sensitive the hydrogen-bond polarization and electrostatic screening by counterions, induces the hydrophobic collapse of the polymer and aggregation into nano- and microscale particles or nanostructured thin films. The coiling, forced planarization, and supermolecular agglomeration of the polymer was measured by UV-VIS spectroscopy, dynamic light scattering, bright field microscopy, and scanning electron microscopy. This novel biosensor fabrication method requires no harsh reagents or acidic conditions that can contribute to enzyme denaturation. Once an implant is in place, continuous data could be collected non-invasively using wavelengths of light that pass readily through living tissue, without requiring needles or optical fibers to penetrate the skin. This information may be sent wirelessly, in real-time, to an insulin pump, audible alarm, and/or health care provider. We also demonstrate generalizations of this method using two other oxidoreductase enzymes, choline oxidase and uricase, to realize a robust biosensing platform.