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Fluorescence Quenching of Polypyrrole Polymer Immobilized Glucose Oxidase Usage of biosensing systems is the promising, rapid and accurate method for detection and analysis of various compounds concentration. Using the conducting polymers, it is possible to design sensitive and biologically compatible biosensors. Nowadays the best-known and the most common biosensors are amperometric glucose biosensors. They can detect blood’s free glucose in a short amount of time – it is very important for people with diabetes. The active part of the biosensor is glucose oxidase (GOx) enzyme immobilized on the surface of the electrode. The conducting polymers are used as a matrix for immobilization that facilitates electrons transfer from enzyme to electrically conductive surface of the electrode. When constructing biosensor, one of the most important aims is to create enzyme and polymer system, which would ensure the thermal stability and retain the catalytic activity of the enzyme. The main goal of this study is to investigate spectroscopic properties of the glucose oxidase self-encapsulated within polypyrrole shell and to determine the pH and glucose influence to enzyme spectroscopic properties. Tasks: 1. To measure absorption, fluorescence spectra and decay kinetics of the integrated solution. To analyze experimental data. 2. To determine the impact of glucose to the solution system. 3. To compare the spectroscopic properties of integrated compound in different pH environments. 4. To determine the polypyrrole shell influence to fluorescence decay times of GOx. During this study, we found that GOx enzyme fluorescence band at 530 nm belongs to coenzyme FAD fluorescence. In 29 day measurement period, the increase of GOx fluorescence, which is due to the fact that the enzyme has changed its structure and denatured, was visible. GOx enzyme selectively catalyzes the oxidation of D-glucose. Products of the reaction change the stability of the solution system. This leads to changes of GOx spectroscopic properties, which are associated with changes in the structure of the enzyme, during which the FAD is more quickly released from the active center of the enzyme. GOx enzyme is sensitive to changes of temperature and pH. In the optimum pH (in GOx case is 5,5-6,0) activity of the enzyme is at maximum, and thus, the fluorescence intensity is the highest. Using a buffer solution with pH 7, the enzyme activity decreases and also fluorescence diminishes. Higher influence of pH changes is observed in fluorescence spectra of FAD: using a buffer with pH 7, the maximum fluorescence intensity was weakened by 9 times and the increase of fluorescence disappeared in the 29-day period. To stabilize GOx and reduce FAD’s release, the enzyme was immobilized in the polypyrrole matrix. In a solution of polymerization, which consisted of GOx, pyrrole, glucose and dissolved oxygen, were proceeded redox reactions which produced hydrogen peroxide (H2O2). Hydrogen peroxide lead to pH drop near enzyme’s active center and then the chemical polymerization of pyrrole could begin. The polypyrrole encapsulated GOx and formed shell that inhibited the release of FAD. The exploration of GOx and pyrrole solution confirms that the encapsulated GOx enzyme has become more stable. It was found that polypyrrole shell reduces the fluorescence quenching twice. Changes of FAD and pyrrole average fluorescence relaxation times, showed that FAD was also encapsulated within polypyrrole. The quenching times of FAD decreased and their changes in the 29-day period have become more even. Usage of a buffer solution with higher pH (pH 7) also decreases the maximum intensity of spectral bands values and the fluorescence relaxation times. |
