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Lasermessverfahren für die zeitaufgelöste Quantifizierung von Konzentrationsprofilen in hydrogel matrices

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Publikationsserver der RWTH Aachen University
Keywords
  • Info:Eu-Repo/Classification/Ddc/530
  • Konfokale Mikroskopie
  • Immobilisiertes Enzym
  • Mehrphotonen-Spektroskopie
  • Hydrogel
  • Laserinduzierte Fluoreszenz
  • Physik
  • Flim
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Abstract

Immobilization of enzymes in aqueous hydrogel beads which are suspended in organic solvents is a promising technique for the production of hydrophobic fine chemicals. For the rational design of such enzyme immobilizates, the complex interaction between mass transfer across the interface, diffusion in the hydrogel bead and enzyme reaction has to be investigated in detail. It is obvious that the development of such a complex model can only be based on highly significant and precise measurement data. An established procedure is to take samples from the organic phase for concentration measurements. It was shown that the performance of the classical approach of taking samples from the organic phase is insufficient for a mechanistically correct understanding of the process. The approach of this work is to develop new methods for the detection of spatially and temporally resolved concentration gradients at a microscopic scale in hydrogel beads by means of laser scanning microscopy. In this work a new method for optical pH monitoring using pH-sensitive dyes is demonstrated. As a first step, calibration curves of two different indicator dyes are recorded. For the calibration of the correlation between the pH-value and fluorescence lifetime the decay of the fluorescence intensity after the excitation pulses is recorded and measured by time-correlated single photon counting with a temporal resolution of 815 fs using a confocal laser scanning microscope. The acidic and basic forms of the dyes have different pH-independent lifetimes. At a defined pH-value the decay curve is composed of lifetime components of both forms. The proportion of the acidic and basic components changes with variation of the pH value and a biexponential intensity decay over time will yield lifetimes and their proportion. The calibration curves are evaluated concerning pH-range, dynamic range and measurement uncertainty. After choosing Resorufin as pH indicator dye, the diffusion of propionic acid into the Ca-alginate hydrogel bead is investigated optically. The change in pH-value over time resulting from the diffusion of propionic acid into the hydrogel bead is observed in the centre of the hydrogel bead with a temporal resolution of one second. As competing model assumptions the Fickian and Nernst--Planck diffusion laws are considered and compared with the measured pH-progress. To our knowledge lifetime confocal laser scanning microscopy is used for the first time in this study for the quantification of dynamic pH-changes in macroscopic particles with microscopic resolution. In the second part of this thesis, multiphoton microscopy is shown as a promising technique to detect spatially and temporally resolved concentration gradients of chemical compounds, e.g. reactants in hydrogel-encapsulated biocatalysts. In contrast to current methods, this method has an improved spatial and temporal resolution and gives the possibility to measure educt or product concentrations in hydrogel beads and hence facilitates the identification of various kinetic phenomena. In a first step, the phenomena diffusion, coupled diffusion and mass transfer through the phase boundary are investigated in the bead center. Finally, the complete system – consisting of diffusion, mass transfer and enzymatic reaction – is observed spectroscopically by measuring concentration gradients along the bead radius with temporal and spatial resolution. This measurement technique enables a subsequent mechanistic model identification, which in turn leads to an enhanced knowledge of reaction kinetics and supports the design of biotechnological processes. This task was only possible due to excellent spatial (25 µm) and temporal (5 sec) resolution and the accuracy (±1 %) achieved by using a multiphoton microscopy set up.

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