New pH sensitive sensor materials. Luminescent fiber-optic dual sensors for non-invasive and simultaneous measurement of pH and pO2 (dissolved oxygen) in biological systems

摘要

This thesis describes the development and characterization of novel, pH-sensitive, optical sensor materials. Special attention is given to the development of dual optical chemical sensors for non-invasive determination of pH and dissolved oxygen (DO) in biological systems. A new measurement scheme is introduced to evaluate and calculate the data for these two parameters via dual luminophore referencing (DLR). An application example for simultaneous monitoring of pH and DO in bioprocessing is given and discussed.udChapter 1 gives an introduction to the importance of optical pH and DO sensors. Furthermore, the overview of existing planar dual sensor materials and dual microsensors was presented. Referencing methods for measurement with luminescent optical sensors were described. udChapter 2 describes the application and spectral properties of a new type of fluorescent pH indicators with different dynamic pH ranges. The indicators used (ACIB, ACIDA and ACISA) are based on the luminescent dye iminocoumarin and were covalently immobilized on the surface of amino-modified polymer microbeads and incorporated into a hydrogel matrix to obtain novel pH-sensitive materials. Due to self-referencing of the ACISA-based microbeads, the ACISA-containing sensor membrane can be read out via ratiometric dual wavelength referencing. A membrane capable of optical pH sensing over a very wide pH range (pH 1 � pH 11) by using a mixture of two different microbeads was also developed. udIn Chapter 3, pH-sensitive sensor membranes for the DLR scheme, based on carboxyfluorescein, dichlorocarboxyfluorescein and iminocoumarin are presented. The membranes are characterized with respect to their cross-sensitivity towards ionic strength at 25 and 140 mM. The sensor membrane based on iminocoumarin is found to possess no cross-sensitivity towards ionic strength at all, while for the other two a small effect is observed, which, however, was small compared to other pH indicators like HPTS. All three membranes showed an excellent photostability. The indicators immobilized on the microbeads are suitable for DLR measurements using polyacrylonitrile-derived nanoparticles incorporating Ru(dpp)32+ as reference standard. It was found that the variety of pH indicators in the DLR scheme results in pH sensors with pKa values from 4 to 8.udIn Chapter 4, a novel modified dual lifetime referencing method (m-DLR) for simultaneous sensing of pH and pO2 with a single fiber-optic sensor is introduced. Three different dual sensor materials were investigated. The dual sensor membrane consisted of fluorescent indicators immobilized in different kinds of organic polymer microbeads, which in turn were contained in a single polyurethane-type hydrogel matrix. The phase-modulated light of an LED excites the luminescence of the indicators, and average decay times or phase shifts served as the analytical information. Data were evaluated by the m-DLR method which relates the phase shift (as measured at two different frequencies) to pH and to oxygen partial pressure. The working range of the dual sensor (pH 4 - pH 9 and 0 � 21.3 kPa pO2) meets the requirements for application in biological systems.udA fiber-optic microsensor for minimal-invasive measurements is presented in Chapter 5. The tip of the optical fiber with a diameter of ~ 140 µm was covered with a sensor composition based on luminescent microbeads (HPTS/p-HEMA microbeads as pH-sensing system and Ru(dpp)32+/ormosil microbeads as oxygen probe) dispersed in a hydrogel polymer matrix. Measurements and evaluation were performed according to the method introduced in chapter 4. The optimal working range of the sensor was from pH 5 to pH 10 and for pO2 0 � 21.3 kPa. The sensor was characterized with respect to its photostability, reproducibility and reversibility.udChapter 6 describes a new approach towards simultaneous, non-invasive and continuous measurements of pH and DO in 24-well microbioreactors. The performance of the system was demonstrated by monitoring pH and DO kinetics during cultivation of Pseudomonas putida and Escherichia coli. The results obtained in microbioreactors were compared with a conventional shake flask. The technique can be used for high-throughput bioprocess optimization.