AbstractsPhysics

The thesis describes the development of a novel reversible H2O2 fluorescent probe comprising the ternary complex of europium(III), tetracycline and hydrogen peroxide; and its application in the assays of H2O2, of H2O2 producing oxidases and their substrates, and of H2O2 consuming catalase and its inhibitors. The probe is applied in steady-state intensity-based, time-resolved �gated�, or lifetime-based detection modes both for microplate fluorescence measurement and imaging. The fluorescent probe�s advantages include the reversibility of the EuTc-HP system, the possibility of a kinetic real-time detection of the production and the consumption of H2O2, and the system works best at pH 6.9 - 7.0. It also exhibits the typical spectral characteristics of a ligand-to-europium energy transfer system which include a Stokes shift of ~210 nm, line-like emission, excitation at 380-420 nm (e.g. by the 405-nm blue diode laser), and a µs decay time (~60 µs) facilitating time-resolved fluorometry and imaging. For the probe study, Chapter 1 gives a overview of the state of art of the H2O2 measurements. In the following first part of Chapter 2 is presented the characterization of the fluorescent EuTc-HP probe for its absorbance, circular dichroism and fluorescence spectra, fluorescence lifetime and decay profile, optimal pH and stability, and the influence of temperature, buffers, quenchers and interferents. In the last part of Chapter 6, the peculiar molar ratio of the EuTc-HP probe is further investigated for its possible structure and a com-binatorial approach for discovery of new lanthanide probes is preliminarily proceeded as well. Different assays have been developed for H2O2, glucose, glucose oxidase and catalase, as examples for the detection of enzyme substrates and enzymes. Furthermore, different fluorometric schemes have been applied, such as the steady-state intensity-based detection (Chapter 5, catalase), the time-resolved gated detections (Chapter 3, glucose), the rapid lifetime determination method (Chapter 2, H2O2, novel on microplate) and the time-correlated single photon counting method of the lifetime-based detection (Chapter 2, H2O2), for both microplates and cuvettes, compatible with high-throughput screening. The µs range lifetime of the EuTc-HP probe has greatly facilitated fluorescence imaging, a means for visualization and mapping of the analyte with multiple chemical information. Four schemes of imaging, those are the conventional fluorescence intensity imaging (FII), the time-resolved ("gated") imaging (TRI), the phase delay ratioing imaging (PDI), and the rapid lifetime determination imaging (RLI), have been tested for the quantitative analysis. Hydrogen peroxide, glucose, and glucose oxidase have been determined by the fluorescent imaging system, with Chapter 4 highlighting the fluorescence imaging of glucose oxidase. There are possible further applications in perspective for the EuTc-HP probe. Chapter 6 summarizes the initial attempts, such as glucose oxidase based ELISA, the coupled catalase/glucose…