Development of a high-sensitive three-dimensional fluorescence stopped-flow

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Fortified with an image-intensifier in front of the detector and using a 150 W Xenon lamp for excitation, an instrument that can record 512 fluorescence spectra with a time resolution of 10 ms was constructed. By introduction of a Xenon flash lamp (Hamamatsu Photonics, L-4633,1 microsecond pulse width), the time resolution was improved to 1 msec. This allows the excitation of a sample solution at pre-programd times with a 1-ms time interval at least after initiation of the reaction, and consecutive 173 fluorescence spectra are recorded. This instrument can be set up to follow the fluorescence change with time at a fixed wavelength, and the software developed determines the kinetic parameter easily.Recorded fluorescence spectra can be displayd three-dimensionally with wavelength, reaction time, and fluorescence intensity as orthogonal axs, respectively. The fluorescence intensity can be converted into pseudo-colors and expressed by contours, too. The softwares for data analysis and disp … More lay were developed by using various software tools for numerical analysis, especially MATLAB.Usually 512*512 data points collected as 512 consecutive spectra are processed by the SVD analysis to obtain the minimal number of the component spectra. With an assumed reaction model that descrives all of the participating reaction species and the initial values for microscopic rate constants for the model, the formation and decay of the reaction intermediates are derived by solving differential equations, and stored in a matrix. The spectra of the reaction intermediates are derived by using pseudoinverse, and the predicted spectral changes are constructed using the time matrix as well as the derived spectra. Optimization is then carried out to minimize the residuals between the experimental and calculated spectra to derive the kinetic constants and the spectra of the intermediates.This technnique can be applied universally to time-resolved spectral change as proved in this study. An application of this technique to the antigen-antibody reaction will be promoted by selecting appropriate reactions. Less