New Solution of Eliminating the Inner Filter Effect in Fluorescent Measurements

Abstract

A method of correction and normalization of the experimentally recorded fluorescence intensity allowing to exclude the influence of the so called inner filter and to select an informative component that is the product of absorbance and fluorescence quantum yield of fluorophore in the under test solution is proposed: F0 = F/kW = Afl qfl. The value of the correction factor W = (1-10-AΣ) /AΣ is determined solely by the absorbance of the solution AΣ, and does not depend on the relative contribution to the absorption of fluorescing and non-fluorescing components of the solution. The normalization factor k can be determined from the measurement (in the same conditions in which the fluorescence intensity of the sample is measured) of reference fluorescence, i.e. the solution of the substance with a known fluorescence quantum yield.It is shown that the proposed method of fluorescence intensity correction and normalization allows determining undistorted fluorescence excitation spectra of solutions with high optical density and 3D-fluorescence spectra, correcting Stern-Volmer dependences if a quencher absorbs at the wavelength of the exciting light. Registration of the fluorescent dye-receptor system fluorescence intensity on the concentration of the dye allows determining the binding constant and the number of binding sites of the dye to the receptor if all of the bound dye molecules, which are responsible for fluorescence, belong to a single binding mode. The complete information about the parameters of the dye-receptor interaction, as well as absorption spectra of dye bound to receptor can be obtained by spectrophotometry using a sample solution and reference solution prepared by an equilibrium microdialysis. Registration of the fluorescence intensity of these solutions also allows determining the fluorescence quantum yield of dye molecules bound to the sites belonging to different binding modes.