Application of numerical analysis of fluorescence spectrum to identify properties of substances associating with Congo red micelle

The particles of Congo red (CR), a bis-azo dye, associate in aqueous solutions, forming ribbon-like micelles. Supramolecular dyes, as a result of their molecular size, have a limited capability of penetrating into the interior of the majority of native proteins and do not enter living cells surrounded by an intact phospholipid membrane. They may however, bind to proteins with structure destabilized by unfolding (during denaturation) or function-related changes (e.g. surface cell receptors). Congo red absorbs visible light with a maximum absorption at 500 nm, in aqueous solutions at neutral pH. After CR bind to the polymers (such as cellulose) or certain proteins, dye’s behaviour changes remarkably. These binding affects spectral properties of the dye: the absorption spectrum of the Congo red shifts towards longer wavelengths and receives a yellow-red fluorescence. A numerical analysis of the graphic data obtained from fluorescent microscope (division into channels corresponding to constituent colours and with the noise separated using a graphical program) allowed to understand better the causes of movement of the CR fluorescence spectrum and to identify the properties of substances associating with the CR micelle. By means of a statistical analysis of the data it was possible to observe that wavelength of emission and intensity of fluorescence is not only associated with the polarity of the environment but mainly with the strength of the association between the Congo red micelle and the matrix.