H-aggregates of the cyanine dye Cy5 are formed during covalent linkage to the cationic macromolecule Poly(allylamine) (PAH). The nonfluorescent H-aggregates strongly restrict the usage of the dye for analytical purposes and prevent a quantitative determination of the labeled macromolecules. The behavior of the H-aggregates has been studied by investigation of the absorption and fluorescence spectra of the dye polymer in dependence on solvent, label degree and additional sulfonate groups. H-aggregate formation is caused by an inhomogeneous distribution of the Cy5 molecules on the polymer chain. The H-aggregates can be destroyed by conformational changes of the PAH induced by interactions with polyanions or in organic solvents. It has been found that the polymer labeling process in high content of organic solvents can prevent the formation of H-aggregates. The results offer a better understanding and improvement of the use of the Cy5 dye for labeling purposes in fluorescence detection of macromolecules. 1. Introduction The labeling of biological or technical materials with fluorescent dyes has gained a great importance in connection with the development of new modern fluorescence-based analytical techniques such as Fluorescence Correlation Spectroscopy (FCS) [1, 2], Single Molecule Spectroscopy (SMS) [3], Confocal Laser Scanning Microscopy (CLSM) [4], Fluorescence Resonance Energy Transfer (FRET) [5], Fluorescence Recovery after Photobleaching (FRAP) [6], or time resolved spectroscopic methods down to femtoseconds [7, 8]. For all of these processes the spectroscopic parameters of the dye molecules such as excitation and emission spectra, lifetime and fluorescence quantum yield are important but often change in an unpredictable way by the labeling procedure [9–12]. Especially in the case of proteins high label degrees or label sites in close neighborhood lead to a strong decrease of the fluorescence quantum yield as well as a shift of absorption and fluorescence energy [13–15]. We investigated these processes on the well-known chromophore Bis(indolenyl)pentamethincyanine that is commercially available under the label name Cy5 (Figure 1). Strong changes in the spectroscopic properties of the dye have been observed earlier by simple adsorption of the positively charged chromophore to negatively charged macromolecules, namely, polyanions [16, 17]. In this paper we studied the spectroscopic behavior of the dye after covalent linkage to the cationic macromolecule PAH. The polyelectrolyte PAH of molecular weight 70？kDa can not only serve as a simple model for
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