Photophysic implications of multiple dye-labelling to a polyelectrolyte

GABRIELA SARMIENTO; NICOLÁS I. KRIMER

Villa Carlos Paz

Congreso; XIII ELAFOT; 2017

Polyelectrolytes have attracted increasing interest in the last decades due to its assiduous use in layer by layer self-assembled structures, which have multiple applications spanning surfaces modification (electrodes or nanoparticles),[1] controlled drug-delivery[2] and formation of microcapsule structures,[3] among others. Commonly the polyelectrolytes can be modified or functionalized with photochemically active molecules to confer additional photophysical properties of interest. Dyes attached to polyelectrolytes have been used for energy transfer processes in layer-by-layer self-assembled nanostructures that mimic antenna systems,[4] as gold nanoparticle coatings in fluorescence enhancement experiments by plasmon interaction,[5] and for the controlled release of genes.[6] However, despite the multiple use of polyelectrolytes covalently labeled with dyes, the literature on the physicochemical and photochemical behavior of these systems is scarce. More importantly, there is little information about how the intermolecular forces beween multiple dye molecules, or between dye molecules and the polymer backbone, affect the labelled polymer conformation in solution, or when it is deposited on a substrate. The present study aims to establish the influence of some chemical and physicochemical parameters - such as the degree of substitution in the polymer chain, hydrophobicity and charge of the dyes - on the conformational and photophysical properties of the macromolecules. For these purposes, the synthesis of a dye (rhodamine B isotiocyanate ? RITC) with a polyelectrolyte (poliallylamine hidrochloride, 450 kD ? PAH) was carried out under an initial stechiometric dye to monomer relationship of 0.02. The concentration of PAH for the synthesis (~0.01M in monomers) was carefully selected by means of Dynamic Ligth Scatering (DLS) measurements to ensure the presence of isolated chains in solution. Samples were purified by dialisis and exclusion chromatography using Sephadex? G-25 M pre-packed colums. After purification, the dye content or polymer molecule was rougly estimated, by absorption spectroscopy, as ~96.UV-absorption and steay-state fluorescence studies on water RITC-PAH solutions indicate that the dye aggregation (with non-fluorescent aggregates) far exceeds 50%. The fluorescence anisotropy of PAH-RITC is ~ 0, showing free rotation of the dye in the polyelectrolyte structure. Fluorescence microscopy experiments on dry RITC-PAH on a coverslip shows approximately 6 molecules of dye, on average, for each polimer unit. Probably, dried condition strongly increase the dye aggregation compared with aqueous enviroment. Fluorescence correlation studies (FCS) of the labelled polymer solutions show the presence of free rodamine-B dye (54 μs and hydrodynamic radius of 3.25 Å) and coiled RITC-PAH molecules (5000 μs and hydrodynamic radius of 55.7 nm). A comparison of the hydrodynamic radius for pure PAH obtained by DLS ( ~80 nm) with the results of FCS allows us to infer that the aggregation of the dye, due to hydrophobic interactions, plays a key role reducing the radius of the coiled conformation of the polielectrolyte. We posit that the dye-to-dye interaction is responsible of intramolecular label aggregation which forces the polymer backbone to a structure much more compact than that for an unlabelled polymer.