Photophysic implications of multiple dye-labelling to a polyelectrolyte

KRIMER, N.; GOLDGERG, T. J.; SARMIENTO, G.P.; MIRENDA, M.; HODAK, J.; BOSSI, M. L.

Villa Carlos Paz - Cordoba

Encuentro; XIII ENCUENTRO LATINOAMERICANO DE FOTOQUIMICA Y FOTOBIOLOGIA; 2017

Polyelectrolytes haveattracted increasing interest in the last decades due to its assiduous use in layerby layer self-assembled structures, which have multiple applications spanning surfacesmodification (electrodes or nanoparticles),[1] controlled drug-delivery[2] andformation of  microcapsule structures,[3]among others. Commonly the polyelectrolytes can be modified or functionalizedwith photochemically active molecules to confer additional photophysicalproperties of interest. Dyes attached topolyelectrolytes have been used for energy transfer processes in layer-by-layerself-assembled nanostructures that mimic antenna systems,[4] as goldnanoparticle coatings in fluorescence enhancement experiments by plasmoninteraction,[5] and for the controlled release of genes.[6] However, despitethe multiple use of polyelectrolytes covalently labeled with dyes, theliterature on the physicochemical and photochemical behavior of these systemsis scarce. More importantly, there is little information about how theintermolecular forces beween multiple dye molecules, or between dye moleculesand the polymer backbone, affect the labelled polymer conformation in solution,or when it is deposited on a substrate.   The present study aims toestablish the influence of some chemical and physicochemical parameters - suchas the degree of substitution in the polymer chain, hydrophobicity and chargeof the dyes - on the conformational and photophysical properties of themacromolecules. For these purposes, the synthesis of a dye (rhodamine Bisotiocyanate ? RITC) with a polyelectrolyte (poliallylamine hidrochloride, 450kD ? PAH) was carried out under an initial stechiometric dye to monomerrelationship of 0.02. The concentration of PAH for the synthesis (~0.01M inmonomers) was carefully selected by means of Dynamic Ligth Scatering (DLS)measurements to ensure the presence of isolated chains in solution. Sampleswere purified by dialisis and exclusion chromatography using Sephadex? G-25 M pre-packed colums. Afterpurification, the dye content or polymer molecule was rougly estimated, byabsorption spectroscopy, as ~96. UV-absorption and steay-statefluorescence studies on water RITC-PAH solutions indicate that the dyeaggregation (with non-fluorescent aggregates) far exceeds 50%. The fluorescenceanisotropy of PAH-RITC is ~ 0, showing free rotation of the dye in the polyelectrolytestructure. Fluorescence microscopy experiments on dry RITC-PAH on a coverslipshows approximately 6 molecules of dye, on average, for each polimer unit.Probably, dried condition strongly increase the dye aggregation compared withaqueous enviroment. Fluorescence correlation studies (FCS) of the labelledpolymer solutions show the presence of free rodamine-B dye (54 μs and hydrodynamicradius of 3.25 Å) and coiled RITC-PAH molecules (5000 μs and hydrodynamicradius of 55.7 nm). A comparison of the hydrodynamic radius for pure PAHobtained by DLS ( ~80 nm) with the results of FCS allows us to infer that theaggregation of the dye, due to hydrophobic interactions, plays a key role reducingthe radius of the coiled conformation of the polielectrolyte. We posit that thedye-to-dye interaction is responsible of intramolecular label aggregation whichforces the polymer backbone to a structure much more compact than that for anunlabelled polymer.  [1] G. Decher, Science 1997, 277, 1232.[2] J. Best, M.Neubauer, S. Javed, H. Dam, A. Fery, F. Caruso, Langmuir 2013, 29, 9814.[3] R. Luo, S. S.Venkatraman, B. Neu, Biomacromolecules2013, 14, 2262.[4] Z. Dai, L.Dahne, E. Donath, H. Mohwald, J. Phys. Chem. B 2002, 106,11501.[5] K. Kim, Y. M. Lee, J.W. Lee, K. S. Shin, Langmuir 2009, 25, 2641.[6] J. Santos, A. Nouri, T. Fernandes, J. Rodrigues,H. Tomas, Biotechnol. Prog. 2012, 28, 1088