Quantifying the presence of unwanted fluorescent species in the study of pyrene-labeled macromolecules

In order to mimic the effect that unwanted fluorescent species have on the process of excimer formation between pyrene labels covalently attached onto macromolecules, the steady-state fluorescence spectra and time-resolved fluorescence decays were acquired for mixtures of pyrene monolabeled and doubly end-labeled 2K poly(ethylene oxide) referred to as Py(1)-PEO(2K) and Py(2)-PEO(2K), respectively, and mixtures of 1-pyrenebutyric acid (PyBA) and a fourth generation dendron end-capped with pyrene (Py(16)-G4-PS). Monolabeled polymers like Py(1)-PEO(2K) and unattached fluorescent labels like PyBA are among the most typical fluorescent impurities that are encountered in the study of fluorescently labeled macromolecules. Our fluorescence experiments revealed that addition of minute amounts of Py(1)-PEO(2K) or PyBA to, respectively, Py(2)-PEO(2K) or Py(16)-G4-PS solutions induced a dramatic reduction of the ratio of the fluorescence intensity of the pyrene excimer to that of the pyrene monomer, namely the I(E)/I(M) ratio. Although the extreme sensitivity of fluorescence in general and the I(E)/I(M) ratio in particular to the presence of fluorescent impurities is a great concern, it is nevertheless reassuring that this effect can be quantitatively accounted for by analyzing the fluorescence decays of the pyrene monomer and excimer globally, according to a protocol which is described in detail in this study. The experiments presented herein demonstrate the importance of studying fluorescently labeled macromolecules that are of the highest purity when probing the rapid internal dynamics of a macromolecule by fluorescence.