Ensemble and Single Particle Fluorescence Characterization of Dye-Labeled Cellulose Nanocrystals

Cellulose nanocrystals (CNCs) have been covalently labeled with both fluorescein and rhodamine and studied by a combination of UV-vis absorption spectroscopy and ensemble and single molecule fluorescence spectroscopy. For all samples, the fluorescence anisotropy and lifetimes were consistent with effects expected for covalently bound dye molecules. Low dye loading levels (similar to 0.1 dye/particle) were estimated for the fluorescein-labeled CNC which coupled with the strong pH dependence make this a less suitable fluorophore for most applications. Rhodamine-labeled CNCs were prepared from both sulfated and carboxylated CNCs and had loading levels that varied from 0.25 to similar to 15 dye molecules/CNC. For the sulfated samples, the absorption due to (nonfluorescent) dimeric dye increased with dye loading; in contrast, the carboxylated sample, which had the highest rhodamine content, had a low dimer yield. Single particle fluorescence studies for two of the rhodamine-labeled CNCs demonstrated that individual particles are readily detected by their stepwise blinking/bleaching behavior and by polarization effects. Overall, the results indicate the importance of understanding the effects of loading on dye photophysics to select an optimal dye concentration to maximize sensitivity while minimizing the effect of the dye on the CNC behavior. The results also demonstrate that CNCs with relatively low dye loadings (e.g., similar to 1 dye/particle) are readily detectable by fluorescence and should be adequate for use in fluorescence-based biological assays or to probe the distribution of CNCs in composite materials.