Method for Improved Fluorescence Corrections for Molar Mass Characterization by Multiangle Light Scattering

Multiangle light scattering (MALS) was used to determine the absolute molar mass of fluorescent macromolecules. It is standard protocol to install bandwidth filters before MALS detectors to suppress detection of fluorescent emissions. Fluorescence can introduce tremendous error in light scattering measurements and is a formidable challenge in accurately characterizing fluorescent macromolecules and particles. However, we show that for some systems, bandwidth filters alone are insufficient for blocking fluorescence in molar mass determinations. For these systems, we have devised a correction procedure to calculate the amount of fluorescence interference in the filtered signal. By determining the intensity of fluorescent emission not blocked by the bandwidth filters, we can correct the filtered signal accordingly and accurately determine the true molar mass. The transmission rates are calculated before MALS experimentation using emission data from standard fluorimetry techniques, allowing for the characterization of unknown samples. To validate the correction procedure, we synthesized fluorescent dye-conjugated proteins using an IR800CW (LI-COR) fluorophore and Bovine Serum Albumin protein. We successfully eliminated fluorescence interference in MALS measurements using this approach. This correction procedure has potential application toward more accurate molar mass characterizations of macromolecules with intrinsic fluorescence, such as lignins, fluorescent proteins, fluorescence-tagged proteins, and optically active nanoparticles.

Automated summary

In this study, we used multiangle light scattering (MALS) to determine the absolute molar mass of fluorescent macromolecules. We found that bandwidth filters alone are insufficient for blocking fluorescence in some systems. To address this issue, we developed a correction procedure to accurately determine the true molar mass by calculating the amount of fluorescence interference in the filtered signal. We successfully eliminated fluorescence interference in MALS measurements using this approach, demonstrating its potential application for characterizing macromolecules with intrinsic fluorescence.