(438d) Improved Characterization of Fluorescent Macromolecules Using Light Scattering

Multi-angle light scattering (MALS) is a powerful technique that can be used to determine molar mass, shape parameters, and size from anisotropic macromolecules. However, many fields employ optically active materials that fluoresce, which can corrupt light scattering measurements. Fluorescence poses a unique challenge to characterization via light scattering due to fluoresced photons being measured as scattered photons. In most systems, species fluoresce significantly more than they scatter, resulting in enormous overestimations in molar mass and obscuring particle properties. To mitigate such interference, it is standard protocol to install bandwidth filters before MALS detectors to suppress detection of fluorescent emissions. However, due to practical limitations of instrumentation it is not possible to design bandwidth filters that block all the fluorescence without also blocking scattered light. Instead, broad filters are used that allow all scattered light through and block most – but not all – of the fluorescence. However, for intense fluorescers, the transmitted fluorescence is still significant enough to invalidate measurements. For these systems we have devised a procedure to calculate the amount of fluorescence that is not blocked by the bandwidth filters. By determining the intensity of fluorescent emission not blocked by the bandwidth filters, we can correct the filtered signal accordingly and accurately elicit the true molar mass and particle properties. The transmission rate of unblocked fluorescence is calculated before MALS experimentation using standard fluorimetry techniques, allowing for the characterization of unknown samples. To validate the correction procedure, we synthesized custom fluorescent dye-conjugated proteins with well-known emission characteristics to purposefully interfere with light scattering measurements. 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 of all shapes and sizes.