A FRAP-based evaluation of protein diffusion in polyelectrolyte multilayers

• FRAP-based evaluation of molecular diffusion.
• Protein (cytochrome C) diffusion coefficient in HA/PLL multilayers ∼1 μm2/s.
• Effect of pinhole and scanning modes to the FRAP evaluation.

The evaluation of molecular diffusion by fluorescence recovery after photobleaching (FRAP) gives an option to examine properties of various soft materials aiming at material design and development. Biofunctional polyelectrolyte multilayer films can be fabricated using the layer-by-layer technique that offers easy tunability of the film chemical composition, thickness, and mechanical properties. Here the mobility of a model protein cytochrome C loaded into the multilayer film consisting of hyaluronic acid and poly-l-lysine is investigated. An approach based on analytical solution of Fick’s law is used for evaluation of the protein diffusion coefficient from FRAP measurements. The FRAP measurement procedure has been optimised to ensure a high quality of the recorded data and to improve sensitivity of the measurements. For this purpose, various settings of the confocal laser scanning microscope have been examined and changing of the pinhole size has been found the most appropriate way to reduce signal noise and improve sensitivity. This study opens up a possibility of the fundamental understanding of intermolecular interactions within the polyelectrolyte multilayers as well as applicability of FRAP for the evaluation of diffusion of various molecules.

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