Real time observation of diffusion and bioaffinity binding processes in single polyelectrolyte-coated microcapsules: A fluorescence-based approach

We report on using fluorescence microscopy to study, visualize and determine the diffusion phenomena into and bioaffinity binding within single microcapsules in real time by using biotin–fluorescein as diffusive species and encapsulated avidin as binding partner. Microcapsules were constructed by entrapment of avidin within an agarose matrix and encapsulated with polyelectrolyte layers by Layer-by-Layer (LbL) polyelectrolyte self assembly. A “ring” of high fluorescence intensity advancing with time towards the capsule centre was observed during incubation of capsules with fluorescent-labeled biotin. Fluorescence intensity was build up in capsule areas where binding to avidin occurred and was visualized in real time. A model for the diffusion process in microcapsules was developed and experimental data was plotted and fitted well with trends predicted by the model. The value of the diffusion coefficient for biotin–fluorescein was determined to be 3.5 × 10−8 cm2/s, which is comparable to literature values of similar sized molecules.