Chitosan surface interaction platform for protein binding quantification by fluorescence microscopy, application to the specificity of CBD proteins

Abstract

Surface-proteins interactions play key roles in many domains such as biomedicine, nanotechnology and the biology of plant-insect interactions. This article proposes a platform that allows to quantify surface interaction of proteins with chitin and chitosans to further discriminate and study specific interactions of proteins with chitin-binding domains (CBD). The platform consists in covalently grafted chitosan thin films of various degrees of acetylation (DA) through surface silanisation, spin-coating and water-temperature treatment. The obtained films were thoroughly characterized by infrared spectroscopy, contact angle measurements, atomic force microscopy and wide and small-angle X-ray scattering. Protein affinity to coated surfaces of reacetylated chitosans with degrees of acetylation ranging from 0.5 % to 76 % was evaluated by fluorescence microscopy. The specific affinity of lectins with a CBD was evidenced in comparison to proteins without CBD. As expected, the affinity was stronger at higher DAs, suggesting that the acetylation pattern play a part in specific lectin binding. In conclusion, chitosan films were fully characterized, and the elaborated platform shows promising results in screening protein interactions to chitin. This protein interaction platform is reportedly the first method able to differentiate the interactions of proteins containing a CBD and proteins which do not contain one, with whole chain “chitin-like” chitosans, by means of a simple and direct fluorescent microscopy quantification. This platform could further be used for other types of chitin-binding proteins or applied to other polysaccharide-protein interactions.