Preparation And Multifunctional Application Of Two-dimensional Protein-based Ultra-large Thin Films Based On Thiol-disulfide Bond Exchange Reaction

The design and synthesis of robust self-supporting protein films with functional properties play a role in the development and application of biological material surface and interface.However,the advanced synthesis and functionalization of materials remains a key challenge in biomaterials research,typically including difficulties as(1)limited potential for scale-up fabrication and application;(2)the use of complex process and toxic reagent;(3)easy protein deactivation during film fabrication;(4)insufficient functions especially lacking robust adhesion on a surface and on-demand actuation.The research outline is as follows:(1)The potential of thiol-disulfide exchange reaction and the mechanism of partial unfolding of proteins.In the present work,we propose that the differentiated redox potentials of disulfide bonds among different proteins determine the noticeable reactivity of cysteine towards specific proteins(e.g.,lysozyme)and inertness to other common proteins.It is further deduced that the site-specificity of the reaction may lie in the Cys6-Cys127 disulfide bond of native lysozyme,which has the longest distance from(and thus minimized influence on)the active cleft of native lysozyme,resulting in the formation of partially unfolded lysozyme-cysteine conjugate monomers and subsequent aggregation at the air/water or solid/liquid interface to form an amyloid-like 2D lysozyme-cysteine nanofilm.Moreover,we found that time,lysozyme concentration,cysteine concentration and pH have a great impact on morphology of protein nanofilm and clearly explains the formation mechanism of the nanofilm.By IR,CD,ThT staining and Congo red staining,it is proved that the secondary structure of proteins inside the nanofilm has changed from ?-helix to ?-sheet during the reaction of lysozyme and cysteine,and the nanofilm presents amyloid-like structures.The robust proteinaceous macroscale nanofilm,which is colorless,transparent and reliably adhered onto virtually arbitrary material surfaces(polymer,metal and inorganic)to resist the disturbance from adhesive tape peeling,organic solvent or extreme pH.Therefore,the lysozyme-cysteine nanofilm provides a robust platform for material surface/interface modification.(2)A strategy to immobilize and release active proteins from proteinaceous nanofilmIn contrast to the coloring issue in existing universal adhesive materials,such as black or brown polydopamine or tannic acid/Fe complex coating,we demonstrate that the nanofilm coating could be used as a tunable platform for the controllable encapsulation of molecules and colloids.The one-step encapsulation of functional blocks(small molecules,polypeptides,proteins and colloids)in the nanofilm was feasibly initiated by mixing lysozyme,cysteine and functional blocks at given concentrations.By BET and PEG test,it is further proved that the pore size of nanofilm is 2 nm,and the nanofilm presents the controlled release of active molecules and colloids with cross-linking,so the nanofilm plays a key role in a wide range of applications such as biomedicine,energy,catalysis and environmental protection.