Preparation And Properties Of Protein-based 3D Microstructures Based On Two-Photon Polymerization Microfabrication Technology

Bovine serum albumin(BSA)as one of the native proteins has congenital advantages,such as nontoxicity,biodegradability and biocompatibility.And it has been widely studied due to its low cost,stimuli-responsive property and similar nature to human serum albumin.BSA-based tunable,dynamic and responsive “smart” threedimensional(3D)microstructures with well-defined geometries and repeatability are crucial aspects of protein application.The 3D microstructures with high resolution,repeatability,controllability and diversity can be fabricated by two-photon polymerization(TPP)microfabrication technology which is an advanced 3D printing technology.In recent years,this technique has been widely used in fabricating proteinbased novel and functional micro/nanostructures,devices and integrated systems.However,the ability to control and quantify the properties of BSA microstructures including both morphology and pH response more accurately remains unexplored.To solve these problem,we prepared BSA-based 3D microstructures with tunable surface morphology and pH-responsive properties via TPP microfabrication technology in this study.Firstly,processing parameters is optimized.Optimized initiator concentration is 8.5 mM.The laser processing parameters are optimized for fabricating well-defined 3D BSA microstructures.The suitable laser processing parameters with laser power of 18.0 mW,scanning speed of 77 ?m/s and layer distance of 200 nm are chosen from the processing window for the subsequent processing.Surface morphology of microstructures with different BSA concentrations is investigated.The average surface roughness(Ra)of the protein cuboid shaped microstructures in air is confirmed by AFM.The Ra of microstructures with different BSA concentrations(20 wt%,30 wt% and 40 wt%)is all below 30 nm,which guarantees the surface quality of microstructures.Meanwhile,40 wt% BSA microstructures have smooth surface morphology with Ra less than 2 nm.Microstructures like Roma relief are involved to investigate the morphology of complex microstructures further.This result indicates that the surface morphology of the complex microstructures with patterns can be controlled from rough to smooth obviously with increasing BSA concentrations.Secondly,the pH-responsive properties of microstructures with different BSA concentrations are investigated.Cuboid-shaped microstructures are used as a basic model to investigate pH-responsive properties of BSA microstructures with different BSA concentrations in different pH solutions and a wide range of pH response corresponding to the swelling ratio of 1.08-2.71 has been achieved.A panda faceshaped BSA microrelief with high BSA concentration and reversible pH responsive properties is fabricated and exhibits unique “facial expression” variations in pH cycle.We further design a mesh sieve-shaped microstructure as a functional device for promising microparticle separation.The pore sizes of microstructures can be tuned by changing the pH values.The arrays and biocompatibility of protein-based microstructures are investigated.The 4×4 and 6×6 protein-based microstructural arrays with homogeneity and highly repeatability are achieved by TPP microfabrication technology.The result of co-cultivation of L929 cells and microstructural arrays indicates the protein-based microstructures have good biocompatibility.