Protein Bioactive Interface Based On Polyphenols And Zwitterionic Polymers

Protein bioactive interface is of great importance in numerous applications,including proteomics,biosensor,drug screening and biocatalysis.Therefore,the fast and highly active immobilization of protein on the surface of materials plays a crucial role in biotechnology and medical testing.In general,proteins are often immobilized onto a surface via nonspecific adsorption,however,nonspecific protein immobilization may lead to partial or complete loss of protein activity resulting from random orientation and structural deformation of the immobilized proteins.In addition,the alien non-specific adsorption of protein on the surface of the material can cover the activie site and further reduce the activity of the immobilized protein.In this paper,a new method based on polyphenols and zwitterionic polymers is proposed,which is simple and suitable for constructing protein-active surfaces.This low-cost and green protein-oriented immobilization strategy may serve as a versatile platform for a range of applications,such as biomaterials,biocatalysis,sensors,drug delivery,and so on.The research contents of this paper include the following aspects:（1）The surface of the nonfouling zwitterionic compound containing catechol groups was prepared by one-pot reaction.The optimum conditions were 24 h of reaction time at 65^oC and pH=5.The results of quartz crystal microbalance with dissipative（QCM-D）showed that the adhesion amount of BSA was much smaller than that of the surface of the unmodified material,showing a good protein resistance property.In addition,the sulfhydryl groups-functional surface was prepared by an addition of cysteine.This method provided a new way for the construction of protein bioactive interface.（2）A green and facile protocol to oriented immobilization of histidine-tagged（His-tagged）proteins based on tannic acid（TA）is described.This is the first time that TA has been applied as ionic chelators to immobilize His-tagged proteins.To reduce the nonspecific interactions between the TA and immobilized His-tagged proteins,we assembled nonfouling zwitterionic poly（sulfobetaine methacrylate）（PSBMA）on the TA surface.The use of PSBMA could maintain the high activity of the His-tagged proteins and inhibit the adsorption of untagged protein to the TA surface.Subsequently,the obtained TA/PSBMA film was further chelated with Co^Ⅱ for specific binding to a His-tagged protein.The loading capacity of the film for the His-tagged Chitinase can reach～4.0μg/cm².As Co^Ⅲ is more stable and inert than Co^Ⅱ,the chelated Co^Ⅱ was oxidized to Co^Ⅲ.The surface of the oxidized TA/PSBMA/Co^Ⅲ is more stable for the binding of the His-tagged protein,which is not easy to be washed out by the imidazole.Besides,this method is suitable for many kinds of material surfaces.（3）The activity and stability of immobilized chitinase were evaluated.Moreover,The effects of the type of nonfouling polymer and the molecular weight of nonfouling polymer on the activity of immobilized protein were studied.The enzyme activity of the chitinase immobilized on the surface of the TA/PSBMA/Co^Ⅱ can be kept at about 95%,which was higher than the immobilized enzyme activity prepared by the covalent method.Among the various highly-hydrophilic polymers,the protein immobilized on the surface of PSBMA-modified material has the best activity.Furthermore,the oriented immobilized chitinase had an excellent thermal and storage stability as well as being more resistant to proteolytic digestion by papain.