Structural Design And Fabrication Of Electrospun Nanofibrous Materials For High-performance Protein Adsorotion And Purification

As an essential biological macromolecular,protein has been widely applied in variuous fields including food engineering,daily chemical industry,life science research,biomedicine,and so on.In order to guarantee the effectiveness and safety of protein products,a high purity is always required by their application fields especially for the biomedicine and pharmaceutical fields.Commonly,the steps of isolation and purification directly determine the purity and biological activity of target protein;meanwhile,they usually consume nearly 80%of the total cost.Therefore,the realization of efficient,rapid,low-cost separation and purification of protein is the key to the development of protein products.Among various separation and purification methods,adsorptive separation has been used most widely due to its high efficiency,convenient operation,large processing capacity and continuous production.Currently,the protein adsorption and separation processes are mainly performed by using micro-particle media-packed chromatographic column.However,due to the internal pore structure of the micro-particle media,a longer contact time is usually required to enable protein molecules to transfer mass and be fully adsorbed into the particle media,thus leading to a relatively low processing rate.Besides,under accelerated liquid velocity,the gradually compact accumulation of particle media would also result in a rapid increase of column pressure and energy consumption,restricting the further development of protein separation and purification fields.Therefore,it is urgent to develop new type of highly efficient protein adsorption and separation materials.As a newly-developed high-tech material,electrospun nanofibers with favorable structural characteristics of large specific surface area and tunable fiber assembly structure,as well as technical features of controllable spinning process and abundant raw materials,have presented huge prospects in preparing high-performance protein and adsorption materials.Therefore,a large number of researchers have made efforts to fabricate nanofibrous protein adsorption materials.Despite the improvement of adsorption capacity at a certain degree,some pivotal bottlenecks still remain unresolved.Firstly,the structure and modification methods of nanofibrous membranes have not been synergistically optimized,meanwhile,the relationship between physicochemical structure and application performance of the nanofibrous membranes is not clear,thus resulting relatively poor performance.Additonally,nearly all of the current nanofiber-based protein adsorbents exhibit2D membranes structure,the intrinsic limitations of membranes including relatively dense stacking status of fibers and the relatively small pore diameter result in a relatively large biomolecules diffusion hindrance and high liquid flow resistance,which caused it difficult to synchronously improve the protein adsorption capacity and processing flux.In this paper,we prepared carboxylated nanofibrous membranes via surface and blend modification by optimizing the modification method and process parameters,therefore effectively improving the protein adsorption performance of nanofibrous membranes.Further,aiming at the structural bottleneck of the current nanofiber-based protein adsorbents,for the first time,we introduced the 3D reconstruction of nanofibrous aerogels into the preparation of protein adsorption and separation materials.Highly carboxylated and phosphately nanofibrous aerogels-based protein adsorbents with synchronously improved protein adsorption capacity and treatment flux were obtained by integrating nanofibrous aerogels forming technique and in-situ modification approaches.The specific research works are summarized as follows:?1?A carboxylated nanofibrous protein adsorbent was fabricated by the organic combination of electrospun ethylene-vinyl alcohol copolymers?EVOH?nanofibrous membrane and surface modification with citric acid?CCA?.The influences of CCA modification on morphologic structure,surface chemical properties,mechanical properties and wettability of the membranes were investigated.Meanwhile,the effect of CCA loading on the protein adsorption properties of carboxylated membrane was also analyzed.Subsequently,the intrinsic relationship between initial protein concentration,adsorption time,buffer properties,adsorption and separation performance of resulting carboxylated membranes were explored.The results show that,the surface wettability and tensile strength of the nanofibrous membrane were significantly improved after surface modification.The optimal protein adsorption capacity of the carboxylated fibrous membrane reached up to 284mg g^-1,and the saturated dynamic adsorption capacity reached up to 250 mg g^-1.Additionally,the carboxylated membrane also exhibited good selectivity and recycling performance.?2?A new type of butanetetracarboxylic acid?BTCA?functionalized EVOH?BTCA@EVOH?nanofibrous protein adsorbents was fabricated,for the first time,by combining electrospinning technique with in situ blend modification technology.The influences of BTCA loading amounts on the morphology,chemical structure,wettability,and protein adsorption properties of the carboxylated membranes were studied.The static and dynamic protein adsorption performance of carboxylated fibrous membrane were systematically studied;meanwhile,the effect of buffer properties on the protein adsorption and separation properties of fiber membrane was deeply analyzed.The results showed that the morphology,structure,surface wettability,mechanical strength and of the membranes could reach the optimally synergistic effect at the BTCA loading amount of 3 wt%,which possessed the optimal protein adsorption capacity of 716 mg g^-1.Additionally,the BTCA@EVOH nanofibrous membranes can directly extract lysozyme from egg white with a relatively large capture capability of 353 mg g^-1.Besides,the carboxylated membranes also presented a distinctive selectivity towards positively charged proteins,excellent acid and alkaline resistance,as well as good reusability.?3?For the first time,highly carboxylated nanofibrous aerogels-based protein adsorbents with anisotropic honeycomb-like structure were prepared by integrating nanofibrous aerogels forming technique and in situ modification approach,which was realized by taking flexible silicon dioxide?SiO₂?nanofibers as building blocks,poly?vinyl alcohol??PVA?and CCA as the functional organic binder and modifier,respectively.The obtained carboxylated nanofibrous aerogels exhibited an unique cellular structure consisted by flexible SiO₂ nanofibers as well as carboxylated and crosslinked PVA wrapping layer,endowing them with ultralight feature and outstanding underwater superelasticity?nearly no plastic deformation after 1000 compressive cycles?.Meanwhile,the hydrated carboxylated nanofibrous aerogels presented an intriguing shape-memory feature and could be directly cut at different angles,indicating their good structural stability.Benefiting from the interconnected nanofibrous cellular structure,good hydrophilicity,high carboxylation,and excellent mechanical properties,the carboxylated nanofibrous aerogels exhibited synchronously promoted static(2.9×10³ mg g^-1)and dynamic(1.7×10³ mg g^-1)lysozyme adsorption capacities as well as improved buffer flux(2.2×10⁴ L m^-2 h^-1,gravity-driven),which were superior to the reported nanofibrous materials and commercial ion-exchange membranes.The carboxylated aerogels also possessed outstanding peformance stability,easy operation,and excellent regenerability.Moreover,the carboxylated aerogels packed column could directly applied in protein chromatography system,and continuously extract lysozyme from egg white solely driven by gravity,highlighting its great prospect of practical application.?4?A novel type of highly phosphorylated nanofibrous aerogels with isotropic structure was fabricated,for the first time,by combining electrospinning,cryogenic induce phase separation regulation,and in situ phosphorylation modification,which was realized by taking flexible SiO₂ as the main building blocks,EVOH as polymer binder,and polyphosphoric acid?PPA?as the phosphate esterification modifier.The influence of loading ratio of PPA to EVOH on the phase separation path of EVOH during the aerogel freezing molding process was studied.The forming mechanism of isotropic pore structure and the underwater compressibility of the phosphorylated nanofibrous aerogels were explored.The internal relationship between physicochemical structure and application performance of the phosphorylated nanofibrous aerogels was clarified,and its practical application performance was preliminarily evaluated.The results showed that the obtained phosphorylated nanofibrous aerogels exhibited an outstanding superelasticity,excellent compression fatigue resistance?almost 0%plastic deformation after compression cycles?,and good shape-memory property in an underwater environment.Amazingly,those excellent mechanical properties could remain in the air or even in the ultra-cold liquid nitrogen.The phosphorylated nanofibrous aerogels exhibited showed an outstanding saturated static and dynamic protein adsorption capability of 3.3×10³ and 1.8×10³ mg g^-1,respectively;meanwhile,the processing flux reached up to 1.5×10⁴ L m^-2 h^-1 solely driven by a small gravity.Moreover,the phosphorylated nanofibrous aerogels also possessed good performance stability,outstanding reusability,easy assembly,and excellent practicability of directly extracting lysozyme from egg white,highlighting their potential actual application.