Preparation And Application Of Hydrogel Composite Membranes For Biomolecular Crystallization

Biomolecules are characterized by complex structure and sensitivity to the environment.It is extremely difficult to control the preparation of biomolecular crystal products with complete crystal faces and large particle size.In recent years,membrane interface shows benefits of efficient preparation for specified crystal products.Among them,hydrogel composite membranes(HCMs)are materials with highly controllable interface properties,which is expected to achieve interface-induced nucleation and efficient crystallization regulation.Therefore,a series of NIPAM-PEGDA HCMs were innovatively prepared,then applied to morphology regulation of lysozyme crystals and the crystals transformation process of urate.The regulatory mechanism and application of HCMs in continuous membrane crystallization process of biomolecules were investigated.Firstly,a “sandwich structure” composed of base membrane-hydrogel prepolymer-glass plate was used to establish a novel preparation method of HCMs by allowing the prepolymer to be interposed between the glass plate and the base membrane in an oxygen-insulating environment,so that the polymerization rate and the stability of the composite layer structure were improved.The "fingerprint" folds were shown on the swelling composite membrane.This biomimetic structure can control the contraction and expansion of the network according to the change of environment,then regulate the aggregation of biomolecules and salt ions,and make the crystallization micro environment of the hydrogel interface adjustable.Through the copolymerization of NIPAM and PEGDA,HCMs have the ability of temperature-response,pHresponse as well as solvent mass transfer.As the temperature and pH decrease,the contact angle of HCMs decreases,corresponding heterogeneous nucleation Gibbs free energy decreases so that the controllable heterogeneous nucleation can be achieved at a low supersaturation concentration,which effectively increases the controllable precision and improves the specified regulation of crystal nucleation.Through the adsorption-recrystallization experiment of salt solution on hydrogel layer,it is proved that with increase of average size of the meshes,the average size of the salt crystals increases,and the crystal morphology tends to multidirectional growth,which reveals the important regulation potential of the hydrogel interface to nucleation crystallization.HCMs were applied to the membrane crystallization process of the model protein lysozyme.Different crystal shapes were regulated by controlling the temperature,pH and concentration of stripping solution.The experiment results show that the prepared crystals inclined to produce rod-like,long hexagonal and other undesirable forms,and the particle size is small(about 20 ?m)and not uniform in the membrane crystallization process of PP.While PEGDA HCM can effectively reduce the proportion of rod-like crystals and increase the selectivity of hexagonal and polyhedral crystals.In the crystallization process of PEGDA:NIPAM HCMs,hexagonal crystals with high selectivity(97%)and complete crystal faces are achieved,and the “flower-shaped” crystals with multidirectional nucleation growth characteristics were prepared with an average particle size of 120 ?m and selectivity of 98% by regulating the temperature and pH.The results fully demonstrate that PEGDA-NIPAM HCMs have ability to regulate the nucleation of multidirectional crystals.In addition,the crystal growth time can be shortened by more than 30% through controlling the concentration of stripping solution while ensuring high morphology selectivity.The HCMs were further applied to the crystal transformation process of urate crystals,and a bionic interface based on HCMs was established to realize the research platform for the urate conversion mechanism and process.The experiment results show that the gel tablets have an obvious influence on the formation of sodium urate crystals.As mass of the gel tablets increases,the urate crystals have experienced the transition of "flower cluster"," sphere " and "sticky sphere".The 3D mesh structure on gel interface promotes the dehydration of uric acid dihydrate crystals and the adsorption of sodium ions,so that the spiculate crystals disappear and the spherical sodium urate crystals appear.The crystal morphology of sodium urate is actively controlled.The above findings provide new ideas and potential research platform for the treatment and mechanism research of gout.