Visible Light Induced Control/living Radical Polymerization For The Immobilization Of Enzymes And Encapsulation Of Cells

Enzymes and cells after immobilization and.encapsulation could significantly enhance their stabilities,improve their tolerances against the environment,reduce the production costs,and broaden their applications,which exhibits the potential in the field of industry,energy,medicine,and biology science.Currently,the immobilization methods based on the conventional radical polymerization are relied on the harsh initiating conditions,including high temperature,UV irradiation,and oxidizer,which will cause seriously loss of viabilities.Therefore,it is an urgent need for a mild strategy to initiate the radical polymerization to immobilize enzymes and cells without compromising with the viabilities.Visible light is a kind of mild initiating condition to initiate radical polymerization,with the advantages of low emissivity,biocompatibility,tolerances with oxygen,and low temperature initiation,which is suitable for the in situ immobilization of enzymes and cells by radical polymerization as the irradiation resource.Based on the consideration,we conducted the studies focus on the immobilization of enzymes and encapsulation of cells relied on the visible light induced controlled/living radical polymerization,and the contexts and results were listed here.1.A new strategy to encapsulate papain by visible light induced inverse emulsion polymerization was developed.In this study,ITX/EDAB was taken as the initiator to initiate the inverse emulsion polymerization for the immobilization of papain with PEGDA as the monomer under the irradiation of LED lamp.Firstly,we investigated the polymerization behaviors of the visible light induced inverse emulsion polymerization,where the water phase composed by PBS buffer and PEGDA,and the oil composed by liquid paraffin,ITX/EDAB,and the emulsifier,and proved the feasibility of the initiation of ITX/EDAB.Secondly,papain and PEGDA were dissolved in PBS buffer as the water phase,and liquid paraffin was taken as the oil phase with ITX/EDAB dissolved in it.When the emulsion was irradiated by LED lamp,papain would be encapsulated inside of the cross-linked PEG microspheres.FTIR and fluorescence microscope gave a clearly prove that papain was embedded successfully with the immobilization yield above 90%.Finally,casein was used as the substrate to determine the activity of immobilized papain.From the results,papain could hydrolyze casein effectively after immobilization,and the recovery of papain is 67%.Thermal stability of papain was improved significantly after immobilization,and 54%of the primary activity remained after the immobilized papain immersed in PBS buffer at 70 ? for 2 h,while just 14%of the primary activity remained compared with that of native papain.Moreover,immobilized papain showed a good reusability with 61%of the primary activity remained after 10 cycles.In this study,we firstly in situ encapsulated enzymes by the visible light induced inverse emulsion polymerization,and demonstrated the superiority of the initiation irradiated by visible with a 21%relative activity higher than irradiated by UV light.2.?-glucosidase and cellulase were separately immobilized by the strategy of visible light induced controlled/living graft polymerization.Firstly,?-glucosidase was encapsulated in the microsphere synthesized by visible light induced inverse emulsion polymerization with ?-glucosidase and PEGDA dissolved in PBS buffer as the water phase,while the oil phase was composed by liquid paraffin,ITX/EDAB and the emulsifier.Secondly,a hairy microsphere was constructed by the surface initiated graft polymerization of AA initiated by the ITXSP on the surface of the cross-linked PEG micro sphere with(3-glucosidase inside irradiated by LED lamp.Finally,cellulase was covalently bond onto the grafted PAA chain.From the results obtained from XPS,UV-vis spectroscopy,and FTIR,ITXSP could initiate the graft polymerization effectively,followed the rules of controlled/living graft polymerization.The activity of(3-glucosidase was determined with pNPG as the substrate and 87%of its primary activity was kept after the graft polymerization of poly(acrylic acid)chains.After cellulase immobilized onto the grafted chains,the dual enzymes immobilization system was formed,and it has further proved the synergistic effect.Compared with cellulase immobilized only,the dual enzymes immobilization system exhibited a 15%higher yield of glucose after 48 h of the hydrolysis of cellulose.The reusability of immobilized dual enzymes with soluble cellulose of carboxyl methyl cellulose(CMC)salt and insoluble cellulose of filter paper as the substrate were also testified,which showed that 75%of the initial enzymes was remained after 10 cycles for the hydrolysis of CMC and 57%of the initial enzymes was remained after 5 cycles for the hydrolysis of filter paper.In this study,two enzymes were co-immobilized separately,and demonstrated the synergistic effect of immobilized two enzymes,indicating the further applications of this strategy in the field of medicine and biosensors.3.A thickness-controllable polymeric shell was constructed on single living cell by visible light induce controlled/living graft polymerization.Firstly,PEI was absorbed onto the surface of yeast cells by electronic interactions.Secondly,a water-soluble photoinitiator TX-Ct was anchored on the surface of PEI by the interactions of carboxyl group and amino group,composing the TX-Ct/PEI initiating system.Finally,a polymeric shell was synthesized by the polymerization of PEGDA initiated by TX-Ct/PEI initiating system under the irradiation of LED lamp.We have proved that the polymerization initiated by TX-Ct/PEI initiating system belongs to controlled/living radical polymerization.Determined by TEM,the thickness of the shell ranges from 20 to 54 nm,and it could be tuned by the polymerization time.The viabilities of yeast cells were demonstrated to be well maintained after decoration with the poly(PEGDA)network shell(>85%).Moreover,the lag phase of yeast cell division was extended and their stability against lysis was improved.In addition,Staphylococcus aureus cells could also be coated by this cross-linking reaction.This strategy not only achieve the construction of a thickness-controllable polymeric shell by visible light induced controlled/living graft polymerization on single living cells,but also provides great potential for single cell analysis and biosensors in the furture.