The Preparation And Application Of The Group Of Thermo-sensitive PNIPAAm In Affinity Precipitation And Aqueous Two-phase Extraction System

Poly(N-isopropyl acrylamide),short for PNIPAAm,shows temperature-sensitive features in aqueous solution due to its macromolecular chains having both hydrophilic amide group(-CONH-) and hydrophobic isopropyl group(-CH(CH₃)₂).It has aroused great interest that the intelligence and memory effect are produced by the temperature-sensitive polymers.Smart polymer can be designed as chemical controllers,sensors,devices for the intelligent film,memory elements,artificial muscles,drug delivery systems.It becomes a hotpoint in the field of polymer.This paper used an approach of copolymerization-chelated metal ions,which randomly synthesized binary linear polymers using isopropyl acrylamide and acrylic acid monomers.So the polymer not only had the temperature-sensitive character but also obtained functional carboxylic groups(-COOH) chelating metal ions.Then the PNIPAAm-co-AAm binary linear polymer was bonded with metal ions,which obtained both temperature sensitivity and affinity properties simultaneously.We also studied their application in affinity precipitation and aqueous two-phase extraction system.The main contents are as follows:We used water as a polymerization solvent,oxidation - reduction system(APS) as initiator,TEMED as an accelerator to prepare PNIPAAm-co-AAm.The optimal synthetic conditions:the concentration of monomers is 4%(accounting for the total mass of solution),the monomer ratio is each 1.0 g NIPAAm needs to add 200μl AAm, the concentration of APS initiator is 0.24%(accounting for the total mass of solution), the concentration of TEMED is 0.24%(v/v,accounting for the total solution volume). The temperature-sensitive polymer with good physical and chemical properties not only has good thermo-sensitive nature,but also has the pH-sensitive properties,which is suitable as the cartier of protein separation and purification.We chose copper ions as a specific ligand.The results indicated that the optimum condition is that pH 4.5, the initial concentration of copper ions is 2.0 g/L,polymer dosage is 0.20 g,and we found the maximum adsorption capacity of copper ions on PNIPAAm-co-AAm was 63.5 mg/g.PNIPAAm-co-AAm-Cu also has good storage stability.The best adsorption condition of BSA on PNIPAAm-co-AArn-Cu is that:pH 6.0, adsorption temperature of 20℃,BSA initial concentration of 1.0 mg/ml.In the same concentration of BSA,a comparison of three kinds of polymer adsorption of BSA: PNIPAAm-co-AAm＜PNIPAAm＜PNIPAAm-co-AAm-Cu.Experimental results showed that,BSA and PNIPAAm-co-AAm-Cu present the specific interactions.The optimal elution conditions for BSA on PNIPAAm-co-AAm-Cu:pH 4.0 0.05 mol/L Tris-HCl buffer(containing 0.20 mol/L NaCl,and 0.050 mol/L EDTA). Metal-chelated thermosensitive polymer PNIPAAm-co-AAm-Cu not only solves the the separation of the multiphase system with complex natures,but also can be recycled many times,saving a lot of resources.It is an environmental friendly, efficient and excellent performance affinity separation media.We also found the best separation and purification conditions for BSA in the aqueous two-phase system formed by the thermo-sensitive affinity polymer/Dextran 40000 were:pH 6.0, Dextran 40000 concentration of 6.0%(w/w),PNIPAAm-co-AAm-Cu concentration of 3.5%(w/w),BSA initial concentration of 15.0 mg/ml.After fetal calf serum was processed by the affinity thermosensitive aqueous two-phase system,we obtained a deeper color bands for the top phase,while the impurities significantly smeared out.The bottom phase almost dose not contain bands of BSA,which indicated that the temperature-sensitive affinity aqueous two-phase system can be effectively used for the enrichment and separation of BSA.