Study On Separation And Analysis Of Protein By Magnetic Nanomaterials Modified With Deep Eutectic Solvents

Magnetic solid phase extraction（MSPE）is an extraction techniqu e that simplifies the extraction process by introducing magnetic materials on the basis of traditional solid phase extraction.This technique avoids some time-consuming steps such as centrifugation and filtration.Due to the introduction of magnetic materi als,MSPE has the characteristics of high responsivity and fast separation during extraction and separation,and is widely used in sample pretreatment.Deep eutectic solvent（DES）is a mixture of various compounds heated in different proportions.The mixture can form a stable,transparent and homogeneous liquid when heated.DES is in liquid state even at low temperature,and has the advantages of high stability,low volatility,biodegradability,and easy preparation.The excellent properties of DES promote its wide application in various fields of chemistry.Protein is the basic substance for the existence of life and participates in various forms of life activities of the body.Most of the proteins in nature are mixtures,and many important proteins are very low in biological materials.Therefore,it is of great significance to separate and analyze proteins without affecting the structure,composition and activity of proteins.In this study,three different DESs were synthesized and modified on the surface of magnetic nanoparticles for magnetic solid-phase extraction of chymotrypsin（Chy）,lysozyme（Lys）and trypsin（Try）.The main research contents are as follows:（1）Choline chloride/xylitol deep eutectic solvent modified magnetic titanium dioxide for solid-phase extraction of chymotrypsinIn this study,a deep eutectic solvent[Ch Cl][Xyl]composed of choline chloride and xylitol was synthesized and modified on Ti O₂-coated magnetic particles（Fe₃O₄@Ti O₂）to obtain magnetic extraction.Fe₃O₄@Ti O₂@[Ch Cl][Xyl]was applied to solid-phase extraction chymotrypsin.Under the optimized extraction conditions,the extraction capacity of Fe₃O₄@Ti O₂@[Ch Cl][Xyl]for chymotrypsin reached 348mg·g^-1.The adsorption equilibrium relationship between Fe₃O₄@Ti O₂@[Ch Cl][Xyl]and chymotrypsin is in good agreement with the Langmuir adsorption.Fe₃O₄@Ti O₂@[Ch Cl][Xyl]maintained good chymotrypsin extraction ability after being reused six times.The limit of detection（LOD）and limit of quantif ication（LOQ）for this extraction method were 0.0037 mg·m L^-1 and 0.012 mg·m L^-1,respectively.The application of Fe₃O₄@Ti O₂@[Ch Cl][Xyl]to the separation and analysis of actual samples was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）experiments.The results showed that Fe₃O₄@Ti O₂@[Ch Cl][Xyl]was able to extract chymotrypsin from crude extracts of porcine pancreas.The Fe₃O₄@Ti O₂@[Ch Cl][Xyl]magnetic nano-extractant prepared in this study is expected to be an excell ent extractant for chymotrypsin extraction from complex samples.（2）Tetraethylammonium chloride/lactic acid deep eutectic solvent modified magnetic gelatin nanomaterials for solid phase extraction of lysozymeThe magnetic gelatin Fe₃O₄-Gelatin composite material was researched and synthesized,and a deep eutectic solvent[TEAC][Lac]was prepared by mixing tetraethylammonium chloride（TEAC）and lactic acid（Lac）.The surface of Fe₃O₄-Gelatin was modified with[TEAC][Lac]to obtain a composite magnetic extractant Fe₃O₄-Gelatin@[TEAC][Lac],which was used for solid-phase extraction of lysozyme.Under the optimized extraction conditions,the extraction capacity of Fe₃O₄-Gelatin@[TEAC][Lac]for lysozyme reached 137 mg·g^-1.Among the five common protein extraction experiments,Fe₃O₄-Gelatin@[TEAC][Lac]exhibited the highest extraction capacity for lysozyme.In the adsorption fitting experiment,the adsorption equilibrium relationship between Fe₃O₄-Gelatin@[TEAC][Lac]and lysozyme was consistent with the Langmuir monolayer adsorption theory.After being reused 6 times,the extraction capacity of Fe₃O₄-Gelatin@[TEAC][Lac]to lysozyme decreased,but it was still in an acceptable range.The LOD and LOQ of this extraction method were 0.0017 mg·m L^-1and 0.0057 mg·m L^-1,respectively.And the results of SDS-PAGE electrophoresis showed that Fe₃O₄-Gelatin@[TEAC][Lac]could extract lysozyme from egg white.The synthesized magnetic extractant Fe₃O₄-Gelatin@[TEAC][Lac]provides a new idea for the extraction of lysozyme from actual samples.（3）Tetraethylammonium chloride/glycerol deep eutectic solvent modified ferric oxide-humic acid nanomaterials for solid-phase extraction of trypsinA magnetic nanomaterial（Fe₃O₄-HA@[TEAC][G]）was synthesized by modifing a deep eutectic solvent（tetraethylammonium chloride/glycerol deep eutectic solvent,[TEAC][G]）on the surface of ferric oxide-humic acid（Fe₃O₄-HA）nanocomposite,and it was applied to the extraction and separation of trypsin.Under the optimized extraction conditions,the extraction capacity of Fe₃O₄-HA@[TEAC][G]for trypsin could reach more than 1300 mg·g^-1.In the adsorption isotherm fitting experiment,the relationship between trypsin and Fe₃O₄-HA@[TEAC][G]conformed to the Langmuir adsorption form,which was a monolayer adsorption.After the extractant w as reused 8times,it still had a good extraction capacity for trypsin.The LOQ and LOD of the method were 0.11 mg·m L^-1 and 0.033 mg·m L^-1,respectively.In addition,the methodological results showed that the extraction method had good stability,instrumental precision and repeatability.The results of SDS-PAGE electrophoresis showed that the extractant could extract trypsin from the crude extract of bovine pancreas.In conclusion,the magnetic extractant prepared in this study has potential application value in the pretreatment of trypsin samples.