Imprinted Polymers And Magnetic Extractants Constructed By Deep Eutectic Solvents For Separation And Analysis Of Proteins

The isolation and purification of proteins is an important topic in the proteomics and biopharmaceutical industries,and with the increasing market opportunities and the continuous development of medical technology,the global demand for protein therapeutics and the food industry is increasing.Therefore,the construction of a mild,simple and efficient method for protein separation and analysis is still a research hotspot in this field.Molecular imprinting technology（MIT）is a technique for preparing molecularly imprinted polymers（MIPs）to mimic the characteristic binding of antibodies.MIPs are comparable to natural receptors due to their comprehensive advantages of excellent mechanical properties,physical durability,and chemical stability in acid and alkali solutions.Due to its high affinity and selectivity,it is favored in the field of protein separation.Magnetic solid-phase extraction（MSPE）is a new development direction of solid-phase extraction,which is developed using magnetic materials as extractants in the adsorption process.MSPE has the advantages of high enrichment efficiency,good concentration effect and fast separation speed,and is one of the most commonly used sample pretreatment techniques.Green solvents are a class of environmentally friendly solvents that have been widely used in separation technology in recent years.Among them,deep eutectic solvents（DESs）prepared from hydrogen bond interactions between hydrogen bond acceptors（HBA）and hydrogen bond donors（HBD）are considered to be the most promising green solvents.In this study,different types of DESs were designed and prepared,and they were applied to MIT and MSPE techniques,and a method for the separation and analysis of trypsin（Try）and bovine hemoglobin（BHb）was constructed.The main research contents are as follows:（1）Specific recognition of bovine hemoglobin by deep eutectic solvent-based magneticβ-cyclodextrin molecularly imprinted polymerSuccessfully constructed allyl trimethyl ammonium chloride/ethylene glycol deep eutectic solvent was used as the functional monomer,methacryloyloxyethyl trimethylammonium chloride/itaconic acid deep eutectic solvent was used as the cross-linker,BHb as template molecule,magneticβ-cyclodextrin（MCD）as carrier surface molecularly imprinted polymer（MCD@DES-MIP）.MCD@DES-MIP was analyzed by thermogravimetric analyzer（TGA）,X-ray diffractometer（XRD）,transmission electron microscope（TEM）,vibrating sample magnetometer（VSM）and Fourier transform infrared spectrometer（FT-IR）.The composition,crystal structure,morphology,magnetic properties and functional groups were characterized.Under the optimized conditions,the maximum adsorption capacity of BHb by MCD@DES-MIP was 195.94mg·g^-1,and the imprinting factor（IF）was 4.68.Among the binary mixed proteins of bovine serum albumin（BSA）and BHb,MCD@DES-MIP exhibited excellent selectivity for BHb.Analysis of real samples showed that MCD@DES-MIP can effectively separate BHb from complex biological samples（calf blood）.After 5 cycles of MCD@DES-MIP,it still maintained 71.63%of the initial adsorption capacity.The results of circular dichroism analysis showed that the adsorption and desorption process of MCD@DES-MIP did not destroy the secondary structure of BHb.MCD@DES-MIP can be used as a novel imprinting material for the separation and purification of BHb.（2）Extraction and separation of trypsin with ethylene glycol class deep solvent functionalized magnetic polydopamineFive kinds of ethylene glycol class DESs-functionalized magnetic polydopamine（MPDA）composites were designed and synthesized by ultrasound-assisted method,and combined with MSPE technology for efficient extraction of Try.Among them,the magnetic extractant（MPDA@[ChCl][EG]）functionalized with a deep eutectic solvent（[ChCl][EG]）constructed with choline chloride and ethylene glycol had the best extraction efficiency for Try.The morphology,magnetic response and functional group distribution of MPDA@[ChCl][EG]were characterized by TEM,VSM and FT-IR.Through single factor optimization experiments,the factors affecting the extraction process of Try were explored.Under the optimized conditions,the extraction amount of Try by MPDA@[ChCl][EG]was 338.99 mg·g^-1,and the extraction equilibrium time was 60 min.Langmuir and pseudo-second-order kinetic equations fit the adsorption behavior of Try on MPDA@[ChCl][EG]well,and the results show that the adsorption behavior of Try on MPDA@[ChCl][EG]is a monolayer chemisorption.MPDA@[ChCl][EG]was successfully used for the extraction and separation of Try in complex biological samples（bovine pancreas crude extract）.The idea of using green DESs and MPDA to construct magnetic extractants provides more options for protein extraction in mild environments.（3）Extraction and separation of trypsin by poly-eutectic solvent-functionalized magnetic graphene oxideThree polymethacrylic acid class DESs-functionalized magnetic graphene oxide composites（MGO@PDES）were designed and synthesized by radical polymerization method for selective solid-phase extraction of Try.Among them,the magnetic extraction（MGO@PDES₂）functionalized with poly-eutectic solvent（PDES₂）constructed with tetraethylammonium chloride and methylpropionic acid with a molar ratio of 1:2 has the highest extraction efficiency for Try.Based on the high specific surface area of MGO and the abundant carboxyl functional groups in PDES₂,the prepared MGO@PDES₂ has greater advantages and better stability for the extraction of Try.Under the optimal conditions,the extraction amount of Try by MGO@PDES₂was 708.85 mg·g^-1.The extraction driving force between MGO@PDES₂ and Try is hydrogen bonding interaction as well as electrostatic interaction.Among the seven biomacromolecules,MGO@PDES₂ exhibited excellent selectivity for Try.In addition,the results of cycling experiments show that the DESs grafted by chemical method are not easy to fall off during the five cycles of adsorption-desorption,which is beneficial to the recycling of MGO@PDES₂.MGO@PDES₂ is a promising magnetic extractant,which is expected to provide new ideas for the extraction and separation of proteins.