Molecular Imprinting And Magnetic Solid Phase Extraction Technology Based On Deep Eutectic Solvents For Separation And Analysis Of Protein

As the coming era of green chemistry,researchers are increasingly concerned about the environmental problem and sustainability of the research process.As a new green solvent in the 21st century,Deep Eutectic Solvents(DESs)has the characteristics of low volatility,well chemical stability,good biocompatibility and simple synthesis methods,and it has extensive used in all separation and analysis fields.DESs is composed by a certain proportion of hydrogen bond donor(HBD)and hydrogen bond acceptor(HBA),and its structure is designable.Therefore,designing different types of DESs according to the characteristics of the analyte to meet target needs is a hot focus and it is deeply favored by researchers.As a technology based on the"lock-key"theory,Molecular Imprinting Technology(MIT)can separate target analyte specifically by using molecularly imprinted polymers(MIPs).MIPs is a good substitute for natural and specific identification materials such as antibodies.Replacing the traditional crosslinkers and functional monomers by different types of DESs can provide more interactions in polymers and target molecules and provide new ideas for biomacromolecule imprinting.Magnetic Solid Phase Extraction(MSPE),as a sample preparation technology,it is committed to using magnetic substance as extractants to pre-concentrate target analytes from complex samples.It has the advantages of simple operation and rapid separation.Designing suitable DESs to modify the MSPE agent is beneficial to improve the extraction capacity of the target analyte and increase the extraction efficiency.In this study,different types of novel DESs were designed and synthesized.Combined with molecular imprinting and magnetic solid phase extraction techniques,a selective separation and analysis method for bovine hemoglobin,lysozyme and trypsin was established.The main contents of this research are listed as follows:(1)Magnetic molecularly imprinted polymers based on deep eutectic solvents for separation and analysis of bovine hemoglobinA novel magnetic molecularly imprinted polymer based on deep eutectic solvents(DESs-MMIP)was successfully synthesized.The DESs-MMIP was constructed by using 2-hydroxyethyl methacrylate/tetrabutylammonium chloride deep eutectic solvent(DES₁)as functional monomer,arylamide/(3-acrylamidopropyl)trimethylammonium chloride deep eutectic solvent(DES₂)as cross-linker and bovine hemoglobin(BHb)as template through surface imprinting technology.DESs-MMIP was characterized by fourier transform infrared spectrometry(FT-IR),X-ray diffractometer(XRD),transmission electron microscope(TEM),thermal gravimetric analyzer(TGA)and vibration sample magnetometer(VSM).By optimizing the adsorption conditions,the imprinting factor and the maximal absorptive quantity of DESs-MMIP on BHb reached up to 21.89 and 229.5 mg·g^-1,respectively.Compared with seven references,DESs-MMIP showed significant selectivity for BHb.The new-type DESs-MMIP exhibited higher adsorption capacity and imprinting factor on BHb than molecularly imprinted polymers constructed with traditional functional monomer and cross-linker in reported methods.The recognition of BHb by DESs-MMIP in calf blood samples demonstrated the practicality of the particles.The DESs-MMIP with excellent selectivity is expected to become an ideal candidate for selective recognition of BHb in complicated samples.(2)Graphene molecularly imprinted polymers based on deep eutectic solvents for separation and analysis of lysozymeA molecularly imprinted polymer(DESs-MGO@MIP)with benzyltriethylammonium bromide/?-methacrylic deep eutectic solvent as functional monomer,hydroxyethyl methacrylate/allyltrimethylammonium chloride deep eutectic solvent as crosslinker,lysozyme(Lyz)as template,magnetic graphene oxide as carrier was designed and synthesized.The size,morphology and magnetic properties of DESs-MGO@MIP were analyzed in detail by TEM,FT-IR,TGA,VSM,XRD and so on.The results of adsorption kinetics and thermodynamic experiments indicated that the adsorption process of DESs-MGO@MIP for Lyz was more consistent with the Langmuir isothermal adsorption model and the second-order kinetic model.The adsorption process tends to be uniform single-layer adsorption,mainly chemical adsorption.The process tended to be uniform single-layer adsorption,with chemical adsorption dominated.Under the optimal experimental conditions,the maximum adsorption quantity for Lyz was 174.6 mg×g^-1,which was much higher than the other eight comparative analytes such as bovine hemoglobin,bovine serum albumin,ovalbumin and so on.DESs-MGO@MIP could successfully separate Lyz from the actual egg white samples,and the proposed method had a good application prospect for the separation and purification of Lyz.(3)Magnetic titanium dioxide functionalized with deep eutectic solvents for the separation and analysis of trypsinA methacrylic acid/choline chloride deep eutectic solvent was synthesized,and it was modified on magnetic titanium dioxide nanoparticles by free radical polymerization to obtain a new magnetic solid phase extractant(Fe₃O₄@Ti O₂@DES)for high-efficiency extraction of trypsin(Try).The properties,composition and microstructure of Fe₃O₄@Ti O₂@DES were characterized by TGA,FT-IR,XRD,VSM and TEM.The maximum extraction adsorption capacity of trypsin by Fe₃O₄@Ti O₂@DES under the optimal conditions was 475.7 mg×g^-1.Adsorption kinetics and adsorption isotherm fitting data suggested that the adsorption procedure of Fe₃O₄@Ti O₂@DES for Try tended to be uniform monolayer adsorption,mainly chemical adsorption process.The results of repeated utilization experiments showed that after four times of reuse,the adsorption capacity remained at about 87%of the original.Methodological data showed that the separation analysis system established in this research had good precision,repeatability and stability.The proposed method could be used for the extraction and separation of Try from bovine pancreatic crude extract and had satisfying experimental results.