| Biomacromolecules such as protein and nucleic acid are the material basis of life and are closely related to various life activities.The research on the structure and function of protein and nucleic acid undoubtedly plays a decisive role in understanding the laws of life activities,guiding industrial production and medical practice.However,all biochemical studies require the analyte to have a sufficiently high purity.Therefore,the development of more efficient,simple and green methods for the separation of biomacromolecules is of great significance.Deep eutectic solvent is a novel type of green solvent.Due to its extraordinary advantages of abundant raw materials,simple synthesis and no further purification of products,deep eutectic solvent has been widely used in combination with other separation technologies for the purification of protein and nucleic acid.Deep eutectic solvent/aqueous two-phase system constructed by using deep eutectic solvent as a phase forming agent is a kind of extraction system with excellent bi ocompatibility,which effectively combines the advantages of deep eutectic solvent and aqueous two-phase extraction technology.Magnetic solid-phase extraction based on deep eutectic solvent is a kind of solid-phase extraction technology that uses deep eutectic solvent modified magnetic material as extractant.It owns the superiority of simple operation,rapid separation and easy to automate.Deep eutectic solvent-based molecular imprinting technology is a technique for synthesizing a polymer that matches the spatial structure and binding sites of a template molecule by taking deep eutectic solvent as functional monomer.The prepared polymer has high mechanical strength,good stability and long lifetime.In this study,a series of deep eutectic solvents were synthesized and applied for the extraction of proteins,enzymes and DNA coupled with aqueous two-phase extraction technology,magnetic solid-phase extraction technology and molecular imprinting technology.The main contents are as follows:（1）Choline chloride-based deep eutectic solvent aqueous two-phase system for protein extractingFour kinds of choline chloride（ChCl）-based deep eutectic solvent（DES）were synthesized and coupled with inorganic salt to form aqueous two-phase system（ATPS）for the extraction of proteins.Bovine serum albumin（BSA）was chosen as the model protein,and ChCl-glycerol（ChCl-G）was selected as the optimal extractant.Single factor experiments were carried out to investigate the influence of amount of DES,concentration of salt,mass of protein,shaking time,temperature and pH value on the extraction efficiency.Under the optimum conditions,the extraction efficiency of BSA was up to 98.16%and an extraction efficiency of 94.36%was achieved for trypsin（Try）at the same time.The back-extraction experiment showed that the back-extraction efficiency of BSA could reach to 32.96%.Methodological research demonstrated the precision,repeatability and stability of the proposed method were excellent.UV-vis,fourier transform infrared spectrometry（FT-IR）and circular dichroism spectra（CD）spectra verified the conformation of protein was not changed during the process of extraction.Measurement of conductivity,dynamic light scattering（DLS）and transmission electron microscopy were further conducted to explore the mechanisms of the extraction.The results suggested the formation of DES–protein aggregates play a significant role in the separation process.T he ATPS method based on DES provides a new idea for the extraction of proteins.（2）Magnetic solid-phase extraction of protein with deep eutectic solvent immobilized magnetic graphene oxide nanoparticlesFour kinds of ChCl-based DES were synthesized and modified on the surface of magnetic graphene oxide（Fe3O4-NH2@GO）to form Fe3O4-NH2@GO@DES for the magnetic solid-phase extraction of protein.The characteristic results of vibrating sample magnetometer（VSM）,X-ray diffraction（XRD）,FT-IR,thermal gravimetric analysis（TGA）and field emission scanning electron microscopy（FESEM）indicated the successful preparation of Fe3O4-NH2@GO@DES.BSA was chosen as the model protein to study the extraction performance of Fe3O4-NH2@GO@DES.The concentration of BSA in the supernatant was determined by UV-vis spectrophotometer at 278 nm.Compared with extractants without modification by DES,the Fe3O4-NH2@GO@DES has a higher extraction capacity of 44.59 mg/g for BSA.Single factor experiments were carried out to investigate the influence of pH value,temperature,extraction time,concentration of protein and amount of Fe3O4-NH2@GO@DES on the extraction capacity.Desorption experiment showed98.73%of BSA could be eluted from the solid extractant by aqueous solution of 1mol/L NaCl-0.1 mol/L Na2HPO4.CD spectra verified the secondary structure of protein was not changed during the process of elution.Additionally,according to the result of real sample analysis experiment,the Fe3O4-NH2@GO@DES was proven to be able to successfully extract BSA from bovine whole blood.Methodological research demonstrated the repeatability,precision and stability of the proposed method were excellent.The MSPE method based on DES provides a new idea for the extraction of proteins.（3）A novel poly（deep eutectic solvent）-based magnetic silica composite for solid-phase extraction of trypsinNovel poly（deep eutectic solvent）grafted silica-coated magnetic microspheres（Fe3O4@SiO2-MPS@PDES）were prepared by polymerization of choline chloride-itaconic acid（ChCl-IA）and 3-（trimethoxysilyl）-propyl methacrylate（γ-MPS）modified magnetic silica composites.The characteristic results of VSM,FT-IR,XPS,TGA and TEM indicated the successful preparation of Fe3O4@SiO2-MPS@PDES.Try was chosen as the model protein to study the extraction performance of Fe3O4@SiO2-MPS@PDES.The concentration of Try in the supernatant was determined by UV-vis spectrophotometer at 278 nm.Single factor experiments were carried out to investigate the influence of concentration of Try,ionic strength,pH value,extraction time and temperature on the extraction capacity.Under the optimum conditions,the extraction capacity of Fe3O4@SiO2-MPS@PDES for Try was up to287.5 mg/g.Compared with extractants without modification by DES,the Fe3O4@SiO2-MPS@PDES displayed higher extraction capacity and selectivity for Try.Regeneration experiment showed the Fe3O4@SiO2-MPS@PDES retained a high extraction capacity of 233 mg/g for Try after eight cycles.Besides,the activity analysis indicated the activity of the extracted trypsin retained 96%of initial activity.Additionally,according to the result of real sample analysis experiment,the Fe3O4@SiO2-MPS@PDES was proven to be able to successfully extract Try from bovine pancreas crude extract.Methodological research demonstrated the precision,repeatability and stability of the proposed method were excellent.T he MSPE method based on DES provides a new idea for the extraction of protein.（4）Solid-phase extraction of DNA by using a composite prepared from multiwalled carbon nanotubes,chitosan,Fe3O4 and a poly（ethylene glycol）-based deep eutectic solventEight kinds of poly（ethylene glycol）-based deep eutectic solvent were synthesized and modified on the surface of chitosan-modified Fe3O4-magnetized multi-walled carbon nanotubes to form DES-mCS/MWCNT for the magnetic solid-phase extraction of salmon sperm DNA.The characteristic results of XRD,VSM,FT-IR,TGA and TEM indicated the successful preparation of DES-mCS/MWCNT.The concentration of DNA in the supernatant was determined by UV-vis spectrophotometer at 260 nm.Single factor experiments were carried out to investigate the influence of concentration of DNA,ionic strength,pH value,temperature and extraction time on the extraction capacity.Under the optimum conditions,the extraction capacity of DES-mCS/MWCNT for DNA was up to 178mg/g.Compared with extractants without modification by DES,the extraction capacity of DES-mCS/MWCNT for DNA turned out to be higher.Mixed sample experiment verified that the DES-mCS/MWCNT could selectively extract DNA from binary mixture of DNA and bovine hemoglobin（BHb）.Desorption experiment revealed that 79%of DNA could be eluted from the solid extractant by aqueous solution of 1 mol/L NaCl.The conformation of the extracted DNA remained unchanged as evidenced by CD spectra.According to the result of regeneration experiment,the DES-mCS/MWCNT could be recycled six times without significant loss of extraction capacity.Additionally,the DES-mCS/MWCNT was proven to be able to successfully extract DNA from bovine whole blood.Methodological research demonstrated the precision,repeatability and stability of the proposed method were excellent.The MSPE method based on DES provides a new idea for the extraction of DNA.（5）Preparation of magnetic molecularly imprinted p olymers based on a deep eutectic solvent as the functional monomer for specific recognition of lysozymeA magnetized molecularly imprinted polymer（MIP）was prepared via a surface-imprinting technique for the selective recognition of lysozyme（Lys）.The DES-Fe3O4@SiO2-MIP was synthesized by using magnetic silica as the support material,DES as the functional monomer and Lys as the template molecule.The characteristic results of XRD,FT-IR,TEM,TGA and VSM indicated the successful preparation of MIP.The influence of type and amount of functional monomer on the adsorption performance of MIP was evaluated,and the result showed the selection of DES as the functional monomer with amount of 100μL was the optimal formulation.According to the result of thermodynamic adsorption and kinetic adsorption experiments,the DES-Fe3O4@SiO2-MIP could reach the equilibrium adsorption at the optimum Lys concentration of 1.2 mg/mL within 7 h.Besides,the maximum adsorption capacity was up to 108 mg/g and the adsorption isotherm fitted well with Langmuirmodel.Selectiveadsorptionexperimentrevealedthatthe DES-Fe3O4@SiO2-MIP could capture Lys specifically with an imprinting factor of2.82,which is higher than other reference proteins（cytochrome C,BHb and BSA）.Additionally,the DES-Fe3O4@SiO2-MIP was proven to be able to selectively recognize Lys from binary proteins mixture and chicken egg white.Regeneration experiment showed the DES-Fe3O4@SiO2-MIP could be recycled four times without significant loss of adsorption capacity.Methodological research demonstrated the precision and repeatability of the proposed method were excellent.T he MIT method based on DES provides a new idea for the specific separation of proteins. |
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