The Research On Magnetic Solid Phase Extraction Of Proteins Based On Green Solvents

Magnetic solid phase extraction ?MSPE? is a kind of dispersive solid phase extraction technology that uses magnetic materials as adsorbent. The key of the technology is designing and synthesizing functional adsorbent. As a new type of green solvent, deep eutectic solvent ?DES? and ionic liquid ?IL? have the advantages of designable and functional. Immobilize deep eutectic solvent or ionic liquid on magnetic particles will obtain supported deep eutectic solvent or supported ionic liquid. Using them as adsorbent for MSPE can take the full advantage of deep eutectic solvent or ionic liquid and magnetic particle. Therefore, there will be wider application prospect if we combine deep eutectic solvent or ionic liquid with MSPE.In this study, two types of green solvents, DES and IL, were synthesised. In combination with magnetic solid phase extraction, a method for protein extraction is established. The main contents of the present study are as follows:?1? Magnetic solid phase extraction of protein based on magnetic graphene oxide modified with choline cholride-based deep eutectic solventFour kinds of green deep eutectic solvents ?DESs? based on choline chloride ?ChCl? have been synthesized and coated on the surface of magnetic graphene oxide ?Fe₃O₄@GO? to form Fe₃O₄@GO-DES for the magnetic solid-phase extraction of protein. X-ray diffraction ?XRD?, vibrating sample magnetometer ?VSM?, Fourier transform infrared spectrometry ?FTIR?, field emission scanning electron microscopy ?FESEM? and thermal gravimetric analysis ?TGA? were employed to characterize Fe₃O₄@GO-DES. Bovine serum albumin ?BSA? was chosen as research object. Effects of the types of DESs, solution temperature, solution ionic strength, extraction time, protein concentration and the amount of Fe₃O₄@GO-DES were studied by a single factor experiments. Under the optimum extraction conditions, the extraction amount could reach 100.2 mg/g. After extraction,0.005 mol/L Na2HPO4-1 mol/L NaCl aqueous solution was selected as eluent to release BSA from the solid extractant, the obtained elution efficiency was about 90.9%. Circular dichroism spectra ?CD spectra? have been used to determine the secondary structure of BSA eluted from the solid extractant. The obtained result suggests that the secondary structures of BSA eluted from the solid extractants are not changed. Moreover, the proposed Fe₃O₄@GO-DES-MSPE has been applied in extracting ovalbumin ?OVA?, lysozyme ?Lys? and bovine hemoglobin ?BHb?. The proposed method provides new opportunities in separating and purifying proteins.?2? Magnetic solid phase extraction of protein based on betaine-based ionic liquid coated magnetic particlesFe₃O₄ was prepared by co-precipitation method, then modified with 3-aminopropyltriethoxysilane ?APTES?. The obtained Fe₃O₄/APTES is combined with grahene oxide ?GO? to form magnetic graphene oxide ?Fe₃O₄/APTES/GO? through electrostatic interactions. The synthetic betaine-based ionic liquids have been used to modify the as-prepared Fe₃O₄/APTES/GO to get Fe₃O₄/APTES/GO/IL, and then used to extract bovine serum albumin ?BSA?. Vibrating sample magnetometer ?VSM?, X-ray diffraction ?XRD?, Fourier transform infrared spectrometry ?FTIR?, thermal gravimetric analysis ?TGA?, field emission scanning electron microscopy ?FESEM?, transmission electron microscopy ?TEM? and Zeta potential analyzer were used to characterize Fe₃O₄/APTES/GO/IL. Single factor experiments were carried out to study the influence of types of ionic liquids, solution temperature, extraction time, concentration of BSA, ionic strength and pH value on the extraction amount. Under the optimum extraction conditions, the extraction amount could reach 139.1 mg/g. Comparison experiment proved that the proposed Fe₃O₄/APTES/GO/IL is more superior to bare Fe₃O₄ nanoparticles and Fee3O4/APTES/GO in the extraction of BSA. 0.8 mol/L K2HPO4-1 mol/L NaCl aqueous solution was selected as eluent to release BSA from the solid extractant, the obtained elution efficiency was about 95.5%. CD spectra showed that the selected eluent could not change the secondary structure of BSA. Additionally, the extraction process of protein from bovine calf whole blood by the prepared Fee3O4/APTES/GO/IL had been preformed to evaluate the practical applicability of the proposed method and the result was satisfactory.?3? Magnetic solid phase extraction of trypsin based on polymeric ionic liquid modified magnetic particlesWith an ionic liquid containing unsaturated bond in anion as monomer, supported polymeric ionic liquid ?Fee3O4/SiO2/y-MPS/PIL? was prepared by grafting ionic liquid onto 3-?trimethoxysilyl?-propyl methacrylate ?y-MPS? modified Fe₃O₄ particles. Vibrating sample magnetometer ?VSM?, X-ray diffraction ?XRD?, field emission scanning electron microscopy ?FESEM?, Fourier transform infrared spectrometry ?FTIR?, thermal gravimetric analysis ?TGA?, and Zeta potential analyzer were applied to characterize Fee3O4/SiO/y-MPS/PIL. A series of single factor experiments were carried out by using Fee3O4/SiO2/y-MPS/PIL as magnetic solid phase extractant and trypsin as research object. Under the optimum extraction conditions, the extraction amount could reach 182.7 mg/g. A comparison experiment has been performed to study the superiority of Fee3O4/SiO2/y-MPS/PIL to Fe₃O₄/SiO2 and Fee3O4/SiO/?-MPS in trypsin extraction. The elution of magnetic solid phase extractant containing trypsin was performed. Determination of the activity of trypsin ?in ultrapure water and in eluent? was carried out. The obtained relative activity ?Ur? was 1.0 and 0.83, respectively. The result showed that the activity of trypsin eluted from solid extractants is lower than that in ultrapure water, but still have certain activity. The proposed method provides a new clew for the extraction of other bioactive materials.