Yanhua Huang

A green deep eutectic solvent-based aqueous two-phase system for protein extracting.

As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n=3), 1.6057% (n=3) and 1.6132% (n=3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV-vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES-protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins.

Development of green betaine-based deep eutectic solvent aqueous two-phase system for the extraction of protein.

Six kinds of new type of green betaine-based deep eutectic solvents (DESs) have been synthesized. Deep eutectic solvent aqueous two-phase systems (DES-ATPS) were established and successfully applied in the extraction of protein. Betaine-urea (Be-U) was selected as the suitable extractant. Single factor experiments were carried out to determine the optimum conditions of the extraction process, such as the salt concentration, the mass of DES, the separation time, the amount of protein, the temperature and the pH value. The extraction efficiency could achieve to 99.82% under the optimum conditions. Mixed sample and practical sample analysis were discussed. The back extraction experiment was implemented and the back extraction efficiency could reach to 32.66%. The precision experiment, repeatability experiment and stability experiment were investigated. UV-vis, FT-IR and circular dichroism (CD) spectra confirmed that the conformation of protein was not changed during the process of extraction. The mechanisms of extraction were researched by dynamic light scattering (DLS), the measurement of the conductivity and transmission electron microscopy (TEM). DES-protein aggregates and embraces phenomenon play considerable roles in the separation process. All of these results indicated that betaine-based DES-ATPS may provide a potential substitute new method for the separation of proteins.

Preparation of magnetic chitosan and graphene oxide-functional guanidinium ionic liquid composite for the solid-phase extraction of protein

A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been synthesized, and then magnetic chitosan graphene oxide (MCGO) composite has been prepared and coated with these functional guanidinium ionic liquids to extract protein by magnetic solid-phase extraction. MCGO-functional guanidinium ionic liquid has been characterized by vibrating sample magnetometer, field emission scanning electron microscopy, X-ray diffraction spectrometer and Fourier transform infrared spectrometer. After extraction, the concentrations of protein were determined by measuring the absorbance at 278 nm using an ultra violet visible spectrophotometer. The advantages of MCGO-functional guanidinium ionic liquid in protein extraction were compared with magnetic chitosan, graphene oxide, MCGO and MCGO-ordinary imidazolium ionic liquid. The proposed method has been applied to extract trypsin, lysozyme, ovalbumin and bovine serum albumin. A comprehensive study of the adsorption conditions such as the concentration of protein, the amount of MCGO-functional guanidinium ionic liquid, the pH, the temperature and the extraction time were also presented. Moreover, the MCGO-functional guanidinium ionic liquid can be easily regenerated, and the extraction capacity was about 94% of the initial one after being used three times.

Magnetic graphene oxide modified with choline chloride-based deep eutectic solvent for the solid-phase extraction of protein

Four 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, and the results indicated the successful preparation of Fe3O4@GO-DES. The UV-vis spectrophotometer was used to measure the concentration of protein after extraction. Single factor experiments proved that the extraction amount was influenced by the types of DESs, solution temperature, solution ionic strength, extraction time, protein concentration and the amount of Fe3O4@GO-DES. Comparison of Fe3O4@GO and Fe3O4@GO-DES was carried out by extracting bovine serum albumin, ovalbumin, bovine hemoglobin and lysozyme. The experimental results showed that the proposed Fe3O4@GO-DES performs better than Fe3O4@GO in the extraction of acidic protein. Desorption of protein was carried out by eluting the solid extractant with 0.005 mol L(-1) Na2HPO4 contained 1 mol L(-1) NaCl. The obtained elution efficiency was about 90.9%. Attributed to the convenient magnetic separation, the solid extractant could be easily recycled.

Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein.

A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by (1)H nuclear magnetic resonance ((1)H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV-vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV-vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins.

Magnetic solid-phase extraction of protein with deep eutectic solvent immobilized magnetic graphene oxide nanoparticles

As a new type of green solvent, four kinds of choline chloride (ChCl)-based deep eutectic solvents (DESs) have been synthesized, and then a core-shell structure magnetic graphene oxide (Fe3O4-NH2@GO) nanoparticles have been prepared and coated with the ChCl-based DESs. Magnetic solid-phase extraction (MSPE) based Fe3O4-NH2@GO@DES was studied for the first time for the extraction of proteins. The characteristic results of vibrating sample magnetometer (VSM), X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), thermal gravimetric analysis (TGA) and field emission scanning electron microscopy (FESEM) indicated the successful preparation of Fe3O4-NH2@GO@DES. The concentrations of proteins in studies were determined by a UV-vis spectrophotometer. The advantages of Fe3O4-NH2@GO@DES in protein extraction were compared with Fe3O4-NH2@GO and Fe3O4-NH2, and Fe3O4-NH2@GO@ChCl-glycerol was selected as the suitable extraction solvent. The influence factors of the extraction process such as the pH value, the temperature, the extraction time, the concentration of protein and the amount of Fe3O4-NH2@GO@ChCl-glycerol were evaluated. Desorption experimental result showed 98.73% of BSA could be eluted from the solid extractant with 0.1 mol/L Na2HPO4 solution contained 1 mol/L NaCl. Besides, the conformation of BSA was not changed during the elution by the investigation of circular dichromism (CD) spectra. Furthermore, the analysis of real sample demonstrated that the prepared magnetic nanoparticles did have extraction ability on proteins in bovine whole blood.

Partition of proteins with extraction in aqueous two-phase system by hydroxyl ammonium-based ionic liquid

A series of hydroxyl ammonium ionic liquids (ILs) have been designed and synthesized. An ionic liquid aqueous two-phase system based on N,N-dimethylethanolamine propionate ([DMEA][Pr]) ionic liquid was studied for the extraction of proteins. Based on the single-factor experiment, an initial serial investigative test was used to identify the optimal conditions. Five factors and four levels of orthogonal experiments were used to verify the optimum extraction conditions. The results show that under the optimum conditions, the extraction efficiency could reach up to 99.50%. The RSD of extraction efficiencies in precision experiment, repeatability experiment and stability experiment were 0.3% (n = 5), 1.1% (n = 5) and 1.5% (n = 5), respectively. The conformation of the proteins was not affected after extraction into the IL-rich phase in terms of the determination by UV-vis and FT-IR spectra. According to the determination of conductivity, dynamic light scattering, and transmission electron microscope images, the microstructure of the IL-rich phase and the possible mechanism for the extraction are investigated. Hydrogen bonding interaction, salt out effect and the aggregation phenomenon played important roles in the extraction. The circular dichroism spectral experiment analysis indicated that the secondary structures of the protein were unchanged after extract. Based on these findings, it is suggested that the method of hydroxyl ammonium-based ILs-ATPs have the potential to offer new possibility in the extraction of bio-analytes.

Ionic liquid-coated Fe3O4/APTES/graphene oxide nanocomposites: synthesis, characterization and evaluation in protein extraction processes

A magnetic solid-phase extraction (MSPE) procedure with a betaine-based ionic liquid (IL) coated 3-aminopropyltriethoxysilane (APTES)–Fe3O4 grafted graphene oxide (GO) nanocomposite (Fe3O4/APTES/GO/IL) as a magnetic adsorbent has been developed for protein extraction. A series of environmentally friendly ionic liquids (ILs) based on betaine and carboxylic acids have been prepared, and their structures have been confirmed by 1H nuclear magnetic resonance (1HNMR). Then the synthetic ionic liquids have been used to modify magnetic graphene oxide nanoparticles (Fe3O4/APTES/GO) to form Fe3O4/APTES/GO/IL. The as-prepared Fe3O4/APTES/GO/IL has been used to extract bovine serum albumin (BSA). After extraction, the concentration of protein in the supernatant was measured by UV-vis spectrophotometry. Several significant factors that affect the extraction amount, such as solution temperature, extraction time, protein concentration, ionic strength and pH value, were investigated. Experimental results show that the extraction amount could reach 139.1 mg g−1. The advantages of Fe3O4/APTES/GO/IL in protein extraction were compared with bare Fe3O4 nanoparticles and Fe3O4/APTES/GO. Moreover, the extraction process of protein from bovine calf whole blood by Fe3O4/APTES/GO/IL has been performed to evaluate the practical applicability of the proposed method.

Magnetic solid-phase extraction of proteins based on hydroxy functional ionic liquid-modified magnetic nanoparticles

Hydroxy functional ionic liquids have been first designed and modified on the surface of silica-coated magnetic Fe3O4 nanoparticles (MNPs) for the investigation of the extraction performance of proteins by magnetic solid-phase extraction (MSPE) and characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). BSA was chosen as a model protein to investigate the effect of extraction parameters. Based on the single-factor experiment, an initial serial investigative test was used to identify the optimal conditions of the extraction, and the extraction efficiency could reach up to 86.92%. The RSD of extraction efficiencies in the precision, repeatability and stability experiments were 1.17% (n = 3), 3.79% (n = 3) and 4.82% (n = 3), respectively. Compared with the other four types of ionic liquids, hydroxy functional ionic liquids-modified magnetic nanoparticles have the highest extraction rates. The desorption of proteins was up to 94.91% when the concentrations of NaCl were greater than 1.1 mol L−1. Nearly 95% of MNPs could be recovered from each run, and extraction rates decreased significantly after five runs.

Design of guanidinium ionic liquid based microwave‐assisted extraction for the efficient extraction of Praeruptorin A from Radix peucedani

A series of novel tetramethylguanidinium ionic liquids and hexaalkylguanidinium ionic liquids have been synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids were confirmed by (1)H NMR spectroscopy and mass spectrometry. A green guanidinium ionic liquid based microwave-assisted extraction method has been developed with these guanidinium ionic liquids for the effective extraction of Praeruptorin A from Radix peucedani. After extraction, reversed-phase high-performance liquid chromatography with UV detection was employed for the analysis of Praeruptorin A. Several significant operating parameters were systematically optimized by single-factor and L9 (3(4)) orthogonal array experiments. The amount of Praeruptorin A extracted by [1,1,3,3-tetramethylguanidine]CH2CH(OH)COOH is the highest, reaching 11.05 ± 0.13 mg/g. Guanidinium ionic liquid based microwave-assisted extraction presents unique advantages in Praeruptorin A extraction compared with guanidinium ionic liquid based maceration extraction, guanidinium ionic liquid based heat reflux extraction and guanidinium ionic liquid based ultrasound-assisted extraction. The precision, stability, and repeatability of the process were investigated. The mechanisms of guanidinium ionic liquid based microwave-assisted extraction were researched by scanning electron microscopy and IR spectroscopy. All the results show that guanidinium ionic liquid based microwave-assisted extraction has a huge potential in the extraction of bioactive compounds from complex samples.