Feng Qu

Superparamagnetic lysozyme surface-imprinted polymer prepared by atom transfer radical polymerization and its application for protein separation

Molecular imprinting as a promising and facile separation technique has received much attention because of their high selectivity for target molecules. In this study, the superparamagnetic lysozyme surface-imprinted polymer was prepared by a novel fabricating protocol, the grafting of the imprinted polymer on magnetic particles in aqueous media was done by atom transfer radical polymerization (ATRP), and the properties of the imprinted polymer were characterized in detail. Its high selective adsorption and recognition to lysozyme demonstrated the separation ability of the magnetic imprinted material to template molecule, and it has been used for quick and direct separation of lysozyme from the mixture of standard proteins and real egg white samples under an external magnetic field. Furthermore, the elution of lysozyme from the imprinted material was achieved by PEG/sulphate aqueous two-phase system, which caused lysozyme not only desorption from the imprinted materials but also redistribution in the top and bottom phase of aqueous two-phase system. The aqueous two-phase system exhibited some of the extraction and enrichment effect to desorbed lysozyme. Our results showed that ATRP is a promising method for the protein molecularly imprinted polymer preparation.

The preparation of bovine serum albumin surface-imprinted superparamagnetic polymer with the assistance of basic functional monomer and its application for protein separation

Currently, small proteins imprinting are more reported since large proteins molecular imprinting faces challenge due to their bulk size and complex structure. In this work, bovine serum albumin (BSA) surface-imprinted magnetic polymer was successfully synthesized based on atomic transfer radical polymerization (ATRP) method in the presence of common monomer (N-isopropylacrylamide) with the assistant of basic functional monomer (N-[3-(dimethylamino)propyl]-methacrylamide), which provides a achievable attempt for imprinting larger target proteins based on the ATPR with the mild reaction conditions. The BSA-imprinted polymer exhibited higher adsorption capacity and selectivity to BSA over the non-imprinted polymer. Competitive adsorption tests indicated the BSA-imprinted polymer had better selective adsorption and recognition properties to BSA in the mixture. The obtained BSA-imprinted polymer was applied to bovine serum, which also showed selectivity to BSA. In addition, a conventional aqueous two-phase solution of PEG/sulphate was used as elution for adsorbed BSA, which was compared with common NaCl elution.

Integration of carboxyl modified magnetic particles and aqueous two-phase extraction for selective separation of proteins.

Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation.

The Preparation of BHb-Molecularly Imprinted Gel Polymers and Its Selectivity Comparison to BHb and BSA

Molecularly imprinted gel polymer (MIP) for the selective imprinting of bovine hemoglobin (BHb) was prepared in aqueous media by bulk polymerization using polyacrylamide matrix. The synthesis conditions of BHb-MIP were investigated, which involved the interaction of functional monomers and template protein in different molar ratio, solution pH, and ionic strength. The adsorption experiments indicated that BHb-MIP had a high affinity for BHb over the non-imprinted polymers. The selectivity of BHb-MIP for BHb and bovine serum albumin (BSA) with similar molecular weight was compared. It was demonstrated that BHb-MIP had better selective adsorption and recognition properties to BHb especially in the presence of BSA as competing protein. It might be helpful for selectively separating template protein with MIP from the proteins mixture with similar molecular weight.

Interaction evaluation of bacteria and protoplasts with single-stranded deoxyribonucleic acid library based on capillary electrophoresis.

For whole-cell aptamers selection, cells surface situation has great impact on single-stranded (ssDNA) binding and aptamers selection. In this work, both Lactobacillus acidophilus and Escherichia coli as well as their protoplasts were as cells targets, their interaction with ssDNA library were evaluated based on capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE) with UV and LIF detection. Our results demonstrated that protoplasts without cells wall had apparently stronger interaction with ssDNA library than bacteria, the protoplasts-ssDNA complex could be observed clearly with CZE-LIF. Furthermore, E. coli pretreated by four organic solvents (methanol, ethanol, formaldehyde and glutaraldehyde) showed binding difference with ssDNA library, which could be identified with ACE-UV. Binding constants indicated the interaction of E. coli with ssDNA library were in the order of E. coli protoplasts>methanol (ethanol) treated E. coli>formaldehyde (glutaraldehyde) treated E. coli≈E. coli. Above results suggest that cells surface situation determines their binding affinity with ssDNA, which should be considered in whole-cell aptamers selection and aptamers further application. Capillary electrophoresis is a preferable technique for interaction evaluation of composite targets binding with ssDNA library.

Ionic liquid-assisted SDS-PAGE to improve human serum protein separation

Ionic liquid (IL)‐assisted sodium dodecyl sulfate polyacrylamide gel electrophoresis (ILs‐SDS‐PAGE) was presented to improve protein separation. ILs were employed during the preparation process of polyacrylamide gel, then the modified gel was used for commercial protein marker, binary bovine serum albumin/lysozyme (BSA/Lyz) and human serum separation. The influence of ionic liquid concentration, cation alkyl chain length, cation and anion types on proteins separation were investigated. The results showed that ILs played a role in improving some protein separation, and ILs‐SDS‐PAGE provided higher resolution and separation efficiency than ordinary SDS‐PAGE for low and middle relative molecular mass proteins in human serum. In addition, the principle of ILs‐SDS‐PAGE was discussed and the comparison of ILs‐SDS‐PAGE with ordinary SDS‐PAGE and Native PAGE was made.

The application of capillary electrophoresis for assisting whole-cell aptamers selection by characterizing complete ssDNA distribution

Whole-cell SELEX faces more difficulties than SELEX against purified molecules target. In this work, we demonstrate the application of capillary electrophoresis for assisting whole-cell aptamers selection by characterizing complete ssDNA distribution. We chose three cancer cell lines U251, Hela and PC3 as target, FAM labeled Sgc8c (a 41mer aptamer) and FAM labeled 41mer random ssDNA library as ssDNA model. CE conditions of running buffer and capillary length and inner diameter as well as UV and LIF detection were optimized. The distribution percentage of Sgc8c and ssDNA library against U251, Hela and PC3 was demonstrated, the relative peak area of their complex is 8.94%, 1.05% and 0.44% for Sgc8c and 9.03%, 1.04% and 0.12% for ssDNA library respectively. Under the chosen experimental conditions, binding ability comparison of three cell lines was U251>Hela>PC3, which was validated by laser confocol microscope. For each cell, distribution percentage of ssDNA library was compared with that of Sgc8c. Finally, whole-cell complex of U251-Sgc8c was confirmed by increase incubation time and fraction CE analysis.

Low pH capillary electrophoresis application to improve capillary electrophoresis-systematic evolution of ligands by exponential enrichment.

In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) as a CE-SELEX variant is proposed. Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein are incubated with a FAM labeled ssDNA library, respectively. Incubation mixture is separated in low pH CE (pH 2.6), where positively charged protein, protein-ssDNA complex and negatively charged ssDNA library migrate oppositely without EOF driven. Analysis of protein-ssDNA complex under positive voltage and unbound ssDNA library under negative voltage by CE-UV are applied for interactive evaluation. By increasing injection time, larger amount protein-ssDNA complex can be collected conveniently at the cathode end whereas ssDNA migrates to anode. Finally, stability of protein-ssDNA complex in low pH CE separation is discussed.

Capillary zone electrophoresis‐based cytotoxicity analysis of Caco‐2 cells

A capillary zone electrophoresis‐based method to evaluate the cytotoxicity of substances to Caco‐2 cells was established. The estimation of the injected cell number (500–5000) and the minor effect of injection condition on cytotoxicity determination were investigated. Caco‐2 cells the best model of the intestinal absorptive epithelium, were treated with substances and then stained with Trypan Blue and fixed with paraformaldehyde. The treated Caco‐2 cells were detected simultaneously at 590 nm and 214 nm, and the absorbance ratio of the two wavelengths (R590/214) can reflect simultaneously the loss of cell membrane integrity and the degradation/leak of intracellular components and indicate the cytotoxicity of substances. The cytotoxicity of the four substances sodium sulfite (Na2SO3), methyl mercury (MeHg), paclitaxel (PTX), and cadmium chloride (CdCl2) were determined and compared. There was no obvious cytotoxicity caused by 20 μM Na2SO3 for 24 h treatment, and the toxicity of the other three toxicants was sequenced as: CdCl2 > MeHg> PTX. The results are in good agreement with the references and the conventional Trypan Blue exclusion counting assay.

Multiple modes of capillary electrophoresis applied in peptide nucleic acid related study

Peptide Nucleic Acid (PNA) is a nucleic acid analogue, whose neutrally charged and hydrophobic backbone makes it more stable in vivo, so it might act as a potentially better protein probe as compared to aptamer. Currently the investigation of PNA and protein interaction is scarce. In this research, multiple modes of capillary electrophoresis were established and applied for PNA characterization and its interaction with ssDNA and protein. A 15-mer PNA having the same nucleobase sequence as 15-mer anti-thrombin DNA aptamer was chosen as PNA model for this study, its pI (7.71) was estimated by capillary isoelectric focusing (cIEF). Due to its neutral charge and strong hydrophobicity, three micellar electrokinetic chromatography (MEKC) modes containing (a) SDS, (b) Triton X-100 and (c) CTAB were compared for PNA related analysis. CTAB was not applicable for PNA analysis, while in 4mM SDS or 2mM Triton X-100, PNA and PNA-ssDNA complex can be identified directly. The significant peak of PNA-ssDNA complex helped in validating the two MEKC modes for PNA and target interaction study. Furthermore, the effect of SDS and Triton X-100 concentrations in the two MEKC modes on the protein target thrombin analysis was investigated by capillary zone electrophoresis (CZE). 4mM SDS caused thrombin denaturation. So in 2mM Triton X-100, interactions of PNA with thrombin, PNA with RNase A and a non-aptameric PNA (n-PNA) with thrombin were compared. PNA with thrombin exhibited strongest binding. In summary, cIEF mode for PNA pI determination, MECK mode for direct PNA, PNA-ssDNA and PNA-protein complex identification and CZE mode for the effect of surfactant in MEKC modes on protein target thrombin analysis were applied. Above results showed that multiple modes of CE provide rapid and very low-sample cost methods for PNA related studies.