Siming Wang

Synthesis of teicoplanin-modified hybrid magnetic mesoporous silica nanoparticles and their application in chiral separation of racemic compounds

Teicoplanin-conjugated mesoporous silica magnetic nanoparticles (TE-MSMNPs) were fabricated as novel chiral magnetic nano-selectors. Successful preparation of the functional magnetic mesoporous materials was achieved by grafting teicoplanin on N-(2-aminoethyl)-3-aminopropyltrimethoxysilane-modified mesoporous silica Fe3O4 magnetic nanoparticles (AEAPTMS-MSMNPs), and this was confirmed by various characterization techniques. The synthesized magnetic nanoparticles were regularly spherical and uniformly mesoporous with an average diameter of around 600 nm and a mean pore size of about 3.9 nm, respectively. These versatile magnetic nanoparticles were effective in a direct chiral separation of five racemic compounds in phosphate buffer. Much stronger interactions were observed with the (+)-enantiomers than with the (-)-enantiomers. After washing with water and ethanol by sonication, TE-MSMNPs could be reused at least three times with little efficiency loss. The functional magnetic mesoporous nanoparticles were easily separated from the racemic solutions using an external magnetic field. These magnetic nano-materials are suitable for enantiomer separations.

Magnetic nanoparticles coated with immobilized alkaline phosphatase for enzymolysis and enzyme inhibition assays

Magnetic nanoparticles are potentially useful as supports for biomacromolecules because of their biocompatibility, low toxicity and easy separation. In this study, alkaline phosphatase (ALP) was used as a model enzyme, and a new type of immobilized ALP was prepared on superparamagnetic nanoparticles and confirmed by various characterization techniques. X-ray diffraction (XRD), scanning electron microscopy (SEM) and vibrating sample magnetometry (VSM) results present that the synthesized nanoparticles possess a clear three-dimensional core-shell architecture with an average diameter of about 390 nm and a high saturation magnetization of 86.7 emu g-1. Fourier-transform infrared spectra (FTIR) and thermogravimetric analysis (TGA) results show that ALP was successfully attached to the surface of magnetic nanoparticles via a crosslinking technique. An enzyme inhibition study was performed on the immobilized ALP magnetic nanoparticles using theophylline, l-tryptophan and d-tryptophan as model inhibitors. The enzyme kinetics indicate that dl-tryptophan possesses chiral discrimination inhibition and l-tryptophan exhibits uncompetitive inhibition for ALP, compared with no obvious inhibition observed with its enantiomer. The results also show that theophylline is a noncompetitive inhibitor and has a markedly higher inhibitory effect than l-tryptophan. The protocol described allows easy manipulation, reduces procedural time and can be adapted to high-throughput screening of enzyme reactions and inhibitors.

Polyamidoamine dendrimer as a spacer for the immobilization of glucose oxidase in capillary enzyme microreactor.

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups. In this study, different generations of PAMAM (G0-G4) were introduced onto the inner wall of fused-silica capillaries by microwave irradiation and a new type of glucose oxidase (GOx) capillary enzyme microreactor was developed based on enzyme immobilization in the prepared PAMAM-grafted fused-silica capillaries. The optimal enzymolysis conditions for beta-d-glucose in the microreactor were evaluated by capillary zone electrophoresis. In addition, the enzymolysis efficiencies of different generations of PAMAM-GOx capillary enzyme microreactor were compared. The results indicate that enzymolysis efficiency increased with increasing generations of PAMAM. The experimental results provide the possibility for the development and application of an online immobilized capillary enzyme microreactor.

Online immobilized enzyme microreactor for the glucose oxidase enzymolysis and enzyme inhibition assay.

An online immobilized glucose oxidase (GOx) capillary microreactor was developed based on an enzymatic redox reaction with 1,4-benzoquinone as an acceptor of electrons, replacing the molecular oxygen typically used in a GOx reaction to achieve direct ultraviolet detection without derivation. A high efficiency of enzymolysis was obtained at 1 mg ml⁻¹ 1,4-benzoquinone for 5 min of incubation at 25°C, and baseline separation of the substrate and product could be achieved with a resolution of 3.85 by employing 20mM phosphate buffer (pH 8.0) containing 40 mg ml⁻¹ sulfated β-cyclodextrin as an additive, a constant voltage of 15 kV, and a detection wavelength of 220 nm. In addition, an online enzyme inhibition study was performed on the immobilized GOx microreactor with metal ions Ag⁺ and Cu²⁺ used as model inhibitors. The results indicate that Ag⁺ (IC₅₀=69.16 μM) has a markedly higher inhibitory effect than Cu²⁺ (IC₅₀=1.33 mM). The protocol described can be applied in high-throughput screening of enzyme reactions and inhibitors.

Dendrimer modified magnetic nanoparticles for immobilized BSA: a novel chiral magnetic nano-selector for direct separation of racemates

Nanomaterials capable of performing highly efficient chiral separations represent promising materials with potential applications in biomedical research. In this paper, we report the development of a new microwave-assisted method for the preparation of polyamidoamine (PAMAM)-magnetite nanoparticles (PMNPs) and their use in the immobilization of BSA to form Fe3O4@SiO2@PAMAM-BSA (BSA-PMNPs) chiral magnetic nanoparticles. The binding capacity of BSA to PMNPs increases with each generation of growth, which can enhance the chiral discrimination of magnetite prepared in this way. UV-vis spectrophotometry and confocal laser scanning microscopy were used to evaluate the binding capacity of BSA. The activity of the BSA on the PMNPs was tested using an immunoaffinity approach, with the eluent being assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The functional magnetic nanoparticles were then used for the direct separation of chiral amino acids, and could be readily separated from the reaction mixtures via the application of a magnetic field. The separation results indicated that the BSA-PMNPs showed much greater affinities for the (-)-enantiomers than the (+)-enantiomers, and effectively demonstrated the potential utility of this material for chiral separations.

Polyethyleneimine as a novel desorbent for anionic organic dyes on layered double hydroxide surface

Polyethyleneimine (PEI) is a positively charged polymer with hydrogen-bonding sites and hydrophobic chains. Therefore, it has been clearly established as an efficient adsorbent by means of these native properties in the literatures. However, there is apparently no good reason to disregard the use of PEI as a desired desorbent. Herein, using methyl orange as a model anionic dye, we investigated the desorption performances of PEI toward anionic dyes adsorbed on the surface of CO3-layered double hydroxides (LDHs) in a wide range of pH values. The experiment results showed that the positively charged PEI had very strong desorption capacity for anionic dyes at low pH values (<9.5) through electrostatic attraction between PEI and methyl orange because of the high degree of protonation of PEI. At high pH values (>9.5), PEI existed as neutral molecule, it could desorb methyl orange via hydrogen bonding between the amino groups of it and sulfonate group of methyl orange; simultaneously, the anion-exchange process occurred between abundant hydroxyl anions and anionic methyl orange. The adsorption capacity of the used LDH adsorbent was about 80% after five cycles of adsorption-desorption-regeneration, which was much higher than that conducted by 0.1M NaOH solution. These findings suggested that PEI could be regarded as a promising desorbent for enriching anionic dyes in wastewater and regenerating LDHs through surface adsorption-desorption cycles.

DNA-directed trypsin immobilization on a polyamidoamine dendrimer-modified capillary to form a renewable immobilized enzyme microreactor

A novel type of trypsin capillary microreactor was developed based on a DNA-directed immobilization (DDI) technique applied to a fused-silica capillary modified with polyamidoamine (PAMAM) dendrimers. Trypsin binding to the inner wall of the capillary was confirmed by confocal laser scanning microscopy. The properties of the trypsin-DNA conjugated, PAMAM-modified capillary microreactor were investigated by monitoring hydrolysis of Nα-benzoyl-L-arginine ethyl ester. Through the hybridization and dehybridization of the DNA, the inner wall of the capillary functionalized with trypsin can be regenerated, thus indicating the renewability of this enzyme microreactor. In addition, these results demonstrated that introduction of PAMAM enabled higher amounts of trypsin to be immobilized, markedly improving the enzymolysis efficiency, compared with traditional modified capillaries. The digestion performance of the trypsin capillary microreactor was further evaluated by digesting cytochrome C, and a peptide numbers of 8, and a sequence coverage of 59% were obtained. This renewable and efficient immobilized trypsin capillary microreactor combines advantages of both DDI technology and PAMAM, and is potentially adaptable to high-throughput enzyme assays in biochemical and clinical research.

IMMUNOEXTRACTION OF TESTOSTERONE AND EPITESTOSTERONE FROM HUMAN URINE SAMPLE BASED ON POLYAMIDOAMINE MODIFIED SILICA

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups. The polyamidoamine modified silica was synthesized with microwave assisted protocol. Anti-epitestosterone monoclonal antibodies were immobilized onto the PAMAM grafted silica and prepared an off-line immunoextraction column that applied in the extraction of testosterone and epitestosterone. The results showed that the affinity activity of the anti-epitestosterone monoclonal antibodies was remained at high level after immobilization. It was satisfactory to apply this new type of immunoextraction column to analyze testosterone and epitestosterone in spiked urine sample.

Analysis of C5 fraction and extractive distillation solvent by two-dimensional gas chromatography with heart-cutting technique

Two-dimensional gas chromatography using the heart-cutting technique was applied to the analysis of the C5 fraction and extractive distillation solvent. High-resolution separation of the C5 fraction and N-methylpyrrolidone with water was obtained under optimized conditions. The separated components were identified by mass spectroscopy using standard samples. The recoveries of standard compounds were 99.5–100.6%, and the relative standard deviations were all less than 3%. It took only 30 min to finish a determination. Industrial samples from Yanshan Petrochemical Industry Corporation were also analyzed using the method. The experimental results indicate that the method is specific, sensitive and suitable for the determination of the C5 fraction and the extractive distillation solvent. Thus, the method is expected to be widely applicable and will be valuable in industrial applications.