Co-reporter:Jiayi Song;Ping Su;Ye Yang
New Journal of Chemistry (1998-Present) 2017 vol. 41(Issue 14) pp:6089-6097
Publication Date(Web):2017/07/10
DOI:10.1039/C7NJ00284J
Enzymes usually have poor thermal and operational stability as well as limited reuse cycles, which greatly limit their practical applications. This study reports a novel strategy for enzyme immobilization based on toehold-mediated DNA strand displacement on modified magnetic nanoparticles using alkaline phosphatase (ALP) and horseradish peroxidase (HRP) as different model enzymes. The immobilized enzyme procedure by DNA strand displacement exhibited high reversibility and reproducibility, and the mild, convenient approach achieved successive enzyme immobilization through triggering a toehold-mediated DNA strand displacement reaction. The immobilized HRP exhibited promising thermal stability after 50 min incubation at 50 °C and 60 °C, which were about 6.5- and 9.7-fold greater than the free enzyme, respectively. Notably, the immobilized enzyme exhibited excellent long-term storage stability, and the enzymatic activities were about 34.4- and 24.9-fold greater than free HRP after storage at 4 °C for 68 days and at room temperature for 9 days, respectively. The immobilized enzyme also preserved high performance towards long-term incubation stability, compared to the free enzyme, and excellent reusability, which showed a high degree of activity (more than 73%) after 5 cycles. Thus, the developed strategy exhibited a promising alternative platform with high magnetic responsiveness and significantly enhanced properties for the immobilization of important enzymes and their broad applications.
Co-reporter:Kangle Zheng, Liqing Wu, Zhangjing He, Bin Yang, Yi Yang
Measurement 2017 Volume 112(Volume 112) pp:
Publication Date(Web):1 December 2017
DOI:10.1016/j.measurement.2017.08.013
•A biuret method to measure the total protein in serum.•Instruments were certificated and the data used in calculation of uncertainty.•Two known concentration standard protein to evaluate the method performance.•Two serum samples given by RELA were measured.•The calculation of the uncertainty here developed is detailed.As recommended by IFCC, the biuret method is generally used to determine the total protein as a reference method. In order to achieve accurate measurement of the total protein by biuret method, a detailed description of establishment, validation and uncertainty evaluation of this method is proposed in the metrological view in this paper. Then the method was used to determine the total protein in RELA serum samples and the results of 2015-RELA sample A and B were 73.9 g·L−1 and 58.9 g·L−1, with the RSD of 1.18% and 1.07%, respectively. The uncertainty was evaluated in detail fully following the Guide to the Expression of Uncertainty in Measurement, and the expanded uncertainty of RELA sample A and B were 2.0 and 1.2 g·L−1, respectively. The results have good agreement with those from other research institutions. The proposed procedure gives a good example to realize an accurate reference method with strict metrological control, which can benefit not only total protein measurement but also other biological measurement by reference methods.Download high-res image (161KB)Download full-size image
Co-reporter:Ye Yang, Ping Su, Kangle Zheng, Ting Wang, Jiayi Song and Yi Yang
RSC Advances 2016 vol. 6(Issue 43) pp:36849-36856
Publication Date(Web):07 Apr 2016
DOI:10.1039/C6RA01621A
This study focused on the development of a common method for the reversible and self-directed immobilization of enzymes based on a DNA-directed immobilization (DDI) technique. The successful anchoring of alkaline phosphatases to the surfaces of magnetic nanoparticles was confirmed using confocal laser scanning microscopy, thermogravimetric analysis and a vibrating sample magnetometer. The length of the DNA linker was optimized, with a base number of 24 providing maximum efficiency for the enzymolysis. Notably, the surface of alkaline phosphatase-functionalized magnetic nanoparticles could be regenerated using a mild dehybridization process of DNA. Furthermore, the resulting single-stranded probe DNA-modified magnetic nanoparticles could be reused to immobilize the alkaline phosphatase, which suggested that the DNA-functionalized surface of magnetic nanoparticles exhibited good reversibility. The biological activity of anchored alkaline phosphatases is evaluated in an enzyme inhibition assay. The results revealed that theophylline exhibited greater inhibitory activity than L-phenylalanine. The proposed protocol demonstrates a simple, mild and economic pathway for fabricating enzyme modified nanomaterial and can be applied in the high-throughput screening of inhibitors.
Co-reporter:Jiale Li;Liqing Wu;Youxun Jin;Ping Su
Analytical and Bioanalytical Chemistry 2016 Volume 408( Issue 13) pp:3485-3493
Publication Date(Web):2016 May
DOI:10.1007/s00216-016-9424-0
Isotope dilution mass spectrometry (IDMS), an important metrological method, is widely used for absolute quantification of peptides and proteins. IDMS employs an isotope-labeled peptide or protein as an internal standard although the use of a protein provides improved accuracy. Generally, the isotope-labeled protein is obtained by stable isotope labeling by amino acids in cell culture (SILAC) technology. However, SILAC is expensive, laborious, and time-consuming. To overcome these drawbacks, a novel universal SI-traceable IDMS method for absolute quantification of proteins in a matrix is described with human transferrin (hTRF). The hTRF and a human serum sample were labeled with different tandem mass tags (TMTs). After mixing the TMT-labeled hTRF and serum sample together followed by digestion, the peptides were separated by nano-liquid chromatography and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the signature peptides, we calculated the ratios of reporter ions from the TMT-labeled peptides which, in turn, allowed determination of the mass fraction of hTRF. The recovery ranged from 97 % to 105 % with a CV of 3.9 %. The LOD and LOQ were 1.71 × 10−5 g/g and 5.69 × 10−5 g/g of hTRF in human serum, respectively, and the relative expanded uncertainty was 4.7 % with a mass fraction of 2.08 mg/g. For comparison, an enzyme-linked immunosorbent assay (ELISA) method for hTRF yielded a mass fraction of 2.03 mg/g. This method provides a starting point for establishing IDMS technology to accurately determine the mass fractions of protein biomarkers in a matrix with traceability to SI units. This technology should support the development of a metrological method useful for quantification of a wide variety of proteins.
Co-reporter:Gehui Liu;Ping Su;Lu Yang
Journal of Separation Science 2015 Volume 38( Issue 22) pp:3936-3944
Publication Date(Web):
DOI:10.1002/jssc.201500477
Ionic liquids immobilized on magnetic nanoparticles were prepared by an efficient microwave-assisted synthesis method, and the properties of the ionic liquids were tuned based on the aromatic functional modification of its anion through a simple metathesis reaction. The novel as-synthesized magnetic materials were characterized by various instrumental techniques. The magnetic nanoparticles have been utilized as adsorbents for the extraction of four sulfonylurea herbicides in tea samples, in combination with high-performance liquid chromatography analysis. Significant extraction parameters, including type and volume of desorption solvent, extraction time, amount of adsorbent, and ionic strength were investigated. Under the optimum conditions, good linearity was obtained in the concentration range of 1–150 μg/L for metsulfuron-methyl and bensulfuron-methyl, and 3–150 μg/L for sulfometuron-methyl and chlorimuron-ethyl, with correlation coefficients R2 > 0.9987. Low limits of detection were obtained ranging from 0.13 to 0.81 μg/L. The relative standard deviations were 1.8–3.9%. Comparisons of extraction efficiency with conventional solid-phase extraction equipped with a commercial C18 cartridge were performed. Results indicated that magnetic solid-phase extraction is simple, time-saving, efficient and inexpensive with the reusability of adsorbents. The proposed method has been successfully used to determine sulfonylurea herbicides from tea samples with satisfactory recoveries of 80.5–104.2%.
Co-reporter:Jun Huang, Ping Su, Bingjie Zhao and Yi Yang
Analytical Methods 2015 vol. 7(Issue 6) pp:2754-2761
Publication Date(Web):12 Feb 2015
DOI:10.1039/C5AY00013K
β-Cyclodextrin-polymer-modified Fe3O4 microspheres were synthesized using a one-pot hydrothermal method, with a β-cyclodextrin polymer as the surfactant. Investigation of the effects of the reaction time and the amount of β-cyclodextrin polymer on the formation of functionalized microspheres suggested that the main factor affecting the microsphere size was the amount of β-cyclodextrin polymer, rather than the reaction time. The obtained β-cyclodextrin-polymer-modified Fe3O4 microspheres were characterized using various methods. The results indicated that the functionalized magnetic microspheres were spherical, dispersible and had high saturation magnetization. The β-cyclodextrin-polymer-modified Fe3O4 microspheres were used for stereoselective absorption of DL-tryptophan enantiomers. The results suggested that the functionalized magnetic microspheres absorbed more L-tryptophan and had chiral discrimination ability, and can therefore be used as a stereoselective absorbent for chiral analysis.
Co-reporter:Lu Yang, Ping Su, Xiaonan Chen, Ruizhe Zhang and Yi Yang
Analytical Methods 2015 vol. 7(Issue 7) pp:3246-3252
Publication Date(Web):03 Mar 2015
DOI:10.1039/C4AY02830A
Poly(ionic liquid) (PIL) was synthesized and immobilized onto prepared silica-coated magnetic nanoparticles, which was used as a magnetic solid-phase (MSPE) sorbent and applied to the extraction of sulfonylurea herbicides (SUHs) in soil samples prior to high-performance liquid chromatography analysis. The type and volume of desorption solvent and quantity of the sorbent were optimized for the extraction process. Under the optimized conditions, the matrix matched calibration curves were linear (R2 > 0.999) for sulfonylurea herbicide concentrations in the range of 10–1500 ng mL−1. The relative standard deviation range for repeated determination of the analytes was 3.2–4.5%. The ranges for the limits of detection and quantification were 1.62–2.94 and 5.4–9.8 ng mL−1, respectively. These results demonstrate that the Fe3O4@SiO2@PIL provides efficient extraction of sulfonylurea herbicides from soil samples.
Co-reporter:Yun Shi, Jun Huang, Jiangning Wang, Ping Su, Yi Yang
Talanta 2015 Volume 143() pp:457-463
Publication Date(Web):1 October 2015
DOI:10.1016/j.talanta.2015.05.025
•Fe3O4/Pβ-CD was fabricated by a simple and general one-pot solvothermal method.•Sensitive and selective detection of glucose in human serum was achieved.•Fe3O4/Pβ-CD composite showed good reusability for H2O2 detection.•Possible catalytic mechanism of Fe3O4/Pβ-CD composite was proposed.Magnetic polymer particles with different surface functionalizations were prepared by a simple one-pot solvothermal method and characterized as peroxidase mimetics. The fluorescence enhancement obtained by attaching a β-cyclodextrin polymer (Pβ-CD) to the surfaces of Fe3O4 magnetic microspheres (Fe3O4 MMs) to generate a Fe3O4/Pβ-CD composite allowed the rapid, sensitive and selective analysis of glucose. The catalytic activity of the Fe3O4/Pβ-CD composite was evaluated with regard to the effects of catalyst particle size and species, pH value, level of catalyst, benzoic acid concentration and reaction time. Detection limits of 0.015 μM for H2O2 and 0.03 μM for glucose were determined when measuring at the 10-min mark. The presence of other saccharides, ion species, amino acids and proteins, had little effect on the results, and this technique was found to allow the analysis of glucose in human serum with high accuracy. The reusability of the Fe3O4/Pβ-CD composite was also investigated after 10 successive runs. A possible mechanism is proposed in which Fe3O4 plays a significant role in inducing fluorescence and Pβ-CD enhances the fluorescence signal, acting as both a stabilizer and a phase transfer agent.Magnetic polymer particles with different surface functionalizations were prepared by a simple one-pot solvothermal method and characterized as peroxidase mimetics. Due to the fluorescence enhancement of β-CD polymer (Pβ-CD) on the surface of the Fe3O4 magnetic microsphere (Fe3O4 MMs), the Fe3O4/Pβ-CD composite has been composed for the rapid, sensitive, and selective fluorescence detection of glucose. The catalytic activity of the Fe3O4/Pβ-CD composite was evaluated in view of catalyst particle size and species, pH value, catalyst addition, benzoic acid concentration and time. The detection limits of 0.015 μM for H2O2 and 0.03 μM for glucose were obtained for 10 min. Other saccharides, such as fructose, galactose, sucrose, and maltose, have little influence on the detection of glucose and the glucose in human serum was detected with high accuracy. The reusability of the Fe3O4/Pβ-CD composite was also investigated after 10 successive runs. Possible mechanism was proposed that the Fe3O4 played a significant role in inducing fluorescence and the Pβ-CD enhanced the fluorescence signals, acting as stabilizer and phase transfer agent.
Co-reporter:Jingwei Wu, Ping Su, Ye Yang, Jun Huang, Yingying Wang and Yi Yang
Journal of Materials Chemistry A 2014 vol. 2(Issue 7) pp:775-782
Publication Date(Web):28 Nov 2013
DOI:10.1039/C3TB21340D
Magnetic microspheres containing dendrimers and chiral selectivity show great performance for applications in pharmacology and biomedicine. In this study, a novel chiral magnetic nano-selector was prepared by immobilizing human serum albumin (HSA) on polyamidoamine dendrimer (PAMAM)-modified magnetic silica microspheres (PMSMs). The functionalized magnetic microspheres had a core–shell structure, with an average diameter of 600 nm. They exhibited strong magnetic response, with a high magnetization saturation of 46 emu g−1. UV-vis spectrophotometry and confocal laser scanning microscopy indicated that the HSA binding capacity on PMSM increased with increasing PAMAM generation. An immunoaffinity assay indicated that HSA retained a high level of activity, after immobilization on PMSM. HSA-immobilized PMSM (HSA–PMSM) was applied in the direct chiral separation of three biological racemates. The separation results show that HSA–PMSM selectively interacted with the racemates, and exhibited different activity toward each enantiomer. The results obtained by CD and CE indicated that HSA immobilized on third-generation PMSM possessed excellent chiral separation capability and biocompatibility. The chiral recognition capability of HSA–PMSM increased with increasing PAMAM generation. The chiral magnetic nano-selector is effective for the enantiomeric separation of chiral drugs, and has potential application in pharmacological and biomedical research.
Co-reporter:Jingwei Wu, Ping Su, Danhua Guo, Jun Huang and Yi Yang
New Journal of Chemistry 2014 vol. 38(Issue 8) pp:3630-3636
Publication Date(Web):16 Apr 2014
DOI:10.1039/C4NJ00030G
Chirally selective functionalized magnetic microspheres show great potential in enantiomeric separations. In this study, a novel class of chiral magnetic selectors was developed by immobilization of vinylimidazolium-β-cyclodextrin chloride (VIMCD-Cl) on 3-methacryloxypropyltrimethoxysilane-modified iron oxide magnetic microspheres through a radical polymerization. The prepared chiral materials have regular three-dimensional core–shell architectures with an average particle size of about 580 nm and a high magnetization saturation of about 51 emu g−1. Fourier transform-infrared spectra (FT-IR), thermogravimetric analysis (TGA) and elemental analysis confirmed that VIMCD-Cl was successfully polymerized on the surface of the magnetic microspheres. The prepared functional magnetic materials were then applied in the selective chiral absorption of three dansyl amino acids using microbatch technology. The results indicated that VIMCD-Cl immobilized magnetic microspheres (VIMCD-MNPs) possessed good enantioselectivities toward the three dansyl amino acids, and showed stronger interactions with the L-enantiomers during the chiral adsorption process. Furthermore, these functionalized chiral magnetic materials possess an excellent recyclability and can be used as effective chiral magnetic selectors for chiral separations.
Co-reporter:Wang Yang, Wu Liqing , Duan Fei, Yang Bin , Yang Yi and Wang Jing
Journal of Agricultural and Food Chemistry 2014 Volume 62(Issue 14) pp:3073-3080
Publication Date(Web):March 14, 2014
DOI:10.1021/jf4054337
β-Lactoglobulin (β-LG) is one of the major allergenic proteins in milk. There is an urgent demand for an accurate and traceable method to develop β-LG certified reference material (CRM). In this work, β-LG was enzymatically digested and a specific peptide was chosen for quantitation by isotope-dilution mass spectrometry (IDMS). With amino acid CRMs as standards, the results could be traced to SI unit. By the proposed method, the recovery ranged from 86.0% to 118.3% with CVs <9.0%. The LOD and LOQ were 4.8 × 10–5 g/g and 1.6 × 10–4 g/g of β-LG in milk powder, respectively. Ten samples from domestic market were analyzed with CVs <5.6%, and the relative expanded uncertainties ranged from 4.2% to 5.9% (k = 2). With the CRMs, it is expected that the comparability of β-LG quantitation results will be improved among different laboratories.
Co-reporter:Ruizhe Zhang;Ping Su
Journal of Separation Science 2014 Volume 37( Issue 22) pp:3339-3346
Publication Date(Web):
DOI:10.1002/jssc.201400767
Here in, magnetic nanoparticles combined with graphene oxide adsorbent were fabricated via a microwave-assisted synthesis method, and used in the solid-phase extraction of three phenolic compounds (phenol, 4-nitrophenol, and m-methylphenol) in environmental water samples. Various instrumental methods were employed to characterize the magnetic nanoparticles modified with graphene oxide. The influence of experimental parameters, such as desorption conditions, amount of adsorbent, extraction time, and pH, on the extraction efficiency was investigated. Owing to the high surface area and excellent adsorption capacity of the prepared material, satisfactory extraction was achieved. Under optimum conditions, a linear response was observed in the concentration range of 1.000–100.0 μg/L for phenol, 0.996–99.6 μg/L for 4-nitrophenol, and 0.975–97.5 μg/L for m-methylphenol, with correlation coefficients in the range of 0.9995–0.9997. The limit of detection (signal-to-noise ratio of 3) of the method varied between 0.5 and 0.8 μg/L. The relative standard deviations were <5.2%. The recovery percentages of the method were in the range of 89.1–104.3%. The results indicate that the graphene oxide-modified magnetic nanoparticles possess high adsorptive abilities toward phenolic compounds in environmental water samples.
Co-reporter:Ruizhe Zhang;Ping Su;Lu Yang
Journal of Separation Science 2014 Volume 37( Issue 12) pp:1503-1510
Publication Date(Web):
DOI:10.1002/jssc.201400125
Novel poly(ionic liquids) were synthesized and immobilized on prepared magnetic nanoparticles, which were used to extract pesticides from fruit and vegetable samples by dispersive solid-phase extraction prior to high-performance liquid chromatography analysis. Compared with monomeric ionic liquids, poly(ionic liquids) have a larger effective contact area and higher viscosity, so they can achieve higher extraction efficiency and be used repeatedly without a decrease in analyte recovery. The immobilized poly(ionic liquids) were rapidly separated from the sample matrix, providing a simple approach for sample pretreatment. The nature and volume of the desorption solvent and amount of poly(ionic liquid)-modified magnetic material were optimized for the extraction process. Under optimum conditions, calibration curves were linear (R2 > 0.9988) for pesticide concentrations in the range of 0.100–10.000 μg/L. The relative standard deviations for repeated determinations of the four analytes were 2.29–3.31%. The limits of detection and quantification were 0.29–0.88 and 0.97–2.93 μg/L, respectively. Our results demonstrate that the developed poly(ionic liquid)-modified material is an effective absorbent to extract pesticides from fruit and vegetable samples.
Co-reporter:Jun Huang, Ping Su, Jingwei Wu and Yi Yang
RSC Advances 2014 vol. 4(Issue 102) pp:58514-58521
Publication Date(Web):27 Oct 2014
DOI:10.1039/C4RA12133C
Multifunctional magnetic microspheres have enormous potential in diverse fields. In this work, surface chiral-modified magnetic microspheres as chiral selectors, are prepared by polymerizing maleic anhydride-β-cyclodextrin (MAH-β-CD) for the enantioselective absorption of four enantiomers. The successful grafting of MAH-β-CD onto the surface of magnetic microspheres is confirmed by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and thermogravimetric analysis. The prepared functional microspheres have a three-ply structure with an average particle size of 550 nm and a high saturation magnetization of 60 emu g−1. The amount of MAH-β-CD modified on the P(MBAAm)@Fe3O4 was about 149.1 mg g−1. The analysis results of specific rotation and capillary electrophoresis reveal that the MAH-β-CD-modified Fe3O4 microspheres show stronger complexation of (−)-enantiomers than (+)-enantiomers. In addition, the MAH-β-CD-modified Fe3O4 microspheres have stronger enantioselective absorption for double-ring chiral compounds. These chiral-functionalized magnetic microspheres are therefore expected to be an efficient and economical chiral separation method for use in further research.
Co-reporter:Ping Su, Ruoyu Wang, Yang Yu and Yi Yang
Analytical Methods 2014 vol. 6(Issue 3) pp:704-709
Publication Date(Web):16 Dec 2013
DOI:10.1039/C3AY41771A
In this work, N-methylimidazolium ionic liquid-modified silica was synthesized using microwave irradiation. The modified silica was characterized by Fourier transform infrared analysis, elemental analysis, energy spectrum analysis, thermogravimetric analysis, and scanning electron microscopy. The synthesized material was used as a sorbent for the solid-phase extraction of four phenolic compounds from water. The extraction conditions including the type and consumption of eluent, the amount of sorbent, the pH, and the flow rate of the water samples were optimized. Satisfactory extraction recoveries were achieved using 200 mg of functionalized silica, 5 mL of methanol as eluent, a sample pH of 5, and a flow rate of 3 mL min−1. The analytes were separated and detected using high-performance liquid chromatography. Analyte recoveries were found to be between 88% and 102% under optimized conditions with a relative standard deviation of ≤7%. The method was successfully applied for the extraction of phenolic compounds from water samples, indicating that ionic liquid-functionalized silica cartridges performed better than bare silica cartridges in the extraction of these analytes.
Co-reporter:Yun Shi, Ping Su, Yingying Wang, Yi Yang
Talanta 2014 Volume 130() pp:259-264
Publication Date(Web):1 December 2014
DOI:10.1016/j.talanta.2014.06.053
•A general H2O2-involved fluorescence system using Fe3O4 as peroxidase mimetics.•Glucose and p-nitrophenol were regarded as models.•Glucose detection had performances of good sensitivity and selectivity.•p-Nitrophenol detection had advantage in simple procedure with high sensitivity.•The system was used for sensing the glucose and p-nitrophenol in real samples.Enzyme mimetics have recently attracted considerable interest because of their high stability and low cost. We developed a general H2O2-involved fluorescence system using Fe3O4 magnetic microspheres as peroxidase mimetics and benzoic acid (BA) as indicator. Glucose and p-nitrophenol were used as models to determine the characteristics and effectiveness of the system. Glucose oxidase hydrolyzes glucose, in the presence of oxygen, to H2O2 followed by the activation of Fe3O4 MMs, resulting in the catalyzed oxidation of benzoic acid. Glucose can be determined by the quantitative fluorescence production. p-Nitrophenol is determined as model compounds which competes with benzoic acid for H2O2 resulting in the decreased catalytic oxidation of benzoic acid with the Fe3O4 MMs. The detection limit of the Fe3O4/H2O2/BA system is 0.008 μM for H2O2, 0.025 μM for glucose and 0.05 μM for p-nitrophenol. Furthermore, the system had high sensitivity, good selectivity and was capable of sensing glucose in human serum and p-nitrophenol in water samples. The proposed system has great potential in the chemical/biological sensing of a variety of analytes associated with reactions that produce or consume H2O2.
Co-reporter:Siming Wang, Ping Su, Jun Huang, Jingwei Wu and Yi Yang
Journal of Materials Chemistry A 2013 vol. 1(Issue 12) pp:1749-1754
Publication Date(Web):29 Jan 2013
DOI:10.1039/C3TB00562C
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.
Co-reporter:Yingying Wang, Ping Su, Siming Wang, Jingwei Wu, Jun Huang and Yi Yang
Journal of Materials Chemistry A 2013 vol. 1(Issue 38) pp:5028-5035
Publication Date(Web):30 Jul 2013
DOI:10.1039/C3TB20889C
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.
Co-reporter:Ruoyu Wang, Ping Su and Yi Yang
Analytical Methods 2013 vol. 5(Issue 4) pp:1033-1039
Publication Date(Web):13 Dec 2012
DOI:10.1039/C2AY26313K
In this work, a simultaneous ionic liquid-based microwave-assisted dispersive liquid–liquid microextraction (IL-based MADLLME) method was developed for the determination of plasticizers in water using high-performance liquid chromatography (HPLC) with ultraviolet detection. Several parameters affecting the extraction efficiency, such as type and volume of ionic liquid and disperser solvent, microwave time and temperature, were investigated by single factor experiments. Then, the microwave irradiation time and temperature, and the volumes of extraction and disperser solvents were optimized using response surface methodology (RSM). The optimal values were determined to be within an extraction volume of 110 μL, a dispersive solvent volume of 0.26 mL, and a microwave irradiation temperature and time of 60 °C and 2 min, respectively. The calibration curves were linear in the range of 23–230 μg L−1 for dimethyl phthalate (DMP), 23.8–238 μg L−1 for diethyl phthalate (DEP), 16.5–165 μg L−1 for benzylbutyl phthalate (BBP), 19.5–195 μg L−1 for dibutyl phthalate (DBP), and 18.6–186 μg L−1 for diethylhexyl phthalate (DEHP). Under the optimum conditions, the limits of detection (LODs) of these plasticizers were in the range of 0.71–1.94 μg L−1. Validation of the methodology was carried out by the method of standard addition at two concentration levels for three water samples. The recoveries of the analytes were in the range of 85.2–103.3%, with the relative standard deviations (RSDs, n = 6) ≤ 5.9%. The results show that IL-based MADLLME is a suitable method for the determination of these five plasticizers in water.
Co-reporter:Yao Zhang, Ruoyu Wang, Ping Su and Yi Yang
Analytical Methods 2013 vol. 5(Issue 19) pp:5074-5078
Publication Date(Web):16 Jul 2013
DOI:10.1039/C3AY40803E
In this article, a method of ionic liquid-based solvent bar microextraction combined with high performance liquid chromatography was developed to measure organophosphorus pesticides (OPPs) in water samples. The solvent bar microextraction was based on applying hydrophobic ionic liquid to the lumen and wall pores of a hollow fiber. The main factors influencing the extraction including types of ionic liquid, stirring speed, extraction time, extraction temperature, and salt effect were investigated. The proposed method was successfully applied in the analysis of real environmental water samples under the optimal extraction conditions and good spiked recoveries of 86.71–103.7% were obtained. The linear range was 1–200 μg L−1 and the detection limits were 0.015–0.026 μg L−1 for the four OPPs analyzed. These results demonstrated that solvent bar microextraction was a simple and accurate technique with a high enrichment factor.
Co-reporter:Qiu Zhong, Ping Su, Yao Zhang, Ruoyu Wang and Yi Yang
Analytical Methods 2012 vol. 4(Issue 4) pp:983-988
Publication Date(Web):16 Mar 2012
DOI:10.1039/C2AY05769G
The analysis of triazine and phenylurea herbicides in soil by ionic liquid based microwave-assisted extraction (IL-MAE) coupled with high-performance liquid chromatography was studied. The operational IL-MAE parameters were optimized, including types and concentration of ionic liquid, microwave power, irradiation temperature, extraction time and extractant volume. The results obtained under the optimized conditions were compared with methanol-MAE and IL-heating extraction, and it was determined that the IL-MAE method was rapid (4 min compared with 2 h for the heating extraction), efficient (recoveries were 1.6–12.2% higher than the other methods) and environmentally friendly (no volatile organic solvents were used). The proposed analytical method was evaluated by considering the average spike recoveries (84.0–101%) and relative standard deviations (2.8–6.0%), all of which indicate that the IL-MAE method is accurate, reproducible, and could be applied to real sample analyses.
Co-reporter:Siming Wang, Ping Su, Yi Yang
Analytical Biochemistry 2012 Volume 427(Issue 2) pp:139-143
Publication Date(Web):15 August 2012
DOI:10.1016/j.ab.2012.05.014
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 20 mM 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.
Co-reporter:Qiu Zhong;Ping Su;Yao Zhang;Ruoyu Wang
Microchimica Acta 2012 Volume 178( Issue 3-4) pp:341-347
Publication Date(Web):2012 September
DOI:10.1007/s00604-012-0847-9
We report on the determination of the triazine herbicides ametryne, prometryne, terbuthylazine and terbutryn in water samples. The herbicides are extracted by in-situ ionic liquid-based microwave-assisted dispersive liquid-liquid microextraction and then determined by high-performance liquid chromatography. This is a new method for extraction that has the advantages of requiring less volume of ionic liquid (IL) than other methods and at the same time is quite fast. The type and volume of IL, the type and volume of disperser, irradiation temperature, extraction time and salt concentration were optimized. Figures of merit include linear regression coefficients between 0.9992 and 0.9995, acceptable recoveries (88.4–114 %), relative standard deviations of 1.6–6.2 %, and limits of detection between 0.52 and 1.3 μg L−1.
Co-reporter:Hui Gong, Ting Huang, Yi Yang, Haifeng Wang
Talanta 2012 Volume 101() pp:96-103
Publication Date(Web):15 November 2012
DOI:10.1016/j.talanta.2012.09.012
Folic acid is one of the most important nutrient substances for human beings, especially for the pregnant women and infants. Therefore the purity determination of folic acid is particularly important. The mass balance method was employed to determine the purity of folic acid, by using the measures of high performance liquid chromatography (HPLC), Karl Fischer titration and other conventional approach. The moisture quantification of folic acid was a major problem since it is a thermally unstable substance and it is apt to contain crystal water. Therefore, a novel improved Karl Fischer method was established for accurate determination of the water content in folic acid, whose repeatability (RSD=2.9%) was significantly better than that of the original direct injection method (RSD=12%). The purity of folic acid certified reference material (CRM) determined by mass balance method was 90.9% with an expanded uncertainty of 0.35%, and the content of water (the major impurity) was 8.5%, with an expanded uncertainty of 0.32%.Highlights► This research established an improved Karl Fischer titration method for folic acid. ► RSD (2.9%) of the new KF method was significantly better than the old one (12%). ► New KF method could protect the sample from the moisture of air effectively. ► New KF method is suitable for thermally unstable or water-soluble substances. ► A mass balance method was built to determine the purity of folic acid.
Co-reporter:Siming Wang, Ping Su, Hongjun E, Yi Yang
Analytical Biochemistry 2010 Volume 405(Issue 2) pp:230-235
Publication Date(Web):15 October 2010
DOI:10.1016/j.ab.2010.06.014
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 β-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.
Co-reporter:Chunting Zhang, Ping Su, Muhammad Umar Farooq, Yi Yang, Xiang Gao, E. Hongjun
Reactive and Functional Polymers 2010 70(2) pp: 129-133
Publication Date(Web):February 2010
DOI:10.1016/j.reactfunctpolym.2009.11.005
Co-reporter:Fan Zhang;Ping Su;Zhenku Guo
Phytochemical Analysis 2009 Volume 20( Issue 1) pp:33-37
Publication Date(Web):
DOI:10.1002/pca.1088
Abstract
Introduction
Euonymus alatus (Thunb.) has been used as one of traditional Chinese medicines for several thousand years. Conventional methods for the extraction of rutin and quercetin from E. alatus, including solvent extraction, Soxhlet extraction and heat reflux extraction are characterised by long extraction times and consumption of large amounts of solvents.
Objective
To develop a simple and rapid method for the extraction of rutin and quercetin from the stalks of Euonymus alatus (Thunb.) Sieb using microwave-assisted extraction (MAE) technique.
Methodology
MAE experiments were performed with a multimode microwave extraction system. The experimental variables that affect the MAE process, such as the concentration of ethanol solution, extractant volume, microwave power and extraction time were optimised. Yields were determined by HPLC. The results were compared with that obtained by classical Soxhlet and ultrasonic-assisted extraction (UAE).
Results
From the optimised conditions for MAE of rutin and quercetin it can be concluded that the solvent is 50% ethanol (v/v) solution, the extractant volume is 40 mL, microwave power is 170 W and irradiation time is 6 min. Compared with Soxhlet extraction and ultrasonic extraction, microwave extraction is a rapid method with a higher yield and lower solvent consumption.
Conclusion
The results showed that MAE can be used as an efficient and rapid method for the extraction of the active components from plants. Copyright © 2008 John Wiley & Sons, Ltd.
Co-reporter:E. Hongjun;Ping Su;Wendi Zhang
Journal of Analytical Chemistry 2009 Volume 64( Issue 4) pp:393-397
Publication Date(Web):2009 April
DOI:10.1134/S1061934809040133
The capillary column modified by sulfated β-cyclodextrin intercalated in layered double hydroxides was prepared for open tubular capillary electrochromatography and was applied to separate the racemoid of 1-phenyl-1,2-ethanediol under the optimized conditions.
Co-reporter:Yi Yang, Fan Zhang
Ultrasonics Sonochemistry 2008 Volume 15(Issue 4) pp:308-313
Publication Date(Web):April 2008
DOI:10.1016/j.ultsonch.2007.05.001
An ultrasonic method for the extractions of rutin and quercetin from Euonymus alatus (Thunb.) Sieb was investigated. The influence of four extraction variables on extraction yield of rutin and quercetin was discussed. The optimum extraction conditions found were: 70% aqueous ethanol; solvent: sample ratio 40:1 (v/w); extraction time 3 × 30 min. The recovery of rutin and quercetin and the reproducibility of the extraction method were determined. The optimized ultrasonic extraction conditions were applied to extract rutin and quercetin from dried stalks of E. alatus (Thunb.) Sieb. The application of sonication method was shown to be highly efficient in the extraction of rutin and quercetin from E. alatus (Thunb.) Sieb, compared with classical methods. The scanning electron microscopy (SEM) micrographs provided evidence of more rapid opening of plant cells treated by UAE in contrast to maceration.
Co-reporter:Siming Wang, Ping Su, Fangyu Ding, Yi Yang
Journal of Molecular Catalysis B: Enzymatic (May 2013) Volume 89() pp:35-40
Publication Date(Web):1 May 2013
DOI:10.1016/j.molcatb.2012.12.011
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, and is suitable to carry biomolecules due to its affinity and biocompatibility. In this study, PAMAM was grafted onto the surface of silica by microwave irradiation. A novel media was developed through immobilizing cellulase onto the prepared PAMAM-grafted silica by adsorption and crosslinking methods and applied in hydrolysis of carboxymethyl cellulose. The results demonstrate that the enzyme binding capacity and enzymolysis efficiency increased with generations of PAMAM. The properties of the immobilized cellulase-PAMAM-grafted silica were investigated, which possessed high enzymatic activity and exhibited better stability with respect to pH, temperature compared with free enzyme. The optimal immobilization conditions for adsorption and crosslinking method were respectively obtained at 5 and 4 mg ml−1 cellulase for 2 h of immobilization. A high enzymolysis efficiency was achieved by employing pH 4.8 and 5.8 substrate solution at 60 °C for adsorbed and crosslinked cellulase, respectively. After repeated three run cycles, the retained activities were found to be 75% and 82%. The results indicate that the PAMAM has a good performance as a carrier, and can be potentially adapted to support other biomacromolecules.Graphical abstractDownload full-size imageHighlights► A new type of immobilized cellulase was developed based on PAMAM-grafted silica. ► The enzyme binding capacity increased with rising PAMAM generations. ► The properties of adsorption and crosslinking immobilization methods were compared. ► The prepared media possessed high enzymatic activity and exhibited better stability. ► The protocol can be potentially adapted to support other biomacromolecules.
Co-reporter:
Analytical Methods (2009-Present) 2014 - vol. 6(Issue 3) pp:NaN709-709
Publication Date(Web):2013/12/16
DOI:10.1039/C3AY41771A
In this work, N-methylimidazolium ionic liquid-modified silica was synthesized using microwave irradiation. The modified silica was characterized by Fourier transform infrared analysis, elemental analysis, energy spectrum analysis, thermogravimetric analysis, and scanning electron microscopy. The synthesized material was used as a sorbent for the solid-phase extraction of four phenolic compounds from water. The extraction conditions including the type and consumption of eluent, the amount of sorbent, the pH, and the flow rate of the water samples were optimized. Satisfactory extraction recoveries were achieved using 200 mg of functionalized silica, 5 mL of methanol as eluent, a sample pH of 5, and a flow rate of 3 mL min−1. The analytes were separated and detected using high-performance liquid chromatography. Analyte recoveries were found to be between 88% and 102% under optimized conditions with a relative standard deviation of ≤7%. The method was successfully applied for the extraction of phenolic compounds from water samples, indicating that ionic liquid-functionalized silica cartridges performed better than bare silica cartridges in the extraction of these analytes.
Co-reporter:
Analytical Methods (2009-Present) 2013 - vol. 5(Issue 19) pp:
Publication Date(Web):
DOI:10.1039/C3AY40803E
In this article, a method of ionic liquid-based solvent bar microextraction combined with high performance liquid chromatography was developed to measure organophosphorus pesticides (OPPs) in water samples. The solvent bar microextraction was based on applying hydrophobic ionic liquid to the lumen and wall pores of a hollow fiber. The main factors influencing the extraction including types of ionic liquid, stirring speed, extraction time, extraction temperature, and salt effect were investigated. The proposed method was successfully applied in the analysis of real environmental water samples under the optimal extraction conditions and good spiked recoveries of 86.71–103.7% were obtained. The linear range was 1–200 μg L−1 and the detection limits were 0.015–0.026 μg L−1 for the four OPPs analyzed. These results demonstrated that solvent bar microextraction was a simple and accurate technique with a high enrichment factor.
Co-reporter:
Analytical Methods (2009-Present) 2013 - vol. 5(Issue 4) pp:NaN1039-1039
Publication Date(Web):2012/12/13
DOI:10.1039/C2AY26313K
In this work, a simultaneous ionic liquid-based microwave-assisted dispersive liquid–liquid microextraction (IL-based MADLLME) method was developed for the determination of plasticizers in water using high-performance liquid chromatography (HPLC) with ultraviolet detection. Several parameters affecting the extraction efficiency, such as type and volume of ionic liquid and disperser solvent, microwave time and temperature, were investigated by single factor experiments. Then, the microwave irradiation time and temperature, and the volumes of extraction and disperser solvents were optimized using response surface methodology (RSM). The optimal values were determined to be within an extraction volume of 110 μL, a dispersive solvent volume of 0.26 mL, and a microwave irradiation temperature and time of 60 °C and 2 min, respectively. The calibration curves were linear in the range of 23–230 μg L−1 for dimethyl phthalate (DMP), 23.8–238 μg L−1 for diethyl phthalate (DEP), 16.5–165 μg L−1 for benzylbutyl phthalate (BBP), 19.5–195 μg L−1 for dibutyl phthalate (DBP), and 18.6–186 μg L−1 for diethylhexyl phthalate (DEHP). Under the optimum conditions, the limits of detection (LODs) of these plasticizers were in the range of 0.71–1.94 μg L−1. Validation of the methodology was carried out by the method of standard addition at two concentration levels for three water samples. The recoveries of the analytes were in the range of 85.2–103.3%, with the relative standard deviations (RSDs, n = 6) ≤ 5.9%. The results show that IL-based MADLLME is a suitable method for the determination of these five plasticizers in water.
Co-reporter:
Analytical Methods (2009-Present) 2012 - vol. 4(Issue 4) pp:
Publication Date(Web):
DOI:10.1039/C2AY05769G
The analysis of triazine and phenylurea herbicides in soil by ionic liquid based microwave-assisted extraction (IL-MAE) coupled with high-performance liquid chromatography was studied. The operational IL-MAE parameters were optimized, including types and concentration of ionic liquid, microwave power, irradiation temperature, extraction time and extractant volume. The results obtained under the optimized conditions were compared with methanol-MAE and IL-heating extraction, and it was determined that the IL-MAE method was rapid (4 min compared with 2 h for the heating extraction), efficient (recoveries were 1.6–12.2% higher than the other methods) and environmentally friendly (no volatile organic solvents were used). The proposed analytical method was evaluated by considering the average spike recoveries (84.0–101%) and relative standard deviations (2.8–6.0%), all of which indicate that the IL-MAE method is accurate, reproducible, and could be applied to real sample analyses.
Co-reporter:Jingwei Wu, Ping Su, Ye Yang, Jun Huang, Yingying Wang and Yi Yang
Journal of Materials Chemistry A 2014 - vol. 2(Issue 7) pp:NaN782-782
Publication Date(Web):2013/11/28
DOI:10.1039/C3TB21340D
Magnetic microspheres containing dendrimers and chiral selectivity show great performance for applications in pharmacology and biomedicine. In this study, a novel chiral magnetic nano-selector was prepared by immobilizing human serum albumin (HSA) on polyamidoamine dendrimer (PAMAM)-modified magnetic silica microspheres (PMSMs). The functionalized magnetic microspheres had a core–shell structure, with an average diameter of 600 nm. They exhibited strong magnetic response, with a high magnetization saturation of 46 emu g−1. UV-vis spectrophotometry and confocal laser scanning microscopy indicated that the HSA binding capacity on PMSM increased with increasing PAMAM generation. An immunoaffinity assay indicated that HSA retained a high level of activity, after immobilization on PMSM. HSA-immobilized PMSM (HSA–PMSM) was applied in the direct chiral separation of three biological racemates. The separation results show that HSA–PMSM selectively interacted with the racemates, and exhibited different activity toward each enantiomer. The results obtained by CD and CE indicated that HSA immobilized on third-generation PMSM possessed excellent chiral separation capability and biocompatibility. The chiral recognition capability of HSA–PMSM increased with increasing PAMAM generation. The chiral magnetic nano-selector is effective for the enantiomeric separation of chiral drugs, and has potential application in pharmacological and biomedical research.
Co-reporter:Yingying Wang, Ping Su, Siming Wang, Jingwei Wu, Jun Huang and Yi Yang
Journal of Materials Chemistry A 2013 - vol. 1(Issue 38) pp:NaN5035-5035
Publication Date(Web):2013/07/30
DOI:10.1039/C3TB20889C
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.
Co-reporter:Siming Wang, Ping Su, Jun Huang, Jingwei Wu and Yi Yang
Journal of Materials Chemistry A 2013 - vol. 1(Issue 12) pp:NaN1754-1754
Publication Date(Web):2013/01/29
DOI:10.1039/C3TB00562C
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.
Co-reporter:Jiayi Song, Ping Su, Ye Yang, Ting Wang and Yi Yang
Journal of Materials Chemistry A 2016 - vol. 4(Issue 35) pp:NaN5882-5882
Publication Date(Web):2016/08/03
DOI:10.1039/C6TB01857B
The development of new strategies for stabilizing or improving the activities of enzymes has attracted considerable interest because of the wide range of potential applications and high cost of the native enzymes. In this study, a novel trypsin immobilization procedure in which the enzyme was immobilized using polyamidoamine (PAMAM) dendrimer modified magnetic nanoparticles as carriers through DNA-directed immobilization was developed for the first time. The optimal DNA base pairs and the optimal generation of PAMAM on the enzymatic activity were 24 bases and G3.0PAMAM, respectively. The trypsin binding capacity of the immobilized trypsin was 74.6 mg g−1, and the Km and Vmax values were 1.23 mM and 468.35 μmol min−1 mg protein−1, respectively. The immobilized trypsin reactor exhibited excellent reusability and stability properties without significant loss in enzymatic activity. Notably, the high level of enzymatic activity remained at more than 63% after 82 cycles, with only a slight decrease (above 88%) after 14 weeks of continuous use at one-week intervals. The high reversibility and reproducibility of this trypsin dynamic immobilization strategy were also investigated. The significantly improved digestion performance of the immobilized trypsin composites was further demonstrated by digesting cytochrome C, myoglobin and glycated hemoglobin, with sequence coverages of 78%, 99% and 88%, respectively, which were higher than those obtained for the free enzyme. This system therefore shows great potential in high throughput enzymatic assays and proteome analysis.
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