Biofunctionalization of silica microspheres for protein separation

Mercapto-silica (SiO₂–SH) microspheres were prepared via direct hydrolysis of 3-mercaptopropyltrimethoxysilane (MPS) in a basic aqueous solution. The content of surface thiol group (SH) of SiO₂–SH microspheres was measured by Ellman's reagent method and X-ray photoelectron spectroscopy (XPS) and the content of surface thiol group of SiO₂–SH microspheres is strongly dependent on the reaction conditions. The thermal stability of SiO₂–SH microspheres was evaluated by thermogravimetric (TG) analysis, which tended to reduce with the increase of content of surface thiol groups. SiO₂–SH microspheres can be easily modified with reduced glutathione (GSH) to generate SiO₂–GSH microspheres for the affinity separation of Glutathione S-transferase (GST). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to examine the validity of the separation procedure. The results showed that SiO₂–GSH microspheres were efficient in GST affinity separation from mixed proteins.     

The preparation and properties of monodisperse core-shell silica magnetic microspheres

The monodisperse core-shell silica magnetic microspheres (MMS) were synthesized by sol–gel method gelling in the emulsion. Optical microscope (OM), field emission scanning electron microscope (FESEM), nitrogen adsorption and desorption Brunauer Emmett Teller Procedure (BET) isotherms and Barrett-Joyner-Halenda (BJH) pore size distribution measurements, X-ray diffraction (XRD), energy dispersive spectrometer (EDS) and vibrating sample magnetometer (VSM) were used to characterize the appearance, size distribution, phase, specific surface area, chemical composition and magnetic property of silica MMS. The results showed that silica MMS prepared through sol–gel method with acid-alkali two-step catalyze and gelling in emulsion exhibited the superior core-shell structure and size distribution of the microspheres concentrated in about 20 μm. The main phase of microspheres was amorphous silica and spinel ferroferric oxide. Meanwhile, the microspheres remained the superparamagnetic behavior and could be used as biomaterials. This work is supported by both National Science Foundation 50572072 and Science & Technology Commission of Shanghai Municipality (STCSM) 0452nm059.     

Acceleration of microwave-assisted enzymatic digestion reactions by magnetite beads

In this study, we demonstrated that microwave-assisted enzymatic digestion could be greatly accelerated by multifunctional magnetite beads. The acceleration of microwave-assisted enzymatic digestion by the presence of the magnetite beads was attributable to several features of the beads. Their capacity to absorb microwave radiation leads to rapid heating of the beads. Furthermore, their negatively charged functionalities cause adsorption of proteins with opposite charges onto their surfaces by electrostatic interactions, leading to a concentration on the surfaces of the beads of proteins present in trace amounts in the solution. The adsorbed proteins are denatured and hence rendered vulnerable to enzymatic digestion and are digested on the beads. For microwave heating, 30 s was sufficient for carrying out the tryptic digestion of cytochrome c, in the presence of magnetite beads, while 1 min was adequate for tryptic digestion of myoglobin. The digestion products were characterized by MALDI-MS. This rapid enzymatic digestion allowed the entire time for identification of proteins to be greatly reduced. Furthermore, specific proteins present in trace quantities were enriched from the sample on the magnetite beads and could be rapidly isolated from the sample by employing an external magnetic field. These multiple roles of magnetite beads, as the absorber for microwave irradiation, the concentrating probe, and the agent for unfolding proteins, contributed to their capability of accelerating microwave-assisted enzymatic digestion. We also demonstrated that trypsin immobilized magnetite beads were suitable for use in microwave-assisted enzymatic digestion.     

Concentration of Bacillus spores by using silica magnetic particles

Silica particles are mainly used for the concentration of nucleic acid for diagnostic purposes. This is usually done under acidic or chaotropic conditions that will demolish most of the living organisms and prevent the application of other diagnostic tests. Here we describe the development of a method for the capturing and concentration of Bacillus spores using silica magnetic particles to enable fast and sensitive detection. We have shown that capturing various Bacilli spores via silica magnetic particles is limited, with large differences between spore batches (42 +/- 25%). The hydrophobic exosporium layer of spore limits the capture by the hydrophilic silica beads. Partial removal of Bacillus exosporium increases capture efficiency. To increase capturing efficiency without harming the spores' viability, a cationic lipid, didecyldimethylammonium bromide (DDAB), was used as a coat for the negatively charged silica particles. DDAB treatment increased capture efficiency from 42% to more than 90%. Using this method, we were able to capture as few as 100 Bacillus anthracis spores/mL with 90% efficacy. Release of captured spores was achieved by the addition of albumin. The capture and release processes were verified by plating and by flow cytometry using light scatter analysis. The method is simple, efficient, easy to operate, and fast.     

Quantitative mass spectrometry combined with separation and enrichment of phosphopeptides by titania coated magnetic mesoporous silica microspheres for screening of protein kinase inhibitors

We describe herein the development of a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) approach for screening of protein kinase inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed products of nonphosphorylated substrates, is a great challenge due to the ion suppression effect of highly abundant nonphosphorylated peptides in enzymatic reaction mixtures. To address this issue, a novel type of titania coated magnetic hollow mesoporous silica spheres (TiO(2)/MHMSS) material was fabricated for capturing phosphopeptides from the enzymatic reaction mixtures prior to MS analysis. Under optimized conditions, even in the presence of 1000-fold of a substrate peptide of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated substrates at the femtomole level can be detected with high accuracy and reproducibility. With a synthetic nonisotopic labeled phosphopeptide, of which the sequence is similar to that of the phosphorylated substrate, as the internal standard, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides in peptide mixtures over the range of 0.1 to 4 with r(2) being 0.99. The IC(50) values of three EGFR inhibitors synthesized in our laboratory were then determined, and the results are consistent with those determined by an enzyme-linked immunosorbent assay (ELISA). The developed method is sensitive, cost/time-effective, and operationally simple and does not require isotope/radioative-labeling, providing an ideal alterative for screening of PKIs as therapeutic agents.     

SiO2微球的制备与应用

综述了致密和多孔SiO2微球的制备方法．重点描述了近年来SiO2微球的一些新应用。     

Bifunctional magnetic silica nanoparticles for highly efficient human stem cell labeling

A superparamagnetic iron oxide (SPIO) nanoparticle is emerging as an ideal probe for noninvasive cell tracking. However, its low intracellular labeling efficiency has limited the potential usage and has evoked great interest in developing new labeling strategies. We have developed fluorescein isothiocyanate (FITC)-incorporated silica-coated core-shell SPIO nanoparticles, SPIO@SiO2(FITC), with diameters of 50 nm, as a bifunctionally magnetic vector that can efficiently label human mesenchymal stem cells (hMSCs), via clathrin- and actin-dependent endocytosis with subsequent intracellular localization in late endosomes/lysosomes. The uptake process displays a time- and dose-dependent behavior. In our system, SPIO@SiO2(FITC) nanoparticles induce sufficient cell MRI contrast at an incubation dosage as low as 0.5 microg of iron/mL of culture medium with 1.2x105 hMSCs, and the in vitro detection threshold of cell number is about 1x104 cells. Furthermore, 1.2x105 labeled cells can also be MRI-detected in a subcutaneous model in vivo. Labeled hMSCs are unaffected in their viability, proliferation, and differentiation capacities into adipocytes and osteocytes which can still be readily MRI detected. This is the first report that hMSCs can be efficiently labeled with MRI contrast nanoparticles and can be monitored in vitro and in vivo with a clinical 1.5-T MRI imager under low incubation concentration of iron oxide, short incubation time, and low detection cell numbers at the same time.     

采用模块式消化、全自动凯氏定氮仪测定食品中蛋白质

采用模块式消化、全自动凯氏定氮仪测定食品中的蛋白质含量,对于食品中蛋白质含量3%-80%范围均能获得良好的结果。与经典凯氏法比较,测定结果令人满意,且该方法快速、准确。     

ATRP法制备磁性高分子微球及表征

通过化学共沉淀法制备了Fe3O4纳米磁性粒子作为内核,在其表面枝节一层引发剂,引发苯乙烯和丙烯酸的原子转移自由基聚合(ATRP)反应。通过SEM,TEM,IR,化学滴定法,表征,制备得到了粒径分布在40～50纳米,磁饱和强度为47.105emu/g,表面羧基含量在0.742mmol/g的磁性高分子复合微球。     

Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion

Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.     

Cavity QED with diamond nanocrystals and silica microspheres

Normal mode splitting is observed in a cavity QED system in which nitrogen vacancy centers in diamond nanocrystals are coupled to whispering gallery modes in a silica microsphere. The composite nanocrystal-microsphere system takes advantage of the exceptional spin properties of nitrogen vacancy centers as well as the ultrahigh quality factor of silica microspheres. The observation of the normal mode splitting indicates that the dipole optical interaction between the relevant nitrogen vacancy center and whispering gallery mode has reached the strong coupling regime of cavity QED.     

微波辅助polyol法制备纳米金属镍及磁性研究

采用微波辅助polyol法成功地制备了直径范围在5～10nm、100～180nm的单分散Ni球,对其磁性进行了测量分析.用XRD和EDAX,接着用TEM和MFM分别对制备的样品进行测试,并用VSM和SQUID进一步讨论了铁磁/反铁磁的界面耦合效应.XRD显示该样品是面心立方结构,EDAX数据表明制备过程中镍球被轻微氧化,MFM和TEM观察结果显示样品金属镍是比较理想的球型,VSM测试结果表明Ni纳米球具有典型的铁磁性.     

SiO2空心微球的制备表征及摩擦性能

以分散聚合法制备的聚苯乙烯(PS)微球为有机模板, 以正硅酸四乙酯(C₈H₂₀O₄Si)为无机前躯体物料, 通过静电吸附作用成功地制备了纳米 PS-SiO₂ 复合微球和SiO₂单分散空心结构。通过红外(FTIR)、热重(TG)、透射电镜(TEM)、扫描电镜(SEM) 和热重分析仪(TGA)等手段对纳米复合材料进行了表征, 并对产物的抗磨性能进行了测试。结果表明, 该方法可一次性制备大量的复合微球, 这些微球的直径约为0.7 μm, 分散性能良好。在煅烧去除模板后, 得到了保持完整的空心纳米 SiO₂ 结构, 微球的球壳稳定性较好。摩擦实验表明, 添加了2 wt%空心微球的植物油在较低载荷工况下具有优异的减磨性能, 摩擦系数可低至0.058。      

磁性高分子微球的制备及应用

本文对新型功能材料磁性高分子微球的组成、制备方法、应用及其发展前景进行了简要介绍     

Immunophenotyping of acute leukemias using a quartz crystal microbalance and monoclonal antibody-coated magnetic microspheres

Immunophenotyping, which utilizes panels of lineage-associated monoclonal antibodies to recognize clusters of differentiation (CD) antigens expressed on various leukocytes, plays a key role in the clinical diagnosis of acute leukemias. In this paper, a rapid, simple, and automatic immunophenotyping technique for acute leukemias has been initially proposed by incorporating immunomagnetic separation and quartz crystal microbalance (QCM) measurement. Core-shell paramagnetic microspheres of silica-coated ZnFe2O4 were newly fabricated following a W/O microemulsion route and further functionalized for anchoring separately CD antibodies of lineage-different leukocytes. The resultant immunomagnetic microspheres were introduced, in turn, to capture target leukocytes from the samples and were magnetically accumulated onto the protein A-modified QCM to be analyzed. The response characteristics of the developed QCM system for immunophenotyping various lineage-defined leukocytes were investigated in detail. Results indicate that this new technique can allow for easy and clear identification of acute leukocytes of lymphoid and myeloid origins as well as their subsets. It may also permit the quantitative determination of acute leukocytes with cell concentration down to approximately 10(3) cells mL(-1). Moreover, the applicable feasibility of the QCM immunophenotyping method was validated through assessing a number of clinical specimens, which phenotypes are in acceptable agreement with those obtained by the immunoenzyme assay clinically used for this purpose.     

Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultra fast, highly efficient proteome digestion, facile 18O labeling and absolute protein quantification

In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile (18)O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using (18)O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification.     

磁性聚乙烯醇缩丁醛微球的制备和性质

运用乳化复合技术制备出磁性聚乙烯醇缩丁醛微球。该微球呈珠状 ,平均粒径为10～ 2 0nm ,具有较大的蛋白质固载量 ,可作为载体对多种酶及蛋白质加以固定。并研究了磁性聚乙烯醇缩丁醛微球制备条件、吸附时间、pH值等对磁性聚乙烯醇缩丁醛微球固定蛋白载量的影响     

Synthesis and structural evolution of hollow flower-type mesoporous silica microspheres

Hollow flower-type mesoporous silica microspheres (designated as HFMSs) with tunable smaller pores and lager pores in the shell have been successfully synthesized via a structural difference-based selective etching treatment of solid silica core/mesoporous silica shell (sSiO₂@mSiO₂) microspheres. The structure of sSiO₂@mSiO₂ microspheres could evolve to rattle-type, hollow structure, flower-type structure, and peanut-type structure from core/shell structure just by varying the etching agent from Na₂CO₃ solution to ammonia solution and adjusting the concentration of etching agent, respectively. Transmission electron microscopy, scanning electron microscopy, and nitrogen adsorption–desorption analysis are applied to characterize the synthesized samples. The mechanism of such a structural evolution is explained in this work.     

Enhanced pervaporative desulfurization by polydimethylsiloxane membranes embedded with silver/silica core-shell microspheres

Pervaporative desulfurization based on membrane technology provides a promising alternative for removal of sulfur substances (as represented by thiophene) in fluid catalytic cracking (FCC) gasoline. The present study focused on the performance enhancement of polydimethylsiloxane (PDMS) membrane by incorporation of core-shell structured silver/silica microspheres. A silane coupling agent, N-[3-(trimethoxysily)propyl]-ethylenediamine (TSD), was used to chelate the Ag(+) via its amino groups and attach the silver seeds onto the silica surface via condensation of its methoxyl groups. The resultant microspheres were characterized by Zeta-positron annihilation lifetime spectroscopy (ZetaPALS), inductively coupled plasmaoptical emission spectrophotometer (ICP), transmission electron microscopy (TEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). The Ag(+)/SiO(2)-PDMS composite membranes were prepared by blending PDMS with the as-synthesized silver/silica microspheres. PALS analysis was used to correlate the apparent fractional free volume with permeation flux. The sorption selectivity towards thiophene was enhanced after incorporation of silver/silica microspheres due to the π-complexation between the silver on the microsphere surface and the thiophene molecules. The pervaporative desulfurization performance of the composite membrane was investigated using thiophene/n-octane mixture as a model gasoline. The composite membrane exhibited an optimum desulfurization performance with a permeation flux of 7.76 kg/(m(2)h) and an enrichment factor of 4.3 at the doping content of 5%.