球形纤维素固定化DNA制备免疫吸附剂

以球形纤维素为载体,经环氧氯丙烷活化后共价键联小牛胸腺DNA,制备DNA免疫吸附剂,通过血液灌流能够治疗系统性红斑狼疮.对病人血清的吸附实验结果表明,每毫升吸附剂与3mL病人血清混合,于37℃保温1 h,可吸附除去40%～70%致病抗体     

Monosize microbeads for pseudo-affinity adsorption of human insulin

Affinity adsorption technique is increasingly used for protein purification, separation and other biochemical applications. Therapeutic molecules such as antibodies, cytokines, therapeutic DNA and plasma proteins must be purified before characterization and utilization. The aim of this study was to prepare micronsized spherical polymeric beads and to investigate the extent of their human insulin adsorption capability. Monosize poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-histidine) [poly(EDMA-MAH)] beads were prepared by modified suspension copolymerization. Functional monomer (MAH) was synthesized using methacryloyl chloride and L-histidine. The beads were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, swelling test and elemental analysis. MAH incorporation into monosize polymeric beads, having an average size around 2-3 μm, was estimated as 55.3 μmol MAH/g bead. Equilibrium swelling ratios of poly(EDMA-MAH) and poly(EDMA) beads were 65% and 55%, respectively. Adsorption experiments were performed under different conditions (i.e., pH, temperature, protein concentration and ionic strength). It was found that adsorption characteristics are strongly depend on these conditions. Maximum insulin adsorption capacity was achieved as 24.7 mg insulin/g poly(EDMA-MAH) beads. Results were well fitted to the Langmuir isotherm model. Compared with poly(EDMA-MAH), nonspecific insulin adsorption onto poly(EDMA) beads was very low (0.61 mg insulin/g bead) and can be negligible. It was observed that insulin could be repeatedly adsorbed and desorbed (at least 10 times) without significant loss in adsorption capacity.     

Effect of reaction solvent on the preparation of thermo-responsive stationary phase through a surface initiated atom transfer radical polymerization

Poly(N-isopropylacrylamide) (PIPAAm) brush grafted silica beads, a thermo-responsive chromatographic stationary phase, were prepared through a surface-initiated atom transfer radical polymerization (ATRP) using 2-propanol, N,N-dimethylformamide (DMF), and water as reaction solvents. The rate of grafting PIPAAm on silica bead surfaces was different and found to be dependent on the reactivity of reaction solvent. Temperature-dependent elution profiles of hydrophobic steroids from the prepared-beads-packed columns were found to be different, although the graft amounts of PIPAAm were similar on silica bead surfaces. Especially, prepared beads using 2-propanol exhibited a higher resolution than those using DMF. Calibration curves using glucose and pullulan suggested that beads prepared using DMF prohibited analytes to diffuse into the pores. On the contrary, beads prepared using 2-propanol allowed analytes to diffuse into the pores. The pore diameter of the prepared beads, measured by N(2) adsorption-desorption measurement, suggested that beads using 2-propanol has relatively larger pore diameter than those using DMF. Thus, the reaction solvent in surfaces-initiated ATRP affected the grafting configuration of PIPAAm on porous silica-bead surfaces, leading to the different separation efficiency of stationary phase for bioactive compounds.     

Polymeric thiols as enzyme activators of serum creatine phosphokinase

Several hydrophilic polymeric thiols were prepared from aminoactivated polymeric supports by reaction with N-acetylhomocysteinethiolactone. Supports include agaroses, cellulose, Glycophase™ controlled-pore glass, and Matrex™ acrylic beads. Thiol content in these polymers was 3–72 μmol SH/g dry polymer. Several were effective solid-phase activators of the sulfhydryl-dependent enzyme creatine phosphokinase at concentrations comparable to that of monomeric thiol required for enzyme activation. The kinetic activation curves for the polymeric and the monomeric (thioglucose) activators were similar, suggesting unhindered interaction of the enzyme with the polymeric activator.     

基于自动磁珠转运的转基因蛋白Cry1Ab检测

基于自动磁珠转运,建立了转基因蛋白Cry1Ab免疫检测的新方法.利用水热法制备了粒径约400 nm的纳米磁球,并进行电镜表征,通过溶胶法对磁球表面进行氨基修饰,采用戊二醛偶联对磁珠实现抗体包被,在核酸提取仪中进行酶联免疫反应,采用分光光度法进行检测.本方法对转基因蛋白Cry1Ab的检出限低于1 μg/L,与商品化酶联免...     

新型吲唑类化合物的合成及其抗肿瘤活性

以2,6-二氟苯腈与吗啉反应制得2-氟-6-吗啉-苯腈(1); 1与水合肼在N-甲基吡咯烷酮中通过环合反应制得3-氨基-4-吗啉-1H-吲唑(2); 2与不同羧酸经缩合反应合成了8个新型吲唑类化合物(4a~4h),其结构经¹H NMR, IR和HR-ESI-MS表征。抗肿瘤活性测试结果表明：3,4,5-三甲氧基-氮-(4-吗啉-1H-吲唑-3-基)苯甲酰胺（4a）的抗肿瘤活性最好,对K-562, SMMC7721和T-47D肿瘤细胞有明显抑制作用,IC₅₀分别为0.056 μmol·L^-1, 0.062 μmol·L^-1和0.078 μmol·L^-1。     

负载高密度乙肝检测探针磁珠的制备及性能

本文介绍了一种负载高密度乙肝检测探针磁珠的制备技术,并对其在乙肝检测中的应用性能进行了研究,开辟了一种乙肝检测的新方法。首先通过化学键连接的方法制备出表面偶联乙肝抗体的磁性复合微球（亦称“乙肝抗原检测免疫磁珠”）,之后将其用于乙肝检测研究表现出了较好的效果,为了进一步提高其检测准确性及灵敏性,对乙肝免疫磁珠的制备过程进行了优化,包括磁珠的胺化工艺及抗体的偶联工艺。通过优化得到氨基磁性复合微球的氨基含量为2.71 mmol/g、单位磁珠抗体偶联量为108.36 ug/mg、偶联效率为77.40%的负载高密度乙肝检测探针的磁珠。并在此过程中采用类“双抗原夹心酶联免疫法”对乙肝抗体的活性及优化效果进行了检测。通过性能检测比较,磁珠法检测灵敏度高于普通酶联免疫法。     

Preparation of Macroporous Poly(vinyl alcohol-co-triallyl isocyanurate)Beads Bearing Aminocarboxylic Acid as Functional Groups by Suspension Polymerization

Macroporous poly(vinyl acetate-co-triallyl isocyanurate)beads were prepared with suspension polymerization method.The copolymer beads were then transformed into poly(vinyl alcohol-co-triallyl isocyanurate)by ester exchange reaction.Aminocarboxylic acids were immobilized on the copolymer beads by the esterification of hydroxyl groups with diethyl-lenetriaminepentaacetic bisanhydride,The weak acid exchange capacities,specific surface areas and mean pore diameters of the rsultant resin beads were measured.     

SYNTHESIS, CHARACTERIZATION AND CATALYTIC ACTIVITY OF N,N''''-BIS(3-ALLYL SALICYLIDENE)ETHYLENEDIAMINE COBALT(Ⅱ) SCHIFF BASE COMPLEX ANCHORED ON A NEW POLYMER SUPPORT

INTRODUCTIONAlthough homogeneous catalysts have higher selectivity and act under mild experimental conditions in variousreactions they have problems of separation and recovery from reaction products. Hence current research activitiesare aimed at developing catalysts of coordination compounds anchored on suitable supports[1,2]. This techniqueof immobilization of catalyst on an inert support increases the catalytic activity, selectivity, efficiency[3],operational flexibility and stability. …     

Model filled polymers. IX. Synthesis of uniformly crosslinked polystyrene microbeads

Monodisperse sized crosslinked polystyrene (PS) beads prepared by reaction of styrene (S) and divinylbenzene (DVB), in batch emulsion copolymerization in the absence of emulsifiers, are not uniformly crosslinked, because DVB is more reactive than S. For copolymerization of 1 to 10 mol % DVB and S, within each crosslinked PS microbead, the crosslink density varies by a factor exceeding two and decreases with increased conversion. A semicontinuous copolymerization, involving incremental additions of DVB, produces uniformly crosslinked PS beads. For both copolymerization techniques, T_g correlates well with crosslink density and PS beads are spherical and monodisperse in size. © 1992 John Wiley & Sons, Inc.     

SYNTHESIS, CHARACTERIZATION AND CATALYTIC ACTIVITY OF N,N''-BIS(3-ALLYL SALICYLIDENE)ETHYLENEDIAMINE COBALT(II) SCHIFF BASE COMPLEX ANCHORED ON A NEW POLYMER SUPPORT

A new chelating polymer support has been prepared by suspension copolymeriz a tion of synthesized N,N'-bis(3-allyl salicylidene)ethylenediamine monomer Schiff base (N,N'-BSEDA) with styrene (St) and divinylbenzene (DVB) using azobisisobutyronitrile (AIBN) as initiator in the presence of poly(vinyl alcohol). The content and complexation ability of monomer Schiff base (N,N'-BSEDA) for cobalt(II) ions in prepared crosslinked polymer beads have shown dependence on the amount of DVB used in reaction mixture. The amount of monomer Schiff base (N,N'-BSEDA) in crosslinked beads showed a substantial decreasing trend at high concentration of DVB in the reaction mixture (＞ 1.5 mol dm-3), hence the efficiency of complexation (EC%) and cobalt(II) ion loading (EL%) of polymer beads showed a decreasing trend. The structure of monomer Schiff base (N,N'-BSEDA) and its cobalt(II) complex on polymer support was elucidated by IR, UV and magnetic measurements. The catalytic activity of polymer bound cobalt(Ⅱ) Schiff base complex was evaluated by analyzing kinetic data of decomposition of hydrogen peroxide in the presence of either supported cobalt (II) complex or free cobalt(II) complex. The activation energy for the decomposition of hydrogen peroxide by polymer supported cobalt(II)complex was found to be low (33.37 kJ mol-l) in comparison with unsupported cobalt(II) complex (56.35 kJ mol-1). On the basis of experimental observations, reaction steps are proposed and a suitable rate expression derived.     

Use of a fluorosurfactant in micellar electrokinetic capillary chromatography

Beads prepared from a thermosensitive polymer, hydroxypropylcellulose, exhibit temperature-dependent porosity. At temperatures below 40°C the beads are swollen having large pores, while at temperatures above 45°C the beads are in a shrunken state having smaller pores. In the presence of 1 M NaCl the transition temperature decreased to about 30°C. In a swollen state the size of pore is large enough to accommodate lysozyme (mol. mass 14 400) and -chymotrypsin (mol. mass 21 600) but not bovine serum albumin (mol. mass 67 000). When the beads are shrunken, all the proteins are eluted from the column packed with hydroxypropylcellulose beads in the volume close to the void volume of the column.     

Design of luminescent biochips based on enzyme,antibody, or DNA composite layers

The use of beads bearing bioactive molecules to develop generic biochips based on chemi- and electro-chemiluminescent detection was evaluated. The biochips were composed of arrayed biosensors, including enzyme-charged beads, antigen-charged beads, or oligonucleotide-charged beads, entrapped in poly(vinyl alcohol) (PVA-SbQ) photopolymer. In each case the sensing layers were spotted at the surface of a glassy carbon electrode as 0.3 µL drops, generating 500–800 µm spots. The luminescent reactions were either catalysed by horseradish peroxidase or triggered by application of a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference. Enzyme biochips were designed for the concomitant detection of choline, glucose, glutamate, lactate, lysine, and urate, based on the corresponding oxidase-charged beads and the electro-chemiluminescent (ECL) reaction with luminol-immobilised beads of the hydrogen peroxide produced. Limits of detection of 1 µmol L^–1 for glutamate, lysine and uric acid, 20 µmol L^–1 for glucose, and 2 µmol L^–1 for choline and lactate were found with detection ranging over three decades at least. Use of the electro-chemiluminescent biochip was extended to a tri-enzymatic sensing layer based on kinase-oxidase activity for detection of acetate. A reaction sequence using acetate kinase, pyruvate kinase, and pyruvate oxidase enabled the production of H₂O₂ in response to acetate injection in the range 10 µmol L^–1 to 100 mmol L^–1. Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realised for the model detection of human IgG. Biotin-labelled anti-human IgG were used in a competitive assay, in conjunction with peroxidase-labelled streptavidin. Free antigen could then be detected with a detection limit of 25 pg (10⁸ molecules) and up to 15 ng. In a similar way, the use of oligonucleotide-immobilised beads enabled the realisation of DNA-sensitive biochips which could be used to detect a biotin-labelled sequence al a level of 5×10⁸ molecules.     

Covalently Bound Antibody on Polystyrene Latex Beads: Formation, Stability, and Use in Analyses of White Blood Cell Populations

CD4 or CD8 antibodies were covalently bound to latex beads by reaction of activated CD4 or CD8 monoclonal antibodies with 2-μm-diameter, 1,3-diaminopropane (DAP) coupled, polystyrene aldehyde/sulfate latex beads. Spectrophotometric analyses of the filtrates of the antibody-bead conjugation mixtures for unreacted antibody allowed construction of binding curves of antibody for the polystyrene bead surface and evaluation of binding constants for association of antibody with bead, ranging from 1.5x10(7) to 1.6x10(7) M(-1) for CD4 and CD8 antibodies. The reaction of the antibody thiol group with the activated maleimide group on the bead at pH 7.2-7.3 was complete within 10-15 min. The kinetics of CD4 or CD8 monoclonal antibody displacement from the surface of covalently conjugated antibody-polystyrene latex beads was followed as a function of temperature (5, 22, and 37 degrees C) and the nature of the final diluent for the antibody-coated beads by measuring the concentration of antibody in the filtrates of conjugated beads by an ELISA (enzyme-linked immunosorbent assay). The displacement reaction showed a pseudo-zero-order dependence of the rate, with constants, k(1), ranging from 0.65x10(-17) to 270x10(-17) M s(-1). The functionality of antibody-coated beads suspended in various media was also monitored in a biological cell assay with whole blood. The cell assay depends on forming a layer of beads around targeted lymphocytes to distinguish them from nontargeted lymphocytes by differences in dc or rf conductivity or median angle light scatter. Covalently bound CD4 and CD8 antibody beads stored in one set of media at 5, 22, and 37 degrees C over a period of 16 weeks showed excellent results in the STKS assay with various blood donors, which correlated well (correlation coefficients of 0.99 for CD4 data and 0.93 for CD8 data) with reference results obtained with fluorescent markers by flow cytometry. Covalently bound CD4/CD8 beads stored for 2 weeks in BSA buffer at 5-37 degrees C performed equally well in providing accurate values of the percentage of CD4- or CD8-positive cells in the total white blood cell population, whereas the same beads stored in the 47-50 degrees C range showed some failures in performance. Comparison with antibody concentrations in filtrates of adsorbed antibody-bead suspensions showed 2- to 10-fold greater amounts of free antibody at comparable elapsed time, media, and temperature conditions. A threshold of 1-2 μg/mL of free antibody was necessary before adverse effects on the biological cell assay were noticeable. Copyright 2001 Academic Press.     

新型2-氨基-呋喃并[2,3-d]嘧啶-4(3H)-酮衍生物的合成及抗肿瘤活性

报道应用aza-Wittig反应,采用易得的原料,在温和的条件下,以78%～90%的产率有效的合成了新型2-氨基-呋喃并[3,2-d]嘧啶-4(3H)-酮衍生物5,其结构经IR,1H NMR,MS和元素分析确认.为进一步得到结构确认,化合物5c经X射线衍射分析证实.运用噻唑蓝(MTT)标准法对化合物5进行了体外抗肿瘤活性的测定,其中5f对肺癌细胞A459的IC50值为18.4μmol/L,显示出潜在良好的抗肿瘤活性.     

磁珠富集与液相色谱-质谱联用结合筛选枳壳中脂肪酶抑制剂

提出了一种磁珠收集与液相色谱-质谱联用(LC-MS)结合用于快速筛选中药提取物中的脂肪酶抑制剂的方法. 通过制备键合脂肪酶的磁珠, 将其与枳壳总黄酮孵育后, 筛选出枳壳中脂肪酶的配体. 通过LC-MS分析发现, 4个化合物均是潜在的脂肪酶抑制剂, 鉴定其分别为柚皮苷、 新橙皮苷、 橙皮苷和枸橘苷. 对4个化合物进行了体外脂肪酶抑制活性验证. 研究结果表明, 新橙皮苷、 橙皮苷和枸橘苷具有显著的脂肪酶抑制活性, IC₅₀分别为48.04, 52.45和46.18 μg/mL, 其中枸橘苷具有脂肪酶抑制活性为首次报道. 结果表明, 磁珠富集与LC-MS集成技术能够用于快速发现中药活性成分.     

基于适配体技术对止咳平喘健康产品中非法添加的沙丁胺醇进行可视化快速检测

基于偶联作用和ssDNA结合蛋白与适配体的结合作用制备检测复合物MB-aptamer～SiO₂@SSB@HRP,利用目标物沙丁胺醇与适配体之间的亲和性比ssDNA结合蛋白与适配体亲和性更高的原理,以沙丁胺醇为"钥匙"将适配体与ssDNA结合蛋白构成的"开关"打开,经过磁性分离,最终以辣根过氧化物酶催化四甲基联苯胺产生的显色反应作为信号输出,建立了沙丁胺醇的可视化快速检测方法。在优化实验条件下,沙丁胺醇浓度与显色反应的吸光度在0.1～10μmol/L之间呈良好线性关系(r²=0.995 9),检出限为0.1μmol/L。对中成药样品进行沙丁胺醇加标回收实验,回收率为86.0%～106%,相对标准偏差为3.9%～12%。该方法能够满足非法添加实际检测需求。     

伊马替尼衍生物的合成及其抗肿瘤活性

以4-硝基-2-氨基甲苯为起始原料,经加成、缩合、环化和还原反应制得中间体N-(2-甲基-5-氨基苯基)-4-(3-吡啶基)嘧啶-2-胺(4),再与取代酰氯反应,合成了7个新型伊马替尼衍生物（5a~5g）,其结构经¹H NMR, ¹³C NMR和HR-MS 表征。采用四甲基偶氮唑盐(MTT)法考察了5对人肝癌细胞(HepG₂)、子宫颈癌细胞（Hela）、肺癌细胞（H460）和乳腺癌细胞（MCF-7）体外抑制活性。结果显示：5e体外抑制活性最优,其IC₅₀分别为10.90±1.00 μmol·L^-1; 8.51±0.90 μmol·L^-1; 13.15±1.11 μmol·L^-1; 14.75±0.78 μmol·L^-1。     

SYNTHESIS, CHARACTERIZATION AND CATALYTIC ACTIVITY OF N,N'-BIS(3-ALLYL SALICYLIDENE)ETHYLENEDIAMINE COBALT SCHIFF BASE COMPLEX ANCHORED ON A NEW POLYMER SUPPORT

 A new chelating polymer support has been prepared by suspension copolymerization of synthesized N,N'-bis(3-allyl salicylidene)ethylenediamine monomer Schiff base (N,N'-BSEDA) with styrene (St) and divinylbenzene (DVB) using azobisisobutyronitrile (AIBN) as initiator in the presence of poly(vinyl alcohol). The content and complexation ability of monomer Schiff base (N,N'-BSEDA) for cobalt(Ⅱ) ions in prepared crosslinked polymer beads have shown dependence on the amount of DVB used in reaction mixture. The amount of monomer Schiff base (N,N '-BSEDA) in crosslinked beads showed a substantial decreasing trend at high concentration of DVB in the reaction mixture (> 1.5 mol dm^-3), hence the efficiency of complexation (EC%) and cobaltⅡion loading (EL%) of polymer beads showed a decreasing trend. The structure of monomer Schiff base (N,N'-BSEDA) and its cobalt(Ⅱ)complex on polymer support was elucidated by IR, UV and magnetic measurements. The catalytic activity of polymer bound cobalt(Ⅱ)Schiff base complex was evaluated by analyzing kinetic data of decomposition of hydrogen peroxide in the presence of either supported cobalt(Ⅱ)complex or free cobalt(Ⅱ)complex. The activation energy for the decomposition of hydrogen peroxide by polymer supported cobalt complex was found to be low (33.37 kJ mol^-1) in comparison with unsupported cobalt(Ⅱ)complex (56.35 kJ mol^-1). On the basis of experimental observations, reaction steps are proposed and a suitable rate expression derived.     

Isonitrile derivatives of polyacrylamide as supports for the immobilization of biomolecules

Isonitrile derivatives of crosslinked polyacrylamide beads (Biogell P-100) were prepared by a two-step procedure: a. N-hydroxymethylation (methylolation) of amide groups on the polymer by treatment with formaldehyde; and b. Attachment of side chains, containing isonitrile functional groups by a displacement reaction involving 1-tosyloxy-3-isocyanopropane (p-CH₃-C₆H₄·SO₂·O·(CH₂)₃ NC) and alkoxide ions generated on methylolated polyacrylamide by treatment with a strong base in a polar aprotic solvent. The modified polyacrylamide beads were tested as support for the immobilization of proteins, and low mol wt ligands by four component condensation (4CC) reactions. Trypsin-polyacrylamide acting on N-benzoyl-L-arginine ethylester exhibited nonlinear Michaelis Menten kinetics and distorted pH activity profiles. The kinetic anomalies could be reduced by increasing the concentration of buffer. The data were consistent with a model assuming “buffer facilitated proton transport” in a diffusionally constrained system.