Preparation of magnetic microspheres with thiol-containing polymer brushes and immobilization of gold nanoparticles in the brush layer

Narrow-disperse magnetic microspheres were prepared by alkaline coprecipitation of Fe²⁺ and Fe³⁺ ions within poly(acrylic acid–divinylbenzene) microspheres that were prepared by distillation–precipitation copolymerization. Magnetic microspheres with polymer brushes that contain epoxy groups were prepared by graft copolymerization of glycidyl methacrylate and glycerol monomethacrylate via atom transfer radical polymerization (ATRP) from the magnetic microsphere surfaces. Subsequently, magnetic microspheres with thiol-containing polymer brushes were prepared by treating the epoxy group-containing magnetic microspheres with sodium hydrosulfide. Gold nanoparticles were immobilized in the brush layer of the thiol-containing magnetic microspheres through Au–S coordination. The catalytic activity of the gold nanoparticle-immobilized magnetic microspheres was investigated using the reduction of 4-nitrophenol to 4-aminophenol with sodium borohydride as a model reaction. The catalyst could be reused for over 10 cycles without noticeable loss of catalytic activity.     

含氨基和环氧基双功能基的聚合物刷磁性微球的制备及对青霉素G酰化酶的固定化

在内部分散超顺磁性Fe3O4纳米粒子的二乙烯苯交联聚丙烯酸微球表面引入原子转移自由基聚合(ATRP)引发剂,引发聚合向微球表面分别引入P(GMMA-r-DMAEMA-r-GMA)、P(GMMA-r-DMAEMA)和P(GMMA-r-GMA)无规共聚物刷(GMMA为甲基丙烯酸甘油单酯,DMAEMA为甲基丙烯酸-N,N-二甲氨基乙酯,GMA为甲基丙烯酸缩水甘油酯),聚合物刷中GMMA链节的作用是使聚合物刷具有亲水性,DMAEMA引入氨基,GMA引入环氧基.研究了青霉素G酰化酶在这些载体上的固定化和其酶活性.结果表明,同时引入环氧基和氨基的P(GMMA-r-DMAEMA-r-GMA)刷磁性微球固定化青霉素G酰化酶的活性和活性收率都最高,其固定化动力学比只含环氧基P(GMMA-r-GMA)刷磁性微球的好.固定化酶比自由酶更耐热,固定化酶的最佳pH值比自由酶的略高,固定化酶重复使用10次后其活性保留70%.     

青霉素G酰化酶在含环氧基团的顺磁性聚合物微球上的固定化（英文）

青霉素G酰化酶(PGA)是一种重要的工业生物催化剂,常用于以青霉素G为底物生产7-氨基去乙酰氧基头孢烷酸(7-ADCA)和6-氨基青霉烷酸(6-APA)等半合成β-内酰胺类抗生素.然而,PGA较差的稳定性和可重复使用性能限制了其在工业上的广泛应用.因此,将PGA固定在固体载体上是很有必要的,可以形成一种可重复使用的高性能的多相催化剂.用于生物酶固定化的良好载体应具备以下条件:(1)载体表面具有可用于与生物酶多点结合的高密度的官能团;(2)载体具有较大的比表面积以固定更多的生物酶.通常情况下,可以通过减小载体的粒径来增加其比表面积,然而,小粒径的载体很难从反应混合液中分离出来,造成固定化酶回收使用困难.为了将聚合物微球的优异固定化性能与磁性纳米粒子的独特顺磁性结合起来,我们制备了一种含环氧基团的顺磁性聚合物微球作为PGA的固定化载体.但由于Fe_3O_4纳米颗粒具有较高的表面能,在反相悬浮聚合反应过程中容易团聚成大颗粒,从而导致制备的顺磁性聚合物微球的磁体含量、表面形貌和粒径分布存在差异.此外,Fe_3O_4纳米颗粒与聚合反应单体之间的相容性不好,使得部分磁性颗粒不能很好地包埋于聚合物微球内部,影响固定化酶的活性和操作稳定性.本文以N,N′–亚甲基双丙烯酰胺为交联剂,以甲基丙烯酸缩水甘油酯和烯丙基缩水甘油醚为功能性单体,用反相悬浮聚合方法在SiO_2包覆的Fe_3O_4纳米颗粒表面成功制备出含环氧基团的顺磁性聚合物微球.用SEM,FT-IR,XRD,VSM和低温氮气吸附等手段对含环氧基团的顺磁性聚合物微球进行了表征.研究了SiO_2对Fe_3O_4纳米颗粒的包覆和Fe_3O_4/SiO_2纳米颗粒的数量对于固定化酶的初始活性和操作稳定性的影响.SiO_2在反相悬浮聚合过程中发挥重要作用,用SiO_2对Fe_3O_4纳米颗粒进行亲水性改性,有效改善了Fe_3O_4纳米颗粒与聚合反应单体的相容性,将其引入反相悬浮聚合体系中,可以制备得到球形度好、粒径分布均匀和超顺磁性的含环氧基团的顺磁性聚合物微球,其中当Fe_3O_4/SiO_2纳米颗粒的质量比为7.5%时制备的含环氧基团的顺磁性聚合物微球具有最好的PGA固定化性能.PGA通过其活性非必需侧链基团–氨基与顺磁性聚合物微球表面的环氧基团的共价结合来制备顺磁性固定化酶,该固定化PGA的初始活性为430 U/g(wet),在外加磁场的作用下容易回收使用,重复使用10次后可保留99%的初始活性,具有良好的热稳定性和酸碱稳定性,具有较好的工业应用前景.     

Surface molecularly imprinted magnetic microspheres for the recognition of albumin

A new approach, combining metal coordination with the molecular imprinting technique, was developed to prepare affinity materials. Magnetic poly(glycidyl methacrylate) microspheres in monosize form were used for specific recognition toward the target protein. The magnetic poly(glycidyl methacrylate) microspheres were prepared by dispersion polymerization in the presence of magnetite nanopowder. Surface imprinted magnetic poly(glycidyl methacrylate) microspheres based on metal coordination were prepared and used for the selective recognition of human serum albumin. Iminodiacetic acid was used as the metal coordinating agent and human serum albumin was anchored by Cu²⁺ ions on the surface of magnetic poly(glycidyl methacrylate) microspheres by metal coordination. The magnetic poly(glycidyl methacrylate) microspheres were coated with a polymer formed by condensation of tetraethyl orthosilicate and 3‐aminopropyltrimethoxysilane. The human serum albumin imprinted magnetic poly(glycidyl methacrylate) microspheres were characterized by scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy and particle size analysis. The maximum adsorption capacity of human serum albumin imprinted magnetic poly(glycidyl methacrylate) microspheres was 37.7 mg/g polymer at pH 6.0. The selectivity experiments of human serum albumin imprinted magnetic poly(glycidyl methacrylate) microspheres prepared with different concentrations in the presence of lysozyme, bovine serum albumin and cytochrome C were performed in order to determine the relative selectivity coefficients.     

Covalent immobilization of albumin on micron‐sized magnetic poly(methyl methacrylate‐divinylbenzene‐glycidyl methacrylate) microspheres prepared by modified suspension polymerization

Micron‐sized magnetic poly(methyl methacrylate‐divinylbenzene‐glycidyl methacrylate) microspheres were prepared by a modified suspension polymerization in the presence of oleic acid‐coated magnetite nanoparticles. The magnetic microspheres were functionalized by reacting the epoxy groups with ammonia solution to provide amino groups. After activated with glutaraldehyde (GA), bovine serum albumin was covalently immobilized on these magnetic microspheres. The influence of initial protein concentration, pH and ionic strength of the protein solution on covalent immobilization was studied. Scanning electron micrographs showed that the magnetic microspheres had an average size of 6.4 µm and relative narrow size distribution. Magnetic measurement revealed the magnetic microspheres were superparamagetic with saturation magnetization of 7.32 emu/g. The successful amination of the magnetic microspheres was confirmed by Fourier transform infrared spectroscopy (FT‐IR). Copyright © 2005 John Wiley & Sons, Ltd.     

亲水性含环氧基磁性聚合物微球的制备与性能表征

选择甲酰胺作磁性Fe3O4微晶的分散剂,通过设计反相悬浮聚合体系,合成了粒径分布窄、球状亲水性含环氧基磁性聚合物(MGM).利用扫描电子显微镜(SEM)、红外光谱(FT-IR)、X射线粉末衍射仪(XRD)、振动样品磁强计(VSM)和低温N2吸附以及化学分析方法对聚合物进行了性能表征.结果表明,合成的MGM呈球形,且粒度分布较窄,粒径为0.13~0.28 mm的粒子占91%;甲酰胺分散Fe3O4,微晶表面的亲水性进一步增强,单体甲基丙烯酸缩水甘油酯和N,N′-亚甲基双丙烯酰胺交联共聚生成的胶粒能够包埋Fe3O4微晶形成胶核,胶核聚集形成均匀、稳定的MGM微球.MGM中Fe3O4含量为6.17%时,比饱和磁化强度σs达6.5 emu/g;其比表面积、平均孔径和孔容分别为117.6 m2/g,15.6 nm和0.46 cm3/g,表面环氧基团含量为0.53 mmol/g.MGM借助自身的活性环氧基团在十分温和的条件下共价偶联青霉素酰化酶(penicillin G acylase EC 3.5.1.11,简称PGA),制备的固定化酶在37℃下催化水解青霉素G钾生成6-氨基青霉烷酸(6-APA)的表观活性达502IU/g,并且在使用过程中没有出现磁聚集现象.     

亲水梳状环氧聚合物载体柔性固定化脂肪酶

以氯乙酰化聚苯乙烯微球载体为大分子引发剂,以甲基丙烯酸缩水甘油酯和亲水性丙烯酰胺为共聚接枝单体,以氯化亚铜及2,2'-联吡啶为催化体系,采用原子转移自由基聚合法接枝合成了具有柔性链的亲水梳状环氧聚合物载体PS-acyl-P(AM-co-GMA),并将其用于耐有机溶剂YCJ01脂肪酶的共价柔性固定化.结果表明,固定化酶催...     

Efficient synthesis of cefadroxil in [Bmim][NTf2]‐phosphate cosolvent by magnetic immobilized penicillin G acylase

For the first time, cefadroxil was synthesized from 7‐Amino‐3‐desacetoxycephalosporanic acid and d ‐hydroxyphenylglycine methyl ester in [Bmim][NTf₂]‐phosphate cosolvent capable of dissolving the substrates using the penicillin G acylase (PGA) immobilized on the micrometer‐size magnetic polymer microspheres having high activity of 2,083 U/g. The high synthesis/hydrolysis (S/H) ratio of 1.12 was achieved with 79.0% yield, where only the S/H ratio of 0.19 and yield of 20.0% was obtained using free PGA under the identical optimum reaction conditions. Cefadroxil had been synthesized efficiently in [Bmim][NTf₂]‐phosphate cosolvent by the magnetic immobilized PGA, which illuminated that there are two very critical and essential designs, that is, effective support and suitable solvent system by PGA, in enzymatic synthesis of cefadroxil. Obviously, there is great potential for the magnetic immobilized PGA and ionic liquid solvent in application to biocatalysis.     

Synthesis and characterization of spherical and hemispherical polyepoxide micrometer‐sized particles of narrow size distribution by a single‐step swelling of uniform polystyrene template microspheres with glycidyl methacrylate

Polystyrene template microspheres of narrow size distribution were prepared by dispersion polymerization of styrene in a mixture of ethanol and 2‐methoxy ethanol. Spherical and hemispherical polystyrene/poly(glycidyl methacrylate) microspheres of narrow size distribution were prepared by a single‐step swelling of the polystyrene template microspheres with the swelling solvent monomer glycidyl methacylate, followed by polymerization of the monomer within the swollen template microspheres at 73 °C. Uniform polystyrene/poly(glycidyl methacylate‐ethylene glycol dimethacrylate) polyepoxide composite microspheres were synthesized similarly, substituting glycidyl methacylate for glycidyl methacylate and ethylene glycol dimethacrylate. Uniform crosslinked poly(glycidyl methacylate‐ethylene glycol dimethacrylate) polyepoxide microspheres have been prepared by dissolution of the PS template polymer of the former composite microspheres. Particles with different properties, for example size, size distribution, shape, surface morphology, surface area, and so forth, were prepared by changing various parameters belonging to the swelling and/or polymerization steps, for example, volume of the swelling monomer/s and/or the swelling solvent dibutyl phthalate. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4612–4622, 2007     

Hydrogels of poly(hydroxyethyl methacrylate) and hydroxyethyl methacrylate—glycerol monomethacry-late copolymers

Hydrophilic three-dimensional polymer networks (hydrogels) were prepared from hydroxyethyl methacrylate (HEMA) and the copolymer HEMA and glycerol monomethacrylate (GMMA). The equilibrium water content of the hydrogels in water was investigated as a function of the initial dilution of the polymerization mixture, the type of solvent, and the hydrophilicity of the polymer. The initial dilution was found to have a decisive effect on the swelling or deswelling of hydrogels after the completion of the gel formation. With relatively less hydrophilic hydrogels, there is a critical initial dilution to produce the gel which does not swell or deswell in water after the gel formation. This “isovolumic” initial dilution shifts toward a higher dilution as the hydrophilicity of the hydrogels increases; however, when hydrophilicity of the polymer rises above a certain point, gels always swell in water. Permeability of oxygen through hydrogels was also studied.     

Synthesis and characterization of magnetic and non-magnetic core-shell polyepoxide micrometer-sized particles of narrow size distribution

Core polystyrene microspheres of narrow size distribution were prepared by dispersion polymerization of styrene in a mixture of ethanol and 2-methoxy ethanol. Uniform polyglycidyl methacrylate/polystyrene core-shell micrometer-sized particles were prepared by emulsion polymerization at 73 degrees C of glycidyl methacrylate in the presence of the core polystyrene microspheres. Core-shell particles with different properties (size, surface morphology and composition) have been prepared by changing various parameters belonging to the above seeded emulsion polymerization process, e.g., volumes of the monomer glycidyl methacrylate and the crosslinker monomer ethylene glycol dimethacrylate. Magnetic Fe(3)O(4)/polyglycidyl methacrylate/polystyrene micrometer-sized particles were prepared by coating the former core-shell particles with magnetite nanoparticles via a nucleation and growth mechanism. Characterization of the various particles has been accomplished by routine methods such as light microscopy, SEM, FTIR, BET and magnetic measurements.     

An efficient synthesis of ampicillin on magnetically separable immobilized penicillin G acylase

<正>Penicillin G acylase(PGA) was immobilized on the magnetic hydrophilic polymer microspheres with average pore size of 17.1 nm,specific surface area of 128.2 m~2/g and saturate magnetization of 6.4 emu/g.The 96.7%ampicillin yield with 1.60 of the synthesis/hydrolysis(S/H) ratio from 6-aminopenicillanic acid(6-APA) and D-(-)-alpha-phenylglycine methyl ester(D-PGME) can be achieved using the resultant magnetic biocatalyst in ethylene glycol,where only 82.1%yield with 1.40 of the S/H ratio was obtained using the free PGA under the identical reaction conditions.The immobilized PGA can be separated magnetically and recycled for five times without obvious loss of its catalytic activity.     

Superparamagnetic Polymer Emulsion Particles from a Soap-Free Seeded Emulsion Polymerization and their Application for Lipase Immobilization

Using emulsion copolymer of styrene (St), glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) as seed latexes, the superparamagnetic polymer emulsion particles were prepared by seeded emulsion copolymerization of butyl methacrylate (BMA), vinyl acetate (VAc) and ethylene glycol dimethacrylate in the presence of the seed latexes and superparamagnetic Fe₃O₄/SiOx nanoparticles (or Fe₃O₄-APTS nanoparticles) through a two-step process, without addition of any emulsifier. The magnetic emulsion particles named P(St-GMA-HEMA)/P(BMA-VAc) were characterized by transmission electron microscope and vibrating sample magnetometry. The results showed that the magnetic emulsion particles held a structure with a thinner shell (around 100 nm) and a bigger cavity (around 200 nm), and possessed a certain level of magnetic response. The resulting magnetic emulsion particles were employed in the immobilization of lipase by two strategies to immobilized lipase onto the resulting magnetic composites directly (S-1) or using glutaraldehyde as a coupling agent (S-2), thus, experimental data showed that the thermal stability and reusability of immobilized lipase based on S-2 were higher than that of S-1.     

Preparation and surface modification of magnetic poly(methyl methacrylate) microspheres

~~Preparation and surface modification of magnetic poly(methyl methacrylate) microspheres~~     

基于超大孔聚合物微球的混合色谱模式层析介质的制备

以甲基丙烯酸缩水甘油酯与乙二醇二甲基丙烯酸酯共聚的超大孔聚合物微球为基质,采用聚乙烯亚胺和丁基缩水甘油醚先后衍生微球的表面,制备成兼具阴离子交换与疏水相互作用的混合色谱模式层析介质。考察离子交换基团、疏水配基密度对蛋白载量、回收率的影响,结果表明,在离子交换容量0.2~0.5 mol/mL范围内,随着介质离子交换容量的增大,蛋白载量及回收率均呈增加趋势,蛋白载量最高值达40 mg/mL,回收率大于90%。当疏水配基的密度大于0.03 mol/mL时,介质开始表现疏水相互作用。此超大孔混合模式色谱介质在大于2000 cm/h 的流速下依然能保持低于2 MPa 的柱背压,同时在高流速下(2880 cm/h)纯化人血清中的抗体应用中表现良好的分辨率。此介质在高通量分离纯化应用方面具有巨大潜力。     

Immobilized trypsin on epoxy organic monoliths with modulated hydrophilicity: novel bioreactors useful for protein analysis by liquid chromatography coupled to tandem mass spectrometry

The development of epoxy organic monoliths with modulated hydrophilicity for the preparation of novel trypsin-based microreactors is reported. Porous polymer monoliths have been prepared using methacrylate chemistry triggered by γ-ray irradiation. In situ polymerization has been optimized and extended to medium and high polymer densities using glycidyl methacrylate (GMA) as reactive monomer as well as to the hydrophilic nature of the co-monomers (glyceryl monomethacrylate, GlyMA and acrylamide, AMD). Enzyme immobilization was smoothly achieved by passing a buffered trypsin solution through the columns kept at room temperature. The activities of the immobilized enzyme were characterized by the apparent Michaelis constant (K(m)) and the apparent maximum velocity (V(max)) of the reaction using a non chromogenic, low-molecular mass substrate N-α-benzoyl-l-arginine ethyl ester (BAEE). For the kinetic constants determination a new off-line chromatographic procedure was developed on purpose. The most efficient IMERs were obtained by immobilizing trypsin on monolithic skeleton prepared with hydrophilic monomers (GlyMA and AMD). One of the most promising bioreactor was applied to the digestion of model proteins with different molecular weight and complexity such as human serum albumin (HSA), β-casein and ribonuclease B (RNase B), and the produced peptides were analyzed by liquid chromatography-mass spectrometry. Using a digestion time of only 25 min the proteins were recognized by the database with satisfactory sequence coverage, which was 78.22, 49.76 and 80.68% for HSA, β-casein and RNase B, respectively.     

Fabrication and functionalization of dendritic poly(amidoamine)-immobilized magnetic polymer composite microspheres

The synthesis of functionalized magnetic polymer microspheres was described by a process involving (1) preparation of the monodisperse magnetic seeds according to a two-step procedure including the preparation of bilayer-oleic acid-coated Fe3O4 nanoparticles followed by soap-free emulsion polymerization with methyl methacrylate (MMA) and divinyl benzene (a cross-linking agent, DVB); (2) seeded emulsion polymerization proceeding under the continuous addition of glycidyl methacrylate (GMA) monomers in the presence of the magnetic PMMA seeds; and (3) chemical modification of the PGMA shells with ethylenediamine (EDA) to yield amino groups. As such, the magnetic poly(MMA-DVB-GMA) microspheres were prepared possessing monodispersity, uniform magnetic properties, and abundant surface amino groups. Then, the dendritic poly(amidoamine) (PAMAM) shells were coated on the magnetic particles on the basis of the Michael addition of methyl acrylate and the amidation of the resulting ester with a large excess of EDA, which could achieve generational growth under such uniform stepwise reactions. For improving the luminescence properties of the composite particles, fluorescein isothiocyanate, which is a popular organic dye, was reacted with the terminal -NH2 groups from the dendritic PAMAM shells, resulting in the formation of multifunctional microspheres with excellent photoluminescence, superparamagnetic, and pH-sensitive properties. In this case, it can be expected that an extension of the functionalization of these microspheres is to immobilize other target molecules onto the PAMAM shells to introduce other desired functions for potential chemical and biological applications.     

含环氧基团的聚合物微球固载2,2,6,6-四甲基哌啶氮氧自由基催化剂的制备及其催化分子氧氧化苯甲醇

以甲基丙烯酸缩水甘油酯(GMA)为单体, 以乙二醇二甲基丙烯酸酯(EGDMA)为交联剂, 采用悬浮聚合法制得交联聚甲基丙烯酸缩水甘油酯(CPGMA)微球, 然后以4-羟基-2,2,6,6-四甲基哌啶氮氧自由基(4-OH-TEMPO)为试剂, 使CPGMA微球表面的环氧基团发生开环反应, 从而制得了TEMPO固载化微球TEMPO/CPGMA, 考察了制备条件对固载化反应的影响, 并采用多种方法对微球TEMPO/CPGMA进行了表征. 将微球TEMPO/CPGMA与CuCl组成共催化体系, 用于分子氧氧化苯甲醇, 考察了反应条件对催化体系性能的影响. 结果表明, 以含环氧基团的聚合物微球CPGMA为载体, 通过开环反应, 可成功地实现TEMPO的固载化, 开环反应属S_N2亲核取代反应, 适宜采用溶剂N,N''-二甲基甲酰胺和反应温度85℃. 非均相催化剂TEMPO/CPGMA与助催化剂CuCl构成共催化体系, 在室温、常压O₂条件下可高效地将苯甲醇氧化为苯甲醛, 产物选择性和产率分别为100%和90%. 主催化剂TEMPO与助催化剂CuCl适宜的摩尔比为1:1.2; 主催化剂适宜用量为0.90 g. 此外, TEMPO/CPGMA固体催化剂具有良好的循环使用性能.     

水相光引发聚合诱导自组装制备嵌段共聚物纳米材料

本文以聚甲基丙烯酸甘油酯(PGMA)作为大分子链转移剂、甲基丙烯酸羟丙酯(HPMA)为单体、苯基钠盐-三甲基苯甲酰亚磷酸盐(SPTP)为引发剂,通过水相光引发可逆加成-断裂链转移(RAFT)分散聚合制备了一系列PGMA-b-PHPMA共聚物纳米材料。考察了水相光引发聚合诱导自组装的反应动力学,在温和条件下(水相、可见光和室温)成功得到不同形貌的聚合物纳米材料(球形、纤维和囊泡),并进一步探究了反应条件对嵌段聚合物形貌的影响。聚合反应的激活或暂停都可以通过对光源的简单"开/关"进行控制。     

Magnetic hydrophilic methacrylate-based polymer microspheres for genomic DNA isolation

Carboxyl groups containing magnetic and non-magnetic microspheres were used in solid-phase reversible immobilization (SPRI) of genomic DNA. Magnetic non-porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)--P(HEMA-co-EDMA), poly(glycidyl methacrylate)--PGMA and P(HEMA-co-GMA) microspheres with hydrophilic properties were prepared by dispersion copolymerization of the respective monomers in the presence of colloidal iron oxides. DNA from chicken erythrocytes and DNA isolated from bacterial cells of Bifidobacterium longum was used for testing of adsorption/desorption properties of magnetic microspheres. The occurrence of false negative results in polymerase chain reaction (PCR) caused by the presence of extracellular inhibitors in DNA samples has been solved using SPRI. The P(HEMA-co-EDMA) and P(HEMA-co-GMA) microspheres were used for isolation of DNA from different dairy products followed by PCR identification of Bifidobacterium strains.