磁性二氧化硅微球的表面修饰及其在植物基因组核酸纯化中的应用

在利用自主研发的专利技术制备球形磁性硅胶微球的基础上,对磁性硅胶微球进行表面改性,使其表面分别键合硅羟基、环氧基、邻二醇基和羧基等官能团,并对表面官能团进行了定量研究。以小牛胸腺基因组脱氧核糖核酸(DNA)为模型化合物,研究了核酸在不同表面官能团的磁性硅胶上的吸附和脱附行为,发现表面具有硅醇基的磁球对DNA的回收率最高。将改性后磁性微球应用于玉米DNA的提取,得到了平均长度大于8kb的高纯度基因组DNA。与传统的有机溶剂抽提法相比,基于磁性微球的核酸固相萃取法具有快速简便、省时省力、易于自动化的特点,适合于大规模植物基因组DNA的样品制备。     

表面电性可控的磁性微球的制备、表征及其在基因组DNA萃取中的应用

运用水热法、St?ber法制备了具有核壳型结构的超顺磁性Fe3 O4@SiO2微球,运用氨丙基三乙氧基硅烷( APTES)对Fe3 O4@SiO2微球表面进行修饰,得到表面电性可控的氨基化Fe3 O4@SiO2磁性微球,运用透射、扫描电镜表征微球形貌,采用Zeta电位研究微球表面的荷电特性。将氨基化Fe3 O4@SiO2微球用于人类全血中基因组脱氧核糖核酸( DNA)的萃取,开发了固相萃取方法,并探究微球与DNA的作用机理,对提取产物进行凝胶电泳和PCR实验。结果表明,自制的微球成功地从全血中提取出纯度较高的基因组DNA,提取率约70%,微球对基因组DNA的饱和吸附量约为40 ng/μg,提取液可直接用于进一步的生物分析。     

磁性壳聚糖硅胶复合微球的制备及其吸附Cu~(2+)的性能研究

以壳聚糖与正硅酸四乙酯为原料,采用溶胶-凝胶法,用戊二醛辅助交联合成了磁性壳聚糖硅胶复合微球。通过红外光谱、扫描电镜、X-射线衍射等方法对磁性壳聚糖硅胶复合微球的形态和组成特性进行分析,制备的磁性复合微球中壳聚糖与硅胶材料复合均匀,材料粒径均一,机械强度较高。考察了制备的磁性壳聚糖硅胶复合微球对Cu~(2+)的吸附性能,结果表明微球对Cu~(2+)具有较好的吸附性能,吸附容量达到98.7mg/g。     

改性果胶-Fe_3O_4磁性微球的制备及对Cu~(2+)吸附性能研究

采用氯化钙、环氧氯丙烷交联改性,制备了改性果胶磁性微粒,分别用红外光谱、扫描电镜、X-射线衍射对样品进行了表征并对实验条件进行了探究。实验结果表明:环氧氯丙烷改性果胶-Fe_3O_4微球吸附剂对Cu~(2+)有较好的吸附。该吸附符合准二级动力学方程,主要为化学吸附。当Cu~(2+)的初始浓度160 mg·L~(-1),吸附剂添加量为20 mg,反应时间为90 min,反应温度为60℃时的单位吸附量为74.89 mg·g~(-1)。研究还表明EDTA对磁性微球的洗脱效果最佳。环氧氯丙烷改性果胶-Fe_3O_4微球吸附剂对香螺、海螺和黄蚬子三种贝类的酶解液中Cu~(2+)进行脱除实验,去除率分别为85.1%,82.4%和83.5%,效果良好。     

表面电性可控磁珠微流控芯片在DNA提取中的应用

制备了表面电性可控的氨基化SiO_2@Fe_3O_4磁性复合微球,采用激光刻蚀和热压键合的方法制作了聚甲基丙烯酸甲酯(PMMA)材质的DNA固相萃取芯片,将磁珠灌注于芯片通道中,借助永磁铁固定并控制磁珠,将磁珠芯片应用于人类全血中的基因组DNA提取,优化了提取实验条件,并对提取产物进行凝胶电泳和PCR分析。实验结果表明,磁珠微流控芯片成功地从全血中提取出纯度较高的基因组DNA,提取效率约35%,提取液的凝胶电泳条带与商品化试剂盒提取的基因组DNA一致,提取液可用于进一步的PCR反应。     

具有高选择性吸附的表面分子印迹磁性纤维素微球的制备与性能

以纤维素和纳米Fe₃O₄为原料制得磁性纤维素微球, 在纤维素微球表面选择合适的模板分子, 以甲基丙烯酸、 丙烯酰胺和N,N'-亚甲基双丙烯酰胺为功能单体, 采用水溶液聚合法制得表面分子印迹磁性纤维素微球. 采用傅里叶变换红外光谱(FTIR)、 X射线衍射(XRD)和振动样品磁强计(VSM)等表征了分子印迹聚合物微球的结构. 以罗丹明B(RhB)为模板分子, 通过吸附动力学与吸附热力学实验研究了表面分子印迹磁性纤维素微球对RhB的吸附性能, 结果表明, 制备的表面分子印迹磁性纤维素微球对罗丹明B具有特异性识别作用, 饱和吸附量达到0.542 mg/mg, 吸附平衡时间为10 h左右. 表面分子印迹磁性纤维素微球大大降低了对吸附环境的依赖, 并可重复利用.     

改性果胶磁性微球的制备及吸附性能研究

为了提高果胶磁性微球的分散性及成球性,采用油酸对磁性微球进行表面改性后,制备改性果胶磁性微球。通过红外光谱、扫描电镜、XRD和磁性分析等对样品进行表征。油酸和果胶在纳米四氧化三铁表面形成了良好的修饰层,且油酸的加入提高了果胶磁性微球的分散性及成球性。研究了改性果胶磁性微球用量和溶液pH值等对其吸附Cu(Ⅱ)性能的影响,考察了其吸附动力学和吸附等温线。25℃时,改性果胶磁性微球对Cu(Ⅱ)的吸附达平衡需要2 h,饱合吸附容量为52.36 mg/g,Freundlich模型和Langmuir模型可以较好的拟合实验结果,最大吸附量为119.05 mg·g~(-1)。     

新型免疫磁性微球的制备及其在α-2b干扰素分离纯化中的应用

采用琼脂糖、纤维素和聚乙烯醇3种材料制备表面带有活性羟基的新型磁性微球,对其性能进行了表征并探讨其致敏条件。通过性能的比较,选择纤维素磁性微球作为分离纯化载体,并建立α-2b干扰素的免疫磁性分离方法。通过对α-2b干扰素发酵菌体裂解液的预处理、上样量和稀释倍数以及洗脱条件进行考察,确立免疫磁性分离条件,用于α-2b干扰素的一步分离纯化,其活性回收率达到88%,平均比活性为1.5×108IU/mg,纯度大于99%。实验结果表明,纤维素磁性微球作为性能优良的新型磁性载体,更适于目的蛋白的免疫磁性分离。     

磁性壳聚糖微球的制备及对Cr(Ⅵ)的吸附性能研究

用壳聚糖包埋磁流体,用戊二醛交联制成磁性壳聚糖微球,并用红外光谱表征其结构。用制备的磁性壳聚糖微球吸附Cr(Ⅵ)离子,考察了其对Cr(Ⅵ)离子的吸附性能;探讨了吸附时间、溶液pH值、吸附剂用量、温度、Cr(Ⅵ)起始浓度以及其他离子存在对Cr(Ⅵ)离子去除率的影响。实验结果表明,磁性壳聚糖微球吸附Cr(Ⅵ)离子的最佳条件为:吸附平衡时间40 min,最佳吸附pH值6左右,磁性壳聚糖微球用量10 mg,温度升高有利于提高磁性壳聚糖微球的吸附效率,Cr(Ⅵ)离子起始质量浓度为12μg/mL,无机盐的存在引起磁性壳聚糖微球的吸附性能降低。并且考察了吸附剂的再生性能,实验结果表明磁性壳聚糖微球具有良好的重复使用性。     

PEI改性磁性壳聚糖微球的制备及其对布洛芬的吸附性能

采用共沉淀法制备磁性Fe_3O_4纳米粒子,然后在乳化体系中,以戊二醛为交联剂,通过席夫碱反应制备了改性磁性壳聚糖微球(Fe_3O_4@CS)以及聚乙烯亚胺(PEI)改性磁性壳聚糖微球(Fe_3O_4@CS/PEI)。采用红外光谱、X射线粉末衍射、磁滞回线测定、扫描电子显微镜和动态光散射对微球的结构、粒径以及磁性进行了表征,通过紫外-可见分光光度计研究了微球对布洛芬的吸附能力和重复利用率。结果表明,在微球制备过程中发生的席夫碱反应不会对纳米Fe_3O_4的晶型产生影响。微球均呈现出规整的球形,分布较窄,且具有一定的磁响应性,对布洛芬的吸附模型符合Langmuir吸附等温模型和二级动力学模型。随着PEI用量的增加,微球对布洛芬的吸附能力增强,经Langmuir吸附方程拟合的最大吸附量为138.63 mg/g。同时,微球具有良好的重复使用效率,重复5次后仍能达到初始吸附量的90%以上。     

Preparation of magnetite-loaded silica microspheres for solid-phase extraction of genomic DNA from soy-based foodstuffs

Solid-phase extraction has been widely employed for the preparation of DNA templates for polymerase chain reaction (PCR)-based analytical methods. Among the variety of adsorbents studied, magnetically responsive silica particles are particularly attractive due to their potential to simplify, expedite, and automate the extraction process. Here we report a facile method for the preparation of such magnetic particles, which entails impregnation of porous silica microspheres with iron salts, followed by calcination and reduction treatments. The samples were characterized using powder X-ray diffractometry (XRD), scanning electron microscopy (SEM), nitrogen adsorption/desorption isotherms, and vibrating sample magnetometry (VSM). XRD data show that magnetite nanocrystals of about 27.2 nm are produced within the pore channels of the silica support after reduction. SEM images show that the as-synthesized particles exhibit spherical shape and uniform particle size of about 3 μm as determined by the silica support. Nitrogen sorption data confirm that the magnetite-loaded silica particles possess typical mesopore structure with BET surface area of about 183 m²/g. VSM data show that the particles display paramagnetic behavior with saturation magnetization of 11.37 emu/g. The magnetic silica microspheres coated with silica shells were tested as adsorbents for rapid extraction of genomic DNA from soybean-derived products. The purified DNA templates were amplified by PCR for screening of genetically modified organisms (GMOs). The preliminary results confirm that the DNA extraction protocols using magnetite-loaded silica microspheres are capable of producing DNA templates which are inhibitor-free and ready for downstream analysis.     

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.     

Triphenylamine-functionalized magnetic microparticles as a new adsorbent coupled with high performance liquid chromatography for the analysis of trace polycyclic aromatic hydrocarbons in aqueous samples

Triphenylamine (TPA)-functionalized magnetic microspheres (Fe(3)O(4)/SiO(2)/TPA) were prepared and applied as solid phase extraction (SPE) adsorbents for the analysis of polycyclic aromatic hydrocarbons (PAHs) in environmental samples in combination with high-performance liquid chromatography (HPLC). The magnetic solid-phase extraction (MSPE) conditions affecting the extraction efficiency were optimized, including elution solvent, standing time, amount of sorbent, and salt concentration. Due to the strong π-π conjugate effect between the benzene rings of TPA and PAHs, high extraction efficiency was achieved with spiked recoveries of 80.21-108.33% and relative standard deviations (RSD) of less than 10%. Good linearities (R(2) > 0.997) for all calibration curves were obtained with low limits of detection (LOD) of 0.25, 0.5, 0.5, 3.75, 0.2 and 0.04 ng L(-1) for anthracene, fluoranthene, pyrene, chrysene, benzo[b]fluoranthene and benzo[k]fluoranthene, respectively. The achieved results indicate the applicability of Fe(3)O(4)/SiO(2)/TPA as MSPE adsorbents.     

Toward a microchip-based solid-phase extraction method for isolation of nucleic acids

A silica-based solid-phase extraction system suitable for incorporation into a microchip platform (nu-total analytical system; nu-TAS) would find utility in a variety of genetic analysis protocols, including DNA sequencing. The extraction procedure utilized is based on adsorption of the DNA onto bare silica. The procedure involves three steps: (i) DNA adsorption in the presence of a chaotropic salt, (ii) removal of contaminants with an alcohol/water solution, and (iii) elution of the adsorbed DNA in a small volume of buffer suitable for polymerase chain reaction (PCR) amplification. Multiple approaches for incorporation of this protocol into a microchip were examined with regard to extraction efficiency, reproducibility, stability, and the potential to provide PCR-amplifiable DNA. These included packing microchannels with silica beads only, generating a continuous silica network via sol-gel chemistry, and combinations of these. The optimal approach was found to involve immobilizing silica beads packed into the channel using a sol-gel network. This method allowed for successful extraction and elution of nanogram quantities of DNA in less than 25 min, with the DNA obtained in the elution buffer fraction. Evaluation of the eluted DNA indicated that it was of suitable quality for subsequent amplification by PCR.     

Preparation of flower-like molybdenum disulfide for solid-phase extraction of N-nitrosoamines in environmental water samples

In this paper, a flower-like molybdenum disulfide material was prepared by hydrothermal method and was first used as adsorbents in the solid-phase extraction process for enriching N-nitrosoamines. Molybdenum disulfide exhibited three-dimensional petal-like microspheres with about 500 nm in diameter. The relevant analyte extraction and elution parameters (sample volumes, solution pH, washing solvents, elution solvents, and elution volumes) were optimized to improve the solid-phase extraction efficiency. The solid-phase extraction process coupled with high-performance liquid chromatography-tandem mass spectrometry for determining N-nitrosoamines in environmental water samples was established. The limits of detection were in the range of 0.01–0.05 ng/mL. The satisfactory recoveries (68.9–106.1%) were obtained at three different spiked concentrations (2, 5, and 8 ng/mL) in water samples, and the relative standard deviations were between 1.96 and 8.38%. This proposed method not only showed high sensitivity and good reusability but also provided a new adsorbent for enriching trace N-nitrosoamines in environmental water samples.     

Isolation of microbial DNA by newly designed magnetic particles

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) microspheres and cobalt ferrite nanoparticles modified with alginic acid (natural carboxylic polysaccharide) were used for isolation of microbial DNA of lactic acid bacteria (LAB) from dairy products, lyophilised cell cultures, and bacterial colonies grown on hard media, and Trichophyton fungi DNA from lyophilised cells. DNA from the samples with lysed cells was reversibly adsorbed to the particles in the presence of high poly(ethylene glycol) (PEG 6000) and sodium chloride concentrations. The optimal final PEG and NaCl concentrations were 9.1 wt.% and 2.0 M, respectively. The adsorbed DNA was released from the particles in low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification. Moreover, PCR amplicons were isolated on cobalt ferrite nanoparticles modified with alginic acid and checked by restriction analysis.     

Cetylpyridinium chloride functionalized silica‐coated magnetite microspheres for the solid‐phase extraction and pre‐concentration of ochratoxin A from environmental water samples with high‐performance liquid chromatographic analysis

A new method based on cetylpyridinium chloride coated ferroferric oxide/silica magnetic microspheres as an efficient solid‐phase adsorbent was developed for the extraction and enrichment of ochratoxin A. The determination of ochratoxin A was obtained by high‐performance liquid chromatography with fluorescence detection. In the presence of cetylpyridinium chloride, the adsorption capacity of ferroferric oxide/silica microspheres was 5.95 mg/g for ochratoxin A. The experimental parameters were optimized, including the amounts of ferroferric oxide/silica microspheres (20 mg) and cetylpyridinium chloride (0.18 mL, 0.5 mg/mL), pH value of media (9), ultrasonic time (5 min), elution solvent and volume [2(1 + 1) mL (washed twice, 1 mL each time) 1% acetic acid acetonitrile]. Under optimal experiment conditions, ochratoxin A had good linearity in the range of 2.5–250.0 ng/L in water samples with correlation coefficient of the calibration curve 0.9995. The limit of detection for ochratoxin A was 0.83 ng/L, and the recoveries were 89.8–96.8% with the relative standard deviation of 1.5–3.5% in environmental water samples. Furthermore, ferroferric oxide/silica microspheres show excellent reusability during extraction procedures for no less than six times.     

Isolation of polymerase chain reaction-ready bacterial DNA from Lake Baikal sediments by carboxyl-functionalised magnetic polymer microspheres

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (P(HEMA-co-EDMA)) microspheres were used for the isolation of polymerase chain reaction (PCR)-ready DNA from samples of Baikal sediments. DNA was isolated using the phenol extraction method or the Soil DNA Isolation Kit. The occurrence of false-negative results in PCR caused by the presence of extracellular inhibitors in DNA samples was solved using solid phase reversible DNA immobilisation. PCR-ready DNA was reversibly adsorbed to the microspheres in the presence of 8.0% (w/v) poly(ethylene glycol) (PEG 6000) and 2.0M sodium chloride concentrations. The adsorbed DNA was released from the microspheres in a low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification.     

Precipitation polymerization in acetic acid: synthesis of monodisperse cross-linked poly(divinylbenzene) microspheres

This paper reports two important results with cross-linked precipitation polymerization. (1) Acetonitrile, a substance harmful to human health, is the most commonly used solvent for the synthesis of cross-linked polymeric microspheres by precipitation polymerization. Here, the much safer acetic acid replaced acetonitrile as a solvent in the precipitation polymerization of monodisperse cross-linked poly(divinylbenzene) (PDVB-55) microspheres. Pumpkin-like particles and microspheres were obtained. XPS results displayed a significant amount of double bonds on the surface of the particles. The effect of monomer content, temperature, and initiator amount on the formed particles were studied. For a DVB loading below 1 vol % at 70 degrees C, monodisperse microspheres with smooth surfaces and narrow diameters were successfully obtained. With a DVB loading of 2 vol % and by observing the shapes of particles obtained with three different temperature(60, 70, and 80 degrees C), we found that more spherical particles were obtained at higher temperatures and pumpkin-like particles were obtained at lower temperatures. No significant differences in morphology or the coefficient of variation (CV) of the particles were obtained for different initiator loadings, whereas the particle diameters could be increased with increased initiator concentrations. (2) In order to obtain a better understanding of the formation mechanism of these particles, time-dependent experiments, for the first time, were conducted in a hydrophobic monomer system. By tracing the whole polymerization process, some important results were found. First, with the polymerization time at 70 degrees C, the particle diameters were found to increase from 800 nm to 3.0 microm, the CV displayed a decrease, and the amount of spheres and the spherical evenness of the particle surfaces improved. Second, by quantitatively calculating the particle number from the yields and diameters data, it is found that starting from 3.1% yield or two hours reaction time the total amount of particles in the system is almost a constant (about 9.6 x 10 (8)/L), which means that no homocoagulation occurred and no new particles were generated after nucleation, and there is a linear relation between cubic diameters and yields. These two results give us a distinct impression that particle growth almost comes from capturing of newly formed oligomers. Based on the above results, a scheme for the particle formation is proposed, which shows that that pumpkin-like particles are caused by a prolonged nucleation including the homocoagulation of primary nuclei. The growth of the particles includes two modes, an in situ surface polymerization of monomer and the adsorption of PDVB-55 oligomers. The differences between results in acetonitrile and in acetic acid (higher yields, smaller size, not spherical but pumpkin-like particles in acetic acid) were due to the lower solubilizability of acetic acid which is the so-called proton-containing solvent with the hydrogen bonding structure.