新型除草剂双甲胺草磷(H-9201)在胡萝卜和土壤中的残留动态研究

建立了新型除草剂双甲胺草磷(H-9201)残留GC FPD检测方法,并对其在胡萝卜田的残留动态进行了研究.结果表明:植物样本的平均添加回收率为89.2%～101.8%,变异系数为:4.3%～5.0%,土壤中的平均添加回收率为90.8%～95.1%,变异系数为:3.0%～16.4%.本方法对双甲胺磷的检出限为:胡萝卜中为0.01 mg/kg,土壤中为0.005 mg/kg.残留动态研究表明:双甲胺草磷土壤中的半衰期为27.8 d,胡萝卜中的最终残留量＜0.02 mg/kg,土壤中的最终残留量＜0.4 mg/kg.     

新城疫病毒流式微球免疫检测新方法

本研究基于新城疫病毒多克隆抗体的制备及优化,以聚苯乙烯微球作为蛋白质载体建立免疫检测体系,建立了快速检测新城疫病毒的流式细胞术新方法。以藻红蛋白荧光染料对新城疫多克隆抗体荧光标记,取1μL荧光微球与100μL新配制的单克隆抗体溶液反应,依次加入一定量待测抗原和藻红蛋白标记的多克隆抗体,漩涡振荡10s,室温振荡反应充分,形成双抗体夹心复合物,用流式细胞仪进行检测。采用ELISA法对免疫试剂进行了匹配性筛选实验,优选了试剂用量,并与传统ELISA方法进行了对比分析。实验结果表明,单克隆抗体350mg/L、多克隆抗体300mg/L时可获得最佳分析效果,与传统的ELISA方法呈现出良好的相关性,不与鸡传染性支气管炎病毒、鸡痘病毒、鸡马立克氏病病毒等发生交叉反应。     

对硫磷化学发光酶联免疫吸附分析方法的建立和评价

建立了基于多克隆抗体的对硫磷间接竞争化学发光酶联免疫吸附分析方法(icCLEIA)。以三氯硫磷为原料,经三步取代反应合成对硫磷半抗原,通过活泼酯法将半抗原分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,制备免疫抗原和包被抗原。经免疫新西兰大白兔,获得对硫磷抗血清。通过优化条件参数,建立了对硫磷的icCLEIA分析方法。本方法的检测线性范围为0.24～15.83!g/L;半抑制浓度IC50为1.14!g/L;检出限为0.09!g/L;对蔬菜样品和水样品的平均添加回收率为74.6%～121.0%。本方法可用于实际样品中痕量对硫磷残留检测。     

胶体金免疫层析法对组织样品中磺胺甲嗯唑残留的快速检测

研究了用于快速检测组织样品中磺胺甲嗯唑残留的胶体金免疫层析检测试剂。采用免疫竞争法,将抗磺胺甲嗯唑多克隆抗体-胶体金复合物包被在胶体金结合垫上,同时将人工合成的磺胺甲嗯唑抗原包被在硝酸纤维素薄膜表面作为检测线（T线）,将抗磺胺甲嗯唑多克隆抗体的羊抗兔二抗包被在硝酸纤维素薄膜表面作为质控线（C线）。T线的人工抗原与待测样品中的磺胺甲嗯唑竞争结合胶体金标记的磺胺甲嗯唑多克隆抗体,通过T线与C线的显色对比读出结果。采用该检测试剂检测组织试样时,定量下限可达20μg／L,整个检测过程只需3—5min,且与磺胺嘧啶、磺胺二甲基嘧啶、盐酸克伦特罗、四环素、青霉素、链霉素无交叉反应。检测试剂具有较高的灵敏度及特异性,操作便捷,稳定可靠,可作为组织中磺胺甲嗯唑残留现场监控的有效筛检手段。     

胶体金免疫层析法对组织样品中磺胺甲噁唑残留的快速检测

研究了用于快速检测组织样品中磺胺甲噁唑残留的胶体金免疫层析检测试剂.采用免疫竞争法,将抗磺胺甲噁唑多克隆抗体-胶体金复合物包被在胶体金结合垫上,同时将人工合成的磺胺甲噁唑抗原包被在硝酸纤维素薄膜表面作为检测线(T线),将抗磺胺甲噁唑多克隆抗体的羊抗兔二抗包被在硝酸纤维素薄膜表面作为质控线(C线).T线的人工抗原与待测样品中的磺胺甲噁唑竞争结合胶体金标记的磺胺甲噁唑多克隆抗体,通过T线与C线的显色对比读出结果.采用该检测试剂检测组织试样时,定量下限可达20 μg/L,整个检测过程只需3 ～5 min,且与磺胺嘧啶、磺胺二甲基嘧啶、盐酸克伦特罗、四环素、青霉素、链霉素无交叉反应.检测试剂具有较高的灵敏度及特异性,操作便捷,稳定可靠,可作为组织中磺胺甲噁唑残留现场监控的有效筛检手段.     

粘着斑激酶的原核表达、纯化及抗体的制备

粘着斑激酶(focal adhesion kinase,FAK)是细胞质内单亚基非受体型酪氨酸激酶,通过各种信号途径参与调节细胞生长、发育、黏附、细胞骨架重组、转化、扩散和迁移等过程.采用PCR方法,从Flag-FAK质粒中克隆编码FAK C端273个氨基酸的基因片段,构建FAK融合蛋白原核表达载体pET28a( )/FAK,进行原核表达与蛋白纯化,取纯化的FAK蛋白免疫小鼠,制备FAK抗血清.结果表明构建的表达FAK C端功能结构域的原核表达质粒pET28a( )/FAK,经过BL21(DE3)大肠杆菌表达、镍亲和层析柱纯化,获得相对分子质量约33 kDa的融合蛋白,并利用小鼠制备了多克隆抗体,EL ISA检测显示该抗体有较高效价.荧光免疫结果显示此多克隆抗体与FAK蛋白特异性结合,为进一步研究神经细胞中FAK的作用机制奠定了基础.     

液相色谱法测定抗体与对硫磷、异丙威及其竞争原的吸附常数

为建立一种测定抗体与小分子化合物吸附性能的色谱分析方法,本研究设计合成了5种对硫磷半抗原和2种异丙威半抗原,将半抗原P1和A1与同一载体蛋白偶联制备"多簇"免疫抗原,免疫动物获得相应的多克隆抗体;采用高效液相色谱结合固相吸附的方法,测定了抗体与对硫磷、异丙威及其竞争原的吸附性能,并通过吸附动力学模型拟合抗体与化合物的吸附行为,获得相应的吸附常数。结果表明抗体与小分子化合物的吸附动力学符合Freundilich模型,抗体与不同化合物的吸附常数为:对硫磷>P1>P4>P3>P2>P5,大小顺序与ELISA亲和常数的测定结果相符,与ELISA灵敏度测定结果有很好的相关性。     

一种检测二恶英的单链抗体方法

本发明公开了一种检测二恶英的单链抗体方法。该单链抗体基因来源于小鼠，克隆到表达载体pET20b，在大肠杆菌BL21中表达，利用表达蛋白上带有的His-tag标记可通过亲和层析纯化单链抗体蛋白，并利用C-myc标记检测单链抗体蛋白。本发明克隆到鼠源的抗二恶英类化学物质的单链抗体，该抗体能应用于检测二恶英类化学物质，检测成本低，应用前景广泛。     

甲氧基有机磷杀虫剂广谱特异性抗体的制备

以通用结构O,O-二甲基硫代磷酸酯为目标检测基团,制备针对甲氧基有机磷杀虫剂的广谱特异性抗体。利用O,O-二甲基硫代磷酸钠和氯乙酸合成半抗原S-羧甲基-O,O-二甲基二硫代磷酸酯(CMP),通过混合酸酐法(MA)和活性酯法(AE)分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联。CMP-MA-BSA、CMP-AE-BSA作为免疫原均获得了免疫应答。其中,CMP-AE-BSA所获得的抗血清效价最高,为256000。研究了有机溶剂种类及含量、pH因素对ELISA曲线的影响,确定CMP酶联免疫分析方法(ELISA)的最佳工作条件,CMP的最低检测浓度为0.076μg/L,IC50为93.97μg/L。以14种常见有机磷杀虫剂为对象,检测抗体对其交叉反应,测定结果表明:抗体对马拉硫磷、稻丰散、乐果、亚胺硫磷、倍硫磷、甲基嘧啶磷、甲基对硫磷、杀螟硫磷及杀扑磷等均有识别作用。IC50分别为69.92、136.90、230.39、416.84、508.57、510.38、607.21、835.30和850.21μg/L。该技术可用于多种甲氧基有机磷杀虫剂的快速定性或半定量检测。     

精吡氟禾草灵酶联免疫吸附分析方法研究

建立了定量测定精吡氟禾草灵的间接竞争酶联免疫吸附分析方法(ic-ELISA)。将精吡氟禾草灵在碱性条件下水解制得半抗原,再将半抗原与蛋白质偶联形成抗原后免疫新西兰大白兔,获得多克隆抗体。对其进行条件优化后,得到了精吡氟禾草灵的ic-ELISA方法的标准曲线。该方法的Ic50为0.553mg/L。检出限为0.0062mg/L。方法对其他芳氧苯氧基丙酸酯类除草剂没有明显交叉反应。精吡氟禾草灵在样品中的回收率为86.80%～103.41%,变异系数为3.96%～12.32%。表明本研究建立的精吡氟禾草灵的ELISA方法符合农药残留分析的要求。     

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin B1 in corn samples: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 levels in corn samples, enforced by European Union legislation. A test portion was extracted with methanol-water (80 + 20); the extract was filtered, diluted with phosphate-buffered saline solution, filtered on a microfiber glass filter, and applied to an immunoaffinity column. The column was washed with deionized water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase LC with fluorescence detection after either pre- or postcolumn derivatization. Precolumn derivatization was achieved by generating the trifluoroacetic acid derivative, used by 8 laboratories. The postcolumn derivatization was achieved either with pyridinium hydrobromide perbromide, used by 16 laboratories, or with an electrochemical cell by the addition of bromide to the mobile phase, used by 5 laboratories. The derivatization techniques used were not significantly different when compared by the Student's t-test; the method was statistically evaluated for all the laboratories. Five corn sample materials, both spiked and naturally contaminated, were sent to 29 laboratories (22 Italian and 7 European). Test portions were spiked with aflatoxin B1 at levels of 2.00 and 5.00 ng/g. The mean values for recovery were 82% for the low level and 84% for the high contamination level. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the values for relative standard deviation for repeatability (RSDr) ranged from 9.9 to 28.7%. The values for relative standard deviation for reproducibility (RSDR) ranged from 18.6 to 36.8%. The method demonstrated acceptable within- and between-laboratory precision for this matrix, as evidenced by the HorRat values.     

Dynamic ultrasound-assisted extraction coupled on-line with solid support derivatization and high-performance liquid chromatography for the determination of formaldehyde in textiles

An on-line method was developed for the extraction, derivatization and determination of formaldehyde in textile samples. Formaldehyde was first extracted with water by ultrasound assisted, and directly introduced into a derivatization column which was packed with a moderately sulfonated cation-exchange resin. The resin used as solid support for the derivatization was charged with 2,4-dinitrophenylhydrazine (DNPH) previously. The formaldehyde DNPH derivative was eluted with the chromatographic mobile phase into an analytical column for the separation, and then monitored by UV detector. The maximum extraction yield was achieved when the extraction vessel was located at 10mm from the ultrasonic source and 10mg textile sample was extracted with 5mL pure water at a flow rate of 1.0mLmin(-1) at 50 degrees C. The detection limit of the proposed method was 0.06mgkg(-1). This method was applied to the determination of formaldehyde in different textile samples, and compared with the state standard method (off-line spectrophotometry) used in China. The similar contents of formaldehyde were obtained for most samples by the two methods, but little higher for some samples obtained by the proposed method. The average relative standard deviation (RSD) obtained by the on-line method was 3.2% which is lower than 29.5% obtained by the standard method.     

胡椒酸己二胺的合成

以胡椒碱为起始原料,与KOH反应生成胡椒酸钾盐,经酸化后制得胡椒酸(2); 2与乙醇经酯化反应制得胡椒酸乙酯(3); 3与己二胺在金属钠催化下经氨解反应合成了胡椒酸己二胺(4),其结构经¹H NMR, ¹³C NMR和IR确证。合成4的最佳反应条件为：催化剂用量2 wt%, n(3):n(己二胺)=0.6, m(乙醇):m(3)=2.5,于65～75 ℃反应48 h,收率83.5%。该工艺在微型实验基础上放大十倍,收率大于79%。     

气相色谱法测定婴幼儿配方谷粉中的碘

建立婴幼儿配方谷粉中碘的气相色谱定量分析方法。样品经α-淀粉酶水解,利用乙酸锌和亚铁氰化钾作为沉淀剂将样品中的蛋白质除去,在硫酸环境中把碘离子氧化成碘,碘与丁酮反应生成3-碘代-2-丁酮,用正己烷萃取,气相色谱法定量检测。碘的含量在0.04~0.48μg/m L范围内与色谱峰面积呈现良好的线性关系,线性相关系数r=0.998 2,方法的检出限为90μg/kg。测定结果的相对标准偏差为3.49%(n=6),样品加标回收率为86.2%~95.6%。该方法具有较高灵敏度和准确度,适用于婴幼儿配方谷粉中碘含量的检测。     

Estimation of the electron-collision initiation rate constant for the plasma polymerization and deposition of ethane using a transport-based model

The plasma polymerization and deposition of ethane in a parallel-plate electrode system was predicted using gas-phase transport equations and published gas-phase kinetic rate constants, with the assumption that the deposition rate was limited by the production of monomer free radicals and their diffusion to the deposition surface. The kinetic rate constant for radical initiation by electron collision with ethane was adjusted to bring the modelpredictionsfor mass deposition into line with experimental measurements reported in the literature for 12 combinations of power, gas fowrate, and pressure. Furthermore, the shapes of the predicted deposition profiles were made to closely match the experimental profiles by introducing an adjustable parameter to account for plasma boundary effects and two coefficients to account for nonradical polymerization (constant for all conditions). The best-fit electron-collision initiation constant was compared with theoretical predictions for oxygen and hydrogen in a manner explainable by bond strengths and vibrational degrees of freedom.     

硅胶自环己烷溶液中吸附苯甲酸和苯的计量置换吸附模型

在293~313 K温度范围, 研究了硅胶在环己烷溶液中对苯甲酸和苯的吸附. 发现苯甲酸能非常好地服从计量置换吸附模型(SDM-A). 在用SDM-A处理苯的吸附时, 出现折线形的吸附等温线, 折线的转折点正好是单分子层吸附与多分子层吸附的分界点. 基于SDM-A, 研究了吸附热力学, 建立了吸附热力学的计算公式. 发现在环己烷溶液中苯甲酸被硅胶吸附是自发的、放热的熵增大过程, 而苯被吸附是自发的放热的熵减少过程, 苯甲酸的吸附自由能大于苯, 而吸附焓小于苯, 这是因为苯甲酸有更大的亲吸附剂作用和疏溶剂作用的结果.     

PURIFICATION AND KINETICS OF MOUSE LIVER FRUCTOSE 6-PHOSPHATE, 2-KINASE

The mouse liver fructose 6-phosphate, 2-kinase was purified by ultracentrifugation, polyethylene glycol precipitation, and subsequently by chromatography on DEAE-Sephadex, Blue-Sepharose and phasphocellulose columnS. Gel filtration and SDS polyacrylamide electrophorcsis showed that the enzyme has a molecular weight of 110,000 with two identical subunits. Mg~(2+) is essential for its activity. The activation of the enzyme by Mg~(2+) showed a positive cooperativity. The substrate saturation curve for fructose 6-phosphate was sigmoidal and for ATP was hyperbolic. The K_m's for ATP increased with decrease in concentrations of fructose 6-phosphate indicating that the sequence for the substrates binding was in an ordered mechanism with respect to fructose 6-phosphate prior to ATP. An ionizable residue at the active site with pKa 9.5 was essential for the ATP binding and the pKa shifted to 9.8 after the binding of ATP.     

微波辅助提取-高效液相色谱法测定苦参中5种苦参生物碱的含量

取粒径在0.18～0.25mm的药材粉末于提取罐中,用水作提取剂,在110℃、压力为15MPa、功率为780W的条件下进行微波提取(PMAE)5min,经离心后取上清液为提取液。提取液经无水乙醇处理提纯后得到待测物的甲醇溶液,从中分取部分溶液按选定条件用液相色谱-质谱(LC-MS)联用做定性分析,用高效液相色谱(HPLC)做定量分析。LC-MS结果表明:提取液中含有氧化苦参碱、氧化槐果碱、槐定碱、苦参碱和槐果碱,与其它3种提取方法比较,PMAE的提取率高且提取时间短。HPLC定量时,采用C18色谱柱,以不同比例的甲醇、乙腈和0.1%氨水溶液(pH 10.3)的混合溶液作流动相进行梯度淋洗,可分离上述5种生物碱,以峰面积和浓度间的线性关系进行定量。5种生物碱的回收率在93.9%～101.0%之间,测定值的相对标准偏差(n=5)在0.53%～2.6%之间。     

气相色谱法测定烟气中拟除虫菊酯杀虫剂在卷烟滤嘴中的残留量

用符合ISO 30008标准要求的吸烟机按标准条件抽吸卷烟样品,抽吸完毕后,取下滤嘴,用乙酸乙酯-环己烷(1+1)混合溶剂超声提取30min。将提取液经无水硫酸钠干燥后旋转蒸至近干,残渣溶于正己烷2mL中,所得溶液经石墨炭黑小柱进行净化。小柱用丙酮-正己烷(2+8)混合溶剂淋洗2次,收集洗脱液,旋转蒸发浓缩后,将溶液用上述混合溶剂定容至1.5mL,供气相色谱分析,采用Agilent HP-5色谱柱进行分离和电子捕集检测。测定了4种拟除虫菊酯,相对标准偏差(n=5)均小于6%,于20个滤嘴样品中加入相当于20μg的4种拟除虫菊酯混合标准溶液,回收率均大于92%。     

泡沫塑料富集–石墨炉原子吸收光谱法测定贝类水产品中痕量铊

采取微波消解的前处理手段消解样品,经泡沫塑料分离富集后,用石墨炉原子吸收光谱法测定贝类水产品中痕量铊。以1.5 mL Fe3+,2 mL H2O2和5%王水介质作为吸附体系将样品中铊分离富集,再以硝酸钯、抗坏血酸作为基体改进剂进行测定。铊的质量浓度在0～50μg/L范围内线性良好,相关系数为0.999 7,方法的检出限可达0.07μg/g。测定结果的相对别准偏差为1.53%～4.01%(n=7),加标回收率为87.1%～98.3%。泡沫塑料富集–石墨炉子吸收光谱法测定贝类水产品中痕量铊是一种准确、安全、便捷的检测方法。