分子生物学手段检测转Bt基因棉花中Cry杀虫蛋白的定性和定量方法

建立了转Bt基因棉花中Cry杀虫蛋白的提取、样品前处理以及酶联免疫(ELISA)定量分析方法,并使用凝胶电泳、普通聚合酶链式反应(PCR)和实时荧光定量PCR等分子生物学手段对转基因棉花中的Bt基因进行定性和定量检测.所建立的苏云金芽孢杆菌杀虫晶体蛋白(Cry1Ab蛋白和Cry1Ac蛋白)标准曲线线性关系良好,相关系数r2均大于0.999,相对标准偏差RSD均小于2.0%.方法简单、快速、重现性和精密度好,可为农业食品行业和环境领域科研人员提供一种简便快速地从转基因棉花中检测Bt毒蛋白的分析方法.     

在线固相萃取-高效液相色谱法测定水体中的多环芳烃

建立了在线固相萃取-液相色谱测定水体残留的多环芳烃的方法,用于测定自来水中的20种多环芳烃( PAHs)。直接进样1 mL经过过滤的水体样品,其中的被测组分富集在SPE柱( Acclaim PA II,50 mm×4.6 mm,3μm)上,在线完成净化和萃取富集;再通过阀切换将它们转移至分析流路,在Hypersil Green PAH色谱柱(150 mm ×3 mm,3μm)上分离检测。在线固相萃取流路以水和乙腈为流动相,0.4和0.6 mL/min流速梯度富集/萃取和洗脱;分析流路亦以水和乙腈为流动相,0.8 mL/min流速梯度洗脱,采用紫外254 nm检测无荧光效应的苊烯和弱荧光效应的萘,其它的多环芳烃化合物则于不同的荧光检测通道里,在其对应的最大激发/发射波长下灵敏测定。整个分析流程32 min即可完成。20种PAHs的保留时间的相对标准偏差均小于0.2％,色谱峰面积的相对标准偏差均小于1.3％(n=7);在3个浓度数量级范围内峰面积与进样质量浓度的线性相关系数均大于0.9910,0.05μg/L的自来水加标样品的回收率为57％~140％,5μg/L的自来水加标样品的回收率为85％~116％;多数有荧光响应的PAHs的方法检出限均小于0.02μg/L (S/N=3)。     

铅镉重金属离子在铋膜电极上的相互影响

采用原位镀铋电极的方式,以玻碳电极为工作电极,通过改变Cd2+和Pb2+的比例,考察不同Pb2+和Cd2+浓度条件下,两者同时存在时的相互影响。实验发现,Cd2+和Pb2+同时存在时,存在相互的影响,特别是对于Cd2+的检测,由于Pb2+较正的析出电位,对于Cd2+的析出有一定的辅助作用。考察了Cd2+和Pb2+单独存在时的分析性能,Pb2+沉积时间为60 s时,在1~80μg/L范围内呈线性关系,检出限为0.5μg/L;Cd2+的沉积时间为120 s,在1~25μg/L和30~200μg/L范围内有良好的线性关系,检出限为1.0μg/L。考察了铋膜电极在不同实际水样中对Pb2+和Cd2+的分析,获得了较好的一致性。     

自动固相萃取-液相色谱-串联质谱法同时测定血液中5种抗抑郁类药物

建立了血液中5种抗抑郁类药物的自动固相萃取-液相色谱-串联质谱(ASPE-LC-MS/MS)分析方法。样品经HLB固相萃取柱提取后,采用LC-MS/MS进行测定,外标法定量。在Waters AtlantisTM dC18反相柱上分离,梯度洗脱,流动相为甲醇-0.1%甲酸(1∶4,V/V)溶液,质谱采集为电喷雾正离子多反应监测模式。采用基质匹配标准溶液校正,5种目标物在1~500μg/L范围内具有良好的线性关系,相关系数(R)大于0.9975,检出限在0.1~0.6μg/L之间;添加浓度水平为10,100和500μg/L时,方法回收率为70.6%~93.8%,相对标准偏差在3.9%~9.2%之间。本方法可用于血液中5种抗抑郁类药物的法庭与临床毒物分析。     

有机氯农药与肿瘤相关基因DNA间的作用机制研究

利用紫外可见(UV-vis)吸收光谱、荧光(FL)光谱和圆二色谱(CD)研究了3种有机氯农药[DDT,DDE和DDD]与人类肿瘤相关基因(p53 DNA和C-myc DNA)的相互作用,阐明有机氯农药的基因毒性。UV-vis和FL光谱实验表明,有机氯农药主要通过嵌插方式与DNA碱基作用,形成非共价复合物。通过FL实验得到的农药分子与p53 DNA的结合能力顺序为:DDE>DDT>DDD,对C-myc DNA为:DDD>DDE>DDT;并通过计算结合过程的热力学常数证实以疏水作用为主要的作用力。CD实验表明,部分有机氯农药能够影响DNA的碱基对堆积和二级结构,很可能进一步造成DNA损伤,并最终导致基因突变。     

基于核酸外切酶Ⅰ催化的核酸降解反应构建乳腺癌细胞电化学传感器

基于核酸外切酶I选择性消化单链核酸的特点,使用MCF-7细胞表面过量表达的肿瘤标志蛋白MUC1的适体,构建了一种灵敏检测乳腺癌细胞的新型电化学传感器。核酸适体链与乳腺癌细胞MCF-7表面过量表达的肿瘤标志蛋白MUC1的结合会阻碍其与互补核酸探针链的杂交,所以电极表面固定的未杂交的核酸探针单链就会被外切酶I选择性消化从而失去末端的亚甲基蓝信号分子。因此,通过检测电化学信号的变化,此传感器在103~106cell/mL细胞浓度范围内线性检测乳腺癌细胞MCF-7,检出限为330 cell/mL,具有高度特异性,可以有效区分对照细胞胰岛β细胞。     

氢化物发生与扇形磁场电感耦合等离子体质谱联用测定天然水中无机硒的价态

通过对仪器参数、质谱干扰、Se同位素选择、NaBH4浓度、样品酸度、Se?还原条件及氢化反应干扰的评估和优化,建立了自制连续流动氢化物发生装置与扇形磁场电感耦合等离子体质谱联用测定天然水中无机硒价态的分析方法,克服了天然水中多种元素对氢化反应的干扰,解决了不同介质样品中Se?还原率保持95%以上时间短的问题。方法检出限为0.002μg/L,灵敏度为215378 cps/(μg/L),优于同类方法,测定河水和海水中Se?和Se?精密度小于4%,加标回收率在97%~103%之间,可应用于准确测定海水、河口区半咸水、河水等天然水中Se?和Se?浓度。     

Synthesis,Crystal Structure and Anticoagulant Activity of 5-Chloro-N-[[（5S）-2-oxo-3-[4-（2- oxopyridin-1 （2H）-yl）phenyl] oxazolidin-5- yl] methyl] thiophene-2-carboxamide①

The title compound(zifaxaban 2, C20H16ClN3O4 S, Mr = 429.87) was synthesized and its crystal structure was determined by single-crystal X-ray diffraction. Zifaxaban crystallizes in monoclinic, space group P21 with a = 5.7900(12), b = 13.086(3), c = 12.889(3) A, β = 100.86(3)°, V = 959.1(3) A3, Z = 2, Dc = 1.489 g/cm3, F(000) = 444, μ = 0.342 mm-1, the final R = 0.0320 and wR = 0.0640 for 2717 observed reflections(I 2σ(I)). The absolute configuration of the stereogenic center in the title compound was confirmed to be S by single-crystal X-ray diffraction. Four existing intermolecular hydrogen bonds help to stabilize the lattice and the molecule in the lattice to adopt an L-shape conformation. Zifaxaban was slightly more active than rivaroxaban 1 in in vitro assay against human FXa and therefore is promising as a drug candidate.     

Syntheses and Crystal Structures of Homleptic Lanthanide Complexes [C_6H_5COCHC(CH_3)N-(p-ClC_6H_4)]_3Ln(THF)_n (Ln =Yb,Y,Nd)

Three homoleptic lanthanide complexes, [C6H5COCHC(CH3)N(p-ClC6H4)]3Ln(THF)n(n = 0, Ln = Yb(1); n = 0, Ln = Y(2); n = 1, Ln = Nd(3)), were synthesized by amine elimination reaction of Ln[N(SiMe3)2]3 with 1-phenyl-3-N-(p-chlorophenylimino)-1-butanone. These complexes crystallize in triclinic, space group P1 with a = 9.805(3), b = 14.831(6), c = 16.075(6) A, α = 111.996(9), β = 91.570(7), γ = 93.744(6)°, V = 2159.4(13) A3, Z = 2, D3 c = 1.515 g/cm, F(000) = 986, μ(MoKα) = 2.396 mm-1, R = 0.0360 and wR = 0.0850 for 9548 observed reflections with I 2σ(I) for complex 1; a = 9.861(5), b = 14.852(9), c = 16.111(9) A, α = 112.362(13), β = 91.949(11), γ = 93.678(14)°, V = 2173(2) A3, Z = 2, Dc = 1.377 g/cm3, F(000) = 924, μ(MoKα) = 1.570 mm-1, R = 0.0735 and wR = 0.1389 for 8015 observed reflections with I 2σ(I) for complex 2; and a = 9.308(3), b = 15.357(3), c = 17.419(4) A, α = 66.493(13), β = 88.61(2), γ = 86.664(19)°, V = 2279.4(9) A3, Z = 2, Dc = 1.499 g/cm3, F(000) = 1046, μ(MoKα) = 1.364 mm-1, R = 0.0843 and wR = 0.2280 for 8433 observed reflections with I 2σ(I) for complex 3. Each central metal in complexes 1 and 2 is six-coordinated by three nitrogen and three oxygen atoms from three β-ketoiminate ligands to give a distorted octahedral geometry, while the central metal in 3 is seven-coordinated by three nitrogen and three oxygen atoms from three β-ketoiminate ligands and one oxygen atom from the solvated THF molecule to complete a distorted monocapped trigonal prism.     

Purification and Characterization of the Glucoside 3-Dehydrogenase Produced by a Newly Isolated Sphingobacterium faecium ZJF-D6 CCTCC M 2013251

A soluble glucoside 3-dehydrogenase (G3DH) was purified from a newly isolated Sphingobacterium faecium ZJF-D6 CCTCC M 2013251. The enzyme was purified to 35.71-fold with a yield of 41.91 % and was estimated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis with a molecular mass of 62 kDa. The sequences of two peptides of the enzyme were all contained in a GMC family oxidoreductase (EFK55866) by mass spectrometry analysis. The optimal pH of the enzyme was around 6.2. The enzyme was stable within a pH range of 5.0–6.6 and was sensitive to heat. G3DH from S. faecium exhibited extremely broad substrate specificity and well regioselectivity to validoxylamine A. The enzyme was completely inhibited by Hg₂Cl₂ and partly inhibited by Cu²⁺, Fe²⁺, Ca²⁺, and Cd²⁺. The apparent K _m values for D-glucose, sucrose, and validoxylamine were calculated to be 1.1, 1.7, and 2.1 mM, respectively. With this purified enzyme, 3-keto sucrose was prepared at pH 5.0, 30 °C for 10 h with a yield of 28.7 %.