禽流感病毒流式微球量子点探针免疫诊断新方法

采用微波法水相中合成羧基化的绿色量子点,通过羧基与禽流感单克隆抗体氨基的共价结合,制备了检测禽流感病毒的探针,并结合流式微球技术,建立了量子点生物探针流式微球免疫检测禽流感病毒的新方法.以聚苯乙烯微球为蛋白质载体,将多克隆抗体包被到荧光微球上,依次加入待测抗原和量子点生物探针,形成双抗体夹心复合物,用流式细胞仪进行检测.实验结果表明,多抗和单抗的最佳质量浓度分别为92和4 mg/L,检测禽流感病毒比双抗体夹心ELISA灵敏16倍,比FITC标记单抗检测方法灵敏4倍.对阳性尿囊液的检测与ELISA呈现良好的相关性,不与鸡传染性支气管炎病毒、鸡马力克氏病毒、新城疫病毒等发生交叉反应.     

基于噬菌体展示抗体的电化学发光免疫传感器检测相思子毒素研究

以多克隆抗体包被磁性微球制备捕获探针,以表面负载大量三联吡啶钌标记物的噬菌体展示抗体为发光探针有效放大信号,成功建立了相思子毒素电化学发光免疫传感检测方法。利用本方法检测相思子毒素,浓度在0.005～100μg/L范围内与电化学发光强度呈良好的对数线性关系,拟合方程为lgY=0.701lgX+1.767(R=0.9964,N=7,p<0.0001),检测限达0.005μg/L(S/N=3)。与采用单克隆抗体制备标记探针相比,检测灵敏度提高20倍,可用于相思子毒素的痕量检测。     

量子点荧光微球免疫层析试纸条定量检测恶性疟原虫

以羧基化Cd Te/Zn Se量子点荧光微球为荧光标记物,采用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)法偶联抗恶性疟原虫富组氨酸蛋白(Pf)单克隆抗体制备荧光探针;以羊抗恶性疟原虫组氨酸多克隆抗体和驴抗鼠二抗分别喷涂硝酸纤维膜,形成试纸条检测线和质控线,建立了免疫层析试纸条定量检测血清中恶性疟原虫的方法。所使用的羧基化量子点荧光微球的荧光强度为单个量子点的2800倍。实验结果表明,该荧光试纸条定量检测血清中恶性疟原虫线性范围为5.8~8010 Parasite/μL,最低灵敏度达到5.8 Parasite/μL,单个样品检测时间只需15 min。加标回收实验显示,试纸条批内回收率为93.0%~111.8%,批间回收率为98.3%~115.1%,且批内、批间的相对标准偏差均小于5%。     

液相芯片技术同时检测莱克多巴胺、盐酸克伦特罗和沙丁胺醇

建立了一种同时检测兽药莱克多巴胺(RAC)、克伦特罗(CL)、沙丁胺醇(SAL)的多残留的新方法.采用液相悬浮芯片技术,基于间接竞争法的原理,将3种兽药抗原分别偶联到不同的荧光微球上作为探针,以藻红蛋白(PE)荧光标记二抗为信号,通过优化实验条件,分别建立3种兽药的标准曲线.基于特异性识别实验,建立3种兽药同时检测的标准曲线.结果表明,同时检测的3条曲线均呈现良好的线性相关,线性相关系数(R2)均大于0.99,RAC,CL和SAL的检测范围分别为1～ 500 μg/L,0.1～500μg/L,1～100μg/L,检出限分别为0.68,0.095和0.88 μg/L.与其它结构类似物的交叉反应率均低于1.5％,实际样品中的加标回收率令人满意.     

基于免疫磁分离的荧光微球免疫层析法检测猪霍乱沙门氏菌

建立了基于免疫磁分离的荧光微球免疫层析法,检测猪霍乱沙门氏菌.待检样品经免疫磁分离富集和热洗脱处理后,用荧光微球免疫层析试纸条进行检测.每毫克纳米磁珠标记30μg抗体制备的免疫磁珠,对浓度为102 ～ 106 CFU/mL的猪霍乱沙门氏菌的捕获率均大于90％,特异性好;在pH=6时,以300μ,g/mg猪霍乱沙门氏菌单抗11D8-D4标记荧光微球,制备免疫荧光微球;以2.0 mg/mL猪霍乱沙门氏菌单抗5F11-B11喷涂检测线(T线),以1.0 mg/mL驴抗鼠IgG喷涂质控线(C线),制备免疫层析试纸条.采用建立的基于免疫磁分离的荧光微球免疫层析方法检测猪霍乱沙门氏菌,在PBS缓冲液中检出限为1.5×105 CFU/mL,牛奶中检出限为7.6×105 CFU/mL,与直接采用荧光微球免疫层析方法检测相比,检出限分别降低了10倍和200倍.本方法可有效富集牛奶中的沙门氏菌,避免了基质干扰,灵敏度大大提高,具有较好的应用前景.     

基于量子点微球免疫层析法快速灵敏检测鸡肉中甲氧苄啶

建立了一种快速灵敏检测甲氧苄啶的量子点微球免疫层析法,对pH、标记抗体浓度、试纸条检测线上的抗原浓度以及试纸条结合垫上的探针体积进行了优化。通过量子点微球试纸条检测仪读取试纸条上检测线和质控线的荧光信号强度,以甲氧苄啶的浓度为横坐标,检测线和质控线荧光信号强度的比值为纵坐标,建立标准曲线。结果表明,方法定量检测范围为1～32μg/L,半抑制率为3.3μg/L,回收率在99.8%～117.4%之间,与12种磺胺类药物均只有不到2%的交叉反应率。该方法适合大批量样品的现场筛查。     

两种基于RAJI细胞的抗CD20单抗新型定量方法的比较及在药物代谢动力学中的应用

建立和比较基于RAJI细胞的流式细胞法(Flow cytometry assay,FCA)和细胞放射免疫法(Immuno-radiametric assay,IRA)测定重组抗CD20人源化单克隆抗体(r-anti-CD20zumAb)的浓度.两种方法均利用标记后抗体和被检测抗体竞争性结合RAJI细胞表面的CD20抗原,由标记后抗体的荧光或放射性变化而间接反应检测抗体的浓度.FCA和IRA定量范围分别为0.1～100 mg/L和0.04～20 mg/L,FCA的日内及日间精密度分别小于4.0%和3.0%,准确度为-1.7%～1.1%,IRA的日内及日间精密度值均小于7.0%,准确度为-8.9%～13.2%.方法学确证研究表明,两种方法均具有良好的特异性、灵敏度、精密度和准确度.血浆样品分析结果显示,两种方法具有良好的一致性,是检测猕猴血浆中人源化单克隆抗体浓度的理想方法.     

转基因作物pat蛋白的双抗体夹心酶联免疫检测方法研究

将克隆到的pat基因构建原核表达载体pET30(+)-pat并在大肠杆菌中进行表达,分离纯化得到高纯度pat蛋白。用所得到的pat蛋白制备了兔多克隆抗体和鼠单抗2F6。多抗效价>8000,单抗效价为5×104;单抗为IgG1类,轻链为κ型。基于抗pat蛋白单克隆抗体和兔多克隆抗体建立了双抗夹心酶联免疫分析方法(Sandwich ELISA)。检测范围为1.6~100μg/L,线性回归方程y=0.6914x-2.572,决定系数为0.9951。用所建立的方法测定了5个转基因抗虫棉和2个非转基因棉花品种叶片中pat蛋白的含量,其中转基因抗虫棉中的3个品种检测出pat蛋白,其余均未检出pat蛋白。     

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。     

基于荧光硅球的克伦特罗快速定量免疫层析试纸条的研制

采用荧光硅球为标记物制备了快速定量检测克伦特罗(CLE)的荧光硅球免疫层析试纸条。通过正交实验得到最优荧光硅球抗体标记量、硅球垫抗体标记物用量以及检测线抗原浓度。在最优条件下,试纸条线性范围为0.28~3.3 mg/L。猪尿样品CLE加标回收率为81.7%~101%,表明此试纸条可实现猪尿中CLE残留的快速定量检测。     

α-Amino acid derived enaminones and their application in the synthesis of N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates and other heterocycles

A new and simple synthesis of novel N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates, which exist in equilibrium with their 4-oxo tautomers, has been developed in two steps starting from N-protected α-amino acids. The key intermediates are enaminones, which can also be isolated, characterized, and used for the construction of other functionalized heterocycles, before they spontaneously decompose to pyrrole products. 4-Hydroxypyrroles are prone to partial aerial oxidation but can be efficiently alkylated or reduced to stable polysubstituted pyrrolidine derivatives.     

Specific intramolecular aromatic CH insertion of diazosulfonamides

The chemoselectivity in the intramolecular CH insertion of various diazosulfonamides has been experimentally studied. The results reveal that the aliphatic 1,4-, 1,5-, or 1,6-C(sp³)?H insertions of diazosulfonamides are not accessible, while the aromatic 1,5-C(sp²)?H insertion can be realized specifically by adjusting the diazo-adjacent group. In addition, the general chemoselectivities in the intramolecular CH insertions of diazosulfonyl compounds are summarized. Generally, diazosulfones undergo both aromatic 1,5-C(sp²)?H and aliphatic 1,5- and 1,6-C(sp³)?H insertions, while diazosulfonates undergo aliphatic 1,5- and 1,6-C(sp³)?H insertions. However, diazosulfonamides only undergo aromatic 1,5-C(sp²)?H insertion.     

N-Heterocyclic carbene-palladacyclic complexes: synthesis,characterization and their applications in the C-N coupling and α-arylation of ketones using aryl chlorides

N-Heterocyclic carbene-palladacyclic complexes 3 were successfully achieved in a one-pot procedure under mild conditions. The structure of 3a was unambiguously confirmed by X-ray single crystal diffraction and it was an active catalyst in the Buchwald-Hartwig amination and α-arylation of ketones even at very low catalyst loadings (0.01?mol%).     

Iodine-mediated oxidative Pictet-Spengler reaction using terminal alkyne as the 2-oxoaldehyde surrogate for the synthesis of 1-aroyl-β-carbolines and fused-nitrogen heterocycles

An efficient iodine-mediated oxidative Pictet-Spengler reaction in dimethyl sulphoxide (DMSO) using terminal alkynes as the 2-oxoaldehyde surrogate for the synthesis of aryl (9H-pyrido[3,4-b]indol-1-yl)methanones is described. The scope of the protocol includes the total synthesis of Fascaplysin, Eudistomins Y₁ and Y₂. The methodology is extended for preparing pyrrolo[1,2-a]-quinoxaline and indolo[1,5-a]quinoxaline derivatives. The utility of 1-aroyl-β-carbolines was demonstrated by performing palladium-catalyzed β-carboline directed ortho-C(sp2)-H functionalization of the phenyl ring with thiomethyl (SMe) group using DMSO as source and for accessing 4-aryl-canthin-6-ones.     

Facile synthesis of bicyclic 1-arylpyrazol-5-ones

In this Letter, we described a facile method for constructing fused bicyclic 1-arylpyrazol-5-one ring system. We employed various methylene-containing carboxylic acids as the substrates and proved that the pyrazolone ring closure requires activated methylene group in intermediate II. Accordingly, a series of structurally diversified, fused bicyclic 1-arylpyrazol-5-ones was prepared in moderate to high yields using the requisite substrates.     

A carbanion induced synthesis of highly congested pyrazole and imidazole containing heterocycles

An efficient approach to the synthesis of highly congested di, penta and hexacyclic pyrazoles as well as imidazole fragment containing novel heterocyclic molecule has been developed through a carbanion induced transformation of suitably functionalized 2H-pyran-2-ones, benzo[h]chromene and thiochromeno[4,3-b]pyrans. Due to the presence of fluorescence, we report their prime application metal sensor as off/on switching in ferric ions.     

Synthesis of pyrrolo[1,2-a]pyrazines and pyrazino[1,2-a]indoles by Curtius reaction in Morita-Baylis-Hillman derivatives

Synthesis of substituted pyrrolo[1,2-a]pyrazines and pyrazino[1,2-a]indoles from the Morita-Baylis-Hillman derivatives of acrylates via saponification followed by Curtius reaction is described.     

正丁胺对激光染料荧光谱影响的研究

用正丁胺作为碳源,采用射频辉光放电制备碳膜,选用激光染料R6G和聚乙二醇混合液作为蒸气源,采用单源热蒸发,在蒸发室与染料同时沉积得到混合膜,用拉曼光谱和红外光谱分析了碳膜的结构和键合方式,分析表明:碳膜中存在胺基团和氢原子.混合膜的荧光谱测量结果表明,认为正丁胺对染料荧光谱的影响是因为胺基和氢原子的存在.     

Selective synthesis of 4,5-dihydroimidazo- and imidazo[1,5-a]quinoxalines via modified Pictet–Spengler reaction

An efficient tandem approach for the selective synthesis of 4,5-dihydroimidazo[1,5-a]quinoxalines 6a–g and imidazo[1,5-a]quinoxalines 7a–h by the reaction of 2-imidazolyl anilines 4a–c with aryl aldehydes 5a–k under mild reaction conditions is described. Introduction of electron releasing alkyl groups in substrates 4a–b was found to be instrumental for the success of the reaction.     

Understanding the Diels-Alder reactivity of 1,2-azaborine analogues

The Diels-Alder reactivity of 1,2-heteroborines (H₄C₄B(H)X, X?=?NH, PH, AsH; O, S, Se) has been computationally explored by means of Density Functional Theory (DFT) calculations. The influence of the HB?=?X fragment on the reactivity of the system has been quantitatively analyzed in detail by means of the so-called Activation Strain Model (ASM) of reactivity. It is found that the interaction between these species and the dienophile is significantly stronger than that computed for their all-carbon isoelectronic counterpart, benzene. In addition, the strain energy plays a key role in the observed reactivity trends. The role of the aromaticity strength of these heteroarenes on the reactivity is also assessed.