基于杂交链式反应信号放大和磁分离技术荧光检测端粒酶活性

端粒酶是由RNA和蛋白质组成的一种核糖核蛋白酶, 它一般在癌细胞中被激活. 它与端粒DNA的不断复制以及癌细胞的不断增殖密切相关. 所以检测端粒酶的活性对癌症的早期诊断以及以端粒酶为靶标分子的抗癌药物的开发具有重要意义. 利用杂交链式反应(HCR)无酶放大检测信号, 建立了一种简单、快速的端粒酶活性检测方法. 端粒酶延伸产物是一条末端具有(ggttag)_n重复序列的DNA. 在实验过程中, 通过链霉亲合素与生物素的特异性作用将端粒酶延伸产物连接在磁性微球上. 设计一条端粒酶延伸产物特异性的DNA探针I作为杂交链式反应的引发探针. DNA探针I的3'-端与端粒酶延伸产物的重复序列匹配, 通过杂交, DNA探针I被固定在磁球上; DNA探针I的5'-端引发DNA探针II和探针III发生杂交链式反应. DNA探针II和探针III上都标记有荧光基团, 可以利用荧光直接进行信号检测. 在反应过程中, 通过磁分离去除多余未反应的三种DNA探针. 在优化条件下, 可以检测到1.0×10⁵个Hela细胞中的端粒酶活性. 该方法简单、快速、检测成本低, 分析全程无酶参与, 在肿瘤或癌症的临床诊断以及以端粒酶为靶标分子的抗癌药物的筛选上具有广阔的应用前景.     

Taqman-MGB实时荧光PCR定量检测转基因玉米MON863

运用实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因玉米MON863进行品种特异性检测和定量分析。通过设计玉米内源基因和外源基因边界序列特异性引物和Taqman-MGB探针,验证了内源基因的物种特异性和外源基因边界序列的品种特异性。利用已知转基因百分含量的MON863玉米作为标准品,进行荧光定量反应,建立定量标准曲线,通过标准曲线对玉米样品MON863玉米成份进行含量分析。结果表明,该方法重复性好,检测特异性强,最低检测限浓度达到0.001 ng/μL,即14个拷贝。由于使用实时荧光PCR技术,检测周期短,操作简便,可广泛运用于转基因玉米MON863的进出口检测和转基因产品的含量分析。     

芝麻致敏原的实时荧光PCR检测

针对芝麻Sesamum indicum 2S albumin mRNA基因设计引物、探针,在实时荧光PCR仪上进行扩增、检测和分析.结果显示:该组探针和引物对芝麻有很强的特异性,除黑芝麻和白芝麻外,其余6种对照材料均未检测到荧光信号,黑白芝麻成分检测灵敏度达到0.1%.该方法具有灵敏度高、快速、简便的特点,可用于芝麻致敏原成分的定量检测.     

巴西果仁致敏原的实时荧光PCR检测

针对巴西果仁2Salbumin mRNA基因设计引物、探针,在实时荧光PCR仪上进行扩增、检测和结果分析.结果显示:该组探针和引物对巴西果仁有很强的特异性,除巴西果仁外,其余8种对照树果材料均未检测到荧光信号,巴西果仁成分检测灵敏度达到0.1%.该方法具有灵敏度高、快速、简便的特点,可用于巴西果仁致敏原成分的定量检测.     

基于引物延伸反应进行SNP基因分型的电化学方法

引物延伸反应的高特异性使其成为单核苷酸多态性(SNP)基因分型的最常用方法. 本文利用引物延伸反应, 通过二茂铁标记的dUTP将二茂铁引入到延伸的产物中, 用一条捕获探针将延伸产物捕获到电极表面, 用差分脉冲伏安法对电极表面的二茂铁进行检测, 从而实现了SNP基因分型. 考察了延伸反应的退火温度、聚合酶用量以及DNA杂交温度等因素的影响. 应用该方法对β-地中海贫血基因密码子28位单碱基突变进行检测, 获得了满意的基因分型结果. 该方法检测限可达到0.86 fmol/L, 是一种简便、快速且灵敏的SNP分型方法.     

基于菲并咪唑的ON⁃OFF⁃ON双比色荧光探针及细胞成像

设计合成了对称型菲并咪唑荧光探针PIP-ph-PIP, 并对其结构进行了表征和确认. 随着探针溶液中水体积分数的增加, 探针的聚集程度逐渐增加, 荧光强度先增强后猝灭. 荧光光谱测试结果表明, 在水相体系中探针PIP-ph-PIP能以ON-OFF-ON的方式分别对Ag⁺和SCN^-, Cu²⁺和PO³₄ ^-进行连续识别, 且连续识别效果可通过裸眼比色观察, 其中对Ag⁺识别具有超低检出限(6.1 nmol/L). 结果表明, 探针PIP-ph-PIP可应用于活体HeLa细胞中Ag⁺和Cu²⁺的定性分析及实际水样中Ag⁺和Cu²⁺的定量检测.     

牛奶α-乳白蛋白基因实时荧光定量PCR检测方法的建立

采用Taqman探针技术,建立食品中牛奶过敏原α-乳白蛋白基因的实时荧光定量PCR检测方法。根据α-乳白蛋白基因序列设计特异性引物及Taqman探针进行PCR扩增,构建质粒,经酶切鉴定测序后,建立拷贝数(copies)-Ct标准曲线。成功克隆α-乳白蛋白目的基因,建立的标准曲线在1.12×103~1.12×108 copies范围内线性关系良好,灵敏度高,液体样品检测限达到1 000copies/mL,特异性强,稳定性好。该法可用于实际商品中牛奶过敏原α-乳白蛋白组分的定量检测。     

TaqMan-分子灯标：一种新型的荧光基因检测探针

在TaqMan及分子灯标的基础上开发了一类新型的均相荧光检测探针—— TaqMan-分子灯标(TaqMan-MB)，该探针集合了分子灯标的发夹结构及TaqMan探针降 解作用的工作原理，使检测效果更好．与实时PCR仪联用，可用于靶基因的定量检 测．     

基于脱氧核酶的无标记端粒酶检测新方法

设计了一种发卡型核酸探针,结合脱氧核酶(DNAzyme)与支点介导链置换技术建立了一种检测端粒酶的新方法.该发卡型探针通过阻碍G-四链体的形成来抑制DNAzyme的过氧化物酶活性.当体系中的端粒酶引物TS被催化延伸后,可以通过链置换反应破坏该发卡结构,从而释放出自由的DNAzyme以催化过氧化氢氧化ABTS2-,产生可被检测的吸收信号变化.实验结果表明,应用该方法可以检测低至500个Hela细胞等当量的端粒酶,且该方法操作简单、不需要荧光标记和复杂的表面修饰,有望在肿瘤细胞端粒酶活性分析中获得广泛应用.     

检测亚硝酸根的近红外荧光探针的设计与合成

亚硝酸根(NO₂)浓度的失控对公共健康和环境的威胁日益严重。因此,开发一种简单有效地检测微量NO₂^-的方法是一个重要课题。本研究以双氰基异膦酮衍生物为荧光团,仲胺为识别单元,合成了一种近红外荧光探针(DIO),并将其用于NO₂的检测。实验结果表明,该探针在乙酸中具有强红色荧光(λ_em=670 nm),加入NO₂后,荧光强度大幅降低,在210 s内完成反应。探针可对不同浓度的NO₂进行定量检测,在0-1μmol·L^-1内具有很好的线性关系和较低的检出限(17 nmol·L^-1)。值得注意地是,当向红色的探针溶液中加入不同浓度的NO₂时,溶液颜色由橘红色逐渐变为黄色,使得该探针还可用于目视检测NO₂^-。此外,该方法对NO₂的检测具有较好的特异性,不受其他常见分析物的干扰。     

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.     

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.     

Cu0-catalysed 1,3-dipolar cycloadditions of α-amino acid derived N,N-cyclic azomethine imines to ynones

A series of 20 CuAIAC reactions between eight 4-acylamino substituted pyrazolidine-3-one-1-azomethine imines and four terminal ynones were performed using Cu⁰ as catalyst. The corresponding fluorescent cycloadducts were obtained in very high yields upon simple workup. Thus, Cu-metal turned out to be a better catalyst than Cu^I in terms of yield and ease of isolation. Availability of azomethine imines, mild reaction conditions, and simple workup enable a “click” access to libraries of densely substituted 2,3-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-ones. Reactivity of differently substituted dipoles was evaluated experimentally and by quantum chemical methods (DFT).     

Synthesis and antioxidative/anti-inflammatory activity of novel fullerene–polyamine conjugates

(E)-4-(Fullerenopyrrolidin-1-yl)-3-methylbut-2-enoic acid and its corresponding succinimidyl ester, readily obtained through Prato-type modification of C₆₀, were used for the selective N-acylation of polyamines. The thus obtained conjugates were evaluated for their antioxidative and anti-inflammatory activity and their cytotoxicity was determined. Members of this family of compounds showed interesting anti-lipid peroxidation, anti-lipoxygenase and anti-inflammatory activity and comparable cytocompatibility to spermidine.