利用无标记的分子信标及核酸染料SYBR Green Ⅰ检测乙肝病毒

本文以野生型的乙型肝炎病毒(HBV)核酸片段为研究对象,利用无标记的分子信标及核酸染料SYBR Green I,建立了一种高灵敏、高选择性的特定序列核酸检测方法.在优化条件下,目标DNA浓度为4×10-11~400×10-11 mol/L之间时,SYBR Green I的荧光强度(ΔI)与目标DNA的浓度(C)具有良好的线性关系,其拟合的回归方程为ΔI=1.9556 C+31.4659(R2=0.9956),方法检测限(3ζ)为2×10-11 mol/L.该方法操作简单、检测速度快、灵敏度高、重现性好、检出限低.利用该方法,结合不对称PCR技术,实现了对HBV的定量检测.     

基于双分子信标对猪圆环病毒Ⅱ型的简单快速检测

根据猪圆环病毒(PCV2)基因组为单链DNA的特点,设计了两条可与PCV2基因组序列特异性杂交的分子信标,建立了基于双分子信标法检测PCV2的方法。实验结果表明,双分子信标法比单分子信标法的灵敏度更高。在10 mmol/L MgCl2、20 mmol/L Tris-HCl(pH=8.0)的杂交缓冲溶液,40℃孵育30 min的优化检测体系下,此方法可实现对检测样本在2~200 nmol/L线性范围内的检测,检出限可达1 nmol/L。将双分子信标检测体系用于18例可疑猪瘟样品病毒检测,其中8例呈PCV2阳性,检测结果与PCR法一致,证明此双分子信标法可用于实际猪感染PCV2的诊断。     

血清样品中乙肝病毒的DNA电化学传感器检测

利用自组装单分子膜技术，将巯己基修饰的具有乙肝病毒(HBV)DNA序列特异性的单链DNA探针固定在金电极表面，制得DNA电化学传感器；以电活性的Hoechst 33258为指示剂，考察了该传感器对血清样品中乙肝病毒DNA的响应；探讨DNA电化学传感器在临床检测中的应用；将传感器法与荧光聚合酶链反应(PCR)法进行对比，两者的分析结果具有一致性。     

分子信标荧光探针用于抑癌基因ING1表达产物的定量测定

根据抑癌基因ING1基因的序列设计并合成了检测ING1转录产物的分子信标核酸探针,发展了一种快速测量从正常细胞系和鼻咽癌肿瘤细胞系提取的ING1转录产物的方法,所得结果与用逆转录结合PCR法(RT-PCR)得到的结果相吻合.并且,将能表达ING1基因的质粒转入肿瘤细胞进行培养后,再将分子信标转入肿瘤细胞,发现转导了质粒的肿瘤细胞比未转导质粒的肿瘤细胞内的荧光明显增强,从而进一步证实了所设计的分子信标核酸探针与ING1转录产物的结合.     

基于磁性颗粒微阵列与双色荧光杂交的单核苷酸多态性分型方法研究

基于磁性颗粒微阵列与双色荧光杂交,建立了单核苷酸多态性(Single nucleoitide polymorphism,SNP)分型方法。将利用不对称扩增得到的含有待检测位点生物素标记的单链PCR产物固定在链亲和素修饰的金磁纳米颗粒(Gold magnetic nanoparticles,GMNPs)表面;将ssDNA-GMNPs混合物点样在底部固定有磁铁的载玻片上构建磁性颗粒微阵列,然后在基因框中与双色荧光探针杂交;杂交完全后,充分洗涤,通过扫描获得分型结果。通过优化不对称PCR的扩增条件,直接扩增出产量较高的单链DNA作为靶序列用于分型。利用本方法对24个样本MTHFR基因的C677T位点多态性进行了检测。实验证明,本方法步骤简单,易实现自动化操作、非常适用于分子诊断与法医鉴定。     

一种基于分子信标荧光探针快速检测烟草花叶病毒的新方法

研究了一种利用新型荧光探针——分子信标进行植物病毒检测的方法，可以不要求对病毒RNA进行严格的分离和纯化，就能快捷地得到检测结果。此方法被用于烟草花叶病毒(tobacco mosaic virus，TMV)基因组RNA的检测，为植物病毒分子生物学的研究提供了新的手段。     

基于滚环合成的聚分子信标用于 凝血酶的靶向检测

采用滚环扩增(RCA)合成得到的DNA长链打开带适配体的分子信标, 由于RCA长链上带有多个与分子信标(MB)互补的重复序列, 其打开分子信标的能力比单一互补短链提高了上百倍. 所形成的聚多价分子信标组装体, 在分子信标浓度相同的情况下, 打开后的荧光强度也大幅上升; 并且由于组装体上多价适配体的存在, 聚分子信标对凝血酶的靶向能力显著增强. 实验结果表明, 聚分子信标结合凝血酶后, 其荧光信号与凝血酶浓度呈线性关系, 检测灵敏度达到0.2 nmol/L, 该体系的构建有利于实现对凝血酶的高灵敏、 特异性检测.     

利用无标记的分子信标及核酸染料Hoechst 33258检测特定序列核酸

利用无标记的分子信标及核酸染料Hoechst 33258建立了一种高灵敏、高选择性的特定序列核酸检测方法,并以野生型乙型肝炎病毒的一段寡核苷酸序列为目标DNA,对这种方法进行了验证。在此体系中,分子信标的茎完全设计成C/G碱基对。在没有目标DNA时,Hoechst 33258与分子信标作用较弱,其荧光信号很弱;当有目标DNA存在时,分子信标与目标DNA杂交形成双链,Hoechst 33258与双链DNA作用后荧光信号显著增强。在优化条件下,目标DNA浓度在2×10-10~2×10-8mol/L范围内时,Hoechst 33258的荧光强度(ΔI)与目标DNA的浓度(C)之间具有良好的线性关系,回归方程为ΔI=3.3439C+18.6949(R2=0.9982),方法检出限(3σ)为9×10-11mol/L。此方法操作简单、检测速度快、灵敏度高、重现性好、检出限低。     

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．     

目视化乙肝病毒基因芯片

优化了基于纳米金标记探针的目视化检测方法,将其用于检测从阳性血清中提取的乙肝病毒(HBV)基因,并与基于荧光素异硫氰酸酯(FITC)标记探针的荧光检测方法进行了比较.结果表明,目视化乙肝病毒基因芯片诊断方法操作简单,成本低廉.此类基因芯片在病毒基因检测领域将会有广泛用途.     

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.