Novel protein detection method based on proximity- dependent polymerase reaction and aptamers

In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics. Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin was used as the model protein, and two aptamer probes with complementary sequence at 3′-end were designed for the two distinct epitopes of the protein. Association of the two aptamers with thrombin resulted in stable hybrids due to the proximity of 3′-end, then polymerase reaction was induced. The amount of obtained dsDNA was indicated using the fluorescence dye Sybr Green I. The results showed that the initial velocity of polymerase reaction had a positive correlation with concentration of thrombin. The advantages of this dual-aptamer-based approach included simple and flexible design of aptamer probes, high selectivity and high sensitivity. The detection limit was 6.9 pmol/L.  

人骨肉瘤细胞适配体的早期筛选

目的 体外随机合成单链寡核苷酸备选文库,以骨肉瘤U-2 OS完整细胞作为靶点,筛选出与其结合的高特异性、高亲和性单链DNA( ssDNA) .方法 将体外合成随机碱基序列的单链DNA 文库与人骨肉瘤U-2 OS细胞相结合,收获与靶细胞结合的单链DNA,通过优化PCR条件进行大量扩增后,应用生物素-链霉亲和素亲和层析柱分离出正链DNA.结果 以10,14以及18个循环时扩增dsDNA产物最理想;应用生物素-链霉亲和素磁珠分离法制备的正链ssDNA纯度高,可有效保证次级文库的筛选.结论 应用Cell-SELEX技术可成功筛选早期ssDNA文库,为后续获得针对人骨肉瘤U-2 OS细胞的高特异性适配体奠定实验基础.  

氧化石墨烯保护的核酸分子探针及其在生物分析中的应用

近年来,氧化石墨烯(graphene oxide,GO)作为石墨烯的一类重要衍生物越来越受到人们的广泛关注.科学家们发现GO具有吸附单链核酸并保护其不受核酸酶降解的能力,该特性引发GO在生物分析领域一系列新的应用:一方面,基于GO能够保护RNA免受环境中普遍存在的核酸酶攻击,发展了稳定RNA探针分子的方法并用于特定目标物的检测、富集及分离;另一方面,基于单链核酸的吸附使其具有抗酶切能力,而形成双链核酸后脱吸附使其失去抗酶切能力,发展了循环酶切放大方法,并用于一系列分析物的高灵敏检测中.此外,功能核酸分子吸附于GO表面后,将具有较高的细胞转染效率、较低的细胞毒性以及较强的生物稳定性,从而被广泛应用于细胞内特定基因及代谢物的检测、成像等.在本篇综述中,首先介绍了GO对单链核酸的保护效果,在此基础上,进一步阐述受保护的单链核酸在生物分析领域的一系列应用.  

Near-infrared fluorescent aptamer-templated silver nanoclusters facilely synthesized for cellular imaging applications

Fluorescent metal nanoclusters(NCs)have received extensive attention for their potential uses in bionanotechnology.Here,we develop a facile strategy to synthesize near-infrared fluorescent silver nanoclusters(Ag NCs)stabilized by MUC1 aptamer.The MUC1-Ag NCs are characterized by UV–Vis absorption spectroscopy,fluorescence spectroscopy,transmission electron microscopy,and fluorescence lifetime.These results indicated that the MUC1-Ag NCs possess bright near-infrared luminescence,high stability,and excellent biocompatibility.The cellular imaging of MUC1-Ag NCs by confocal laser microscopy demonstrated them to be promising candidates as novel fluorescent probes for biomedical application.  

Stable silver nanoparticles–aptamer bioconjugates for cellular prion protein imaging

Combining the unique optical properties of metal nanoparticles and the specific recognition of aptamer,aptamer–nanoparticle conjugates have been extensively used in a wide range of applications,particularly multifunctional nanoparticles for cell detection and molecular imaging.Conventional conjugates prepared by chemisorption of monothiol-modified oligonucleotides onto nanoparticle surfaces suffer from a lack of stability when exposed to a variety of small molecules.If silver is used in place of gold,then this lack of stability is even more pronounced.In this study,we reported here the effective and facile strategy of preparing stable silver nanoparticle–aptamer conjugates by in situ generation of strong metal affinity capping ligands,dithiocarbamates modified anti-prion protein aptamer.The conjugates produced are stable and can withstand NaCl concentration at0.25 mol/L.Meanwhile,they could be applied in the cellular prion protein imaging successfully.  

DNA聚合与RNA转录联用分子机器的改进

建立了一个集成DNA聚合和RNA转录过程,能够重复产生RNA适体片段,并结合孔雀石绿产生荧光信号的分子机器,并探索了此分子机器在检测DNA方面的应用.转录产生的大量孔雀石绿适体序列被释放到溶液中,并可以与孔雀石绿结合产生荧光,实现信号的放大.85μL体系中的聚合转录的最适条件为：DNA聚合酶10 IU（0.118 IU·μL-1）,RNA聚合酶60 IU（0.706 IU·μL-1）,反应时间为3h.在上述条件下,荧光信号随着引发DNA用量的增加而增大.  

新型凝血酶电化学传感器的制备

制备了金纳米粒子-核酸适体/凝血酶/巯基六醇修饰的金电极,测试了该修饰电极检测凝血酶含量的性能指标.利用电化学技术研究了电极的构造对检测凝血酶含量的影响,探究了最佳实验条件.研究结果表明,在最佳的实验条件下构筑的修饰电极检测凝血酶的线性范围为1.0~10.0 nmol/L,检测限为0.2 nmol/L,并模拟了实际样品进行检测.该方法简便迅速,有较高的检测灵敏度、较好的重复性和抗干扰性.  

连接臂对适体与凝血酶相互作用影响的电化学阻抗研究

以5＇-C6-T0-A1,5＇-C6-T5-A1和5＇-C6-T10-A1三条不同长度连接臂修饰的凝血酶适体作为生物传感器的识别分子,结合巯基自组装技术,利用电化学阻抗技术,测定了凝血酶的线性范围和检出限,比较了三者灵敏度的差异。利用浓度平衡法计算了适体与凝血酶作用的结合常数,三条链的结合常数Ka分别为0.32 nM-1,0.84 nM-1和0.67 nM-1,表明,5＇-C6-T5-A1组装的传感器与凝血酶的结合常数最大,灵敏度最好。  

运用SELEX技术筛选嗜水气单胞菌适配子

以嗜水气单胞菌为研究对象,采用适体筛选技术(SELEX,system evolution of ligands by expo-nential enrichment),利用体外合成的长度为78个核苷酸的随机ssDNA文库,以嗜水气单胞菌为靶目标进行12轮的筛选,利用生物素-抗地高辛碱性磷酸酶显色系统检测DNA适配子与嗜水气单胞菌的亲和力.研究结果表明:随着筛选轮数的增加,DNA适配子与嗜水气单胞菌结合后显色,A(absorbance)值逐渐增加,亲和力由最初的0.32升到第9轮的0.72.研究已初步筛选到与嗜水气单胞菌具有高亲和力的DNA适配子,为嗜水气单胞菌的二级结构研究及其检测试剂盒的研发提供了基础.  

生命有机磷化学、生化分析与生物传感研究进展

化学生物学是一门化学与生命科学高度交叉的前沿学科,它利用化学的方法、手段和策略从分子水平认识生命现象的本质,创造对生理或病理过程具有调控作用的活性物质,从而为生物医药、农业和环境科学的发展提供解决方案.本文就厦门大学化学生物学学科近5年在生命有机磷化学、生化分析与生物传感等特色研究领域的进展进行综述.