纳米粒子三明治型传感器的组装及其在BRCA-1检测中的应用

采用纳米磁、DNA、纳米金组装三明治型生物传感器。其中,纳米磁粒子(PMPS)用于捕获并分离目标DNA,而被基因探针序列修饰过的纳米金(Au-NPs)则扮演识别序列及产生信号的角色,从而使目标DNA序列量转化为光信号,继而通过紫外分光光度计进行定量分析。这种基于纳米金修饰的DNA探针的新型基因诊断技术,可以用来定量检测分析目标DNA。结果显示,这是一种非常简单经济且实用的检测单个突变基因序列的新方法。     

盐诱导纳米金基因探针杂交及其应用研究

因为DNA双螺旋结构的稳定性对周围环境介质非常敏感,通过分析不同盐浓度缓冲溶液中纳米金基因探针的杂交效率,发现由于纳米金对于基因探针的末端修饰作用,使得错配DNA链的双螺旋结构更加不稳定,尤其在盐浓度较低的缓冲体系中,不稳定性差异更为明显。通过优选出特定盐浓度值,以稳定性的明显差异来区分完全配对和错配基因序列,通过纳米金探针紫外-可见光谱吸光强度,定量检测分析目标DNA,结果显示,这是一种非常简单经济且实用的检测单个错配基因序列的新方法。     

DNA序列分析

综述了DNA测序技术。对DNA序列分析方法 ,包括 :毛细管电泳、阵列毛细管电泳、芯片毛细管电泳、超薄层毛细管电泳、质谱法、原子探针法、杂交法、流动式单分子荧光检测法 ,作了详细评述。     

聚二甲基硅氧烷表面改性研究进展

聚二甲基硅氧烷（PDMS）具有良好的化学稳定性和透光性,以及易加工,价格低廉等优点,广泛应用于微流控芯片领域。但是由于PDMS自身为表面高度疏水且多孔性材料,导致其对DNA、蛋白质等生物大分子具有强烈的非特异性吸附,限制了它的应用范围,需要对其进行表面修饰。PDMS表面修饰的方法众多,主要分为物理方法、化学方法两大类。概述了目前常用的几种PDMS表面修饰的方法。     

生物化工

纳米科技研究中的新热点 ——纳米生物化学研究 最近,生物大分子DNA、多聚核苷酸被用来作为分子模板,通过改变DNA分子大小、形状和组合来控制半导体纳米材料的大小、形状、取向和组装等,合成半导体纳米线、量子环、量子点。Murply等研究了DNA与CdS纳米族的相互作用,目的是发展DNA探针诊断技术,一旦取得突破将对DNA芯片生物信息技术,建立快速高效的基因疾病诊断技术起到巨大推动作用。(李哲元)     

CHO宿主细胞DNA残留量检测方法的建立

目的建立检测生物制品中CHO宿主细胞DNA残留量的方法,制备检测CHO细胞DNA残留量的内部参考品,为国家相关标准的制定和修订提供依据。方法分别采用酚:氯仿:异戊醇抽提、DNA提取试剂盒和高盐抽提3种方法提取CHO细胞DNA,比较3种方法的提取效果;用地高辛标记最佳方法提取的CHO细胞DNA探针,并优化标记体系,直接法检测探针的标记效率;采用3种方法处理CHO细胞DNA内部参考品(1组不作处理;2组加入牛血清白蛋白和蛋白酶K;3组按2组方法处理后,增加DNA抽提步骤),通过点杂交法,用标记的探针检测上述样品的灵敏度。结果高盐抽提法提取的CHO细胞DNA效果较好;体系4(DNA10μg,Vial416μl,总体积64μl)标记效率最高;经不同方法处理的1、2、3组CHO细胞DNA内部参考品的检测灵敏度分别为1pg、10pg和1ng。结论建立的地高辛标记CHO细胞DNA探针-点杂交检测CHO细胞DNA残留量的方法稳定可靠,可用于生物制品中CHO宿主细胞DNA残留量的检测。     

量子点标记DNA的应用与进展

量子点作为新型纳米微晶,在生物标记方面有着无与伦比的应用前景.综述了量子点标记DNA的方法,以及量子点-DNA探针在检测DNA、蛋白质及DNA之间相互作用等领域的最新应用和进展.并对DNA探针在未来的应用进行了展望.     

DNA分离技术的研究进展

DNA是生物遗传信息的主要携带者,分离和检测DNA是生物学研究的重要组成部分。本文综述了近年来关于DNA的分离方法,如微纳毛细管的流体动力色谱、物理方法富集分离、微纳米磁颗粒分离富集及电泳芯片分离DNA等方法。     

TaqMan MGB探针实时定量PCR检测Vero细胞残余DNA方法的适用性验证及应用

目的对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行适用性验证及应用。方法对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行特异性、灵敏度、精密性、准确性验证,并应用该方法检测3批次脊髓灰质炎病毒浓缩液、纯化液和原液中Vero细胞残余DNA含量,计算Vero细胞残余DNA去除率。结果 Taq Man MGB探针实时定量PCR能够特异性扩增Vero细胞基因组DNA长串连重复序列;DNA浓度在10-1~10-6 ng/μl范围内标准曲线线性良好,相关系数为0.996,扩增效率为93.54%;检测灵敏度为10-6 ng/μl,高于斑点杂交法;检测高(10-2 ng/μl)、中(10-4 ng/μl)、低(10-6 ng/μl)浓度阳性对照DNA模板的试验内变异系数(CV)为0.145%~3.110%,试验间CV为0.624%~2.359%;检测不同浓度阳性对照DNA的回收率在101.5%~109.4%之间,均在定量方法可接受的回收率范围内;在斑点杂交检测灵敏度范围内,同一样品采用Taq Man MGB探针实时定量PCR法与斑点杂交法半定量检测的结果吻合。3批次脊髓灰质炎病毒浓缩液纯化后可有效去除Vero细胞残余DNA,总去除率约为100%。结论 Taq Man探针实时定量PCR法特异性、精密性、准确性良好,灵敏度高,可作为斑点杂交法的替代方法,用于实验室内部的质量控制。     

替换氧桥原子的罗丹明类似物的合成及应用

罗丹明类化合物是一类重要的荧光探针染料,近年已被广泛地应用到生命科学、化学、诊断医学等学科中.目前文献报道了罗丹明类化合物的多种修饰方法,主要对通过替换桥原子氧得到罗丹明类似物这一修饰方法进行综述,重点介绍了该类罗丹明类似物的合成及应用.     

Inhibitor and protein microarrays for activity-based recognition of lipolytic enzymes

Protein and small-molecule microarrays are useful tools for high-throughput analysis of DNA-protein, protein-protein, and protein-small molecule interactions. Here we report on novel microarrays for activity screening of lipases and esterases based on phosphonic acid ester inhibitors. These compounds are activity recognition probes (ARPs) and bind to active serine hydrolases in a stoichiometric and irreversible manner. Protein microarrays were generated by spotting six different lipolytic enzymes onto hydrogel-coated glass slides. The activity of immobilized enzymes was determined after treatment with fluorescently labeled ARPs. Alternatively, biotinylated ARPs were bound to streptavidin slides in order to identify their affinity for enzymes in solution. Both systems, the protein- and ARP microarrays proved to be useful and versatile tools for the rapid identification and characterization of novel and known lipolytic enzymes.     

Tools for glycomics: mapping interactions of carbohydrates in biological systems

The emerging field of glycomics has been challenged by difficulties associated with studying complex carbohydrates and glycoconjugates. Advances in the development of synthetic tools for glycobiology are poised to overcome some of these challenges and accelerate progress towards our understanding of the roles of carbohydrates in biology. Carbohydrate microarrays, fluorescent neoglycoconjugate probes, and aminoglycoside antibiotic microarrays are among the many new tools becoming available to glycobiologists.     

Self-assembled small-molecule microarrays for protease screening and profiling

Small-molecule microarrays are attractive for chemical biology as they permit the analysis of hundreds to thousands of interactions in a highly miniaturized format. Methods to prepare small-molecule microarrays from combinatorial libraries by a self-assembly process based on the sequence-specific hybridization of peptide nucleic acid (PNA) encoded libraries to oligonucleotide arrays are presented. A systematic study of the dynamic range for multiple detection agents, including direct fluorescence of attached fluorescein and cyanine-3 dyes, antibody-mediated fluorescence amplification, and biotin-gold nanoparticle detection, demonstrated that individual PNA-encoded probes can be detected to concentrations of 10 pM on the oligonucleotide microarrays. Furthermore, a new method for parallel processing of biological samples by using gel-based separation of probes is presented. The methods presented in this report are exemplified through profiling two closely related cysteine proteases, cathepsin K and cathepsin F, across a 625-member PNA-encoded tetrapeptide acrylate library. A series of the specific cathepsin K and F inhibitors identified from the library were kinetically characterized and shown to correlate with the observed microarray profile, thus validating the described methods. Importantly, it was shown that this method could be used to obtain orthogonal inhibitors that displayed greater than tenfold selectivity for these closely related cathepsins.     

Fluorescence-based assay formats and signal amplification strategies for DNA microarray analysis

DNA microarrays are powerful tools for the high throughput analysis of nucleic acids due to their parallel detection capabilities. To realize the apparent power of DNA microarray, an efficient assay format is essential and a variety of assay formats have been developed for nucleic acid detection on microarrays. Many of them employ fluorescence-based methods because fluorescence detection is straightforward and easy to implement. Herein we broadly review fluorescence-based assay formats with a focus on PCR-associated target preparation. For the heterogeneous assay of nucleic acid on DNA microarrays, sensitivity is one of the most important factors and special emphasis has been given to recently developed signal amplification strategies aimed at achieving high sensitivity.     

Protein–DNA Arrays as Tools for Detection of Protein–Protein Interactions by Mass Spectrometry

Analysis of multiple protein–protein interactions using microarray technology remains challenging, and site‐specific immobilization of functional proteins is a key step in these approaches. Here we establish the efficient synthesis of protein–DNA conjugates for several members of a small family of GTPases. The family of Rab/Ypt GTPases is intimately involved in vesicular trafficking in yeast and serves as a model for the much larger group of analogous human proteins, the Rab protein family, with more than 60 members. The Ypt–DNA hybrid molecules described here are used for DNA‐directed immobilization on glass‐ and silica‐based microarrays. Methods for the detection of protein–DNA conjugates, as well as approaches for nucleotide exchange and distinguishing between GDP‐ and GTP‐bound Ypts on microarrays, are reported. The high specificity of different Rab/Ypt‐effector interactions, which also depends on the bound nucleotide, is shown by fluorescence readout of microarrays. Furthermore, initial experiments demonstrate that direct readout by mass spectrometry can be achieved with commercially available instruments. These developments will significantly contribute to the elucidation of complex transport networks in eukaryotic cells.     

DNA分子荧光探针

本文综述了各种DNA荧光探针的结构特征荧光性质和与DNA的作用方式，主要涉及了6类化合物：吖啶和菲啶类、菁类染料、荧光素和罗丹明类、噻嗪和恶嗪类染料、BODIPY类染料以及其它类别的探针，概述了DNA探针在生物分子分析方面的应用，并展望了DNA荧光探针的发展趋势和应用前号。有47篇参考文献。     

Protein Microarray Copying: Easy on-Demand Protein Microarray Generation Compatible with Fluorescence and Label-Free Real-Time Analysis

Protein microarrays are essential to understand complex protein interaction networks. Their production, however, is a challenge and renders this technology unattractive for many laboratories. Recent developments in cell-free protein microarray generation offer new opportunities, but are still expensive and cumbersome in practice. Herein, we describe a cost-effective and user-friendly method for the cell-free production of protein microarrays. From a polydimethylsiloxane (PDMS) flow cell containing an expressible DNA microarray, proteins of interest are synthesised by cell-free expression and then immobilised on a capture surface. The resulting protein microarray can be regarded as a “copy” of the DNA microarray. 2 His₆- and Halo-tagged fluorescent reference proteins were used to demonstrate the functionality of nickel nitrilotriacetic acid (Ni-NTA) and Halo-bind surfaces in this copy system. The described process can be repeated several times on the same DNA microarray. The identity and functionality of the proteins were proven during the copy process by their fluorescence and on the surface through a fluorescent immune assay. Also, single-colour reflectometry (SCORE) was applied to show that, on such copied arrays, real-time binding kinetic measurements were possible.     

Single Labeled DNA FIT Probes for Avoiding False‐Positive Signaling in the Detection of DNA/RNA in qPCR or Cell Media

Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real‐time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance‐dependent interaction between a fluorophore and another label. Such duallabeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label–label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, “DNA FIT probes”, that are designed to avoid false‐positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the “TO base” in cis with the disordered nucleobases of the single strand, and 2) intercalation of the “TO nucleotide” with double strands in trans. However, formation of the probe–target duplex enforces stacking and increases the fluorescence of the TO base. We explored open‐chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe–target complexes. DNA and RNA targets provided up to 12‐fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false‐positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.     

Dendrimer-activated solid supports for nucleic acid and protein microarrays

The generation of chemically activated glass surfaces is of increasing interest for the production of microarrays containing DNA, proteins, and low-molecular-weight components. We here report on a novel surface chemistry for highly efficient activation of glass slides. Our method is based on the initial modification of glass with primary amino groups using a protocol, specifically optimized for high aminosilylation yields, and in particular, for homogeneous surface coverages. In a following step the surface amino groups are activated with a homobifunctional linker, such as disuccinimidylglutarate (DSG) or 1,4-phenylenediisothiocyanate (PDITC), and then allowed to react with a starburst dendrimer that contains 64 primary amino groups in its outer sphere. Subsequently, the dendritic monomers are activated and crosslinked with a homobifunctional spacer, either DSG or PDITC. This leads to the formation of a thin, chemically reactive polymer film, covalently affixed to the glass substrate, which can directly be used for the covalent attachment of amino-modified components, such as oligonucleotides. The resulting DNA microarrays were studied by means of nucleic acid hybridization experiments using fluorophor-labeled complementary oligonucleotide targets. The results indicate that the novel dendrimer-activated surfaces display a surface coverage with capture oligomers about twofold greater than that with conventional microarrays containing linear chemical linkers. In addition, the experiments suggest that the hybridization occurs with decreased steric hindrance, likely a consequence of the long, flexible linker chain between the surface and the DNA oligomer. The surfaces were found to be resistant against repeated alkaline regeneration procedures, which is likely a consequence of the crosslinked polymeric structure of the dendrimer film. The high stability allows multiple hybridization experiments without significant loss of signal intensity. The versatility of the dendrimer surfaces is also demonstrated by the covalent immobilization of streptavidin as a model protein.     

Fluorescent Probes for G‐Quadruplex Structures

Mounting evidence supports the presence of biologically relevant G‐quadruplexes in single‐cell organisms, but the existence of endogenous G‐quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure–function relationships of nucleic acids. In this review, we present progress towards the direct detection of G‐quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell‐permeable probes that selectively bind to G‐quadruplex structures with high affinity, these same probes can induce G‐quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as “internal” fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G‐quadruplex, single‐stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G‐quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.