基于氧化石墨烯识别特定双螺旋DNA

依据三螺旋DNA的形成,以氧化石墨烯为基础建立了一种识别特定序列双螺旋DNA的方法。单链探针DNA能够通过静电引力作用吸附在氧化石墨烯表面,标记在单链DNA末端的荧光探针分子TAMRA由于荧光能量共振转移作用使得其荧光发生淬灭。加入目标双螺旋DNA后,单链探针DNA与目标DNA分子形成三螺旋DNA,探针DNA从氧化石墨烯表面脱附,标记在探针DNA上的荧光分子的荧光恢复。在最佳实验条件下,荧光恢复的强度与探针DNA的浓度在20.0～300.0 nmol/L具有良好的线性关系,检出限为16.9 nmol/L。该方法在DNA药物筛选及基因疾病的诊断方面具有一定的应用前景。     

光谱法研究对氨基苯乙酮缩对二甲氨基苯甲酰腙与DNA的相互作用

合成了对氨基苯乙酮缩对二甲氨基苯甲酰腙(AAPABH),采用荧光光谱法和UV-Vis吸收光谱法研究了AAPABH与DNA的相互作用。在pH 7.4Tris-HCl缓冲溶液中,以345 nm激发该化合物发射弱荧光,加入DNA后出现明显的荧光增强现象,表明探针分子与DNA结合形成了稳定配合物,其结合常数为5.48×107L/mol。研究了离子强度对体系荧光强度的影响,结果显示NaCl的加入未引起AAPABH-DNA体系荧光强度明显变化,表明AAPABH和DNA间不存在静电作用;同时DNA对AAPABH吸收光谱的影响表现为减色效应、探针分子与热变性后的DNA作用力明显小于与未变性DNA间的作用力、探针分子与溴化乙锭(EB)竞争结合DNA使得EB-DNA体系荧光猝灭,上述实验结果均表明探针分子主要以嵌插作用方式与DNA作用。     

《化学通报》网络版(Chemistry Online)2004年第9期论文摘要

[w0 6 8 ]DNA分子荧光探针DNA Fluorescence Probes陈秀英　彭孝军 * (大连理工大学精细化工国家重点实验室　大连　 1 1 6 0 1 2 )介绍了各种 DNA荧光探针的结构特征、荧光性质及其与 DNA的作用方式 ,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景。The structure characters,fluorescence properties and the functional ways of the various kinds of probes forDNA,including some applications on the detection of biomolecules,have been reviewed,and the trends andprospect on th…     

基于纳米金探针和基因芯片的DNA检测新方法

运用荧光纳米金探针和基因芯片杂交建立一种新的DNA检测方法. 荧光纳米金探针表面标记有两种DNA探针: 一种为带有Cy5荧光分子的信号探针BP1, 起信号放大作用; 另一种为与靶DNA一部分互补的检测探针P532, 两种探针比例为5∶1. 当靶DNA存在时, 芯片上捕捉探针(与靶DNA的另一部分互补)通过碱基互补配对结合靶DNA, 将靶DNA固定于芯片上; 荧光纳米金探针通过检测探针与靶DNA及芯片结合, 在芯片上形成“三明治”复合结构, 最后通过检测信号探针上荧光分子的信号强度来确定靶DNA的量. 新方法检测灵敏度高, 可以检测浓度为1 pmol/L的靶DNA, 操作简单, 检测时间短. 通过改进纳米金探针的标记和优化杂交条件, 可进一步提高核酸检测的灵敏度, 这将在核酸检测方面具有重要的应用价值.     

罗丹明B-β-环糊精衍生物探针型主-客体分子相互识别作用的研究

应用光谱法研究了生物大分子探针型主体分子罗丹明B一β-环糊精衍生物与DNA的相互作用及温度对该探针特性的影响.探讨了相应的主体分子与DNA分子相互作用的方式、嵌插作用能力大小以及温度对主体分子含客体分子时的影响．进一步研究了探针型主一客体分子相互识别作用及作用能力的大小．探讨了主一客体分子相互识别作用的机制．     

芘二聚体发夹探针用于细胞DNA单链断裂修复能力的评估

基于芘分子二聚体的荧光特性设计了一种新型的发夹探针,用于评估不同细胞的单链断裂修复(SSBR)能力.首先利用芘荧光分子设计一个茎上有缺口的DNA发夹探针,该探针在Bst DNA聚合酶作用下可以水解,使荧光信号“关闭”.如果探针茎上的缺口被DNA修复酶修复,可以阻止Bst DNA聚合酶对探针的消化作用,从而阻止荧光信号的“关闭”.该设计可以用于评估细胞的SSBR能力,并通过提取细胞的核蛋白考察了不同细胞的SSBR能力.研究结果表明,肿瘤细胞及细胞系不具有原代细胞的SSBR能力.利用SSBR的关键酶进一步探索了肿瘤细胞SSBR能力缺失的原因,证明是由于肿瘤细胞中含有可以抑制SSBR过程的活性物质.此外,该方法能够同时进行500个细胞的SSBR能力检测,并用于抗衰老药物筛选.     

亚甲蓝作为光谱探针研究对-二甲氨甲基-杯[8]芳烃与DNA间的作用

亚甲蓝作为光谱探针研究对-二甲氨甲基-杯[8]芳烃与DNA间的作用;对二甲基杯[8]芳烃;小牛胸腺DNA;紫外光谱法;荧光探针     

巯嘌呤金属配合物与小牛胸腺DNA的作用

用溴化乙锭为探针研究了巯嘌呤(mercaptopurine, MP)金属配合物与小牛胸腺 DNA的作用机制，探讨了其作用模式，即巯嘌呤与DNA是非嵌插结合，巯嘌呤金属酴 物与DNA之间的作用为静电方式和一定的嵌入方式。并求得巯嘌呤金属配合物与 DNA的结合常数。     

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.     

基于纳米银-曙红Y-DNA的荧光共振能量转移探针

以曙红Y-DNA为荧光探针,运用紫外可见光谱法、荧光光谱法和电泳法研究了纳米银与曙红Y-DNA之间的共振能量转移,并应用能量共振转移的原理解释了纳米银及曙红Y与DNA之间的相互作用。结果表明,曙红Y与DNA的作用方式为嵌插作用,纳米银与DNA的作用方式为静电作用,曙红Y和纳米银结合到DNA上以后,导致了DNA结构的改变。     

二茂铁标记DNA电化学探针的研制及性质研究

以乙基－（3－二甲基丙基）碳化二亚胺盐酸(EDC)为偶联活化剂,利用缩合反应分别将电化学活性物质氨基二茂铁（Aminoferrocene,AFC)和苯基二茂铁（Ferrocencearboxadehyde,FCA)成功地标记在变性小牛胸腺DNBA片段上,制备成二茂铁标记DNA探针,分别采用电化学,紫外,红外光谱等方法研究了二茂铁标记DAN探针的性质,计算了二茂铁的标记效率,变性小牛胸腺DNA的反应效率以及二茂铁化合物与变性小牛胸腺DNA的反应比率,实验表明,氨基二茂铁和醛基二茂铁与DNA上的磷酸基是以1：1比率进行的反应,该标记反应不影响DNA链碱基的紫外吸收,二茂铁标记DNA探针在石墨电极上有良好的电化学影响,将制备好的二茂铁标记DNA电化学探置于冰箱中冷冻（温度低于－15度）保存3个月后用于测定,峰电流仅下降1．2％。     

QCM DNA biosensor for the diagnosis of a fish pathogenic virus VHSV

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases damaging both fresh and marine fish species. VHS caused by VHSV and diagnosis of VHSV has been dependent on the conventional methods, such as cell culture and RT-PCR, which takes a few days or several hours. This study demonstrates a rapid and sensitive QCM biosensor for diagnosis of VHSV infection in fish. The QCM biosensor was developed to detect a main viral RNA encoding G protein in VHSV using the specific DNA probe. To maximize the sensitivity of the biosensor, we prepared three different DNA probes which modified 3′ end of DNA by thiol, amine, or biotin and compared three different immobilisation methods on quartz surface coated with gold: immobilisation of thiol labelled probe DNA on naked gold surface, immobilisation of amino labelled probe DNA on gold surface prepared as carboxyl chip using MPA followed by EDC/NHS activation, and immobilisation of biotin labelled probe DNA on gold surface after immobilising avidin on carboxyl chip prior to biotin. As a result, immobilisation method using avidin-biotin interaction was most efficient to immobilise probe DNA and to detect target DNA. The QCM biosensor system using biotinylated probe DNA was stable enough to withstand 32 times of repeated regenerations and the detection limit was 0.0016 μM. Diagnosis using the QCM biosensor system was more sensitive and much faster than a conventional RT-PCR analysis in detecting the viral RNA.     

纳米探针芯片技术用于微量乙肝病毒DNA的检测

利用两组探针修饰的微粒:(1)表面标记有可与待测乙肝病毒(HBV) DNA另一端结合的纳米金探针1(信号探针)以及可与信号探针部分结合的纳米金探针2(检测探针);(2)表面标记有可与待测HBV DNA一端结合的磁珠探针(捕捉探针1).检测靶HBV DNA时,磁珠探针与信号探针在液相中可分别与HBV DNA靶序列一端结合最终形成三明治样结构.再以磁场将三明治样复合物从反应液中分离,以DTT溶液将信号探针从纳米金颗粒上洗脱.洗脱后的信号探针数量反映靶基因的多寡,信号探针一段与预先点样的基因芯片上的捕捉探针2结合,检测探针与信号探针另一段相结合,最后用银染液将检测探针显色从而得到靶目标DNA相对定量信息.结果表明,本检测方法的检测灵敏度达到10-15 mol/L水平.检测时间少于1.5 h,检测结果与HBV DNA水平呈现较好的线性关系且无假阳性结果;本方法有望用于乙肝病人血清中HBV DNA的快速筛测及其它微生物基因的检测.     

荧光探针Ru(phen)2(dppx)2+测定H1N1禽流感病毒DNA

利用荧光探针Ru(phen)2dppx2+与ssDNA作用时不产生荧光或荧光很弱,而与dsDNA作用时荧光增强的机理,将H1N1禽流感病毒ssDNA与其完全互补ssDNA杂交形成dsDNA实现Ru(phen)2dppx2+对H1N1禽流感病毒DNA特定序列（5’-CTA CCA TGC GAA CAA TTC AAC CGA CAC TGT T-3’）的定量检测。在优化的实验条件下,测定H1N1禽流感病毒 DNA的线性范围为9.3×10-10～7.4×10-8 mol/L,线性关系：y = 3.3829x + 8.3948,R2 =0.9982,检出限为5.3×10-10 mol/L。该方法具有操作简单,检测快速,灵敏度高和选择好等优点。     

Immobilization of oligonucleotides onto zirconia-modified filter paper and specific molecular recognition

A morphologically complex cellulosic substance (e.g., commercial filter paper) was employed as a substrate for DNA immobilization and successive recognition. A uniform ultrathin zirconia gel film was first deposited on each cellulose nanofiber in bulk filter paper by a facile sol-gel process. Relying on the large surface area of filter paper and the strong affinity of zirconia for the phosphate group, terminal-phosphate probe DNA was abundantly immobilized on the zirconia-modified filter paper so as to convert the composite to a biofunctional material for the sensitive and repetitive recognition of the corresponding complementary target DNA on the nanomolar level. By contrast, in spite of the viability of the immobilization of the probe DNA and the recognition of target DNA on the quartz plate, the amount of captured probe DNA or recognized target DNA on such a flat substrate was much less than that captured or recognized on filter paper, resulting in a relatively insensitive recognition event. Moreover, control experiments on bare filter paper (without a zirconia nanocoating) suggested that the zirconia gel film was essential to probe DNA immobilization and subsequent target DNA recognition.     

基于工具酶信号放大技术电化学发光法检测凝血酶

利用巯基乙胺将合成的金纳米粒子氨基化;基于纳米粒子负载羧基化的联吡啶钌和巯基DNA制得电化学发光信号探针;采用酶循环信号放大技术,获得大量含新增DNA的溶液来捕获信号探针;以金电极为载体,将巯基DNA自组装到电极表面,依次杂交互补DNA和信号探针,构建电化学发光生物传感器.在优化的条件下,此传感器对凝血酶具有良好的响应,在3.0× 10-13～6.0×10-11 mol/L范围内,凝血酶的浓度与发光强度呈良好的线性关系,检出限为1.8× 10-13 mol/L(3a).采用酶切循环放大技术制备的生物传感器具有灵敏度高,选择性和重现性良好等特点.     

One Mismatch Detection of DNA Hybridization Using Fluorescence Polarization

Abstract

The potential of fluorescent polarization analysis as a method for detection of mismatch DNA hybridization was investigated. The dependency of DNA hybridization rate on salt concentration was surveyed. In greater than 0.1 M NaCl, the hybridization of probe and target DNA proceeds rapidly and the reaction is complete within 3 min. Furthermore, the hybridization of probe DNA and one mismatch target DNAs was investigated. It was successfully shown that even one mismatch could be detected using fluorescence polarization analysis if the mismatch position was on the base that pairs with the probe DNA at the 5′ terminus where fluorescein isothiocyanate (FITC) is attached.     

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

We report on a highly sensitive chemiluminescent (CL) biosensor for the sequenc-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H₂O₂ and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L^-1 of target DNA.

Label‐Free Bioelectronic Detection of Point Mutation by Using Peptide Nucleic Acid Probes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes with a label‐free protocol is described. The detection of PNA‐DNA and DNA‐DNA hybridizations were accomplished based on the oxidation signal of guanine by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE). It was observed that the oxidation signals of guanine obtained from the PNA and DNA probe modified CPEs were higher than those obtained from the PNA‐DNA and DNA‐DNA hybrid modified CPEs due to the accessible unbound guanine bases. The detection of hybridization between PNA probe and point mutation containing DNA target sequences was clearly observed due to the difference of the oxidation signals of guanine bases, because the point mutation was guanine nearly at the middle of the sequence. The effect of the DNA target concentration on the hybridization signal was also observed. The PNA probe was also challenged with excessive and equal amount of noncomplementary DNA and also mixtures of point mutation and target DNA.