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1.
基于纳米金探针和基因芯片的DNA检测新方法   总被引:2,自引:0,他引:2  
包华  贾春平  周忠良  金庆辉  赵建龙 《化学学报》2009,67(18):2144-2148
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.  相似文献   

2.
基于纳米金胶标记DNA探针的电化学DNA传感器研究   总被引:6,自引:0,他引:6  
以纳米金胶为标记物,将其标记于人工合成的5-端巯基修饰的寡聚核苷酸片段上,制成了具有电化学活性的金胶标记DNA电化学探针;在一定条件下,使其与固定在玻碳电极表面的靶序列进行杂交反应,利用ssDNA与其互补链杂交的高度序列选择性和极强的分子识别能力,以及纳米金胶的电化学活性,实现对特定序列DNA片段的电化学检测以及对DNA碱基突变的识别.  相似文献   

3.
利用电沉积导电聚合物薄膜,提出了一种对乳腺癌相关的BRCA-1基因的高灵敏检测方法.以Au电极表面自组装DNA捕获探针,利用电沉积在DNA修饰电极表面固定含Ss^2+/3+的导电高分子作为电子传递媒介体.通过夹心法杂交目标靶DNA及辣根过氧化物酶标记的信号DNA探针.靶DNA杂交的信号探针上的辣根过氧化物酶与检测溶液中的过氧化氢反应,采用时间.电流(T-I)法,可以灵敏检测BRCA-1基因,其检测限可以达到10fM.  相似文献   

4.
纳米探针芯片技术用于微量乙肝病毒DNA的检测   总被引:1,自引:0,他引:1  
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.  相似文献   

5.
基于硫化镉纳米团簇标记DNA电化学传感的研究   总被引:5,自引:2,他引:3  
祝宁宁  张爱平  何品刚  方禹之 《化学学报》2003,61(10):1682-1685
合成了表面具有自由羧基的硫化镉纳米团簇,以乙基-(3-二甲基丙基)碳二 亚胺盐酸盐为偶联活化剂,将其标记于人工合成的5'端氨基修饰的寡聚核苷酸片段 上,制备成CdS纳米团簇标记DNA探针,该寡聚核苷酸片段与大肠杆菌肠毒素基因相 关。在一定的条件下,使基与固定晨玻碳电极表面的待测DNA序列进行杂交反应, 利用阳极溶出示差脉冲伏安法(ASDPV)间接测定Cd的量,实现对互补、非互补 DNA片段的识别和电化学检测,从而对大肠杆菌肠毒素基因片段识别和检测。  相似文献   

6.
用模板法在氧化铟锡(ITO)电极上制备具有三维有序多孔结构的金掺杂纳米二氧化钛修饰电极(3DOM GTD/ITO),扫描电镜(SEM)结果表明,制备的修饰电极三维结构规整有序、孔径均一。将标记有二茂铁(Fc)的DNA探针修饰到3DOM GTD/ITO电极上构建了一种新的标记型DNA生物传感器,通过Fc在DNA探针杂交前后的电化学信号变化可识别目标靶序列。采用循环伏安(CV)、示差脉冲(DPV)和交流阻抗(EIS)等方法对DNA探针在电极表面的固定和杂交进行表征。实验结果表明,该DNA生物传感器可以成功地识别乳腺癌基因靶序列,Fc的氧化还原电流与靶序列浓度在8.0×10-7~1.0×10-5 mol/L范围内呈线性关系,线性相关系数为0.9908,检测限为5.2×10-7 mol/L。  相似文献   

7.
Cy-5标记脱氧核糖核酸荧光毛细分析法的研究   总被引:2,自引:0,他引:2  
在荧光毛细分析法(FCA)的基础上开发了一种用于DNA快速检测的DNA-FCA法。在毛细管内表面将DNA探针固定化,制成荧光毛细生物反应器(DNA-CBR)。测定时,用DNA-CBR吸入含Cy-5标记的靶DNA样品液进行杂交反应,然后在646nm激发波长、664nm发射波长下进行荧光测定;Cy-5标记的靶DNA浓度在0.1~1.0μmol/L之间线性良好(y=139.73x+39.613,r=0.9985);RSD〈5.5%;检出限为0.17pmol,样品用量10μL;DNA-CBR能够重复使用6次。本方法可用于靶DNA的定性和定最检测。  相似文献   

8.
基于"核酸外切酶(ExoⅢ)辅助靶序列循环"和"DNA长距自组装"两种信号放大技术研制了一种DNA电化学生物传感器,并将其用于乳腺癌相关靶序列的高灵敏、高特异性检测。通过将发卡型探针固定在金电极表面,当靶序列存在时,在ExoⅢ的辅助下,发生杂交、酶降解、再杂交的第一重信号放大过程。接着在电极表面加入两条辅助探针,即可发生级联式杂交,形成长距超级"三明治"DNA结构。该结构可吸附大量的电活性分子六氨合钌配合物(RuHex),产生很强的电化学信号,从而实现信号的第二重放大。实验结果表明,在最佳条件下,该传感器的线性范围为10 amol/L~10 pmol/L,检出限达到8 amol/L,而且能较好地识别完全互补和错配序列,有望用于临床实际样本中超低含量靶序列的检测。  相似文献   

9.
高秀峰  李永生  姜艳霞 《分析化学》2006,34(Z1):220-222
在荧光毛细分析法(FCA)的基础上开发了一种用于DNA快速检测的DNA-FCA法.在毛细管内表面将DNA探针固定化,制成荧光毛细生物反应器(DNA-CBR).测定时,用DNA-CBR吸入含Cy-5标记的靶DNA样品液进行杂交反应,然后在646 nm激发波长、664 nm发射波长下进行荧光测定;Cy-5标记的靶DNA浓度在0.1~1.0 μmol/L之间线性良好(y=139.73x+39.613,r=0.9985);RSD<5.5%;检出限为0.17 pmol,样品用量10 μL;DNA-CBR能够重复使用6次.本方法可用于靶DNA的定性和定量检测.  相似文献   

10.
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1.0×10~5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.  相似文献   

11.
采用疏基化合的自组装/共价键合反应的逐层固定方法将双链DNA固定到金表面得到DNA修饰电极,并对该电极表面进行了电化学和X射线光电子能谱表征。研究了电极表面固定化DNA的表面分子杂交。对开发电化学基因诊断芯片和基因传感器具有一定意义。  相似文献   

12.
《Electroanalysis》2005,17(22):2001-2017
New strategies for the construction of DNA chips and the detection of DNA hybridization will be discussed in this review. The focus will be on the use of polypyrrole as a linker between a substrate and oligonucleotide probes. The modification step is based on the electrochemical copolymerization of pyrrole and oligonucleotides bearing a pyrrole group on its 5′ end. This strategy was employed for the immobilization of oligonucleotides on millimeter‐sized electrodes, microelectrode arrays, as well as for the local structuring of homogeneous gold surfaces. Our approaches for the localized patterning of gold surfaces will be also discussed. Localized immobilization was achieved by using an electrospotting technique, where a micropipette served as an electrochemical cell where spot sizes with 800 μm diameters were fabricated. The use of a microcell using a Teflon covered metal needle with a cavity of 100 μm resulted in immobilized probe spots of 300 μm. Scanning electrochemical microscopy (SECM) was also used, and surface modifications of 100 μm were obtained depending on the experimental conditions. Different detection methods were employed for the reading of the hybridization event: fluorescence imaging, surface plasmon resonance imaging (SPRI), photocurrent measurements, and voltamperometric measurements using intercalators. Their advantages concerning the various immobilization strategies will also be discussed.  相似文献   

13.
采用巯基化合物自组装 /共价键合反应的逐层固定方法将双链 DNA固定到金表面得到 DNA修饰电极 ,并对该电极表面进行了电化学和 X射线光电子能谱表征 .研究了电极表面固定化 DNA的表面分子杂交 .对开发电化学基因诊断芯片和基因传感器具有一定意义  相似文献   

14.
The potential for a new biochip design based on a continuous gradient of density of immobilized single-stranded DNA oligonucleotide probes (ssDNA) is explored. This gradient resolved information platform (GRIP) can provide sequence identification based on the spatial location and extent of hybridization by a target sequence. Surfaces based on indium-tin oxide (ITO) on glass were first functionalized by 3-aminopropyltriethoxysilane (APTES) followed by attachment of glutaraldehyde, prior to immobilization of oligonucleotide probe that was terminated with amine. The use of Cy3 and Cy5 dye-labelled ssDNA probes and targets allowed estimation of density and correlation of the location of binding of labelled targets. Probe molecules of 20 mer lengths were loaded to produce density gradients in the range of 1.0-200 ng/mm2. The biochips could resolve a mixture of fully complementary five base-pair mismatched targets by the location of binding on the surface. Thermal control provided additional selectivity. Thermal cycling and washing provided for regeneration of the surface, and the fluorescence intensities showed no deterioration in at least five cycles of hybridization reactions.  相似文献   

15.
Sequence-specific detection and quantification of nucleic acids are central steps in many molecular biology procedures which have also been transferred to chip-based procedures. Hybridization-based assays can be used to quantify and discriminate between DNA target sequences down to the level of single base mismatches. Arrays of DNA probes immobilized on a support enable simultaneous testing of multiple sequences of a single sample. DNA arrays can be produced either by in-situ synthesis of oligonucleotides or by immobilization of pre-assembled DNA probes. Covalent and directed immobilization improves the reproducibility and stability of DNA arrays. This is especially interesting with repeated use of transducers or chips. Procedures are described for effective covalent immobilization of pre-assembled amino-linked oligonucleotides, by use of ink-jet techniques, on a modified and heated glass surface, with addressable surface areas ranging from 0.01 mm2 to a few mm2. Almost immediate evaporation of the spotted droplets on the heated surfaces leads to very high surface hybridization capacities. The surfaces are suitable for use with a label-free detection method - reflectometric interference spectroscopy (RIfS). It is shown that hybridization capacity and non-specific interaction at these DNA-surfaces can be characterized by use of RIfS. With a consumption of less than 80 ng mm(-2) oligonucleotide and a specific hybridization capacity of more than 300 fmol mm(-2), the activated aminodextran procedure was usually suitable for setting up a DNA array with label-free detection. Non-specific interactions with random oligomers or protein (ovalbumin) were low. Up to 150 repeated regenerations (stripping) of the surfaces by acid treatment and denaturing agents, and 50 days of storage, have been possible without significant loss of hybridization capacity.  相似文献   

16.
Si/SiO(2) chips were used to detect the hybridization of immobilized Oligo d(T)(20) through impedance measurement. The immobilization procedure involved an aminopropyl silane grafted silicon oxide surface activated by glutaraldehyde and subsequently modified by an aminolinker supporting oligonucleotides. The immobilization procedure was optimized and, in the best conditions, the hybridization of the immobilized oligonucleotide was able to generate a 50 Omega impedance change at an applied dc potential of -300 mV. The optimized DNA sensor was then used to directly determine the immobilized oligonucleotide T(m) via impedance measurement in a continuous temperature control flow system. A reproducible and specific 65 Omega impedance change was observed at 32 degrees C with a step duration as low as 15 min. This value compared well with the 31.4 degrees C theoretical value calculated from the sequence base pair composition and length.  相似文献   

17.
Different methods for the covalent immobilization of specific antibodies and their fragments on a silicon surface with the subsequent formation of immune complexes that consist of an immobilized monoclonal antibody, an antigen molecule, and a molecule of a second monoclonal antibody labeled with gold nanoparticles have been studied. Prostate-specific antigen (PSA), which is a molecular biomarker for prostate cancer, was used as an antigen. A covalent conjugate of the fragments of PSA-specific antibodies with gold nanoparticles has been obtained using the thiol groups of the antibodies. Scanning electron microscopy (SEM) was used for the registration of immune complexes on the surface. The high resolution of the method made it possible to detect individual immune complexes by the presence of gold nanoparticles and to calculate their number. A new method for the chemical modification of silicon by 3-aminopropyltrimetoxysilane (APTMS) and a bifunctional reagent 1,4-phenylene diisothiocyanate (PDITC) has been developed. This method provides a uniform distribution of antigen-binding centers and their availability for the formation of immune complexes. The developed immobilization method is promising for the formation of a biospecific biosensor layer based on silicon nanowires.  相似文献   

18.
玻璃微流控通道中水凝胶固定寡核苷酸探针的方法及应用   总被引:1,自引:0,他引:1  
核酸杂交是分子生物学研究中最常用和最基本的分析方法之一.杂交技术有多种,主要区别在于探针的固定.目前常用的是将探针直接固定在载体表面(尼龙膜或硅烷化的玻片)或用磁珠法和水凝胶法固定,其中水凝胶法兼有三维立体和简单实用的优势,其发展颇为引人注意.微流控芯片技术具有集成化和自动化的优势.将水凝胶和微流控技术相结合,将使核酸分析中的杂交、变性以及重新杂交等操作更为简单、快速、易行.  相似文献   

19.
A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16 INK4A gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed. The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional surface. The p16 INK4A gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes. The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray technology.  相似文献   

20.
A method for the development of continuous density gradients of immobilized oligonucleotide probes (20mer) along the length of microfluidic channels is demonstrated. The development of continuous density gradients was achieved using variable electrokinetic transport of probes in hybrid glass-polydimethylsiloxane microfluidic chips. The probes were terminated with an amine functional group, and were delivered by electrokinetic pumping to the flat glass channel wall after it had been densely coated with covalently immobilized aldehyde groups. This method provided probe immobilization densities ranging from 4.5(±0.8)×10(13) to 2.5(±0.8)×10(11) molecules cm(-2), with longitudinal dilution and differential mass transport of the injected plug of probes being the primary factors responsible for the gradient of density. The utility of the resulting density gradient of immobilized probes to control the selectivity of hybridization was demonstrated at room temperature by discrimination between a fully complementary oligonucleotide target, and a target strand containing 3 base pair mismatches (3 BPM) based on the spatial pattern of hybridization for sub-picomole quantities of targets. Single nucleotide polymorphism (SNP) discrimination was possible when temperature control was implemented to improve resolution of the mismatch discrimination, allowing SNP discrimination at 35 °C with a contrast ratio of almost 5 to 1.  相似文献   

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