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1.
Fouqué B Schaack B Obeïd P Combe S Gétin S Barritault P Chaton P Chatelain F 《Biosensors & bioelectronics》2005,20(11):2335-2340
In microarrays experiments, a serious limitation is the unreliability of low signal intensities data and the lack of reproducibility for the resulting ratios between samples and controls. Most of the light emitted by a fluorophore at the air/glass interface of a glass slide is absorbed by the glass so just a part of the emitted fluorescence is detected. To improve the sensitivity of the fluorescence detection of both common fluorophores Cy3 and Cy5 in DNA microarrays and fluorescent cell analyses, we have designed a multi layer mirror with alternative thin layers of SiO2 and HfO2. This mirror (MOTL) prevents fluorescence absorption, allows the simultaneous enhancement of the fluorescence signals and increases the dynamic range of the slides. Using MOTL slides, Cy3 and Cy5 intensities are enhanced by 5-8-fold, consequently, the fluorescence analysis becomes easier and should allow the detection of low copy number genes or weakly fluorescent cells. With the same approach, other multiple optical thin layer slides could be designed for other series of fluorophores, extending the field of their applications. 相似文献
2.
Increasing the sensitivity of DNA microarrays by metal-enhanced fluorescence using surface-bound silver nanoparticles 总被引:1,自引:0,他引:1
The effects of metal-enhanced fluorescence (MEF) have been measured for two dyes commonly used in DNA microarrays, Cy3 and Cy5. Silver island films (SIFs) grown on glass microscope slides were used as substrates for MEF DNA arrays. We examined MEF by spotting biotinylated, singly-labeled 23 bp DNAs onto avidin-coated SIF substrates. The fluorescence enhancement was found to be dependent on the DNA spotting concentration: below ~12.5 μM, MEF increased linearly, and at higher concentrations MEF remained at a constant maximum of 28-fold for Cy5 and 4-fold for Cy3, compared to avidin-coated glass substrates. Hybridization of singly-labeled oligonucleotides to arrayed single-stranded probes showed lower maximal MEF factors of 10-fold for Cy5 and 2.5-fold for Cy3, because of the smaller amount of immobilized fluorophores as a result of reduced surface hybridization efficiencies. We discuss how MEF can be used to increase the sensitivity of DNA arrays, especially for far red emitting fluorophores like Cy5, without significantly altering current microarray protocols. 相似文献
3.
Dendrimeric coating of glass slides for sensitive DNA microarrays analysis 总被引:7,自引:1,他引:6
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Le Berre V Trévisiol E Dagkessamanskaia A Sokol S Caminade AM Majoral JP Meunier B François J 《Nucleic acids research》2003,31(16):e88
Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive ~100 Å layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 µM with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations. 相似文献
4.
Bras M Dugas V Bessueille F Cloarec JP Martin JR Cabrera M Chauvet JP Souteyrand E Garrigues M 《Biosensors & bioelectronics》2004,20(4):797-806
This paper presents a comprehensive theory and experimental characterisation of the modulation of the fluorescence intensity by the construction of optical interferences on oxidised silicon substrates used for DNA microarrays. The model predicts a 90-fold variation of the fluorescence signal depending on the oxide thickness. For a Cy3 dye, the signal is maximal for a 90 nm oxide thickness corresponding to a 7.5-fold enhancement with respect to a standard glass substrate. For experimental validation of the model, we have prepared Si/SiO2 substrates with different parallel steps of decreasing oxide thicknesses on the same sample using a buffered oxide etch (BOE) etching process after thermal oxidation. The SiO2 surface has been functionalized by a silane monolayer before in situ synthesis of L185 oligonucleotide probes. After hybridisation with complementary targets, the variations of the fluorescence intensity versus oxide thickness are in very good accordance with the theoretical model. The experimental comparison against a glass substrate shows a 10-fold enhancement of the detection sensitivity. Our results demonstrate that a Si/SiO2 substrate is an attractive alternative to standard glass slides for the realisation of fluorescence DNA microarrays whenever detection sensitivity is an important issue. 相似文献
5.
用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因 总被引:35,自引:0,他引:35
以基因表达谱芯片对人正常肝及肝癌组织基因表达的差异性进行了研究比较。奖4096条人cDNA用点样仪点在特制玻片上制备成表达谱芯片;利用肝和肝癌组织的mRNA通过逆转录方法,将Cy3和Cy52种荧光分别标记到两种组织的cDNA上,制备成cDNA探针,并与表达谱芯片进行杂交及扫描,重复4次实验,通过计算机数据处理判定基因是否在上述2种组织中有表达差异,筛选出差异表达的基因共903条。基因芯片技术可同时 相似文献
6.
百合病毒的DNA芯片检测技术研究 总被引:1,自引:0,他引:1
根据已知的黄瓜花叶病毒,百合无症病毒、百合斑驳病毒基因核苷酸序列,设计引物和探针,制备寡核苷酸芯片。用Cy3标记核苷酸引物,不对称RT-PCR扩增产物与芯片上的寡核苷酸探针杂交,荧光扫描仪检测并分析信号。研究制备的基因芯片能够检测侵染百合的3种重要病毒核酸的特异性荧光信号,该项技术具有特异、灵敏、快速的优点。 相似文献
7.
Rajendra J Redkar Niko A Schultz Volker Scheumann Luis A Burzio Daniel E Haines Ezzeldin Metwalli Otmar Becker Samuel D Conzone 《Journal of biomolecular techniques》2006,17(2):122-130
Optical inteference (OI) coated slides with unique optical properties were utilized in microarray analyses, demonstrating their enhanced detection sensitivity over traditional microarray substrates. The OI coating is comprised of a proprietary multilayered, dielectric, thin-film interference coating located beneath the functional coating (aminosilane or epoxysilane). It is designed to enhance the fluorescence in the Cy3 and Cy5 channel by increasing the light absorption of the dyes by about 6-fold and by redirecting emitted fluorescence into the detector during scanning, resulting in a theoretical limit of about 12-fold signal amplification. Two-color DNA microarray experiments conducted on the OI slides showed over 8-fold signal amplification, conservation of gene expression ratios, and increased signal-to-noise ratio when compared to control slides, indicating enhanced detection sensitivity. Protein microarray assays also exhibited over 8-fold signal amplification at three different target concentrations, demonstrating the versatility of the OI slides for different microarray applications. Further, the DNA and protein assays performed on the OI slides exhibited excellent detection sensitivity even at the low target amounts essential for diagnostic applications. The OI slides are compatible with commonly used protocols, printers, scanners and other microarray equipment. Therefore, the OI slides offer an attractive alternative to traditional microarray substrates, where enhanced detection sensitivity is desired. 相似文献
8.
To establish a rapid, sensitive and specific diagnostic assay for Hantavirus with microarray techniques, specific primers and probes were designed according to the conservative and specific DNA sequence of 76-118 strain and R22 strain. The probes were spotted on glass slides to form microarrays.The Cy3-1abled single stranded DNA fragments prepared by dissymmetical PCR were hybridized with the probes on the glass slides. The microarrays were scanned and analyzed with a scanner. The results showed that the DNA microarray could detect the different typed DNA of HTN and SEO with adequate specificity and sensitivity. The developed DNA microarray and techniques might be a very useful method for diagnosis and prevention, and could be widely applied in specific pathogens detection ofinfectious diseases such as hemorrhagic fever with renal syndrome. 相似文献
9.
We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on. 相似文献
10.
人乳头瘤病毒(HPV)基因芯片的研究 总被引:6,自引:0,他引:6
探讨将基因芯片与限制性显示技术相结合对HPV进行基因检测和分型的方法。分离HPV6,11,16和18型的基因片段作为探针,纯化后应用PixSys 5500点样仪将其打印在氨基包被的玻片上制作HPV基因芯片,对HPV样品进行荧光标记后与芯片杂交,经清洗和干燥后对芯片进行扫描和结果分析。对HPV基因检测芯片的制作与检测的实验条件进行了初步研究,并对应用HPV基因芯片进行分型做了初步探讨。建立的检测芯片实验方法可行,并且显示了在HPV分型中的应用前景。 相似文献
11.
Olivier C Hot D Huot L Ollivier N El-Mahdi O Gouyette C Huynh-Dinh T Gras-Masse H Lemoine Y Melnyk O 《Bioconjugate chemistry》2003,14(2):430-439
We describe in this paper the preparation and characterization of semicarbazide glass slides and their use for the fabrication of microarrays using site-specific alpha-oxo semicarbazone ligation. The functional density and homogeneity of the semicarbazide glass slides were optimized by analyzing the reactivity of the layer toward a synthetic glyoxylyl fluorescent probe. Oligonucleotide microarrays were prepared by site-specific immobilization of glyoxylyl oligodeoxynucleotides. The slides were directly used in the hybridization assays using fluorescence detection and displayed a significant gain in sensibility as compared to the aldehyde glass slide/amino oligodeoxynucleotide chemistry. Semicarbazide slides were also used for the immobilization of a biotinylated peptide alpha-oxo aldehyde. The peptide microarrays allowed model interaction studies with streptavidin or an anti-biotin antibody. 相似文献
12.
蛋白质微阵列生产用琼脂糖修饰玻片制备的条件优化 总被引:5,自引:1,他引:4
目的:建立一种以琼脂糖修饰的玻片为载体的蛋白质微阵列制备的优化方法,比较琼脂糖修饰玻片和醛基修饰玻片及氨基修饰玻片对蛋白质固定效率的优劣。方法:将羊IgG固定在载体表面,经过洗涤、封闭,再加入Cy3标记的兔抗羊IgG,孵育,洗涤后用共聚焦激光扫描仪获取图像,检测各点的荧光强度,根据荧光强度确定最佳琼脂糖浓度,最佳NaIO4浓度,最佳固定时间以及封闭时间等实验条件。结果:琼脂糖浓度为1.2%、NaIO4浓度为20mmol/L、固定时间为1h、孵育时间为45min时,蛋白质在载体上的固定效率和反应活性最高。在固定的抗体浓度相同的情况下,琼脂糖修饰玻片荧光强度是醛基修饰玻片的2.6倍,是氨基修饰玻片的9倍。结论:确立了蛋白质微阵列生产用琼脂糖修饰玻片制备的优化条件,用该优化条件制备的琼脂糖玻片更适合用于蛋白质微阵列载体。 相似文献
13.
Malainou A Petrou PS Kakabakos SE Gogolides E Tserepi A 《Biosensors & bioelectronics》2012,34(1):273-281
We demonstrate a method to create high density protein microarrays with excellent spot uniformity using photolithography and plasma processing on low cost commercially available microscope glass slides. Protein deposition and fluorescence signal evaluation on these substrates are performed by standard arrayers and scanners. To this end, spots of commercial photoresists (AZ5214, SU8 and Ormocomp(?)) were defined through lithography on glass substrates followed by short SF(6) plasma treatment and selective protein adsorption on these spots with respect to glass (spot to background fluorescence signal ratios 30:1 to 40:1) was demonstrated using model protein binding assays. Among the photoresists tested, Ormocomp was selected since it provided the highest protein binding capacity. No ageing of Ormocomp/glass substrates in terms of protein binding capacity was observed for at least two months. Besides to protein microarrays, DNA microarrays were also developed by spotting streptavidin-biotinylated oligonucleotide conjugates corresponding to wild- and mutant-type sequences of four deleterious BRCA1 gene mutations. For all of the examined mutations, higher specific hybridization signals (1.5-4 times) and improved discrimination ratios between wild- and mutant-type sequences as well as higher spot uniformity and repeatability were demonstrated on Ormocomp/glass substrates with intra- and inter-spot CVs of 8.0% and 4.5%, respectively, compared to commercial polystyrene (intra- and inter-spot CVs 36% and 18%) and epoxy-coated glass (intra- and inter-spot CVs 26% and 20%) slides. Thus, the proposed substrates can be readily applied to protein and DNA microarrays fabrication and, moreover, the described method for selective protein adsorption can be advantageously implemented in various analytical microdevices for multi-analyte detection. 相似文献
14.
Yuri B. Yurov Ilia V. Soloviev Svetlana G. Vorsanova Bertrand Marcais Gerard Roize Robert Lewis 《Human genetics》1996,97(3):390-398
We tested DNA probes directly labeled by fluorescently labeled nucleotides (Cy3-dCTP, Cy5-dCTP, FluorX-dCTP) for high resolution uni- and multicolor detection of human chromosomes and analysis of centromeric DNA organization by in situ hybridization. Alpha-satellite DNA probes specific to chromosomes 1, 2, 3, 4 + 9, 5 + 19, 6, 7, 8, 10, 11, 13 + 21, 14 + 22, 15, 16, 17, 18, 20, 22, X and Y were suitable for the accurate identification of human chromosomes in metaphase and interphase cells. Cy3-labeled probes had several advantages: (1) a high level of fluorescence (5–10 times more compared with fluorescein-labeled probes); (2) a low level of fluorescence in solution, allowing the detection of target chromosomes in situ during hybridization without the washing of slides; and (3) high resistance to photobleaching during prolonged (1-2 h) exposure to strong light, thus allowing the use of a high energy mercury lamp or a long integration time during image acquisition in digital imaging microscopy for the determination of weak signals. For di- and multicolor fluorescence in situ hybridization (FISH), we successfully used different combinations of directly fluorophorated probes with preservation of images by conventional microscopy or by digital imaging microscopy. FluorX and Cy3 dyes allowed the use of cosmid probes for mapping in a one-step hybridization experiment. Cyanine-labeled fluorophorated DNA probes offer additional possibilities for rapid chromosome detection during a simple 15-min FISH procedure, and can be recommended for basic research and clinical studies, utilizing FISH. 相似文献
15.
Development of a toxicological gene array and quantitative assessment of this technology 总被引:34,自引:0,他引:34
Bartosiewicz M Trounstine M Barker D Johnston R Buckpitt A 《Archives of biochemistry and biophysics》2000,376(1):66-73
High-density arrays of DNA bound to solid substrates offer a powerful approach to identifying changes in gene expression in response to toxicants. While DNA arrays have been used to explore qualitative changes in gene regulation, less attention has focused on the quantitative aspects of this technology. Arrays containing expressed sequence tags for xenobiotic metabolizing enzymes, proteins associated with glutathione regulation, DNA repair enzymes, heat shock proteins, and housekeeping genes were used to examine gene expression in response to beta-naphthoflavone (beta-NF). Upregulation of cytochrome P4501a1 (Cyp1a1) and 1a2 in mouse liver was maximal 8 h after beta-NF administration. Significant upregulation of Cyp1a2 was noted at beta-NF doses as low as 0.62 and 1.2 mg/kg when gene expression was measured by microarray or Northern blotting, respectively. Maximal Cyp1a2 induction is 5-fold by Northern analysis and 10-fold by microarray. Induction of Cyp1a1 was 15- and 20-fold by Northern and microarray analysis, respectively. The coefficient of variation for spot to spot and slide to slide comparisons was <15%; this variability was smaller than interanimal variability (18-60%). Comparison of mRNA expression in control animals indicated that there are differences in labeling/detection associated with Cy3/Cy5 dyes; accordingly, experiments must include methods for establishing baseline signals for all genes. We conclude that the dynamic range and sensitivity of DNA microarrays on glass slides is comparable to Northern blotting analysis and that variability of the data introduced during spotting and hybridization is less than the interanimal variability. 相似文献
16.
17.
蛋白质微阵列检测抗原-抗体相互作用 总被引:2,自引:0,他引:2
为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料(Cy3,Cy5)对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测. 相似文献
18.
Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays 总被引:1,自引:0,他引:1
A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm2. The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray™ slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm2, respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques. 相似文献
19.
Oillic C Mur P Blanquet E Delapierre G Vinet F Billon T 《Biosensors & bioelectronics》2007,22(9-10):2086-2092
To improve the sensitivity of fluorescence detection in DNA microarrays, the use of silicon nanostructures based on chemical vapor deposition (CVD) processes adopted for the growth of rough polycrystalline silicon was investigated. These substrates present advantages of two main properties which could lead to an enhancement of the fluorescence detection, i.e. (i) the increase of the available surface area in order to achieve a high loading capacity of biomolecules and (ii) the optimization of the stack of silicon nanostructures support. Indeed, the structures were elaborated on an initial thermal oxide layer and then covered with a silicon oxide layer, obtained by oxidation and allowing the functionalization for the subsequent grafting of DNA probes. Moreover, these oxide layers play a part in the fluorescence detection. The influence of the silicon oxide layer thickness above and below the silicon grains in close relation with the density of nanostructures on the emitted fluorescence was emphasized. This paper presents an experimental characterization of the fluorescence intensity and the optimization of the different layers that composed the substrate used for DNA microarrays. The performances of the microarrays were investigated by means of hybridization experiments using complementary fluorescent labeled-oligonucleotides targets. Our results indicate that an optimized substrate can be designed and that the use of oxidized silicon nanostructures for support of biochip could be a strategy for improving the sensitivity of fluorescence detection. 相似文献
20.