Functionalized nanocomposite coating of a glass surface for oligonucleotide immobilization

A new type of coating for manufacturing DNA chips was constructed on the basis of an organicinorganic nanocomposite based on the polyvinylbutyral-tetraethoxysilane copolymer. The organosilicon composite was functionalized by introduction of ethanolamine vinyl ether copolymers, which contain amino groups and anchor vinyloxide units capable of reacting with silanol groups of the nanocomposite. The resulting coatings form a film on glass slides with a high surface density of amino groups (up to 700 groups/nm²) suitable for three-dimensional immobilization of oligonucleotides. The use of bifunctional reagents (e.g., phenylene diisothiocyanate) for the attachment of oligonucleotides bearing amino linkers to the amino-containing surface provides an immobilization density of 0.5–1.6 pmol/mm². Immobilization with a higher density (10–12 pmol/mm²) was achieved for attachment to amino-containing glass slides upon the use of oligonucleotides containing a selectively activated terminal phosphate group. The activation of oligonucleotides was carried out with the triphenylphosphine-dithiodipyridine pair in the presence of dimethylaminopyridine N-oxide. The resulting DNA chips were shown to be useful in principle for DNA detection.     

Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms

Different chemical methods used to attach oligonucleotides by their 5′-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5′-specific linkage that satisfyingly withstands PCR conditions and leaves the 3′-ends available for DNA polymerase activity. The best results were obtained with 5′-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers. Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5′-end on a solid support and can be used as an alternative route for producing DNA chips for genomic studies.     

Detection of DNA and proteins using amorphous silicon ion-sensitive thin-film field effect transistors

Amorphous silicon-based ion-sensitive field-effect transistors (a-Si:H ISFETs) are used for the label-free detection of biological molecules. The covalent immobilization of DNA, followed by DNA hybridization, and of the surface adsorption of oligonucleotides and proteins were detected electronically by the a-Si:H ISFET. The ISFET measurements are performed with an external Ag/AgCl microreference electrode immersed in 100mM phosphate buffer electrolyte with pH 7.0. Threshold voltage shifts in the transfer curve of the ISFETs are observed resulting from successive steps of surface chemical functionalization, covalent DNA attachment to the functionalized surface, surface blocking, and hybridization with a complementary target. The surface sensitivity achieved for DNA oligonucleotides is of the order of 1pmol/cm(2). Point-of-zero charge estimations were made for the functionalized surfaces and for the device surface after DNA immobilization and hybridization. The results show a correlation between the changes in the point-of-zero charge and the shift observed in the threshold voltage of the devices. Electronic detection of adsorbed proteins and DNA is also achieved by monitoring the shifts of the threshold voltage of the ISFETs, with a sensitivity of approximately 50nM.     

Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays

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.     

凝胶基片的制备与应用研究

在Bind-Silane^R处理的玻片上交联聚丙烯酰胺凝胶层(15mm×15mm×20μm),戊二醛活化。与末端氨基修饰的寡核苷酸片段共价结合制成芯片。这种芯片能够区分液相中序列不同的Cy3标记的目标核酸。与平面基片相比,凝胶基片具有背景低、固定探针量高、杂交时间短的优点。将细胞因子IL-4、IL-5、IL-6、IL-7、ANG、I-309和VEGF的单克隆抗体加样于凝胶基片上制成蛋白质芯片,对乳腺癌患者和正常人的血清进行检测,发现乳腺癌患者细胞因子IL-4、IL-5、I-309和VEGF的表达量高于正常人的表达量,对临床诊断具有重要的参考意义。     

Developing site-specific immobilization strategies of peptides in a microarray

In peptide-based microarrays, most existing methods do not allow for site-specific immobilization of peptides on the glass surface. We have developed two new approaches for site-specific immobilization of kinase substrates onto glass slides: (1) slides were functionalized with avidin for attachment of biotinylated peptides; and (2) slides were functionalized with thioester for attachment of N-terminally cysteine-containing peptides via a native chemical ligation reaction.     

Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation

We report the synthesis of new phosphoramidite building blocks and their use for the modification of oligonucleotides with hydrazides. The reaction of these hydrazide oligonucleotides with active esters and aldehydes is demonstrated for solution conjugation and immobilization. Compared with the established amino modified oligonucleotides, hydrazides show enhanced reactivity at neutral and acidic buffer conditions. One method to introduce hydrazides is using amidites with preformed, protected hydrazides. A completely novel approach is the generation of the hydrazide functionality during the oligonucleotide cleavage and deprotection with hydrazine. Therefore, building blocks for the introduction of esters as hydrazide precursors are described. For the enhanced attachment on surfaces branched modifier amidites, which introduce up to four reactive groups to the oligonucleotide, are applied. The efficiency of branched hydrazide oligonucleotides compared with standard amino modified oligonucleotides for the immobilization of DNA on active electronic Nanogen chips is demonstrated.     

Oligonucleotide probes containing polylysine residues for fabrication of DNA chips on various solid surfaces

Various materials, such as glass, plastic, metals, etc., are utilized for preparing DNA chips. In each particular case special approaches are used for immobilization of different oligonucleotide derivatives on the solid supports. We describe a general technique for DNA chips preparation on various unmodified surfaces using one type of oligonucleotide derivative, polylysine-oligonucleotide conjugates (PL-oligo). A long polyamine spacer in the PL-oligo conjugates provides a durable irreversible non-covalent immobilization onto a variety of solid supports and enough distance between oligonucleotides and the surface. The resulting DNA chips were shown to be useful for the detection of PCR DNA fragments and to be sensitive to single nucleotide discrepancies. They represent a promising instrument for revealing genetic diseases, genotyping viruses and bacteria, and for displaying their drug-resistant strains.     

Efficient surface patterning of oligonucleotides inside a glass capillary through oxime bond formation

The efficient surface patterning of oligonucleotides was accomplished onto the inner wall of fused-silica capillary tubes as well as on the surface of glass slides through oxime bond formation. The robustness of the method was demonstrated by achieving the surface immobilization of up to three different oligonucleotide sequences inside the same capillary tube. The method involves the preparation of surfaces grafted with reactive aminooxy functionalities masked with the photocleavable protecting group, 2-(2-nitrophenyl) propyloxycarbonyl group (NPPOC). Briefly, NPPOC-aminooxy silane 1 was prepared and used to silanize the glass surfaces. The NPPOC group was cleaved under brief irradiation to unmask the reactive aminooxy group on surfaces. These reactive aminooxy groups were allowed to react with aldehyde-containing oligonucleotides to achieve an efficient surface immobilization. The advantage associated with the present approach is that it combines the high-coupling efficiency of oxime bond formation with the convenience associated with the use of photolabile groups. The present strategy thus offers an alternative approach for the immobilization of biomolecules in the microchannels of "labs on a chip" devices.     

Directed immobilization of nucleic acids at ultramicroelectrodes using a novel electro-deposited polymer

A two-step method for the directed immobilization of nucleic acids at ultramicroelectrodes with micron-size dimensions is described. The approach is based on the immobilization of streptavidin at the surface of carbon or noble metal electrodes within a novel electro-deposited polymer, formed by electropolymerization of the natural compound scopoletin (7-hydroxy-6-methoxy-coumarin) at potentials between 0.4 and 0.7 V vs. Ag/AgCl. Biotin-tagged nucleic acids or proteins are immobilized on top of the modified electrodes in a second step. The new method has some advantages compared to classical electropolymerization approaches (e.g. polypyrrole, polyphenol), because the growing polymer is highly hydrophilic, resulting in efficient incorporation of streptavidin and a high biotin binding capacity of 6 pmol cm(-2). The polymer film seems to be non-conductive but shows good swelling properties in aqueous solutions. The feasibility of the method for the electro-directed biochemical modification of individual microelectrodes has been demonstrated by sequential immobilization of two different single strand oligonucleotides onto interdigitated ultramicroelectrodes. The resulting miniature DNA probe was used for single base mutation detection with two synthetic targets (fluorescence-labeled 17-mer oligomers) by evaluating the fluorescence patterns after hybridisation with the immobilised DNA probes. The new method is useful for the production of microelectrode based DNA chips and for the electro-directed immobilisation of biomolecules at microelectrode structures with high spatial resolution and yield.     

用氨基修饰的载玻片制作cDNA微阵列

cDNA微阵列已在基因差异表达、寻找新基因等研究方面获得广泛应用,但有关cDNA微阵列的制作,目前多采用多聚赖氨酸修饰的载玻片为探针固定载体,固定效果较差.用氨基硅烷处理的载玻片为载体制作cDNA微阵列,然后考察其固定效率、检测灵敏度、稳定性、实用性等指标.结果表明,用氨基硅烷处理的载玻片具有比多聚赖氨酸更令人满意的核酸固定效率、检测灵敏度,且稳定实用.因此,用氨基硅烷修饰的载玻片为探针固定载体制作cDNA微阵列较为理想.     

Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries

In the microarray format of the minisequencing method multiple oligonucleotide primers immobilised on a glass surface are extended with fluorescent ddNTPs using a DNA polymerase. The method is a promising tool for large-scale single nucleotide polymorphism (SNP) detection. We have compared eight chemical methods for covalent immobilisation of the oligonucleotide primers on glass surfaces. We included both commercially available, activated slides and slides that were modified by ourselves. In the comparison the differently derivatised glass slides were evaluated with respect to background fluorescence, efficiency of attaching oligonucleotides and performance of the primer arrays in minisequencing reactions. We found that there are significant differences in background fluorescence levels among the different coatings, and that the attachment efficiency, which was measured indirectly using extension by terminal transferase, varied largely depending on which immobilisation strategy was used. We also found that the attachment chemistry affects the genotyping accuracy, when minisequencing on microarrays is used as the genotyping method. The best genotyping results were observed using mercaptosilane-coated slides attaching disulfide-modified oligonucleotides.     

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis

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.     

Integrated capillary fluorescence DNA biosensor

Covalent attachment of dsDNA molecules inside a glass capillary without the need for hybridization is described. It is shown that the glass capillary has a surface density of 2.5 x 10(13) molecules/cm(2) with specific binding capacity of 62.5%. The resulting substrate was used to develop a biosensor for determining fluorescent organic analytes and metal binding with DNA. The biosensor combines highly specific immobilization chemistry with a capillary-geometry flow cell arrangement. The results show that fluorescent dyes are retained in the dsDNA-modified surface and that exposure to concentrations of nickel and lead ions resulted in a recoverable, highly reproducible diminishment of the fluorescence intensity.     

Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces

A recently described reaction for the UV-mediated attachment of alkenes to silicon surfaces is utilized as the basis for the preparation of functionalized silicon surfaces. UV light mediates the reaction of t-butyloxycarbonyl (t-BOC) protected ω-unsaturated aminoalkane (10-aminodec-1-ene) with hydrogen-terminated silicon (001). Removal of the t-BOC protecting group yields an aminodecane-modified silicon surface. The resultant amino groups can be coupled to thiol-modified oligodeoxyribonucleotides using a heterobifunctional crosslinker, permitting the preparation of DNA arrays. Two methods for controlling the surface density of oligodeoxyribonucleotides were explored: in the first, binary mixtures of 10-aminodec-1-ene and dodecene were utilized in the initial UV-mediated coupling reaction; a linear relationship was found between the mole fraction of aminodecene and the density of DNA hybridization sites. In the second, only a portion of the t-BOC protecting groups was removed from the surface by limiting the time allowed for the deprotection reaction. The oligodeoxyribonucleotide-modified surfaces were extremely stable and performed well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of nucleic acid biomolecular recognition elements to semiconductor materials.     

Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, Z(Taq) and Z(IgA), binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.     

A new heterobifunctional reagent for immobilization of biomolecules on glass surface

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.     

Attachment of benzaldehyde-modified oligodeoxynucleotide probes to semicarbazide-coated glass

Attachment of oligodeoxynucleotides (ODNs) containing benzaldehyde (BAL) groups to semicarbazide-coated glass (SC-glass) slides is described. 5′-BAL-ODNs are prepared using automated DNA synthesis and an acetal-protected BAL phosphoramidite reagent. The hydrophobic protecting group simplifies purification of BAL-ODNs by reverse phase HPLC and is easily removed using standard acid treatment. The electrophilic BAL-ODNs are stable in solution, but react specifically with semicarbazide groups to give semicarbazone bonds. Glass slides were treated with a semicarbazide silane to give SC-glass. BAL-ODNs are coupled to the SC-glass surface by a simple one-step procedure that allows rapid, efficient and stable attachment. Hand-spotted arrays of BAL-ODNs were prepared to evaluate loading density and hybridization properties of immobilized probes. Hybridization to radiolabeled target strands shows that at least 30% of the coupled ODNs were available for hybridization at maximum immobilization density. The array was used to probe single nucleotide polymorphisms in synthetic DNA targets, and PCR products were correctly genotyped using the same macroarray. Application of this chemistry to manufacturing of DNA microarrays for sequence analysis is discussed.     

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm² from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.     

Immobilization of acrylamide-modified oligonucleotides by co-polymerization.

A flexible chemistry for solid phase attachment of oligonucleotides is described. Oligonucleotides bearing 5'-terminal acrylamide modifications efficiently co-polymerize with acrylamide monomers to form thermally stable DNA-containing polyacrylamide co-polymers. Co-polymerization attachment is specific for the terminal acrylamide group. Stable probe-containing layers are easily fabricated on supports bearing exposed acrylic groups, including plastic microtiter plates and silanized glass. Attachment can be accomplished using standard polyacrylamide gel recipes and polymerization techniques. Supports having a high surface density of hybridizable oligonucleotide (approximately 200 fmol/mm2) can be produced.