<i>In situ</i> synthesis and direct immobilization of ssDNA on electron beam patterned hydrogen silsesquioxane

Negrete, Omar D.; Önses, M. Serdar; Nealey, Paul F.; Cerrina, F.

doi:10.1116/1.3263190

Cited by 3 publications

(3 citation statements)

References 30 publications

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“…The microarrays are typically synthesized on standard-format glass microscope slides, but other chemically-compatible substrates with native surface hydroxyl groups, or surfaces which can be functionalized to add hydroxyl groups, can be used. Glassy carbon and nanocrystalline diamond films [ 7 ], carbon-on-metal films [ 8 ], and electron beam patterned hydrogen silsesquioxane [ 9 ] have been successfully used as substrates in MAS.…”

Section: Introductionmentioning

confidence: 99%

Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

et al. 2011

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BackgroundLight-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate.ResultsStepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast.ConclusionsMaskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies.

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Section: Introductionmentioning

confidence: 99%

Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

et al. 2011

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“…The work reported here was motivated by our interests in the design of reactive, polymer-based interfaces − and expanding the range of substrates that are compatible with in situ oligonucleotide synthesis. ,− Polymer-based substrates could present attractive alternatives to glass and carbon-based substrates used in past studies because they are low cost, durable, and easily processed. Approaches based on polymer thin films could also be attractive because they can often be used to functionalize the surfaces of nonplanar (i.e., curved) objects and porous/flexible substrates that could provide practical advantages during synthesis or subsequent screening.…”

Section: Introductionmentioning

confidence: 99%

In situ Synthesis of Oligonucleotide Arrays on Surfaces Coated with Crosslinked Polymer Multilayers

et al. 2011

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We report an approach to the in situ synthesis of oligonucleotide arrays on surfaces coated with crosslinked polymer multilayers. Our approach makes use of methods for the ‘reactive’ layer-by-layer assembly of thin, amine-reactive multilayers using branched polyethyleneimine (PEI) and the azlactone-functionalized polymer poly(2-vinyl–4,4’-dimethylazlactone) (PVDMA). Post-fabrication treatment of film-coated glass substrates with d-glucamine or 4-amino-1-butanol yielded hydroxyl-functionalized films suitable for the Maskless Array Synthesis (MAS) of oligonucleotide arrays. Glucamine-functionalized films yielded arrays of oligonucleotides with fluorescence intensities and signal-to-noise ratios (after hybridization with fluorescently labeled complementary strands) comparable to those of arrays fabricated on conventional silanized glass substrates. These arrays could be exposed to multiple hybridization-dehybridization cycles with only moderate loss of hybridization density. The versatility of the layer-by-layer approach also permitted synthesis directly on thin sheets of film-coated poly(ethylene terephthalate) (PET) to yield flexible oligonucleotide arrays that could be readily manipulated (e.g., bent) and cut into smaller arrays. To our knowledge, this work presents the first use of polymer multilayers as a substrate for the multi-step synthesis of complex molecules. Our results demonstrate that these films are robust and able to withstand the ~450 individual chemical processing steps associated with MAS (as well as manipulations required to hybridize, image, and dehybridize the arrays) without large-scale cracking, peeling, or delamination of the thin films. The combination of layer-by-layer assembly and MAS provides a means of fabricating functional oligonucleotide arrays on a range of different materials and substrates. This approach may also prove useful for the fabrication of supports for the solid-phase synthesis and screening of other macromolecular or small-molecule agents.

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“…Functionalization of the substrate with a two-step linker chemistry led to specific attachment of oligonucleotides to the SiO 2 patterns. Alternatively, oligonucleotides were directly synthesized/immobilized on a nanopatterned negative tone photoresist on a silicon background by multistep functionalization of the substrate. Despite the different techniques developed, the ability to pattern DNA oligonucleotides using lithographic techniques simultaneously meeting sub-50 nm resolution, minimal nonspecific binding to background and high hybridization efficiency remains a challenge.…”

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Localization of Multiple DNA Sequences on Nanopatterns

et al. 2011

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DNA oligonucleotides of different sequences were patterned at the nanoscale. Areas of positive charge were generated by exposure of insulating substrates, spin-on hydrogen silsesquioxane or vapor-deposited SiO(2) on Si, with ionizing radiation sources used in electron beam and extreme ultraviolet lithography. Au nanoparticles (NPs) with a diameter of 15 nm, carrying covalently bound negatively charged single-stranded DNA oligonucleotides, were site specifically immobilized directly on the exposed regions and presented oligonucleotides for subsequent hybridization. Repeated exposure and deposition of NPs allowed for patterning multiple DNA sequences. Patterns with dimensions as small as 15 nm were fabricated using electron beam lithography. The use of DNA-functionalized NPs rather than just DNA facilitates metrology in scanning electron microscopy and improves the hybridization efficiency of the oligonucleotides on the surface.

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In situ synthesis and direct immobilization of ssDNA on electron beam patterned hydrogen silsesquioxane

Cited by 3 publications

References 30 publications

Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

In situ Synthesis of Oligonucleotide Arrays on Surfaces Coated with Crosslinked Polymer Multilayers

Localization of Multiple DNA Sequences on Nanopatterns

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