In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication

Abstract

Glass has become the standard substrate for the preparation of DNA arrays. Typically, glass is modified using silane chemistries to provide an appropriate functional group for nucleic acid synthesis or oligonucleotide immobilization. We have found substantial issues with the stability of these surfaces as manifested in the unwanted release of oligomers from the surface when incubated in aqueous buffers at moderate temperatures. To address this issue, we have explored the use of carbon-based substrates. Here, we demonstrate in situ synthesis of oligonucleotide probes on carbon-based substrates using light-directed photolithographic phosphoramidite chemistry and evaluate the stabilities of the resultant DNA arrays compared to those fabricated on silanized glass slides. DNA arrays on carbon-based substrates are substantially more stable than arrays prepared on glass. This superior stability enables the use of high-density DNA arrays for applications involving high temperatures, basic conditions, or where serial hybridization and dehybridization is desired.

A simple and cost-effective molecular diagnostic system and DNA probes synthesized by light emitting diode photolithography

“…For this, a 0.1% acetic acid in 95% ethanol stock solution was prepared. 10) The chip was immersed in 4% (v/v) N-(3-triethoxysilylpropyl)-4-bydroxy-butyramine (Gelest) in stock solution for 4 h at room temperature. Then, the slides were washed in stock solution for 15 min and rinsed three times in diethyl ether.…”

“…5,6) Then, maskless lithography appeared, where patterns were created by light reflection either from analog or digital mirrors. [7][8][9][10] Direct synthesis of DNA on a platform is important because today real-time PCR and DNA microarrays are the most commonly used molecular diagnostics tools for in-vitro diagnosis (IVD). With the real-time PCR, one can determine the amount of viral particles in blood and quantitatively analyze the number of DNA in samples.…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…A carbon chip is treated with 9-decene-1-ol under UV light to modify the surface with alcohol groups, which can be used directly to synthesise oligonucleotides using photolithographic synthesis. 11 Such arrays are reported to be much more stable, usable under higher temperature and extremes of pH and therefore able to be re-used for longer periods. Oligonucleotides have been attached to glass surfaces by first treating the oligonucleotides with 3-glycidyloxypropyltriethoxysilane and then attaching to glass using silanising chemistry.…”

“…54 It has been proposed that introduction of a positive charge may facilitate double strand invasion. Using the lysine derivative (11) it has been shown that the modified PNA will double strandinvade duplex DNA even at GC-rich sequences. 55 Negative charges have also been introduced into PNA to aid cellular uptake of antisense PNA by addition to the N-terminus of substitutions of either phosphonate glutamine (12) or bis-phosphonate lysine.…”

Parallel DNA Synthesis on Poly(ethylene terephthalate)

“…[5] The oligonucleotide attachment chemistry also proved more stable to elevated temperatures than the silyl-ether bond found in arrays synthesized on silanized glass. [6] However, a limitation of this work was that the polymer bilayers used to install hydroxyl groups on the PET were prone to delamination during the array fabrication. This limited the length of the oligonucleotides which could be synthesized as well as the complexity of the arrays.…”