In situsynthesis of oligonucleotide arrays by using soft lithography

Abstract

This paper describes the in situ synthesis of oligonucleotide arrays on glass surfaces by using soft lithography. In this method, based on the standard phosphoramidite chemistry protocol, the coupling was achieved by the glass slide being printed with a set of polydimethylsiloxane (PDMS) microstamps, on which was spread nucleoside monomer and tetrazole mixed solution. The elastic characteristic of PDMS allowed it to make conformal contact with the glass slide in the printing coupling. With regard to the efficiency of the printing coupling, the hybridization microscope images of 20-mer oligonucleotide synthesized via the directly drip-dropped coupling and the contact coupling were compared; the fluorescence intensities of the two methods showed no significant differences. The coupling efficiency was also investigated via an end-labelled fluorescence nucleotide method and a stepwise yield of 97% was obtained. A high-quality, high spatial resolution and large-scale PDMS stamp, which was developed by integrating 168 different microstamps on one glass substrate for synthesizing oligonucleotide arrays. The stamp was modified to improve the surface wettability by plasma discharge treatments, so that microstamps could be used to fabricate oligonucleotide arrays. A motional printing head was developed to improve the contact effect of the glass slide with different microstamps. A higher boiling point solvent was used in the printing coupling to inhibit solvent volatilization and to maintain the consistency of reagents on different features of the microstamp. An effective method was used to eliminate residual reactive nucleosides on chips with small molecules containing a hydroxyl group. A specific oligonucleotide array of four probes both matched and mismatched with the target sequence was fabricated to identify the perfect match and mismatch sequences.

Perspectives of DNA microarray and next-generation DNA sequencing technologies

“…LU Zuhong's group [11] from Southeast University in China has established another in-situ synthesis of oligonucleotide microarrays on glass surfaces by using soft lithography (called molecular stamp). This method is based on the standard phosphoramidite chemistry protocol.…”

Forced Peptide Synthesis in Nanoscale Confinement under Elastomeric Stamps

“…Microcontact printing has previously been used to couple molecules to a reactive surface, but in all cases a catalyst or activated substrate was used to induce covalentbond formation. [6][7][8][9] Herein we report the formation of new bonds solely as a result of the nanoscale confinement of molecules between stamp and surface.Amide-bond formation is a suitably challenging test case, as such reactions require catalysts (4-(dimethylamino)pyridine (DMAP), N-hydroxybenzotriazole (HOBT), or dicyclohexylcarbodiimide (DCC)) or elevated temperatures and long reaction times when performed in solution. In a typical reaction (Figure 1), we prepared a clean amine-terminated SAM surface on gold.…”

A new approach to fabricating high density nanoarrays by nanocontact printing

“…This method has been used in electronics,10–12 photonics,13 sensors,14–16 catalytic surfaces,17–19 and cell biology 20–22. However, the ability to generate dense patterns at nanoscale using nCP with accurate alignment and overlay between multiple printings has not yet been reported in literature.…”