Automated and Multiplexed Soft Lithography for the Production of Low-Density DNA Microarrays.

Julie Fredonnet,Childérick Séverac,Jean Marie François,Emmanuelle Trévisiol,Julie Foncy,Jean-Christophe Cau

doi:10.3390/microarrays5040025

Abstract

Microarrays are established research tools for genotyping, expression profiling, or molecular diagnostics in which DNA molecules are precisely addressed to the surface of a solid support. This study assesses the fabrication of low-density oligonucleotide arrays using an automated microcontact printing device, the InnoStamp 40®. This automate allows a multiplexed deposition of oligoprobes on a functionalized surface by the use of a MacroStampTM bearing 64 individual pillars each mounted with 50 circular micropatterns (spots) of 160 µm diameter at 320 µm pitch. Reliability and reuse of the MacroStampTM were shown to be fast and robust by a simple washing step in 96% ethanol. The low-density microarrays printed on either epoxysilane or dendrimer-functionalized slides (DendriSlides) showed excellent hybridization response with complementary sequences at unusual low probe and target concentrations, since the actual probe density immobilized by this technology was at least 10-fold lower than with the conventional mechanical spotting. In addition, we found a comparable hybridization response in terms of fluorescence intensity between spotted and printed oligoarrays with a 1 nM complementary target by using a 50-fold lower probe concentration to produce the oligoarrays by the microcontact printing method. Taken together, our results lend support to the potential development of this multiplexed microcontact printing technology employing soft lithography as an alternative, cost-competitive tool for fabrication of low-density DNA microarrays.

Highlights

Immobilization of nucleic acids in array format requires the attachment of DNA molecules at well-defined positions onto the surface of a solid support
We explored the potential of producing an oligonucleotide array using an automated microcontact printing device, the InnoStamp 40®, and compared biochips fabricated by this technology with those using a conventional microarrayer
We showed that the magnetic MacroStampTM composed of 64 pillars, each microstructured at its extremity by a repetition of 50 circular micropatterns of 160 μm diameter, allowed the simultaneous deposition of 64 different molecules in one step at high micropatterned resolutions and without any contamination between oligonucleotide probes inked on adjacent pillars

Summary

Introduction

Immobilization of nucleic acids in array format requires the attachment of DNA molecules (probes) at well-defined positions onto the surface of a solid support. DNA probes onto a solid support using metal pins [8] or microactuated nozzles [9]. Another way to immobilize oligonucleotides on the surface is in situ high-density DNA microarray fabrication, which is based on the step by step direct synthesis of oligonucleotides on the surface using light-activated chemistry combined with photolithographic techniques using pre-made masks [10,11] or moving mirrors, leading to maskless array synthesis and flexibility in array design and manufacturing [7]

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Results

Discussion

Conclusion