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

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.

Lithographic Processes for the Scalable Fabrication of Micro- and Nanostructures for Biochips and Biosensors

“…The stamp was inked by immersion in a microtiter plate, where each pillar fits one well of the microplate and can potentially be inked with a different DNA sample by varying the DNA content of each single well of the plate. 73 Thus far, a resolution of only 160 μm (feature diameter) has been reported, but it is anticipated that greater resolution can be achieved with further optimization.…”

“…(B) Fluorescence microscopy image of multiplexed DNA printing, showing each individual stamp depositing a different ink. Figure adapted from ref ( 73 ). CC BY 4.0.…”

“…From this basic concept, several patterning processes have been developed . Microcontact printing (μCP) is the most mature soft lithography process where the elastomeric stamp is inked with the molecules that, when deposited on the substrate upon stamping, are able to form self-assembled monolayer (SAM) films (Figure D). − In some examples, the stamp has been mounted on a microscope or a motorized system, leading to greater precision, repeatability, and higher throughput.…”

“…Indeed, there are now credible examples of parallelization and automation of μCP to enable the printing of arrays presenting multiple DNA sequences on a single chip. 73 Although its resolution is inferior to that offered by photolithography, the low cost of implementation, together with the fact that the PDMS stamp can be used for multiple times without being re-inked (thus conserving DNA material), are significant advantages. 7,102 NIL-based fabrication processes could offer a low-cost route to the fabrication of high-resolution (down to 100 nm readily achievable) and high-density oligonucleotide arrays.…”

Innovative DendrisChips® Technology for a Syndromic Approach of In Vitro Diagnosis: Application to the Respiratory Infectious Diseases

“…This technology developed more than 25 years ago by Affymetrix [14] has been largely exploited in academic research and is still subjected to several technical developments, including microstamping, nano-tip printing, electro-printing, etc. [15,16]. However, application of microarray technology in medicine, environment or food security is almost non-existent, likely because this technology is apparently complex, not robust and not user-friendly yet [17].…”