1. 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. The array is then hybridized using labeled nucleic acids (DNA or RNA targets) and the matched sequences are identified by position and fluorescent signal intensity. Microarrays are established research tools for genotyping, expression profiling, or molecular diagnostics [1,2]. Different types of patterning methods can be used to fabricate microarrays [3,4,5,6,7] and the production parameters must be adjusted for the desired applications, including the degree of multiplexing, the achievable feature shape, size and pitch, the surface area that can be patterned, and the throughput of the patterning process.
A common approach to produce DNA microarrays is to mechanically spot pre-synthesized 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]. Inkjet printing process could also be used to produce long length 60-mer DNA microarrays by in situ synthesis, one nucleotide at a time [12,13]. All of these technological platforms of production have their own advantages and weaknesses [14] and, until now, the use of DNA microarrays as routine tools in analytical/clinical laboratories for rapid molecular in vitro diagnostics is still limited.
In parallel to these technologies, microcontact printing (µCP) emerged in the literature as an alternative way of deposition of self-assembled monolayers of molecules on a surface [15,16]. This technology is also termed “soft lithography” because it is based on the use of a soft elastomeric stamp, usually made of polydimethysiloxane (PDMS), which is topographically structured by casting a PDMS prepolymer against a silicon mold leading to a structured PDMS stamp. The stamp is then inked with the molecules of interest, blown dry under a stream of nitrogen, and deposited on a solid surface, leading to patterns of molecules that are defined by the topographical structures of the stamp. In this way, microcontact printing offers a simple and low-cost surface patterning methodology with high versatility and sub-micrometer accuracy. This methodology may encompass a large range of possibilities in the patterning of various biomolecules, from nucleic acids to carbohydrate molecules [17,18,19] or proteins, being today the biomolecule of choice to be microcontact printed [20,21,22,23].
The surface of a reticulated untreated PDMS stamp is covered with methyl residues and, consequently, is hydrophobic with a water contact angle of 108° [24]. This property may weaken both inking and transfer efficiency during microcontact printing of biomolecules. Since nucleic acids are negatively-charged polyelectrolytes, electrostatic interactions play a major role in determining adsorption of the nucleic acids to the stamp (inking step) and the transfer of the inked stamp to the surface of the solid support (patterning step). To promote both steps, Lange et al. [4] functionalized the reticulated PDMS stamp and glass slide surfaces with aminosilane, rendering them attractive to DNA by electrostatic reversible binding. Using a similar double-surface functionalization strategy, Xu et al. [25] designed amphiphatic DNA (so-called DNA-surfactant) by attaching a large hydrophobic group to the 3′- or 5′-end of an oligonucleotide, thus forming a molecule able to electrostatically interact with an amino-modified reticulated PDMS stamp and able to be transferred via its hydrophobic moiety to the surface of interest. Rozkiewicz et al. [26] reported the modification of reticulated PDMS stamps with positively-charged dendrimers that can attract nucleic acids and transfer them from the surface of the stamp to a target solid support. All of these processes require the chemical functionalization of the reticulated PDMS stamp surface and are not straightforward. Thibault et al. [27] showed that, contrary to expectation, unmodified reticulated PDMS stamp surface can retain nucleic acids which can then be transferred onto the substrate surface, enabling a facile route for the production of DNA microarrays. To account for this patterning process using unmodified reticulated PDMS surfaces, these authors showed that DNA transferred onto the target surface originated from nucleic acids retained by low molecular weight (LMW) siloxane fragments present within and at the surface of the cured PDMS stamp [28].
Although various biomolecules have been successfully microcontact printed, the production of biomolecule-arrays by microcontact printing remains a challenging task and needs an effective, fast, robust, and low-cost automation process. In this paper, 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. Due to the difference in patterning (i.e., printing DNA under dry conditions versus spotting a biomolecule in solution) we showed that probe and target concentrations, as well as the nature of the surface chemistry onto which the printing is made, strongly impact on the quality and robustness of the printed biochips. Taking into account these parameters, this automated soft-lithography technology can be foreseen as an alternative competitive tool for the fabrication of low-density DNA microarrays.