Direct delivery and submicron patterning of DNA by nanofountain probe

Abstract

We present biomolecular patterning results using a nanofountain probe (NFP) that permits microfluidic direct delivery of oligonucleotides without hydrophilic modification of the tip for coating effectiveness. This is advantageous for integrated probes arrays that may require different functionalization to support writing with different inks, and in which damage of tips is more likely and critical. A linear array of NFPs was used to produce DNA nanoarrays. Routine feature sizes in the submicron range were obtained to prove both microfluidic transport and high-resolution writing. Gold nanoparticles functionalized with complementary sequence were hybridized to the patterned oligonucleotides, to demonstrate the biological activity of the deposited features. The results demonstrated the potential of our device as a production tool for nanofabrication of biomolecular arrays. Nanopatterning of biological materials has high potential in biology and medicine because they allow the selective recognition of biological or chemical species of interest. Central to this technology is the nanofabrication of arrays of DNA and proteins. Such arrays have influenced the areas of genomics and proteomics because of their ability to “multiplex,” i.e., to simultaneously detect a large number of molecular species. The miniaturization of bioassays has been eagerly pursued in an effort to achieve enhanced performance such as shorter response time, smaller sample volumes, and higher sensitivity [1-4]. Dip-pen nanolithography (DPN) was developed to directly write nanoscale patterns on substrates [5, 6]. Among the many applications of these technique, it has been extensively used to pattern modified oligonucleotides on gold and silicon oxide surfaces with feature sizes ranging from a few micrometers to less than 100 nm [7]. In typical DNA patterning experiments with DPN, the surface of silicon nitride AFM cantilevers is chemically modified for increased hydrophilicity of the tip surface. This step is needed to improve the coating effectiveness of the DNA molecules. In principle, tip modification limits the usability of the DPN technique beyond laboratory studies. For example, if nanoarrays of DNA are to be made using an array of DPN tips, much in the same way microarrays are made by robot spotters, the need for tip modification may lower productivity. Furthermore, during the tip modification process, the array can be contaminated or damaged. In other approaches of biomolecular patterning, microfluidic components such as reservoirs, microchannels, and small apertures or slots are commonly adopted to control the delivery of sample solutions [8-16]. Such tools do not require tip coating because samples are directly delivered onto a surface in a solution. However, the resolution of previously reported microfluidic tools is about 1 μm. This is the case because in these devices the geometry of apertures or slots controls the molecular writing mechanisms. We here present patterning of DNA molecules in a submicrometer range using a volcanolike tip. This eliminates the need for the tip modification by implementing direct delivery of a solution containing DNA molecules to the tip. As a microfluidic AFM probe, the nanofountain probe (NFP) was reported to have advantages for the delivery of molecular inks in solution and high writing resolution. A feature size as small as 40 nm was demonstrated with thiol molecules [17, 18]. The NFP was microfabricated to integrate an on-chip reservoir, microchannels, and a volcano-like dispensing tip. Following the development of the first generation NFP, a single-probe system [17, 18], we recently augmented it into a linear array of 12 NFP cantilevers with two on-chip reservoirs on each side of the chip, Figure 1 [19]. The cantilever lengths on each side are 630 μm and 520 μm, respectively. Each reservoir feeds six adjacent cantilevers to achieve simultaneous patterning with two molecular species. The NFP chip is designed to fit com