Direct Delivery and Submicrometer Patterning of DNA by a Nanofountain Probe

Abstract

Nanopatterning of biological materials has significant potential in life sciences and medicine because highly miniaturized structures provide routes to high-density combinatorial libraries and ways of manipulating biological structures (proteins, viruses, and cells) at the single particle level. Central to the development of 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 simultaneously detect a large number of molecular species or ‘multiplex’. The miniaturization of bioassays has been eagerly pursued in an effort to achieve enhanced performance such as shorter response times, smaller sample volumes, and higher sensitivities. Dip-pen nanolithography (DPN) was developed to write nanoscale patterns directly on substrates. Among the many applications of this technology is the ability to pattern modified oligonucleotides on gold and silicon oxide surfaces. Here, feature sizes can range from a few micrometers to less than 50 nm. In a typical DNA-patterning experiment with DPN, the surfaces of silicon nitride atomic force microscopy (AFM) cantilevers are chemically modified for increased tip hydrophilicity. This step is used to improve the coating efficiency of the DNA molecules. A more attractive way of delivering inks in DPN mode is through the use of microfluidics that can deliver different inks to specific tips within a cantilever array. This approach, when it has been successfully developed, will provide greater flexibility and increased utility of DPN for the generation of combinatorial bioarrays. Other recent approaches to biomolecular patterning include the use of microfluidic components such as reservoirs, microchannels, and small apertures or slots to control the delivery of chemical solutions. Such tools do not require tip coating because samples are directly delivered onto a surface in a solution. However, the reported resolution of microfluidic tools is about 1 lm at best because of limitations from the geometries of the apertures or slots that control the molecular writing mechanisms in these devices. Herein, we present the patterning of DNA molecules on the submicrometer length scale using a volcano-like tip. This eliminates the need for tip-surface 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 allow high-resolution writing and have advantages for delivery of molecular inks in solution. A feature size as small as 40 nm was demonstrated with thiol molecules. The NFP was microfabricated to integrate an on-chip reservoir, microchannels, and a volcanolike dispensing tip. Following the development of the first generation NFP, a single-probe system, we made augmentations to produce a linear array of 12 NFP cantilevers with two on-chip reservoirs on each side of the chip, Figure 1. The cantilever lengths on each side were 630 and 520 lm, respectively. Each reservoir fed six adjacent cantilevers to achieve simultaneous patterning with two molecular species. The NFP chip was designed to fit commercially available AFM instruments and utilized the instrument scanner, optical detection, and feedback control to engage surfaces. While one cantilever was selected for the feedback control of the chip, the other cantilevers made passive contact with the surface by adjustment of the tilting angle between the array and the substrate. The multiple-tip, double-ink patterning with this linear-array NFP chip was recently demonstrated. More recently, a third generation of the NFP was fabricated. Modifications for this NFP included the possibility of fabrication on silicon-on-insulator (SOI) wafers, improved channel sealing, deeper channels to allow the delivery of larger particles, and more robust fabrication processes that led to enhanced yield and better control of C O M M U N IC A TI O N