2309 Angew. Chem. Int. Ed. 2003, 42, 2309–2312 DOI: 10.1002/anie. 200351256 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim nanostructures of extraordinary complexity, thus offering routes to important tools in the life sciences such as gene chips and proteomic arrays, as well as templates that could be used by chemists and materials scientists to build ordered two-and three-dimensional functional architectures.[1] Although direct-patterning approaches offer many potential advantages over indirect methods (such as facile processing and fabrication procedures, and elimination of the need for resists), they pose several challenges. Methods must be developed for facilitating the transport of the high-molecular-weight biomolecules from a coated tip to a substrate without sacrificing sub-100-nm resolution and patterning speed. Dip-pen nanolithography (DPN)[2] is a promising tool in this respect. However, in the area of protein arrays it has thus far been used primarily as a tool for making affinity arrays out of small organic molecules that can subsequently direct the assembly of proteins from solution. This indirect approach does not allow one to generate arrays made of more than one protein. Three approaches have been taken in the direct patterning of proteins by DPN. The first involves the use of a chemically tailored form of collagen that has surface-binding groups built into it to help facilitate nanostructure adsorption.[3] This designer-protein approach, although powerful, can not be used generally by researchers interested in proteomic-array research. The second method involves the use of glass pretreated with 3-glycidoxypropyltrimethoxysilane;[4] with this technique, human chorionic gonadotropin could be patterned at the micrometer scale. However, the biological activity of the resulting patterns has not been demonstrated. The third approach involves the use of gold substrates and modified tips.[5] No general methods have been developed for using DPN to generate arrays of protein nanostructures through direct deposition on oxide substrates, the materials of choice in the microarray industry, while preserving the biological activity of the proteins. Herein, we present two strategies that offer patterning capabilities from the 50-to the 550-nm scale. This method complements other approaches to the indirect patterning of proteins on surfaces through techniques such as nanografting [6] and electron-beam lithography [7], as well as larger-scale patterning techniques such as microcontact printing.[8]

The approach described herein relies on the modification of an AFM tip with 2-[methoxypoly (ethyleneoxy) propyl] trimethoxysilane (Si-PEG), which forms a biocompatible and hydrophilic surface layer. This layer inhibits protein adsorption and reduces the activation energy required for protein transport from tip to surface. It also protects the proteins from denaturation on the tip surface.[9] In the absence of this tip coating, the protein inking solutions (500 μg mLÀ1 in phosphate-buffered saline (PBS) at pH 7.3) do not wet the silicon nitride cantilevers. We have found that untreated cantilevers often produce inconsistent or low-density protein patterns. In addition to cantilever modification, we have developed two other strategies to facilitate ink transport and nanostructure formation, both of which involve silicon oxide surfaces. The first such strategy is the creation of a negatively charged surface through treatment with base [10] and the other is the use of an aldehyde-modified surface [11](Scheme1). After the formation of the desired surface the slides were rinsed with