1. Dip-Pen Nanolithography (DPN) DPN is a direct-write scanning-probe-based lithography in which an AFM tip is used to deliver chemical reagents directly to nanoscopic regions of a target substrate Components: Pen: AFM scanning cantilever Ink: the materials that bind to the surface Paper: Substrate such as Au, SiOx, mica Figure 1. Schematic representation of the DPN process. A water meniscus forms between the AFM tip which is coated with “ink” molecules and the solid substrate

2. Direct-Write DNP procedure Prepare the ink (Fe 2 O 3 nanoparticles hydrosol) Prepare the paper (mica & silicon, surface treatment by aqueous HF solution) Coat the cantilever with the ink Scan the area for patterning in the contact mode, with a slower scan rate, 1 µm s -1 For AFM characterization, use the same AFM, scan it at higher scan rate, 10 µm s -1

3. Direct-Write DNP for protein array Prepare the ink (MHA: mercaptohexadecanoic acid) The paper (silicon slides) Coat the cantilever with the ink Scan the area for patterning in the contact mode. Protein adsorption (rabbit IgG ) antibody recognition. The resulting protein arrays were then characterized by AFM. B) Topography image of the protein array

4. Direct-Write DNP for protein array Height profiles of TM- AFM images: rabbit IgG assembled on an MHA dot array generated by DPN before (A) and after (B) treatment with a solution containing lysozyme, goat/sheep anti-IgG, human anti-IgG, and rabbit anti-IgG; a control protein nanoarray before (C) and after (D) exposure to a solution containing lysozyme, retronectin, goat/sheep anti-IgG, and human anti-IgG.

5. Nanostructures patterned by DPN A)Nanoscale dot arrays and B) nanoscale letters written on a Au surface C) TM-AFM image of 25- and 13-nm gold nanoparticles hybridized to surface DNA templates generated with DPN D) Fluorescence image of DPN patterns of fluorescently labeled IgG on SiOx

6. Nanostructures patterned by DPN B) TM-AFM image of 60-nm gold nanolines C)12-nm gold nanogap on a Si/SiOx surface. D) 3D Si(100) nanostructures. TM-AFM image of DNA-modified line (E), features after hybridization with complementary DNA-modified nanoparticles; insert: high-resolution TM-AFM images

7. Advantages It might be the only lithographic technique that offers High resolution High registration With direct-write printing capabilities

8. Limitation of the method More efforts should be put in improving the speed and in transforming it into massively parallel process to be a powerful production tool in both the life science and the semiconductor industry.

9. Conclusions DPN is a unique scanning-probe-based lithographic tool for generating high-resolution patterns of chemical functionality on a nano range of surfaces. The combination of resolution, registration, and direct-write capability offered by DPN distinguishes it from any alternative lithographic strategy and makes DPN a promising tool for patterning soft organic and biological nanostructures.