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This paper combines the topographic imaging capability of the atomic force microscope (AFM) with a compositionally patterned array of immobilized antigenic rabbit IgG on gold as an approach to performing immunoassays. The substrates are composed of micrometer-sized domains of IgG that are covalently linked to a photolithographically patterned array of a monolayer-based coupling agent. The immobilized coupling agent, which is prepared by the chemisorption of dithiobis(succinimidyl undecanoate) on gold, is separated by micrometer-sized grids of a monolayer formed from octadecanethiol (ODT). The strong hydrophobicity of the ODT adlayer, combined with the addition of the surfactant Tween 80 to the buffer solution that is used in forming the antibody-antigen pairs, minimizes the nonspecific adsorption of proteinaceous materials to the grid regions. This minimization allows the grids to function as a reference plane for the AFM detection of the height increase when a complementary antibody-antigen pair is formed. The advantageous features of this strategy, which include ease of sample preparation, an internal reference plane for the detection of topographic changes, and the potential for regeneration and reuse, are demonstrated using rabbit IgG as an immobilized antigen and goat anti-rabbit IgG as the complementary antibody. The prospects for further miniaturization are discussed.Immunoassays play a critical role in clinical, pharmaceutical, and environmental chemistries. [1][2][3] To such ends, a range of different transduction (e.g., optical, 4-6 amperometric, 7 radiochemical, 8 piezoelectric, 9-13 and capacitive 14,15 ) mechanisms have been successfully exploited for the detection of antigen-antibody binding. However, radiochemical, amperometric, and optical

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This paper investigates three different approaches to patterning proteins within ultrathin resist layers formed from self-assembled monolayers using scanning probe lithography (SPL) at the submicrometer length scale. The first approach uses a “nanografting” method to pattern a reactive carboxylic acid terminated thiol into a resist composed of a methyl-terminated monolayer. Rabbit IgG antigen is bound to the patterned region, and an immunoassay utilizing direct readout of the topographic change resulting from specific binding of anti-rabbit IgG antibody is performed using scanning force microscopy. To address issues related to nonspecific protein adsorption, the other two approaches investigated the patterned removal of glycol-terminated monolayers by mechanically “scraping” patterns at high tip−sample forces by SPL. Protein attachment to the scraped regions was achieved either through the chemisorption of a disulfide coupling agent or by the direct adsorption of Fab‘-SH antibody fragments. Results obtained from all approaches are presented and compared, and the strengths and weaknesses of each toward fabricating high-density, multiple protein arrays are discussed.