Streptavidin that has four binding sites arranged in two opposing pairs is known as one of the most important linker proteins for binding the second biotinylated protein. To efficiently locate streptavidins to selective positions without nonspecific binding, we prepared well-controlled arrays of biotins on a gold surface by using a mixed self-assembly process. Two thiol derivatives (11-mercapto-1-undecanol and 11-mercaptoundecanoic-(8-biotinylamido-3,6-dioxaoctyl)amide) were used for preparing the mixed self-assembled monolayers. Fragment antibodies modified with biotin were immobilized on a gold surface covered with streptavidin. This system was applied to gold dot arrays formed by nanosphere lithography. The gold dot arrays were used as the mother structure to construct the array of proteins at the nanometer scale. Selective immobilization of antibodies was characterized by imaging the Substrate with an atomic force microscope and measuring the interaction force between biomaterials by chemical force microscopy. Also, the interaction force between antibodies was compared with the force predicted using the Johnson-Kendall-Roberts theory. (C) 2009 Elsevier Inc. All rights reserved.