Nanometer-Scale Arrangement of Human Serum Albumin by Adsorption on Defect Arrays Created with a Finely Focused Ion Beam Anna A. Bergman, † Jos Buijs,* ,† Jens Herbig, ‡ David T. Mathes, § James J. Demarest, § Christian D. Wilson, | Curt T. Reimann, ‡ Rau ` l A. Baragiola, | Robert Hull, § and Sven O. Oscarsson † Department of Chemical Engineering, Ma ¨ lardalen University, Box 325, S-631 05, Eskilstuna, Sweden, Division of Ion Physics, Ångstro ¨ m Laboratory, Uppsala University, Box 534, S-751 21, Uppsala, Sweden, Laboratory of Atomic and Surface Physics, Engineering Physics, University of Virginia, Charlottesville, Virginia 22901, and Department of Material Science and Engineering, Thornton Hall, University of Virginia, Charlottesville, Virginia 22901 Received June 2, 1998. In Final Form: October 2, 1998 Well-ordered arrays of pits were prepared on gallium arsenide and silicon wafers using a finely focused ion beam (FFIB). The defect pits on gallium arsenide, examined with tapping mode scanning force microscopy (TM-SFM), had a rim diameter of 60 nm and were spaced 185 nm apart. TM-SFM images showed that human serum albumin (HSA) adsorption was highly specific to the inner portion of the rims of the pits on gallium arsenide, while there was no specific adsorption to the rims of pits on silica. This study demonstrates that a controlled spatial distribution of adsorbed proteins can be achieved on a nanometer scale and that the choice of material is of importance. Moreover, surface features such as pits and lines produced by FFIB can serve as a guide to easily reposition the TM-SFM probe tip at a specific location on the surface to within a few nanometers after temporary removal of the sample from the microscope. Introduction An important step toward realizing developments in biotechnology and nanotechnology is gaining control over the spatial distribution of adsorbed proteins at the nanometer scale. In the field of information technology, use of ordered biomolecular arrays can lead to ultrahigh- density nanometer-scale bioelectronic integrated circuits such as memories. 1,2 More immediate applications lie in the area of miniaturized bioanalysis. 3-6 The possibility to identify and discriminate between the individual components of a single molecular immunocomplex and the immunocomplex itself has already been demonstrated by our research group. 7 Control of the spatial position of adsorbed proteins can be achieved by site-selective adsorption of proteins onto surfaces which display a spatially defined heterogeneity on a nanometer scale. The first example of a site-selective adsorption of individual glucose oxidase molecules at step edges on highly oriented pyrolytic graphite (HOPG) was reported by Cullen et al. 8 Similar adsorption of immu- noglobulin E on HOPG step edges has recently been observed in our group. Previous research also demon- strates that artificially created ion impact defects on mica manifest site-selective adsorption of -galactosidase to the hillock-like defects. 9 Nevertheless, these results are examples of site-selective adsorption with a limited control over the spatial distribution of the adsorption sites. On a micrometer scale, however, it has been shown that by using photolithography, a regular array of modified surface sites can be obtained resulting in spatial control of the subsequent process of protein adsorption. 4,5,10 On a 10- 100 nm scale, spatially ordered surface modifications can be produced based on the ability of a scanning probe microscope to mechanically 2,11 or electronically 12-14 “write” on a surface. Alternatively, short-wavelength lithography sources such as electron 15 or ion 16,17 beams can be employed to write small features on a surface. For example, it has been demonstrated that by using a finely focused ion beam (FFIB) ordered patterns of pits each with a 30 nm diameter could be created. 17 A logical next step is to explore the new nanopatterning techniques as a means for realizing site-selective protein adsorption. In this Letter we describe the site-specific immobiliza- tion of human serum albumin (HSA) to an ordered array of nanometer-sized pits drilled in a gallium arsenide * Corresponding author: fax, +46 (0)18 555 736; e-mail, jos.buijs@angstrom.uu.se. † Department of Chemical Engineering, Ma ¨ lardalen University. ‡ Division of Ion Physics, Uppsala University. § Department of Material Science and Engineering, University of Virginia. | Laboratory of Atomic and Surface Physics, University of Virginia. (1) Service, R. F. Science 1996, 274, 723. (2) Sasaki, K.; Ueno, K.; Koma, A. Jpn. J. Appl. Phys. 1997, 36, 4061. (3) Houston, J. G.; Banks, M. Curr. Opin. Biotechnol. 1997, 8, 734. (4) Fodor, S. P. A.; Read, J. 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