Nanometer-Scale Arrangement of Human Serum Albumin by

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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 ultrahighdensity 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 immunoglobulin E on HOPG step edges has recently been * Corresponding author: fax, +46 (0)18 555 736; e-mail, [email protected]. † 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. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767. (5) Dontha, N.; Nowall, W. B.; Kuhr, W. G. Anal. Chem. 1997, 69, 2619. (6) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. Science 1997, 276, 779. (7) Quist, A. P.; Bergman, A. A.; Reimann, C. T.; Oscarsson, S. O.; Sundqvist, B. U. R. Scanning Microsc. 1995, 9, 395. (8) Cullen, D. C.; Lowe, C. R. J. Colloid Interface Sci. 1994, 166, 102.

observed in our group. Previous research also demonstrates that artificially created ion impact defects on mica manifest site-selective adsorption of β-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 10100 nm scale, spatially ordered surface modifications can be produced based on the ability of a scanning probe microscope to mechanically2,11 or electronically12-14 “write” on a surface. Alternatively, short-wavelength lithography sources such as electron15 or ion16,17 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 immobilization of human serum albumin (HSA) to an ordered array of nanometer-sized pits drilled in a gallium arsenide (9) Quist, A. P.; Petersson, Å.; Reimann, C. T.; Bergman, A. A.; Daya, D. D. N. b.; Halle´n, A.; Carlsson, J.; Oscarsson, S. O.; Sundqvist, B. U. R. J. Colloid Interface Sci. 1997, 189, 184. (10) Liu, J.; Hlady, V. Colloids Surf., B 1996, 8, 25. (11) Song, X.; Liu, G.-Y. Langmuir 1997, 13, 127. (12) Takimoto, K.; Kuroda, R.; Shido, S.; Yasuda, S.; Matsuda, H.; Eguchi, K.; Nakagiri, T. J. Vac. Sci. Technol., B 1997, 15, 1429. (13) Hamanaka, H.; Ono, T.; Esaschi, M. J. Vac. Sci. Technol., B 1997, 15, 1414. (14) Ahn, C. H.; Tybell, T.; Antognazza, L.; Char, K.; Hammond, R. H.; Beasley, M. R.; Fisher, Ø.; Triscone, J.-M. Science 1997, 276, 1100. (15) Lercel, M. J.; Redinbo, G. F.; Pardo, F. D.; Rooks, M.; Tiberio, R. C.; Simpson, P.; Sheen, C. W.; Parikh, A. N.; Allara, D. L. J. Vac. Sci. Technol., B 1994, 12, 3663. (16) Perrin, D.; Seifert, W. Solid State Technol. 1994, 37, 95. (17) Harada, K.; Kamimura, O.; Kasai, H.; Matsuda, T.; Tonomura, A.; Moshchalkov, V. V. Science 1996, 274, 1167.

10.1021/la980642o CCC: $15.00 © 1998 American Chemical Society Published on Web 11/04/1998

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Figure 1. Overview of the defect arrays as prepared on a GaAs wafer using a finely focused ion beam (FFIB). (A) Schematic overview of the pattern of square-shaped ion-milled trenches as can be observed in an optical microscope. By use of scanning force microscopy (SFM) software, a specific pit can be located. The black bar at the right side of the image represents the SFM cantilever, under which the tip is mounted. (B) Each square contains three pit arrays which can be located by examining large-scan area TM-SFM images (image size 20 × 20 µm2). (C) In a magnification of image B, the array of pits in the GaAs surface is clearly seen.

(GaAs) wafer by a FFIB. The FFIB patterns and HSA adsorption were studied using scanning force microscopy (SFM). Experimental Section Well-ordered defect arrays were prepared on a GaAs wafer, previously rinsed with ethanol, using a FFIB (FEI 200) producing 30-keV indium ions at a beam current of 2 pA. The beam spot size was 15 nm, and the dwell time during the production of each pit was 0.1 ms. The modified region consisted of 24 identical square-shaped (25 × 25 µm2) milled trenches, each enclosing three 12 × 12 µm2 regions containing 4.2 × 103 pits. The sample surfaces were imaged with a Nanoscope III scanning force microscope (Digital Instruments, Inc.) run in tapping mode (TM-SFM) under ambient conditions. The surfaces were probed using etched silicon tips with a nominal radius of curvature of 5-10 nm as specified by the manufacturer and with oscillation frequencies around 300 kHz. The SFM is accompanied by an optical microscope (Optizoom, Nikon, Inc.) which allows for direct monitoring of the position of the cantilever relative to the sample on the micrometer scale. The milled trenches as well as regions which had been subjected to a continuous scan with the FFIB were visible in the optical microscope, allowing for easy location of the modified surface area. A schematic overview of the modified region and an SFM image of a part of the squareshaped milled trench are shown in Figure 1. HSA was obtained from Sigma and used without further purification. HSA was dissolved at a concentration of 3.7 µg/mL in a 40 mM HEPES (Sigma) buffer, adjusted to pH 7.6 with KOH. Before the adsorption experiment was performed, the cleanliness of the buffer solution was examined by placing 30 µL of pure buffer on fleshly cleaved muscovite green mica (AshevilleSchoonmaker Mica Co.) for 2 min, followed by a rinse with 1 mL of pure buffer whereafter the excess solution was removed from the sample with a flow of nitrogen. Subsequently, the cleanliness of the sample was examined using TM-SFM. Next a 30 µL drop of the HSA solution was placed on the piece of GaAs wafer and adsorption was allowed for 2 min. Then the HSA solution was rinsed off with 1 mL of pure buffer, and excess solution was removed from the sample surface using a flow of nitrogen. Even though the sample was removed from the microscope during the adsorption procedure, the same array area scanned before HSA adsorption could easily be located after the adsorption by visual recognition of the square-shaped milled trenches. The tip was placed roughly in the correct position using the optical microscope. To exactly locate specific pits, Nanoscope III software was employed to navigate the tip position relative to the surface.

Results The modified surface regions containing the wellordered pits were imaged before and after adsorption of HSA (Figure 2). The diameter of the pits at the level of the basal plane was ≈40 nm, but in SFM images this diameter was reduced relative to the true diameter by convolution with the finite-sized probe tip. The pits had slightly elevated rims with measured height values of 0.4 nm and a diameter of 60 nm. The apparent depth of the pits was around 0.8 nm. The spacing between the pits was 185 nm. After adsorption of HSA, the rim of each pit was clearly decorated with features each of which, taking into account broadening of TM-SFM image features caused by a tip with a nominal end radius of 5-10 nm, corresponded to a cluster of several HSA molecules (Figure 2b). These decorated pit features were located on a circular boundary of diameter ≈40 nm. The heights of these features, relative to the flat region between the pits, were about 2.2 nm. There were five to eight discernible features located on the elevated rims of each pit (Figure 3). Thus, in each pit, the area density of HSA molecules was at least ≈5.6 × 103 µm-2. By contrast, in the region between the pits, the area density of features in the size of the order of HSA molecules was only 20 µm-2. A similar amount of such features was also observed on the GaAs surface outside the milled surface, indicating that the HSA molecules adsorb directly to the pit sites rather than diffusing laterally over the surface into the pits. For biosensing applications, it is worthwhile noting that any height alteration occurring at the regularly spaced pits can easily be observed in two-dimensional Fourier transformed (FT) images (insets in Figure 2). In this way, height information at the pit locations is condensed to a few specific spots in the FT image allowing for easy read-out, independent of the precise location of the center of the image. Discussion A highly preferential adsorption of HSA molecules to the well-ordered array of defects is clearly demonstrated. On a per unit area basis, the adsorption enhancement of HSA to the defects is a factor of the order of 300. A previous TM-SFM study of albumin on mica showed that heights for features corresponding to single albumin molecules

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Figure 2. Spatial control of human serum albumin (HSA) adsorption to gallium arsenide (GaAs). Left: TM-SFM images of an array of pits milled by a FFIB of indium ions on a GaAs surface. Small raised rims, with apparent heights ≈0.4 nm, surround each pit. The diameter of each pit from rim to rim is 60 nm, and the depth is g0.8 nm. The pits are situated 185 nm apart. Right: HSA was adsorbed from a solution to the GaAs surface. The same region could be relocated after protein adsorption due to recognition of the array pattern. HSA molecules are clearly observed to preferentially adsorb to the inner portion of the rims of the pits. (Image sizes 1 µm × 1 µm.) Insets: Height alterations occurring at the pits can easily be observed in two-dimensional Fourier transformed (FT) images, where the periodicity of the height information at the pit locations is condensed to a few specific spots in the FT image.

Figure 3. HSA molecules adsorbed on the rim of one pit milled by a FFIB of indium ions in a GaAs surface. The height of the features around the pit is 2.2 ( 0.4 nm. HSA molecules appear to be packed closely together around the inner sides of the rims, and some features corresponding to single HSA molecules can be resolved.

on this substrate measured 0.60 ( 0.12 nm,18 which is considerably lower than heights measured in this study. The molecules decorating the rims of the FFIB pits are not single ones however; probably we observe molecular aggregates. Because the pit rim diameter decreased considerably after HSA adsorption, it is deduced that the HSA molecules are located at the inner part of each pit. It is likely that the pits are filled with HSA molecules and that, due to the tip shape, only the HSA molecules at the elevated outer part of the pit are observed in the images. (18) Quist, A. P.; Bjo¨rck, L. P.; Reimann, C. T.; Oscarsson, S. O.; Sundqvist, B. U. R. Surf. Sci. 1995, 325, L406.

Although the results clearly demonstrate highly preferential adsorption of HSA on pit structures, it is unclear what physical interaction is responsible for this behavior. Several of the surface characteristics of the native oxide layer of GaAs19 can be changed by ion bombardment. First, it is possible that some of the indium ions, which are implanted into the surface, are exposed during the milling procedure. Studies have shown that some protein molecules, including albumin, have enhanced affinity for certain metal chelates.20 Nevertheless, this explanation for the preferential adsorption is highly unlikely as the dose of 30 keV In ions is small (4.4 × 1014 ions/cm2) and they are buried at a penetration depth of 15 nm, with a dispersion of 7 nm.21 A second plausible effect is the generation of defects, like vacancies, steps, etc., in the roughened pit, producing unsaturated bonds which lead to stronger protein-surface interactions.22 Third, the ion impacts might result in an altered chemical composition of the surface at each pit site. Preferential sputtering of As occurs when GaAs is bombarded with energetic heavy ions.23,24 The metalized surface will then be oxidized by exposure to air. The end result is a local enrichment of gallium oxide at the pit sites, which may have properties that cause preferential protein adsorption. At present, the lack of a detailed characterization of the chemical composition of the 60-nm pits does not allow to ascertain which type of interaction is responsible for the preferential adsorption of HSA. This uncertainty is increased by the (19) Song, Z.; Shogen, S.; Kawasaki, M.; Suemune, I. J. Vac. Sci. Technol., B 1995, 13, 77. (20) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature 1975, 258, 598. (21) Ziegler, J. F.; Biersack, P.; Littmark, U. The stopping and range of ions in solids; Pergamon Press: New York, 1985. (22) Rasmusson, J. R.; Erlandsson, R.; Salaneck, W. R.; Schott, M.; Clark, D. J.; Lundstro¨m, I. Scanning Microsc. 1994, 8, 481. (23) Homma, Y.; Ishii, Y. J. Vac. Sci. Technol., A 1984, 3, 531. (24) Singer, I.; Murday, J. S.; Cooper, L. R. Surf. Sci. 1981, 108, 7.

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possibility that under ambient conditions, a thin layer of hydrocarbons can contaminate the surface. From additional experiments, it is clear that the adsorption of HSA is at least specific to the chemical composition of the sorbent surface. No significant sitespecific adsorption of HSA was observed on a silica surface mechanically indented with a diamond tip. Also, silica bombarded with a beam of focused gallium ions to create pit structures did not manifest any site-specific adsorption. Furthermore, it was found that the site-selective adsorption to defects was not molecule-specific for HSA. After decoration of the pits by HSA, when the GaAs sample was further exposed to a solution of anti-HSA (antibody molecules which are specific against HSA), it was observed that large aggregates of anti-HSA were attached to the surface where the indium beam had continuously scanned the sample (data not shown). Conclusion The present study demonstrated that spatial control of HSA adsorption is possible on gallium arsenide. The adsorption is highly specific to the inner parts of 60-nm pits (rim diameter), created with a finely focused ion beam of indium ions. With this technique it might be possible

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that even single molecules can be adsorbed at defined locations since holes with a diameter of molecular dimension can in principle be created. The specific adsorption of HSA at the pits is caused by an alteration of the surface resulting from the ion bombardment, in the form of the generation of active defect sites and/or changes in chemical composition. Furthermore, the creation of well-defined defects allows us to navigate on the surface and repeatedly return to the exact same location with the scanning probe tip, even after temporarily removing the sample from the scanning force microscope. This allows, for example, for future studies of protein adsorption behavior and for specific protein-protein interaction studies on a single molecule basis in multiple step experiments. Acknowledgment. We acknowledge the Swedish Ministry of Education and the Swedish Research Council for Engineering Sciences (TFR) and the Knut and Alice Wallenberg foundation for financial support. Furthermore we thank both Magnus Bergkvist and Greger Ledung for technical support. LA980642O