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Fabrication and Imaging of Nanometer-Sized Protein Patterns Kapila Wadu-Mesthrige, Song Xu, Nabil A. Amro, and Gang-yu Liu* Department of Chemistry, Wayne State University, Detroit, Michigan 48202 Received September 10, 1999 This Letter describes a new method for production of nanometer-sized protein patterns with precise control of the size and geometry. Self-assembled monolayers (SAMs) are first patterned using nanografting, an atomic force microscopy (AFM)-based lithographic method. Methyl-terminated n-alkanethiols serve as matrix layers within which nanopatterns of thiols with carboxylate or aldehyde terminal groups are fabricated. By controlling solution conditions, we selectively immobilize proteins on the patterned areas, through either electrostatic interactions or covalent binding. In our approach, AFM tips are used for both fabrication and characterization. AFM allows patterned SAMs and proteins to be visualized in situ. The individual proteins within the nanopatterns and their orientations can be clearly resolved from the AFM images.
Micrometer-sized protein patterns have been produced previously using self-assembled monolayers (SAMs) in combination with lithographic methods.1-5 Previous approaches include two main steps: (1) producing a patterned substrate such as a self-assembled monolayer which contains spatially separated components terminated with protein resistive and adhesive groups, respectively, and (2) adsorbing proteins selectively onto the patterned substrate.1-5 Microscopic patterns of SAMs are formed using microcontact printing, micromachining, or photolithography. Further miniaturization shall benefit the development of ultrasmall biosensors and biochips by increasing the density of receptor elements, improving detection limits, and controlling the reactivity of the receptor elements by engineering them with molecular precision.5-7 Electron beam lithography can produce smaller metal and semiconductor patterns but requires high-vacuum environments.8 Mixed SAMs formed by the * To whom correspondence should be addressed. E-mail: gyl@ chem.wayne.edu. (1) Lopez, G. P.; Biebuyck, H. A.; Harter, R.; Kumar, A.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10774. (2) Mrksich, M.; Chen, C. S.; Xia, Y.; Dike, L. E.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. USA 1996, 93, 10775. (3) James, C. D.; Davis, R. C.; Kam, L.; Craighead, H. G.; Isaacson, M.; Turner, J. N.; Shain, W. Langmuir 1998, 14, 741. (b) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225. (4) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. USA 1996, 93, 12287. (b) Wollman, E. W.; Kang, D.; Frisbie, C. D.; Lorkovic, I. M.; Wrighton, M. S. J. Am. Chem. Soc. 1994, 116, 4395. (c) Tarlov, M. J.; Burgess, D. R. F.; Gillen, G. J. Am. Chem. Soc. 1993, 115, 5305. (5) Jones, V. W.; Kenseth, J. R.; Porter, M. D.; Mosher, C. L.; Henderson, E. Anal. Chem. 1998, 70, 1233. (6) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580583. (b) Bamdad, C. Biophys. J. 1998, 75, 1989-1996. (c) Vo-Dinh, T.; Alarie, J. P.; Isola, N.; Landis, D.; Wintenberg, A. L.; Ericson, M. N. Anal. Chem. 1999, 71, 358-363. (d) Proudnikov, D.; Timofeev, E.; Mirzabelov, A. Anal. Biochem. 1998, 259, 34-41. (e) Nagayama, K. Nanobiology 1992, 1, 25-37. (7) Menon, V. P.; Martin, C. R. Anal. Chem. 1995, 67, 1920-1928. (b) Galla, H.-J. Angew. Chem., Int. Ed. Engl. 1992, 31, 45-47. (c) Tender, L. M.; Worley.; Fan, H.; Lopaz, G. P. Langmuir 1996, 12, 5515. (d) Haronian, D.; Lewis, A. Appl. Phys. Lett. 1992, 61, 2237-2239. (8) Bergman, A. A.; Buijs, J.; Herbig, J.; Mathes, D. T.; Demarest, J. J.; Wilson, C. D.; Reimann, C. T.; Baragiola, R. A.; Hull, R.; Oscarsson, S. O. Langmuir 1998, 14, 6785-6788. (b) Sondag-Huethorst, J. A. M.; Van Helleputte, H. R. J.; Fokkink, L. G. J. Appl. Phys. Lett. 1994, 64, 285. (c) Allara, D. L.; Tiberio, R. C.; Craighead, H. G.; Lercel, M. Appl. Phys. Lett. 1993, 62, 476.
coadsorption of two alkanethiols can be used to fabricate nanometer-sized protein patterns.9 However, using this approach, it is difficult to precisely control the size and distribution of these patterns. In this Letter, we introduce a new method to produce nanometer-sized protein patterns. Our approach utilizes an atomic force microscopy (AFM)-based lithographic technique to produce welldefined nanometer-sized protein patterns in buffer solutions. The nanostructures produced at each step are also characterized in situ with high spatial resolution using AFM. This new technique includes two main steps: production of nanometer-sized patterns of self-assembled monolayers (SAMs) followed by selective adsorption of proteins onto these patterns. The quality of the protein nanostructures depends on the spatial precision of SAM nanopatterns and on the selectivity of protein adsorption. Patterned SAMs are produced with nanometer precision using nanografting that was developed and reported by our group.10,11 As illustrated in Figure 1, nanografting uses an atomic force microscopy12 (AFM) tip as a “nanoshaver”. The nanoshaver is operated on a matrix SAM immersed in a solution containing a different thiol. As the tip plows through the matrix monolayer, the SAM molecules under contact are removed and replaced by the new adsorbate molecules.10,11 Selectivity of protein adsorption can be achieved with the knowledge of the variation in protein affinity toward different SAMs.13-17 (9) Fang, J.; Knobler, C. M.; Eiserling, F. A.; Gingery, M. J. Phys. Chem. B 1997, 101, 8692. (b) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber, D. E.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 6548. (10) Xu, S.; Liu, G.-Y. Langmuir 1997, 13, 127. (11) Xu, S.; Laibinis, P. E.; Liu, G.-Y. J. Am. Chem. Soc. 1998, 120, 9356. (12) Our atomic force microscope is home-constructed. All cantilevers used for this study are sharpened microlevers from Park Scientific Instrument with a force constant of 0.1 N/m. (13) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (b) Norde, W.; Giesbers, M.; Pingsheng, H. Colloids Surf., B: Biointerfaces 1995, 5, 255. (c) Buijs, J.; Btitt, D. W.; Vladimer, H. Langmuir 1998, 14, 335. (14) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052. (15) Vinckier, A.; Heyvaert, I.; D′Hoore, A.; McKittrick, T.; Haesendonck, C. V.; Engelborghs, Y.; Hellemans, L. Ultramicroscopy 1995, 57, 337. (16) Blawas, A. S.; Oliver, T. F.; Pirrung, M. C.; Reichert, W. M. Langmuir 1998, 14, 4243.
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Figure 1. Schematic diagram of nanografting and subsequent selective adsorption of proteins on Z-terminated patterns instead of the X-terminated SAM matrix. The examples of Xand Z-terminal groups are illustrated in the three experiments described later in this letter.
We first patterned lysozyme (LYZ) as a proof-of-concept experiment. As shown in Figure 2A, two HS(CH2)2COOH nanopatterns, a line and a rectangle, were first grafted within a matrix CH3(CH2)9S/Au(111) SAM (abbreviated as C10S/Au). The patterning and imaging of SAMs were conducted in an aqueous medium containing 1 mM HS(CH2)2COOH. Prior to protein adsorption, the patterned SAM was first washed with deionized water to remove any remaining HS(CH2)2COOH and then washed with 20 mM HEPES buffer (pH 7.0). After injection of a 10 µg/mL LYZ solution,18 proteins adsorbed exclusively onto the two HOOC-terminated areas within 3 min, as shown in Figure 2B. Little adsorption was observed at the methylterminated areas during the experiment (4 h). At first glance, this observation appears to contradict the common knowledge that proteins adsorb on hydrophobic surfaces, including methyl-terminated SAMs. We need to emphasize that LYZ does not adsorb onto alkanethiol SAMs under the specified conditions (e.g. concentration and time) used in our experiment. Higher concentration (10 mg/mL) and longer reaction time (> 5 h) would result in protein attachment to methyl-terminated surfaces. Thus, the control of protein adsorption conditions is vitally important to achieve high selectivity. The observed strong selectivity is also attributed to the electrostatic interaction between LYZ and the surface of HOOC(CH2)2S/Au(111). At neutral pH, LYZ (IEP 11.1) exhibits net positive charges,19 while ∼10% of the carboxylate termini have negative charge (COO-).20 Therefore, electrostatic attraction drives the (17) Frey, B. L.; Jordan, C. E.; Kornguth, S. Anal. Chem. 1995, 67 (7), 4452. (b) Patel, N.; Davies, M. C.; Hartshorne, M.; Heaton, R. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 6485. (18) Lysozyme (LYZ, from hen egg, 95% purity) was obtained from Sigma Chemical Co., St. Louis, MO. (19) Imoto, T.; Johnson, L. N.; North, A. C. T.; Philips, D. C.; Rupley, J. A. In The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, 1972; Vol. 7, p 665. (20) The pKa value for HOOC(CH2)2S/Au is 8 according to: Hu, Kai.; Bard, A. J. Langmuir 1997, 13, 5114.
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selective adsorption of LYZ onto the HOOC-terminated areas instead of the methyl-terminated matrix. Individual LYZ particles within the patterns can be resolved from the AFM image shown in Figure 2B. The corresponding cursor profiles shown in Figure 2C reveal that the immobilized protein molecules exhibit different heights: 4.3 ( 0.2 nm and 3.0 ( 0.2 nm. LYZ molecules are ellipsoidal with dimensions about 4.5 × 3.0 × 3.0 nm3 from X-ray crystallographic studies.21 The variation in heights shown in Figure 2 is consistent with the fact that electrostatic interactions are not specific and often result in various protein orientations with respect to the surface.13,22 The advantage of using this physically mediated protein immobilization is the experimental simplicity. In addition, a larger portion of proteins retain their activity after the adsorption.2,4,23 The immobilization by electrostatic interactions is normally reversible, and proteins can be removed by certain buffers and detergents or can be replaced by other proteins in solutions.13c,24 More stable protein patterns can be produced by formation of covalent bonds or through specific interactions.14-16 Figure 3 includes two examples where nanopatterns of immunoglobulin G (IgG)25 and LYZ were produced through formation of imine bonds.15,26 Figure 3A displays an AFM image of a 40 × 40 nm2 area of HS(CH2)2CHO grafted within a C10S/Au(111) SAM.27 The shorter chain aldehyde pattern appears as a dark square hole in the topographic image. After washing the patterned SAM with water, we injected a 5 µg/mL IgG solution into the liquid cell. Adsorption occurred at both aldehyde- and methyl-terminated areas within 5 min. After subsequent washing with a 1% tween-20 solution, the adsorbed proteins in the methyl-terminated areas were completely detached, while the proteins attached to the HOCterminated area remained, as shown in Figure 3B. The attachment of IgG is therefore weaker on a methylterminated matrix than on aldehyde-terminated areas. The former is due primarily to the hydrophobic interaction, while the latter is attributed to the formation of imine bonds15,26 between the surface aldehyde and the primary amine groups such as lysine in IgG. Since each IgG has 57 lysine groups,28 the Y-shaped IgG may adopt various orientations on the surface. The cursor profile shown in Figure 3C reveals different heights for these immobilized IgG molecules, which correspond well with the orientations shown in the schematics above the cursor plots. Our AFM studies have shown that 50% of IgG molecules within nanopatterns retained their bioreactivity, as they can bind the secondary antibodies. A 340 × 300 nm2 of HS(CH2)10CHO was produced within the same matrix (Figure 3D).27 Due to the low solubility of HS(CH2)10CHO in water, 2-butanol was used as the (21) Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Nature 1965, 206, 757. (22) Thomas, N. H.; Smith, B. L.; Almqvist, N.; Schmitt, L.; Kashlev, M.; Kool, E. T.; Hansma, P. K. Biophys. J. 1999, 76, 1024. (23) Browning-Kelley, M. E.; Wadu-Mesthrige, K.; Hari, V.; Liu, G.Y. Langmuir 1997, 13, 343. (24) Feng, M.; Morales, A. B.; Beugeling, T.; Bantjes, A.; Werf, K. V. D.; Gosselink, G.; Grooth, B. D.; Greve, J. J. Colloid. Interface. Sci. 1996, 177, 364. (25) The polyclonal, rabbit IgG (purity > 95%) was obtained from Sigma Biochemicals Co., St. Louis, MO. The IgG was diluted to the desired concentration with a solution containing 20 mM HEPES and 150 mM KCl (final pH 6.5 ( 0.5). (26) Baker, A.; Zidek, L.; Wiesler, D.; Chmelik, J.; Pagel, M.; Novotny, M. V. Chem. Res. Toxicol. 1998, 11, 730. (27) The compounds HS(CH2)10CHO and HS(CH2)2CHO were synthesized in our laboratory by the oxidation of HS(CH2)10CH2OH and HS(CH2)2CH2OH, respectively, with pyridinium dichromate (PDC). (28) The amino acid sequence of IgG was obtained from the protein data bank at BNL. The PDB ID code is 1IGT.
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Figure 2. Formation of two LYZ nanopatterns through electrostatic interactions: (A) 400 × 400 nm2 topographic image of a C10S/Au(111) SAM, within which a 10 × 150 nm2 line and a 100 × 150 nm2 rectangle of HS(CH2)2COOH were produced using nanografting; (B) the same area imaged after a 4-min immersion in a LYZ solution; (C) corresponding cursor profiles displayed in one coordinate where the possible protein orientation is sketched. The origin of the Y-axis is the gold surface.
Figure 3. Formation of LYZ and IgG nanopatterns through imine bonds: (A) 150 × 150 nm2 topographic image of a C10S/Au(111) SAM, within which a 40 × 40 nm2 area of HS(CH2)2CHO was produced; (B) the same area imaged after a 5-min immersion in a normal rabbit IgG solution (5 µg/mL in HEPES buffer, pH 6.5) followed by washing with a 1% tween-20 solution; (C) corresponding cursor profiles across the IgG nanopattern; (D) 470 × 470 nm2 topographic image of a C6S/Au(111) SAM, within which a 340 × 300 nm2 area of HS(CH2)10CHO was produced using nanografting; (E) the same area imaged after a 5-min immersion in a LYZ solution (10 µg/mL in HEPES buffer, pH 7.0); (F) corresponding cursor profiles across the LYZ nanopattern.
solvent for nanografting. Before injection of LYZ solution, we passed an excess amount of ethanol and then deionized water through the liquid cell to clean the SAM and change the imaging media from 2-butanol to water. At pH 7, LYZ has net positive charges19,20 and, thus, did not adsorb to the methyl-terminated matrix. A near monolayer of LYZ formed exclusively onto the HOC-terminated pattern. AFM allows individual protein molecules to be resolved as shown in Figure 3E. The LYZ molecules in the nanopattern exhibit various orientations as a result of multiple binding sites (e.g. six lysine residues in LYZ).29
The total coverage of the protein can be controlled by varying the exposure time or the protein concentration. In summary, nanometer-sized protein patterns can be produced using nanografting followed by selective adsorption of proteins on SAMs. Individual proteins and their adsorption orientations were clearly resolved in situ and under buffer solutions. Subsequent biorecognition or other bioreactions of these patterned proteins can also be (29) The amino acid sequence for LYZ was obtained from the SWISS-PROT protein data bank, ID P00698.
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monitored in situ using AFM. This new fabrication method is currently tested using various proteins and SAMs. The strength of our approach is the ability to engineer and image SAMs and protein patterns with nanometer precision. These nanostructures shall provide a unique opportunity for exploration of chemical and biochemical reactions under spatially well-defined and controlled environments. Although not yet practical for high throughput applications and manufacturing, the fundamental information obtained from these investigations in research laboratory settings should serve as a useful guide for nanofabrication for biosensors and biochips.
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Acknowledgment. We thank professor C. Chow, J. Garno, J. Oravec, and S.Cruchon-Dupeyrat at WSU for many helpful discussions. G.Y.L. gratefully acknowledges the Camille and Henry Dreyfus Foundation for a New Faculty Award and the Arnold and Mabel Beckman Foundation for a Young Investigator Award. This work was also supported by the National Science Foundation (Career Award CHE-9733400 and IGERT-970952), the ACS Petroleum Research Fund, and the Whitaker Foundation. LA991196N
