NANO LETTERS
Biomimetic Nanostructure Fabrication: Nonlithographic Lateral Patterning and Self-Assembly of Functional Bacterial S-Layers at Silicon Supports
2003 Vol. 3, No. 3 315-319
Erika S. Gyo1 rvary,*,† Alan O’Riordan,‡ Aidan J. Quinn,‡ Gareth Redmond,‡ Dietmar Pum,† and Uwe B. Sleytr† Center for Ultrastructure Research and Ludwig Bolzmann Institute for Molecular Nanotechnology, UniVersita¨ t fu¨ r Bodenkultur Wien, Gregor-Mendel-Str. 33, A-1180 Vienna, Austria, and Nanotechnology Group, NMRC, Lee Maltings, Prospect Row, Cork, Ireland Received December 11, 2002; Revised Manuscript Received January 31, 2003
ABSTRACT Fabrication of spatially well-defined, ordered bacterial S-layer arrays of the protein SbpA of Bacillus sphaericus CCM 2177 at silicon supports is achieved by exploiting the soft lithography technique, micromolding in capillaries, as a nonlithographic patterning tool. Lateral patterning of simple and moderately complex crystalline S-layer array structures ranging in critical dimension from submicron to hundreds of microns is demonstrated. For micron-scale patterned S-layers, the integrity of the native chemical functionality of the protein following patterning is verified by covalent attachment of human IgG antibody and subsequent binding of antihuman IgG antigen. The structural diversity of S-layers combined with their ease of patterning, self-assembly, and surface chemical modification suggest that this approach could be exploited for biomimetic fabrication of a wide range of functional nanostructures.
The ability to pattern small structures is central to the fabrication of a broad range of highly integrated microscale technologies. Photolithography is the most successful technique in microfabrication. However, the continued shrinking of device feature sizes below 100 nm poses new challenges for microfabrication. Fabrication of future nanodevices and nanosystems with applications in areas such as nanoscale electronics, photonics, mechanics, and biotechnology will require development of new processes for high-resolution patterning of many novel nanoscale materials on various unconventional substrates. To this end, bacterial surface layers (S-layers)1 have recently been shown to function as a versatile substrate for templated assembly of organized arrays of molecules as well as metal and semiconductor nanoparticles.2 S-layer proteins form the outermost cell envelope component of a broad spectrum of bacteria and archaea and can be isolated from these cells and reassembled at various technologically important solid supports (e.g., silicon, metals, polymers). S-layers are crystalline two-dimensional (2D) structures composed of a single (glyco-)protein species which exhibit either oblique (p1, p2), square (p4), or hexagonal * Corresponding author. Email:
[email protected] † Universita ¨ t fu¨r Bodenkultur Wien. ‡ NMRC. 10.1021/nl025936f CCC: $25.00 Published on Web 02/15/2003
© 2003 American Chemical Society
(p3, p6) lattice symmetries with unit cell dimensions in the range 3-30 nm depending on the protein strain. The layers are generally 5-10 nm thick with identical pore dimensions (2-8 nm diameter) and morphology. Critically, S-layer lattices also possess a high density of functional groups in well-defined positions and orientations. However, while S-layers show enormous promise as substrates for assembly and organization of functional nanocomponents, to date little work has focused on the development of methods for spatially controlled lateral patterning of crystalline S-layer proteins.3 To address this challenge, we report on the application of a well-known soft lithography technique, micromolding in capillaries (MIMIC),4 to the patterning and self-assembly of 2D S-layer protein arrays at silicon supports. In this letter, we demonstrate lateral patterning of crystalline S-layer arrays across a range of length scales from tens of microns to submicron dimensions using epi-fluorescence and atomic force microscopies to monitor protein layer assembly. Further, patterning is successfully achieved while also preserving the native chemical functionality of the protein as indicated by covalent attachment of human IgG antibody and subsequent recognition-directed binding of FITC-labeled antihuman IgG antigen at assembled S-layer substrates.
Figure 2. Fluorescence image of 6 µm wide FITC-labeled S-layer protein tracks patterned at a plasma-treated native silicon oxide support. S-layer protein: SbpA of B. sphaericus CCM 2177.
Figure 1. Schematic representation of S-layer protein patterning and assembly by MIMIC. (a) Channels are formed when a PDMS mold contacts a silicon wafer support. (b) Channels are filled with a protein solution by capillary forces. (c-d) Following mold removal, crystalline protein patterns are observed on the support surface. (e) S-layer patterns may be labeled with a fluorescence marker or (f) used as substrates for an antibody-antigen immunoassay.
Prior to patterning, batches of S-layer protein SbpA of Bacillus sphaericus CCM2177 are prepared by growth in continuous culture, cell wall preparation, extraction, dialysis, and sedimentation as described previously.5 The concentration of the clear supernatant containing the disassembled S-layer subunits is adjusted with milli-Q water to 1 mg/mL, and the desired subunits are thereafter retained for patterning and self-assembly (recrystallization) experiments. For mold formation, we fabricate 6 µm high mesastructure mold masters in photoresist (AZ 9260; Clariant) on 4-in. silicon wafers using photolithography. A variety of master patterns are employed ranging from straight lines to more complex meandering structures. Poly(dimethylsiloxane), PDMS (Sylgard 184; DOW Corning), is used to fabricate the molds from these masters using the standard procedure.6 Microchannels are formed when the recessed grooves in a PDMS mold are brought into conformal contact with a planar support, typically a native oxide terminated silicon wafer; see Figure 1a. These microchannels are subsequently filled from one end with S-layer protein subunit solution (0.1 mg/mL SbpA in 0.5 mM Tris-HCl buffer, pH 9, 10 mM CaCl2) by capillary action; see Figure 1b. Note that the silicon wafer supports (solvent precleaned) are O2 plasma-treated (Gala Instruments, Germany: cleaning time, 20 s; plasma pressure, 0.01 bar; power density, 70%; high purity grade O2 was used as working gas) before application of the mold in order to increase the wettability of the support surface and to thereby 316
improve channel filling (water contact angles at the planar support surface decrease from 65° to 5° following O2 plasma treatment). Using this method, rapid filling of the micronscale channels may be followed with an optical microscope, and capillaries may be observed to fill even when the solution enters from both ends of the mold. After S-layer selfassembly and crystallization (30 min to 24 h) the PDMS mold is removed under milli-Q water, leaving the patterned S-layer arrays behind on the support; see Figure 1c,d. These protein structures are then labeled with fluoresceinisothiocyanate, FITC (Sigma-Aldrich), which binds to the free amino groups on the S-layer; see Figure 1e. Microscopic imaging is employed for analysis of the fidelity of pattern transfer. Figure 2 shows a fluorescence micrograph of patterns of S-layer protein of B. sphaericus CCM2177 on a hydrophilic native silicon dioxide support surface. Sets of parallel tracks approximately 1 cm in length with track widths of 6, 8, or 10 µm are observed corresponding to the width of the grooves recessed in the PDMS mold. The S-layer protein structures show up as bright green tracks with the fluorescence intensity reflecting the amount of protein successfully assembled on the silicon wafer. The homogeneous fluorescence of the patterns indicates an even adsorption of protein subunits onto the silicon surface and suggests the formation of a crystalline layer. A high signalto-background ratio is also observed, indicating that FITC does not bind to regions of the support between the S-layer tracks. The patterned S-layer tracks therefore appear to be uniform with sharp edge interfaces indicating that the proteins do not diffuse across the oxide support following removal of the mold. These assertions are confirmed by inspection of tapping mode atomic force microscopy (AFM) images taken in air on dried samples (see Supporting Information Figure S1) that show homogeneous coverage over the S-layer track surface and sharp, well-defined edges. Contact mode AFM images acquired under liquid conditions also show welldefined S-layer tracks; see Figure 3. Further, high-resolution contact mode images show the square lattice pattern with a spacing of 13 nm of the morphological units expected for a self-assembled fully crystalline SbpA S-layer; see Figure 3 Nano Lett., Vol. 3, No. 3, 2003
Figure 3. Contact mode AFM image of 6 µm wide S-layer protein tracks patterned and recrystallized on a hydrophilic silicon wafer support (Z-range 50 nm). Inset shows the square lattice symmetry of the SbpA S-layer protein (Z-range 1.2 nm). The images are acquired under 100 mM NaCl. The regions denoted S and P represent S-layer tracks and PDMS residue, respectively (see text for details).
inset. The height of the S-layer structures is measured to be 15 nm. This value is calculated from line scans measured across regions of the sample where only partial channel filling occurs, i.e., where recrystallized S-layer lies adjacent to the bare silicon oxide support (see Supporting Information Figure S2a). This layer thickness is higher than that reported for SbpA monolayers7 (9 nm), indicating bilayer formation within the mold channel. S-layer proteins are known to form bilayers at air-liquid interface8 and in solution where socalled self-assembly products9 are formed (see Supporting Information Figure S2b). Note that height differences measured between the patterned S-layer surface (i.e., area marked S in Figure 3; see figure captions) and the adjoining region of the planar support where the PDMS mold was in contact with the support surface (i.e., area outside the mold channel marked P) are subject to errors since it appears that a residual layer of polymeric material remains on the support surface following removal of the PDMS mold. In fact, AFM measurements on control microchannel structures prepared as described above but filled with milli-Q water instead of S-layer protein subunit solution reveal a homogeneous residual layer, 2-5 nm in thickness, remaining in the regions where the PDMS mold contacts the support. The exact composition of this residual layer is not known; however, O2 plasma treatment as a bonding technique to join PDMS and silicon surfaces is well established in microfluidic applications.10 Current work is focused on investigation of antiadhesion treatments for the PDMS molds as a means of eliminating this residue. The effect of the degree of silicon support hydrophobicity on S-layer patterning and recrystallization may be investigated using silicon supports prepared without O2 plasma treatment. During spatial pattering of S-layers at these more hydrophobic supports, mold microchannel filling is observed to be comparatively slower than on O2 plasma-treated Nano Lett., Vol. 3, No. 3, 2003
Figure 4. Contact mode AFM image of submicron S-layer protein structures patterned using MIMIC along with a line profile indicating the achieved protein layer track widths. The image is acquired under 100 mM NaCl (Z-range 12 nm).
surfaces. Tapping mode AFM imaging in air reveals that the structure of the resulting patterned protein layers is heterogeneous and three-dimensional in nature. This is consistent with previous studies of SbpA S-layer protein reassembly on hydrophobic surfaces where formation of homogeneous and closed protein mosaics consisting of small, randomly aligned, monocrystalline S-layer domains was observed.11 Recrystallization of SbpA S-layer protein was also observed on the hydrophobic PDMS molds (see Supporting Information Figure S3). Further work is in progress on the use of PDMS molds for microcontact printing of S-layers. To explore the possibilities of MIMIC patterning of S-layers at submicron length scales, PDMS molds with channels of submicron dimension are prepared using conventional optical diffraction gratings (Edmund Optics Ltd.) as masters. These gratings have a pitch of 833 nm and, in lateral view, a triangular cross-section with a maximum height of 310 nm. Consequently, the typical channel crosssection of molds prepared from these masters is over 2 orders of magnitude smaller than those of the micron-scale molds used above, and capillary filling is slow even though hydrophilic plasma-treated silicon wafer supports are employed. Despite slow filling, the typical widths of patterned S-layer tracks observed in initial experiments are on the order of 820 nm, reflecting the high fidelity of pattern transfer attainable using the MIMIC method; see Figure 4. (Patterned S-layer lengths are approximately 50-100 µm.) Further, the self-assembled S-layers are crystalline (see Supporting Information Figure S4) clearly demonstrating the suitability of micromolding as a nonlithographic technology for forma317
tion of spatially well-defined crystalline S-layers at submicron dimensions. Since it is intended that patterned S-layers may function as substrates for subsequent template-directed assembly of organized arrays of molecules or other nanoscale devices, it is essential that patterned, recrystallized layers retain the native chemical functionality of the protein as observed in unpatterned recrystallized S-layers.12 To demonstrate the functional integrity of patterned S-layers, we perform a sandwich-type immunoassay based on human IgG antibodyantigen recognition. For this purpose, a more complex PDMS patterning mold consisting of a set of meandering microchannels is used, which serves to demonstrate that capillary filling, and therefore patterning and self-assembly of crystalline S-layers, is not limited to definition of simple structures such as straight channels. Following in-situ S-layer crystallization and mold removal, human IgG is immobilized onto the crystalline S-layer patterns as described previously.13 Briefly, the recrystallized S-layer tracks are first chemically cross-linked with glutaraldehyde followed by activation of S-layer surface carboxylic acid groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Human IgG antibody (Sigma-Aldrich) is then covalently immobilized onto the S-layer pattern during a 2 h incubation at room temperature. FITC-labeled antihuman IgG (Sigma-Aldrich) is subsequently applied to the modified S-layer tracks for a 1 h incubation period followed by washing with PBS buffer and milli-Q water; see Figure 1f for a schematic representation of the resulting complex. The activity and surface density of the IgG antibodies covalently attached to the crystalline S-layer tracks is characterized by epi-fluorescence microscopic imaging of the bound FITC-labeled IgG antigen. Uniform labeled-antigen fluorescence is observed across the entire S-layer pattern, indicating homogeneous covalent attachment of IgG antibodies to the S-layer track surface along with efficient antigen-binding activity; see Figure 5. Although the antibodies are not expected to form an oriented close-packed monolayer,10 it is clear that there is a high level of specific binding of the antibodies to the patterned S-layer. Note that the EDC activation step is critical since nonactivated S-layers show low fluorescence, indicating poor antibody binding. Finally, comparing the fluorescence images of Figures 2 and 5, it is clear that the fluorescence background associated with the region of the planar support adjoining the patterned S-layer surface in the former image, acquired using simple FITC-label S-layer staining, is relatively lower than that observed in the latter image, acquired using FITC-labeled antigen staining. We believe that this is due to nonspecific binding of human IgG antibody and/or FITC-labeled antihuman IgG to the thin polymeric residue remaining on the support following mold removal. In support of this assertion, control fluorescence images of S-layers acquired under identical measurement conditions following layer assembly, antibody attachment, and antigen binding in the absence of MIMIC patterning show significantly lower background fluoresence intensities. 318
Figure 5. Fluorescence images of a SbpA S-layer patterned using a “circuit-like” PDMS mold. Following layer recrystallization and mold removal, human IgG was covalently attached to active carboxylate groups on the S-layer track surface. Subsequent binding of FITC-labeled antihuman IgG enabled fluorescence imaging of the modified S-layer and verified the functional integrity of the patterned layer.
In conclusion, we have applied the soft lithography technique, micromolding in capillaries (MIMIC), to the patterning and self-assembly of ordered two-dimensional S-layer protein arrays at silicon supports. We have demonstrated the utility of the MIMIC technique for lateral patterning of simple and moderately complex crystalline S-layer array structures ranging in critical dimension from submicron to hundreds of microns. Finally, we have demonstrated that MIMIC patterning is compatible with the native chemical functionality of the protein as indicated by attachment of human IgG antibody and subsequent binding of antihuman IgG antigen at patterned S-layer substrates. Future work will focus on extension of this versatile patterning technique to spatial definition of crystalline S-layer substrates for hierarchical self-assembly of nanocrystal based nanoelectronic devices and on the use of patterned fusion protein S-layers incorporating functional domains (e.g., streptavidin)14 for controlled binding of molecular species with well-defined locations and orientations. Acknowledgment. We thank Jacqueline Friedmann for assistance with fluorescence microscopy, Oliver Stein for assistance with AFM, and Angela Neubauer for discussions. This work was supported by the EU Future and Emerging Technologies Initiative (Project No. IST-199911974 “BIOAND”), the Improving Human Potential Program (RIMDAC Project: Novel patterning methods of solid supported biomolecular architectures; Project No. P1), the Austrian Science Fund (project P14419-MOB), and the Competence Center “Biomolecular Therapeutics”. Nano Lett., Vol. 3, No. 3, 2003
Supporting Information Available: Tapping mode AFM image acquired in air of patterned S-layer protein tracks. Contact mode AFM images acquired in liquid of partially crystallized S-layer protein tracks demonstrating bilayer formation; S-layer protein recrystallized on a PDMS mold; and square lattice symmetry of patterned submicron S-layer structures. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Sleytr, U. B.; Messner, P.; Pum, D.; Sa´ra, M. Angew. Chem., Int. Ed. Engl. 1999, 38, 1034-1054. Sleytr, U. B.; Beveridge, T. J. Trends Microbiol. 1999, 7, 253-260. Sa´ra, M.; Sleytr, U. B. J. Bacteriol. 2000, 182, 859-868. Sleytr, U. B.; Sa´ra, M.; Pum, D.; Messner, P. In Nano-Surface Chemistry; Rosoff, M., Ed.; Marcel Dekker: New York, 2001; pp 333-389. (2) Messner, P.; Pum, D.; Sa´ra, M.; Stetter, K. O.; Sleytr, U. B. J. Bacteriol. 1986, 166, 1046-1054. Douglas, K.; Devaud, G.; Clark, N. A. Science 1992, 257, 642-644. Shenton, W.; Pum, D.; Sleytr, U. B.; Mann S. Nature 1997, 389, 585-587. Dieluweit, S.; Pum, D.; Sleytr, U. B. Supramol. Sci. 1998, 5, 15-19. Mertig, M.; Kirsch, R.; Pompe, W.; Engelhardt, H. Eur. Phys. J. 1999, 9, 45-48. Hall, S. R.; Shenton, W.; Engelhardt, H.; Mann, S. ChemPhysChem 2001, 3, 184-186. (3) Douglas, K.; Clark, N. A. Appl. Phys. Lett. 1986, 48, 676-678. Pum, D.; Stangl, G.; Sponer, C.; Fallmann, W.; Sleytr, U. B. Colloids Surf. B 1996, 8, 157-162.
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