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Supramolecular engineering at functionalized surfaces
Synthetic Metals, 61 (1993) 5-11
5
Supramolecular engineering at functionalized surfaces W. Knoll a'b, L. A n g e r m a i e r c, G. Batz c, T. Fritz a, S. F u j i s a w a a'd, T. F u r u n o a, H.-J. G u d e r c, M. H a r a a, M. Liley b, K. Niki d a n d J. S p i n k e b aFrontier Research Program, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-01 (Japan) bMax-Planck-Institut fiir Polymerforschung, Ackermannweg 10, D-55021 Mainz 1 (Germany) CBoehringer Mannheim, Werk Tutzing, Bahnhofstrasse 9-15, D-82327 Tutzing (Germany) dDepartment of Physical Chemistry, Yokohama National University, 156 Tokiwadai, Hodogaya-Ku, Yokohama 240 (Japan)
Introduction
Future device configurations for electronic, photonic or other 'intelligent' applications involving (bio-)organic materials require nano-fabrication,-manipulation and -functionalization techniques of supramolecular aggregates and assemblies, with control of their structure, order and dynamic behavior at the molecular/atomic level. This contribution summarizes a few of our activities aiming at a better understanding of the fundamental structure/order-property/function relations in such complex systems which is needed for the design and preparation of artificial superstructures with tailor-made properties. We focus, in particular, on different levels of organization of interfacial and thin film architectures: The first one, aiming at the 'perfect', molecularly engineered order, employs (organic) molecular beam epitaxy (OMBE) to grow single crystalline lattices of functional thin films. As a key requisite suitable substrates, i.e., solid supports with an appropriate lattice spacing and symmetry and of sufficient planarity at the atomic level, are to be prepared. The fabrication of two-dimensional arrays of biopolymers is the focus of another program that searches for general principles and techniques useful for the controlled organization of functional proteins. Based on general interactions like electrostatic or hydrophobic forces such 2D crystals can be grown by organization at a Langmuir monolayer at the water/air interface. High resolution structural analysis is possible after transfer to a solid support in an electron microscope. The last example concerns principles realized in nature in all biomembranes: no longer crystal formation, only liquid-crystalline ordering for (dynamically) organized functional units. Various schemes for such membrane-mimetic approaches can be envisaged, including the Langmuir-Blodgett-Kuhn technique and
self-assembly systems with a broad flexibility for obtaining different functionalities.
On the way to perfect architectures
Already many decades ago, physicists and materials scientists had learned how to grow electronic materials, e.g. silicon, in single crystalline form and how to treat a sample such that its surface was also nearly perfect, smooth at the atomic level. Thus, one could grow other materials on top, again with an atomically controlled (layered) architecture. That way, completely new artificial systems could be generated with structure-related properties that could be tuned far beyond what was possible with naturally occurring architectures. The appropriate technique to accomplish this was molecular beam epitaxy (MBE) [1]. For the organization of organic materials envisaged, e.g., for future opto-electronic device applications, the same technique is considered to be a very promising approach: the goal is to deposit the functional organic molecules onto a (perfect) substrate in such a gentle way that these materials can organize themselves not only through interaction with each other but also with the substrate without any defects [2] that would deteriorate the material's optical or electrical performance. The combination of various materials then could also lead to new exotic systems with unconventional properties. Unfortunately, there are only a few substrate materials that would be naturally suited to serve as atomically fiat targets for deposition: freshly cleaved mica, e.g., or graphite and MoS2 are systems that do not require the sometimes rather sophisticated, time-consuming annealing procedures known from surface analysis techniques, e.g., in low-energy electron diffraction. For various reasons, we are particularly interested in the preparation of single crystalline noble metal substrates
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with atomically controlled flatness and planarity. First encouraging results were obtained with Au, epitaxially grown onto mica in our MBE system under R H E E D (reflection high energy electron diffraction) control at a deposition rate of approximately 20-30 nm/min. The obtained thin Au samples were then further characterized by STM (Nano Scope II from Digital Instruments). Figure 1 shows an image of an area on the sample of c. 700×700 nm ~- taken in the constant current mode. Clearly, the sample grew in a single crystalline way though not with a perfect surface: the step heights between different terraces are typically 1-3 atomic layers. Nevertheless, if imaged with higher resolution in the constant height mode, individual gold atoms can be identified on the terraces: the area shown in Fig. 2 is c. 2 × 2 nm 2 and demonstrates the sixfold symmetry of the Au (111) surface.
Onto such a Au substrate organic materials could be grown by the organic MBE technique. Copper phthalocyanine (CuPc) sublimed from a Knudsen cell was deposited as a monomolecular layer onto the Au single crystalline substrate and then also investigated by STM [3]. Figure 3(a) shows an image of an area of 12 × 12 nm 2 taken in the constant current mode. If compared to the structural formula of CuPc (Fig. 3(b)) one can identify the individual four-membered rings of the chromophores in a defect-free perfect lattice arrangement with fourfold symmetry. It is interesting to note that, by a careful analysis of the molecular arrangement, e.g., with respect to the orientation and position of the CuPc molecules relative to the Au substrate, one can conclude that the crystalline packing is different from what is found in 'normal' samples grown by three-dimensional crystallization [3]. Since it is known that already subtle changes in the intermolecular interaction distances can modify the electronic coupling of molecular aggregates quite dramatically, it
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Fig. 1. S T M image taken in the constant c u r r e n t m o d e across 7 0 0 × 7 0 0 n m 2 of a A u s a m p l e epitaxially grown on mica.
(a)
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Fig. 2. High resolution S T M image of an atomically flat A u surface of 2 × 2 nm 2 taken in the constant height mode.
Fig. 3. (a) S T M image of a m o n o l a y e r of c o p p e r p h t h a l o c y a n i n e (CuPc) grown by O M B E onto an atomically fiat A u substrate. T h e area of 1 2 × 1 2 n m 2 was scanned in the constant c u r r e n t mode. (b) Structural formula of p h t h a l o c y a n i n e with a central metal ion M.
is to be expected that by the presented nanofabrication of photonic and electronic materials their properties can be tuned over a wide range by purposefully selected substrates.
Organizing functional proteins into two-dimensional arrays
The observation that only few proteins in nature have to crystallize in order to be able to perform their task is certainly related to the fact that most functions of proteins require a fluid environment of a cell membrane. The biological concept of dynamic structure formation for the purpose of bringing together functional units in a transient way (which obviously requires a fluid matrix) at present is difficult to mimic for technological processes because an artificial system lacks the link to the biosynthetic and the metabolic backup by a living organism. This leads to the limited lifetimes of biosystems, not compatible with the stability requirements of bio-electronic circuits. Therefore, the idea developed, also for proteins as functional units to stabilize and functionalize them by organization, i.e., to arrange proteins in a two-dimensional crystalline packing [4]. Our interests concentrate on the development of very general strategies for the crystallization of proteins by means of nonspecific interactions with a suitable substrate. One of the concepts is schematically outlined in Fig. 4. Water-soluble proteins (e.g. ferritin, catalase, streptavidin, etc.) adsorb to a flexible, ultrathin polymer layer, e.g. of poly(benzyl-L-histidine), prepared at a water/air interface. This positively charged monolayer attracts the negatively charged proteins by coulombic and hydrophobic forces and thus helps to organize and eventually crystallize these functional units into regular arrays. A scanning electron microscopy (SEM) picture of a protein monolayer of streptavidin thus grown
(transferred to a solid support by horizontal lifting and stained with uranylacetate) is given in Fig. 5(a). A detailed analysis of the protein arrangement by optical diffraction techniques gives a crystal structure as schematically depicted in Fig. 5(b). It is interesting to note that this packing obtained by streptavidin adsorption to the polymer monolayer is nearly identical to the one reported by Darst et al. [5] for streptavidin crystals grown by specific recognition and binding to a biotinfunctionalized lipid monolayer. Such highly correlated protein aggregates or even crystals are considered to be basic structures for future biomolecular electronics. The possibility to change purposefully the natural properties of proteins by sitedirected mutagenesis opens an unbelievable horizon for the tuning of their structural and functional features by genetic engineering techniques [6].
(a)
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@ Fig. 4. Schematic of the formation of a 2D protein crystal by adsorption from solution to the polymer monolayer prepared at the water/air interface.
(b) Fig. 5. (a) Scanning electron micrograph of a streptavidin 2D crystal grown by adsorption (cf. Fig. 4). (b) Packing of the streptavidin molecules in the crystal shown in (a).
Nano-engineering by specific biorecognition The controlled build-up of supramolecular architectures by specific recognition reactions at functionalized surfaces is currently studied in many laboratories employing a broad range of experimental techniques. In particular, the ligand-receptor pair, biotin-streptavidin [7], is widely used as a model system because: (i) it undergoes a highly specific and strong binding reaction (with a binding constant of K = 10 -~s M); (ii) the complex formed is structurally well characterized by X-ray analysis so that information about the sterical requirements of the binding reaction can be deduced [8, 9]; (iii) biotin (vitamin H) can be easily linked to other functional units, e.g., to phospholipids or self-assembling molecules like alkyl-thiols [10]; (iv) the two biomolecules are easily available and relatively stable to work with. We first concentrate on the functionalization of Au surfaces by self-assembly monolayers composed of thiols, sulfides and disulfides focusing on the question of how to optimize the interface for maximum, molecularly controlled protein binding [11]. The self-assembled monolayers (SAMs) were formed on clean gold films, vacuum-evaporated onto high-index glass substrates. All adsorptions were performed from 5 X 10 - 4 M ethanolic solutions. After rinsing and drying the SAM-coated substrates were mounted to a liquid cell in a surface plasmon spectrometer [12]. Two modes of operation could be used. The first one scans the angle of incidence 0 with the angular position of resonance seen in the reflectivity, R(0), being sensitive to the molecular architecture at the solid/solution interface: any thin film formation, e.g., a monolayer of bound protein, shifts the minimum of R to higher angles. For time-dependent studies, the reflected intensity is measured at a fixed angle of incidence. Thus, kinetic data of monolayer formation can be obtained. First, single-component SAMs were studied. Generally speaking, we found that biotin-functionalized thiols and disulfide systems showed good monolayer formation but with a low streptavidin binding capacity. This could be shown as being due to a steric hindrance for the biotin label to reach the binding pocket inside the protein. This was different for systems like all sulfides that showed only poor monolayer formation (as inferred from a largely reduced layer thickness): their binding capacities were relatively high owing, however, to substantial non-specific binding. (This could be demonstrated with 'complexed streptavidin', i.e., with a protein solution that was saturated with free biotin, thus blocking all binding sites of the protein for specific recognition.)
On the other hand, SAM formation with hydroxyterminated alkylthiols and disulfides showed a complete passivation of the substrate surface for any streptavidin adsorption. These findings led us to a strategy for optimized protein binding even at well-ordered monolayers by using binary mixtures of biotin- and OH-terminated self-assembly systems. Figure 6 shows the surface-plasmon optical results of the thickness determinations for co-adsorbed SAMs and of their streptavidin binding. The striking result is the maximum protein layer thickness at c. 5-10 mol% biotinylated thiol indicating the necessity of a high lateral dilution (in addition to a sufficient spacer length between the biotin group and the alkylthiol) for optimized binding capacity. The thickness d=4.0 nm found for the protein monolayer matches the known protein dimension of c. 4.5 × 4.5 x 5.0 nm 3. One might speculate that the residual binding at high biotin content originates from disordered sites which must exist at these rough evaporated gold surfaces. If instead of biotin the less strongly interacting desthiobiotin (structural formula given in Fig. 7) is linked to a self-assembled monolayer, one has a further degree of flexibility to manipulate the interfacial architecture. This is schematically depicted in Fig. 7. A desthiobiotinylated SAM is first covered with a dense monolayer of streptavidin. After washing the cell, an excess of
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Fig. 6. Top: schematic representation of an optimized selfassembled monolayer (SAM). The lateral dilution of the recognition sites and their steric decoupling by a spacer of sufficient length allows binding of the streptavidin. Bottom: thickness data of the binary SAMs of biotin- and OH-terminated thiols ( 0 ) (structural formulae given in the inset) and of the bound streptavidin monolayer ( 4 ) as a function of biotin content.
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Fig. 7. Schematic of the reaction cycle for the competitive replacement of streptavidin from a desthiobiotin (structural formula given in the inset) matrix by excess free biotin. d/nm 0.46
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Fig. 8. Thickness data as obtained by surface-plasmon spectroscopy for the streptavidin monolayer formation at a desthiobiotinfunctionalized SAM and its disassembly by competitive replacement by free biotin.
free biotin (10 -4 M) is injected which competes now for the binding sites of the streptavidin. Figure 8 shows that, indeed, an almost complete detachment of the streptavidin layer can be achieved though on a very long time scale! The residual coverage after rinsing
amounts to only 0.3 nm. The regenerated free desthiobiotin matrix can be used again for streptavidin binding (cf. Fig. 8), and this cycle can be repeated many times without noticeable loss of binding capacity or monolayer degradation. As is briefly summarized in Fig. 9, this regeneration approach can also be taken for more complex architectures envisaged in biosensor configurations. After binding of the streptavidin layer, a layer of biotinylated Fab (=antibody) fragments of Ad= 2.8 nm is formed followed by a layer of human chorionic gonadotrophin-antigen (HCG-antigen) of Ad= 1.5 nm. Again injection of free biotin removes the whole complexes and regenerates the free desthiobiotin surface (Fig. 9, fight side). Conclusions for future strategies in nano-engineering at surfaces
The examples given may look at first sight somewhat arbitrary and independent of each other. But the various approaches do, in fact, represent alternative, complementary concepts on the way to the controlled buildup of functional architectures at interfaces. This is schematically summarized in Fig. 10 for the special case of the so-called membrane-mimetic approach. The left side (Fig. 10(a)) illustrates the problem that one encounters in trying to fabricate molecular architecture
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/
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(d) Fig. 10. Different strategy concepts for the molecularly controlled build-up of supramolecular architectures at solid supports.
Fig. 11. STM image (constant current mode; 7 x 7 nm z) of a 4mercaptobenzoic acid SAM on epitaxially grown Au (111) substrate.
at general surfaces, controlled at the nanometer scale: the typical dimensions of the natural roughness is of the same order of magnitude as the size of the molecular building blocks that are to be assembled in a LEGO or tinkertoy approach. The cartoon here sketches in this for an amphiphilic molecule with a polar head group and an apolar tail - a molecular unit that is used in nature to build up membranes (hence the term membrane-mimetic). The results given for the optimization of streptavidin binding to biotinylated SAMs have shown that, by a purposefully designed synthetic modification of molecules, the desired surface functionalization can be obtained. This indicates that by a proper balance of the different interaction potentials, e.g., in our case between the chains of the molecules, their head groups, solvent molecules, and between the head groups and the substrate, a reasonable degree of order can be induced (cf. Fig. 10(b)), enough to allow for a well-defined protein monolayer formation by specific binding while simultaneously suppressing any unspecific adsorption. These monolayers could then even be used for more complex multilayer formation. Another idea for allowing these membrane-constituting molecules to self-organize into their usual monomolecular order is to decouple them from the substrate imperfections by a fluid or at least flexible polymer buffer layer [13]. This concept is sketched in Fig. 10(c). This way the molecules can nearly freely assemble, weakly but sufficiently linked to the substrate which then is just a support not interfering too much with the monolayer's own identity. What is shown in Fig. 10(c) is, of course, also the basic principle for the successful transfer of 2D protein crystals grown on the thin polymer layer at the fluid air/water interface to a (rough) substrate without disrupting the order in the thin protein crystal layer as discussed above. The last approach, finally, comes back to the principle of starting with a perfect substrate. If one can combine MBE techniques for the fabrication of, e.g., atomically
11
fiat Au surfaces, then also the self-assembly process of various functional thiols should lead to highly ordered and organized supramolecular architectures. A first example of a monolayer of 4-mercaptobenzoic acid assembled on epitaxially grown Au films and imaged with STM is given in Fig. 11. Several features in the picture showing an area of c. 7 x 7 nm 2, like the 2 x 2 packing relative to the substrate lattice, the formation of dimers, the observation of a dislocation line, etc. indicate that one is, indeed, imaging the thiol layer. With only a little more optimization one should be able to generate the kind of perfect architecture indicated in Fig. 10(d) as the goal of this approach. There is still a long way to go but we think we are on the right tracks.
Acknowledgements
Helpful discussions with H. Ringsdorf, H. Sasabe and J. Yang are gratefully acknowledged.
References 1 M.A. Herman and H. Sitter, Molecular Beam Epitaxy, Springer, Berlin, 1989. 2 M. Hara, H. Sasabe, A. Yamada and A.F. Garito, Jpn. J. Appl. Phys., 28 (1989) L310. 3 T. Fritz, M. Hara, H. Sasabe and W. Knoll, manuscript in preparation. 4 A. Sato, T. Furuno, C. Toyoshima and H. Sasabe, Biochim. Biophys. Acta, 1162 (1993) 54. 5 S.A. Darst, M. Ahlers, P. Meller, E.W. Kubalek, R. Blankenburg, H.O. Ribi, H. Ringsdorf and R.D. Kornberg, Biophys. J., 59 (1991) 387. 6 S.G. Sligar and F.R. Salemme, Current Opinions in Structural Biology 2, 1992, p. 587. 7 M. Wilchek and E.A. Bayer, Methods Enzymol., 184 (1990) 5. 8 W.A. Hendrickson, A. P/ihler, J.C. Smith, Y. Satow, E.A. Merritt and R.P. Phizackerley, Proc. Natl. Acad. Sci. USA, 86 (1989) 2190. 9 P.C. Weber, D.H. Ohlendorf, J.J. Wendoloski and F.R. Salemme, Science, 243 (1989) 85. 10 L. H~iussling, H. Ringsdorf, F.-J. Schmitt and W. Knoll, Langmuir, 7 (1991) 1837. 11 J. Spinke, M. Liley, F.-J. Schmitt, H.-J. Gruder, L. Angermaier and W. Knoll, J. Chem. Phys., in press. 12 W. Knoll, Mater. Res. Soc. Bull., 16 (1991) 29. 13 J. Spinke, J. Yang, H. Wolf, M. Liley, H. Ringsdorf and W. Knoll, Biophys. J., 63 (1992) 1667.
5
Supramolecular engineering at functionalized surfaces W. Knoll a'b, L. A n g e r m a i e r c, G. Batz c, T. Fritz a, S. F u j i s a w a a'd, T. F u r u n o a, H.-J. G u d e r c, M. H a r a a, M. Liley b, K. Niki d a n d J. S p i n k e b aFrontier Research Program, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-01 (Japan) bMax-Planck-Institut fiir Polymerforschung, Ackermannweg 10, D-55021 Mainz 1 (Germany) CBoehringer Mannheim, Werk Tutzing, Bahnhofstrasse 9-15, D-82327 Tutzing (Germany) dDepartment of Physical Chemistry, Yokohama National University, 156 Tokiwadai, Hodogaya-Ku, Yokohama 240 (Japan)
Introduction
Future device configurations for electronic, photonic or other 'intelligent' applications involving (bio-)organic materials require nano-fabrication,-manipulation and -functionalization techniques of supramolecular aggregates and assemblies, with control of their structure, order and dynamic behavior at the molecular/atomic level. This contribution summarizes a few of our activities aiming at a better understanding of the fundamental structure/order-property/function relations in such complex systems which is needed for the design and preparation of artificial superstructures with tailor-made properties. We focus, in particular, on different levels of organization of interfacial and thin film architectures: The first one, aiming at the 'perfect', molecularly engineered order, employs (organic) molecular beam epitaxy (OMBE) to grow single crystalline lattices of functional thin films. As a key requisite suitable substrates, i.e., solid supports with an appropriate lattice spacing and symmetry and of sufficient planarity at the atomic level, are to be prepared. The fabrication of two-dimensional arrays of biopolymers is the focus of another program that searches for general principles and techniques useful for the controlled organization of functional proteins. Based on general interactions like electrostatic or hydrophobic forces such 2D crystals can be grown by organization at a Langmuir monolayer at the water/air interface. High resolution structural analysis is possible after transfer to a solid support in an electron microscope. The last example concerns principles realized in nature in all biomembranes: no longer crystal formation, only liquid-crystalline ordering for (dynamically) organized functional units. Various schemes for such membrane-mimetic approaches can be envisaged, including the Langmuir-Blodgett-Kuhn technique and
self-assembly systems with a broad flexibility for obtaining different functionalities.
On the way to perfect architectures
Already many decades ago, physicists and materials scientists had learned how to grow electronic materials, e.g. silicon, in single crystalline form and how to treat a sample such that its surface was also nearly perfect, smooth at the atomic level. Thus, one could grow other materials on top, again with an atomically controlled (layered) architecture. That way, completely new artificial systems could be generated with structure-related properties that could be tuned far beyond what was possible with naturally occurring architectures. The appropriate technique to accomplish this was molecular beam epitaxy (MBE) [1]. For the organization of organic materials envisaged, e.g., for future opto-electronic device applications, the same technique is considered to be a very promising approach: the goal is to deposit the functional organic molecules onto a (perfect) substrate in such a gentle way that these materials can organize themselves not only through interaction with each other but also with the substrate without any defects [2] that would deteriorate the material's optical or electrical performance. The combination of various materials then could also lead to new exotic systems with unconventional properties. Unfortunately, there are only a few substrate materials that would be naturally suited to serve as atomically fiat targets for deposition: freshly cleaved mica, e.g., or graphite and MoS2 are systems that do not require the sometimes rather sophisticated, time-consuming annealing procedures known from surface analysis techniques, e.g., in low-energy electron diffraction. For various reasons, we are particularly interested in the preparation of single crystalline noble metal substrates
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with atomically controlled flatness and planarity. First encouraging results were obtained with Au, epitaxially grown onto mica in our MBE system under R H E E D (reflection high energy electron diffraction) control at a deposition rate of approximately 20-30 nm/min. The obtained thin Au samples were then further characterized by STM (Nano Scope II from Digital Instruments). Figure 1 shows an image of an area on the sample of c. 700×700 nm ~- taken in the constant current mode. Clearly, the sample grew in a single crystalline way though not with a perfect surface: the step heights between different terraces are typically 1-3 atomic layers. Nevertheless, if imaged with higher resolution in the constant height mode, individual gold atoms can be identified on the terraces: the area shown in Fig. 2 is c. 2 × 2 nm 2 and demonstrates the sixfold symmetry of the Au (111) surface.
Onto such a Au substrate organic materials could be grown by the organic MBE technique. Copper phthalocyanine (CuPc) sublimed from a Knudsen cell was deposited as a monomolecular layer onto the Au single crystalline substrate and then also investigated by STM [3]. Figure 3(a) shows an image of an area of 12 × 12 nm 2 taken in the constant current mode. If compared to the structural formula of CuPc (Fig. 3(b)) one can identify the individual four-membered rings of the chromophores in a defect-free perfect lattice arrangement with fourfold symmetry. It is interesting to note that, by a careful analysis of the molecular arrangement, e.g., with respect to the orientation and position of the CuPc molecules relative to the Au substrate, one can conclude that the crystalline packing is different from what is found in 'normal' samples grown by three-dimensional crystallization [3]. Since it is known that already subtle changes in the intermolecular interaction distances can modify the electronic coupling of molecular aggregates quite dramatically, it
.....
. . . . . . .
. . . . . . .
. . . . . . . . . . . .......... ....
%~!i~ii!i iiiiiilliii!i~..~:~,,~% iiii~0~ ~.~.~.,-~,~,~.~, iiiiiiiiiiiiiiiiiiiiiiiii~i~
Fig. 1. S T M image taken in the constant c u r r e n t m o d e across 7 0 0 × 7 0 0 n m 2 of a A u s a m p l e epitaxially grown on mica.
(a)
(3
(b)
Fig. 2. High resolution S T M image of an atomically flat A u surface of 2 × 2 nm 2 taken in the constant height mode.
Fig. 3. (a) S T M image of a m o n o l a y e r of c o p p e r p h t h a l o c y a n i n e (CuPc) grown by O M B E onto an atomically fiat A u substrate. T h e area of 1 2 × 1 2 n m 2 was scanned in the constant c u r r e n t mode. (b) Structural formula of p h t h a l o c y a n i n e with a central metal ion M.
is to be expected that by the presented nanofabrication of photonic and electronic materials their properties can be tuned over a wide range by purposefully selected substrates.
Organizing functional proteins into two-dimensional arrays
The observation that only few proteins in nature have to crystallize in order to be able to perform their task is certainly related to the fact that most functions of proteins require a fluid environment of a cell membrane. The biological concept of dynamic structure formation for the purpose of bringing together functional units in a transient way (which obviously requires a fluid matrix) at present is difficult to mimic for technological processes because an artificial system lacks the link to the biosynthetic and the metabolic backup by a living organism. This leads to the limited lifetimes of biosystems, not compatible with the stability requirements of bio-electronic circuits. Therefore, the idea developed, also for proteins as functional units to stabilize and functionalize them by organization, i.e., to arrange proteins in a two-dimensional crystalline packing [4]. Our interests concentrate on the development of very general strategies for the crystallization of proteins by means of nonspecific interactions with a suitable substrate. One of the concepts is schematically outlined in Fig. 4. Water-soluble proteins (e.g. ferritin, catalase, streptavidin, etc.) adsorb to a flexible, ultrathin polymer layer, e.g. of poly(benzyl-L-histidine), prepared at a water/air interface. This positively charged monolayer attracts the negatively charged proteins by coulombic and hydrophobic forces and thus helps to organize and eventually crystallize these functional units into regular arrays. A scanning electron microscopy (SEM) picture of a protein monolayer of streptavidin thus grown
(transferred to a solid support by horizontal lifting and stained with uranylacetate) is given in Fig. 5(a). A detailed analysis of the protein arrangement by optical diffraction techniques gives a crystal structure as schematically depicted in Fig. 5(b). It is interesting to note that this packing obtained by streptavidin adsorption to the polymer monolayer is nearly identical to the one reported by Darst et al. [5] for streptavidin crystals grown by specific recognition and binding to a biotinfunctionalized lipid monolayer. Such highly correlated protein aggregates or even crystals are considered to be basic structures for future biomolecular electronics. The possibility to change purposefully the natural properties of proteins by sitedirected mutagenesis opens an unbelievable horizon for the tuning of their structural and functional features by genetic engineering techniques [6].
(a)
I~[ylnsr
monolayer 21:)cryltal
~
.~
water
©
@ Fig. 4. Schematic of the formation of a 2D protein crystal by adsorption from solution to the polymer monolayer prepared at the water/air interface.
(b) Fig. 5. (a) Scanning electron micrograph of a streptavidin 2D crystal grown by adsorption (cf. Fig. 4). (b) Packing of the streptavidin molecules in the crystal shown in (a).
Nano-engineering by specific biorecognition The controlled build-up of supramolecular architectures by specific recognition reactions at functionalized surfaces is currently studied in many laboratories employing a broad range of experimental techniques. In particular, the ligand-receptor pair, biotin-streptavidin [7], is widely used as a model system because: (i) it undergoes a highly specific and strong binding reaction (with a binding constant of K = 10 -~s M); (ii) the complex formed is structurally well characterized by X-ray analysis so that information about the sterical requirements of the binding reaction can be deduced [8, 9]; (iii) biotin (vitamin H) can be easily linked to other functional units, e.g., to phospholipids or self-assembling molecules like alkyl-thiols [10]; (iv) the two biomolecules are easily available and relatively stable to work with. We first concentrate on the functionalization of Au surfaces by self-assembly monolayers composed of thiols, sulfides and disulfides focusing on the question of how to optimize the interface for maximum, molecularly controlled protein binding [11]. The self-assembled monolayers (SAMs) were formed on clean gold films, vacuum-evaporated onto high-index glass substrates. All adsorptions were performed from 5 X 10 - 4 M ethanolic solutions. After rinsing and drying the SAM-coated substrates were mounted to a liquid cell in a surface plasmon spectrometer [12]. Two modes of operation could be used. The first one scans the angle of incidence 0 with the angular position of resonance seen in the reflectivity, R(0), being sensitive to the molecular architecture at the solid/solution interface: any thin film formation, e.g., a monolayer of bound protein, shifts the minimum of R to higher angles. For time-dependent studies, the reflected intensity is measured at a fixed angle of incidence. Thus, kinetic data of monolayer formation can be obtained. First, single-component SAMs were studied. Generally speaking, we found that biotin-functionalized thiols and disulfide systems showed good monolayer formation but with a low streptavidin binding capacity. This could be shown as being due to a steric hindrance for the biotin label to reach the binding pocket inside the protein. This was different for systems like all sulfides that showed only poor monolayer formation (as inferred from a largely reduced layer thickness): their binding capacities were relatively high owing, however, to substantial non-specific binding. (This could be demonstrated with 'complexed streptavidin', i.e., with a protein solution that was saturated with free biotin, thus blocking all binding sites of the protein for specific recognition.)
On the other hand, SAM formation with hydroxyterminated alkylthiols and disulfides showed a complete passivation of the substrate surface for any streptavidin adsorption. These findings led us to a strategy for optimized protein binding even at well-ordered monolayers by using binary mixtures of biotin- and OH-terminated self-assembly systems. Figure 6 shows the surface-plasmon optical results of the thickness determinations for co-adsorbed SAMs and of their streptavidin binding. The striking result is the maximum protein layer thickness at c. 5-10 mol% biotinylated thiol indicating the necessity of a high lateral dilution (in addition to a sufficient spacer length between the biotin group and the alkylthiol) for optimized binding capacity. The thickness d=4.0 nm found for the protein monolayer matches the known protein dimension of c. 4.5 × 4.5 x 5.0 nm 3. One might speculate that the residual binding at high biotin content originates from disordered sites which must exist at these rough evaporated gold surfaces. If instead of biotin the less strongly interacting desthiobiotin (structural formula given in Fig. 7) is linked to a self-assembled monolayer, one has a further degree of flexibility to manipulate the interfacial architecture. This is schematically depicted in Fig. 7. A desthiobiotinylated SAM is first covered with a dense monolayer of streptavidin. After washing the cell, an excess of
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Fig. 6. Top: schematic representation of an optimized selfassembled monolayer (SAM). The lateral dilution of the recognition sites and their steric decoupling by a spacer of sufficient length allows binding of the streptavidin. Bottom: thickness data of the binary SAMs of biotin- and OH-terminated thiols ( 0 ) (structural formulae given in the inset) and of the bound streptavidin monolayer ( 4 ) as a function of biotin content.
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free biotin (10 -4 M) is injected which competes now for the binding sites of the streptavidin. Figure 8 shows that, indeed, an almost complete detachment of the streptavidin layer can be achieved though on a very long time scale! The residual coverage after rinsing
amounts to only 0.3 nm. The regenerated free desthiobiotin matrix can be used again for streptavidin binding (cf. Fig. 8), and this cycle can be repeated many times without noticeable loss of binding capacity or monolayer degradation. As is briefly summarized in Fig. 9, this regeneration approach can also be taken for more complex architectures envisaged in biosensor configurations. After binding of the streptavidin layer, a layer of biotinylated Fab (=antibody) fragments of Ad= 2.8 nm is formed followed by a layer of human chorionic gonadotrophin-antigen (HCG-antigen) of Ad= 1.5 nm. Again injection of free biotin removes the whole complexes and regenerates the free desthiobiotin surface (Fig. 9, fight side). Conclusions for future strategies in nano-engineering at surfaces
The examples given may look at first sight somewhat arbitrary and independent of each other. But the various approaches do, in fact, represent alternative, complementary concepts on the way to the controlled buildup of functional architectures at interfaces. This is schematically summarized in Fig. 10 for the special case of the so-called membrane-mimetic approach. The left side (Fig. 10(a)) illustrates the problem that one encounters in trying to fabricate molecular architecture
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(d) Fig. 10. Different strategy concepts for the molecularly controlled build-up of supramolecular architectures at solid supports.
Fig. 11. STM image (constant current mode; 7 x 7 nm z) of a 4mercaptobenzoic acid SAM on epitaxially grown Au (111) substrate.
at general surfaces, controlled at the nanometer scale: the typical dimensions of the natural roughness is of the same order of magnitude as the size of the molecular building blocks that are to be assembled in a LEGO or tinkertoy approach. The cartoon here sketches in this for an amphiphilic molecule with a polar head group and an apolar tail - a molecular unit that is used in nature to build up membranes (hence the term membrane-mimetic). The results given for the optimization of streptavidin binding to biotinylated SAMs have shown that, by a purposefully designed synthetic modification of molecules, the desired surface functionalization can be obtained. This indicates that by a proper balance of the different interaction potentials, e.g., in our case between the chains of the molecules, their head groups, solvent molecules, and between the head groups and the substrate, a reasonable degree of order can be induced (cf. Fig. 10(b)), enough to allow for a well-defined protein monolayer formation by specific binding while simultaneously suppressing any unspecific adsorption. These monolayers could then even be used for more complex multilayer formation. Another idea for allowing these membrane-constituting molecules to self-organize into their usual monomolecular order is to decouple them from the substrate imperfections by a fluid or at least flexible polymer buffer layer [13]. This concept is sketched in Fig. 10(c). This way the molecules can nearly freely assemble, weakly but sufficiently linked to the substrate which then is just a support not interfering too much with the monolayer's own identity. What is shown in Fig. 10(c) is, of course, also the basic principle for the successful transfer of 2D protein crystals grown on the thin polymer layer at the fluid air/water interface to a (rough) substrate without disrupting the order in the thin protein crystal layer as discussed above. The last approach, finally, comes back to the principle of starting with a perfect substrate. If one can combine MBE techniques for the fabrication of, e.g., atomically
11
fiat Au surfaces, then also the self-assembly process of various functional thiols should lead to highly ordered and organized supramolecular architectures. A first example of a monolayer of 4-mercaptobenzoic acid assembled on epitaxially grown Au films and imaged with STM is given in Fig. 11. Several features in the picture showing an area of c. 7 x 7 nm 2, like the 2 x 2 packing relative to the substrate lattice, the formation of dimers, the observation of a dislocation line, etc. indicate that one is, indeed, imaging the thiol layer. With only a little more optimization one should be able to generate the kind of perfect architecture indicated in Fig. 10(d) as the goal of this approach. There is still a long way to go but we think we are on the right tracks.
Acknowledgements
Helpful discussions with H. Ringsdorf, H. Sasabe and J. Yang are gratefully acknowledged.
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