Layer-by-layer self-assembly of supramolecular and biomolecular films

Reviews in Molecular Biotechnology 90 Ž2002. 55᎐70

Layer-by-layer self-assembly of supramolecular and biomolecular films Tim Salditt a,U , Ulrich S. Schubert b a

b

Experimentalphysik, Uni¨ ersitat Germany ¨ des Saarlandes, Im Stadtwald 38, Postfach 15 11 50, 66041 Saarbrucken, ¨ Laboratory of Macromolecular and Organic Chemistry and Center for Nanomaterials, Eindho¨ en Uni¨ ersity of Technology, P.O. Box 513, 5600 MO Eindho¨ en, The Netherlands

Abstract In this paper, we give a short account on recent studies of layer-by-layer self-assembly of supramolecular and biomolecular films. Such films are built up from layers of macro-ions with opposing charge. A simple film can be obtained by alternating the adsorption of two components: a flexible, synthetic polycation chains and a supramolecular or biomolecular moiety. We focus on three examples, in which the second component consists either of a supramolecular metal᎐organic complex ŽMOC., a nucleic acid, or a biological membrane patch Žpurple membrane.. While the flexible polycation chains Žas well as eventual annealing layers. ensure a uniform build-up of the chain, the second macromolecular component may be used to functionalize the films. The combination of layer-by-layer self-assembly and biotechnologically relevant macromolecules may lead to new devices or biomaterial applications. To this end, precise studies of the deposition process and the film structure are needed. Here, we focus on interface sensitive scattering techniques for the structural analysis. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Supramolecular films; Biomolecular films; Interface sensitive scattering techniques; Layer-by-layer self-assembly

1. Introduction The development of thin films composed of functional molecules for applications in materials science and biotechnology is an active field of research. To exploit the specific properties Želectrical, optical, mechanical, or chemical. of syn-

U

Corresponding author.

thetic or biological macromolecules, it is often advantageous to prepare them in form of thin films. Compared to classical thin film technologies of inorganic solids, thin film growth of macromolecules typically relies on relatively weak noncovalent inter-molecular forces. These interactive forces may lead to the aggregation of molecules, e.g. at the interface between a solid and a solvent phase. The challenges in this field are to, firstly, control the deposition or the self-assembly

1389-0352r02r$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 9 - 0 3 5 2 Ž 0 1 . 0 0 0 4 9 - 6

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T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

process, secondly, to correlate deposition parameters with thin film structure, and thirdly, to correlate the thin film structure to physical and chemical properties. In this very general context, we review some quite specific research which we have carried out recently. The work includes several macromolecular systems, but only one deposition process: sequential adsorption of charged macromolecules from solution, or layer-by-layer self-assembly. The basic deposition process of these self-assembled thin films is as simple as it is versatile, but was discovered and developed only a few years ago by Decher and co-workers Žreviewed in Decher, 1997.. The method relies on the fact that charged surfaces, which are built up by the adsorption of one monolayer, are over-compensated and charge-reversed by the adsorption of a next layer, so that subsequently, alternatively charged layers are deposited. In this way, very rich and versatile structures can be prepared in a multi-layered architecture. Compared to the Langmuir᎐Blodgett technique, the self-assembled molecular films exhibit a much larger thermal and mechanical stability and can be prepared up to hundreds of layers. As a further advantage, the technique is not restricted to substrates, but can be used for coating arbitrarily-shaped objects. Most importantly, the method has been extended from simple model polyelectrolytes to many technologically relevant systems, ranging from semiconducting polymers ŽFou et al., 1996. to biological macromolecules like nucleic acids and proteins ŽLvov et al., 1993; Sukhorukov et al., 1996; Caruso et al., 1997.. An additional degree of complexity, and possibly also functionality, can be achieved if the adsorbed macro-molecules themselves are of a supramolecular nature and governed by non-covalent interactions. At the same time, there are many recent advances in supramolecular chemistry that open up new possibilities for the design and combination of different molecules with new functionalities ŽLehn, 1995.. The preparation of such molecules in the form of thin films might lead to novel devices. In the first part of this review, the combination of supramolecular chem-

istry and thin film technology based on the selfassembly method as presented in a few recent papers ŽSalditt et al., 1998, 1999; Schubert et al., 1999a,b, 2001a. will be discussed, including also some original and hitherto unpublished work on multi-layered thin films. The basic molecular building blocks of supramolecular systems studied here consist of octahedral coordinating metal ions and terpyridine or fused terpyridine ligands, held together by noncovalent interactions ŽHanan et al., 1997a,b,c., in contrast to the linear and covalently bonded metal᎐organic chains deposited previously by Kurth and co-workers by the self-assembly technique ŽSchutte ¨ et al., 1998.. The organic ligands can be arranged in the presence of suitable metal ions into ladders Žw1 = n x arrays., or grid-like architectures Žw n = m x arrays., and can be functionalized with additional groups ŽHanan et al., 1997b.. Among others, we used w1 = 1x complexes with CuŽII., CoŽII., ZnŽII. and HgŽII. metal ions, and w2 = 2x grids of CdŽII. metal ions in the thin film growth and characterization studies Žsee Fig. 1.. These novel thin film materials of metal᎐ organic complexes ŽMOC. could have potential applications in various optical, electronic or magnetic devices, e.g. in form of coordination arrays they could serve as molecular memory units and switches ŽAshoori, 1996; Friedmann et al., 1996; Schubert et al., 2001a,b.. From a more fundamental point of view, important new structural aspects of supramolecular self-assembly, as well as the corresponding microscopic interactions, can be studied in these systems. In the second part of this review, we present two examples of biopolymers self-assembled Žor complexed. with synthetic polyelectrolytes. The first example relates to complexes composed of DNA and synthetic polycations like polyŽethylenimine. hydrochloride ŽPEI., polylysine or polyŽallylamine. hydrochloride ŽPAH.. Such complexes can be prepared not only in solution, but also in the form of thin films by the self-assembly method described above. The structure formation and interaction between such different polymers as DNA on one hand, which is well-known as a strong polyanion and a semi-flexible chain, and

T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

very flexible synthetic polycations, on the other hand, is of fundamental interest. Furthermore, bulk complexes of DNA and PEI are known to exhibit interesting transfection properties for gene therapy applications ŽPollard et al., 1998.. The second example shows that not only globular proteins can be adsorbed and complexed with synthetic polyelectrolytes in thin films, but also na-

57

tive, integral membrane patches with integral membrane proteins in their functional state. The case of bacteriorhodopsin ŽBR. in the purple membrane ŽPM. complexed with PEI illustrates that stable films of membrane proteins and polyelectrolytes can be prepared by the self-assembly method. The structure of the films has been character-

Fig. 1. Ža. The chemical structure of the terpyridine ligand in the w1 = 1x organo᎐metal complexes; Žb. chemical structure of fused terpyridine ligand in the w2 = 2x complex; Žc. atomic arrangement of a w1 = 1x complex consisting of a octahedrally coordinated metal ion in its center and two perpendicularly oriented ligands Žmolecular modeling of the w1 = 1x CoŽII. complex, MacSpartan, level MM2.; Žd. analogous model of a w2 = 2x complex; Že. sketch of the thin film with a polyelectrolyte cushion composed of a PEI and a PSS layer and the organo-metal complexes adsorbed in a monomolecular layer on top Žfrom Salditt et al., 1999..

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T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

ized by synchrotron based X-ray reflectivity, diffraction and fluorescence techniques.

2. Synthesis, sample preparation and X-ray experiment 2.1. Synthesis of MOC The 5,5⬘-dimethyl-2,2⬘:6⬘,2⬘⬙-terpyridine ligand was synthesized according to published procedures and using a new synthetic strategy with organotin intermediates and Stille type coupling procedures ŽSchubert et al., 1998, 1999a,b, 2001a,b,c.. The 4,6-bisw6⬘-Ž2⬘,2⬙-bipyridyl .x pyrimidine ligand was synthesized as described in Schubert et al. Ž1999a,b.. The reaction of suitable quantities of the ligands and the corresponding metal acetates in refluxing methanol or water exclusively leads to the formation of the monomolecular or tetranuclear complexes Žisolated as PF6 salts and recrystallized twice from acetonerether .. The complexes were characterized using UVrVis spectroscopy, elemental analysis, electrospray, and MALDI-TOF mass spectrometry as well as analytical ultracentrifugation. In Fig. 1, the chemical structure of the Ža. terpyridine ligand in the w1 = 1x organo᎐metal complexes and of the Žb. extended ligand in the w2 = 2x complexes are shown, along with the atomic arrangement of a Žc. w1 = 1x complex and the Žd. w2 = 2x complex, respectively. 2.2. Polyelectrolytes PolyŽstyrene. sulfonate PSS Žsodium salt, Mr s 70 000., polyŽethylenimine . PEI Ž50 wt.% in aqueous solution Mr s 2000., and polyŽallylamine. hydrochloride PAH Ž Mr s 17 000, Aldrich 47,913-6. were obtained from Aldrich and used as received. 2.3. Self-assembly of MOC films The silicon or glass substrates were washed extensively in trichloroethylene, methanol, and ultrapure water ŽMillipore.. To render the surface hydrophilic, the substrates were washed in a 5 M

solution of KOH in water Žglass, quartz. and a saturated KOH solution in ethanol Žsilicon., respectively, for approximately 1 min. Subsequently, they were rinsed several times in Millipore water. All samples were then kept in a 0.5-wt.% solution of PEI for 20 min to adsorb a monomolecular layer of PEI. After the PEI adsorption, the samples were rinsed in salt-free Millipore water. In the next step, the surface charge density was reversed by the adsorption of a layer of PSS in a 3-mgrml solution of PSS in water at pHs 2. The samples were again washed thoroughly in ultrapure water. Finally, the organo᎐metal complexes were adsorbed onto the PSS layer by immersing the samples in a 0.5-mgrml solution of the complexes in acetone for approximately 20 min and subsequently rinsing in acetone and water before drying in a stream of nitrogen. Putting the samples in acetone did not destroy the polyelectrolyte layers, as has been shown by ex-situ and in-situ measurements ŽPlech et al., 2000.. Fig. 1e shows a sketch of the thin film with a polyelectrolyte cushion composed of a PEI and a PSS layer and the organo᎐metal complexes adsorbed in a monomolecular layer on top. 2.4. Self-assembly of DNAr polyelectrolyte films Calf thymus DNA ŽSigma D-1501. was dissolved at a concentration of 67 ␮grml in salt free Millipore water and used for adsorption. PAH was dissolved at 20 wt.% in aqueous solution. The PEI solution was prepared and used as in the case of the MOC films. 2.5. Self-assembly of BRr PEI films Films were prepared from purified and extensively washed suspensions of purple membrane ŽPM.. The PM solution was generously given by D. Oesterhelt and his group at the Max Planck Institut fur ¨ Biochemie in Martinsried, Germany. The ratio of the optical absorption coefficients at ␭ s 280 and 568 nm was 8:5. PM suspended in ultra pure water ŽMillipore. at, typically, 0.2 mgrml was used for the sequential adsorption, along with the PEI solution as described above.

T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

2.6. Specular and non-specular X-ray reflecti¨ ity The samples were then routinely characterized by X-ray reflectivity. For this method, the incident beam with wave vector k i has to be collimated to approximately a few hundredths of a degree and directed onto the sample at glancing incident angle ␣ i . The reflected intensity is then measured as a function of ␣ i under specular conditions, e.g. at an exit angle ␣ f s ␣ i , with the wave vector of the exit beam denoted by k f . Thus, the momentum transfer of the elastic scattering q s k f I k i is always along q z , with the z-axis parallel to the sample normal. Contrarily, changing the detector or sample positions to ␣ i / ␣ f Ždiffuse or nonspecular scattering . results in a component q I parallel to the sample surface. In Born’s approximation, the reflectivity is given by ŽTolan, 1999.:

59

mirror to suppress higher harmonics. The station is equipped with a multi-circle z-axis diffractometer, fully motorized slits and a fast NaJ scintillation counter ŽCyberstar, Oxford Instr... The diffractometer is controlled by the TASCOM language. On this platform, macros were developed to allow for automated control of beam attenuators, illumination control, and diffuse background subtraction. Typically, the reflectivity was recorded over eight orders of magnitude, after correction for diffuse scattering background, as measured in a so-called offset-scan. In order to get correct results when fitting the reflectivity, the correction for a diffuse background at higher angles is essential, i.e. the diffuse scattering has to be measured in a longitudinal transverse scan Žor offset scan. and subtracted. 2.7. Non-specular (diffuse) scattering

⬁

1 d␳ Ž z . R Ž qz . s R F Ž qz . exp Ž iq z z . d z ␳⬁ y⬁ d z

H

2

Ž1.

where ␳Ž z . is the electron density profile of the film, averaged in the xy-plane, and ␳⬁ is the electron density of the substrate. R F is the Fresnel reflectivity of the ideal interface between the semi-infinite substrate and air or vacuum. Eq. Ž1. neglects any dynamic effects of multiple scattering in the film structure which typically become important at small angles of ␣ i , in particular for thin film structures with sharp interfaces. Even though we believe the Born approximation sufficient for the type of samples studied here, all of our simulations were carried out within the framework of a fully dynamic Parrat algorithm ŽParratt, 1954; Tolan, 1999; Holy ´ et al., 1999.. For Ž . this purpose, ␳ z is decomposed in a number of different layers, e.g. substrate, PEI, PSS, organo᎐metal complexes, with constant density ␳ i and an error function profile of width ␴i interpolating between the values of ␳ i at the interface positions. The reflectivity measurements presented here were carried out at the D4 bending magnet station at HASYLABrDESY, using a monochromatic beam of 20 or 12.5 keV. Before being monochromatized by a single SiŽ110. crystal monochromator, the beam impinges on a flat Rh

Diffuse X-ray scattering can be employed to investigate the lateral structural parameters of the samples, such as layer inhomogeneity, interface roughness; and at higher values of parallel momentum transfer q I , eventually also short range correlations and conformational properties of the macro-molecules. To investigate the lateral film homogeniety, and also, eventually, packing parameters of the organo᎐metal complexes, the surface diffuse scattering Žnon-specular scattering. has been measured both in the plane of incidence and out of the plane of incidence ŽSalditt et al., 1994, 1996.. In these scattering geometries, small and high q I values can be accessed, respectively. At small q I , the diffuse scattering is dominated by the long wavelength undulations of the silicon or glass substrates, which are copied conformally by the surface of the self-assembled thin films. Since they do not reflect the intrinsic film structure, we do not present the data here, but focus on the out-ofplane measurements, where molecular conformation and ordering can be probed. For the present study, diffuse scattering in the out-of-plane geometry was collected at the D4 bending magnet beamline of the storage ring DORIS in HASYLABrDESY.

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T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

2.8. X-Ray fluorescence analysis

deposited adsorbed on a PSSrPEI polyelectrolyte cushion on silicon, along with the corresponding fits: from top to bottom: w1 = 1x Hg, w1 = 1x Cd, w1 = 1x Co, w1 = 1x Zn ŽAn, 1999.. The curves have been shifted vertically for clarity. The thickness ŽKiessig. oscillations directly evidence the total film thickness and the successful adsorption of the MOC layers onto the oppositely charged polyelectrolyte Žin this case PSS.. The parameters of the simulation are shown in Table 1, providing information on the density profile which has been parameterized by a model of three layers, respectively, of density ␳ i and thickness d i on the silicon substrate. Error-function profiles of width ␴ ŽGaussian interface roughness. have been assumed for the interfaces between layers ŽParratt algorithm.. We note, however, that given the relatively little amount of structure in the data, the model is somewhat over-parameterized. Correspondingly, the density values in particular, as well as the individual layer thicknesses, have to be interpreted with some caution. Contrarily, the total layer thickness D is quite safe and signifi˚ Therefore, the thickness value cant up to 0.5 A. ˚ should be of the fitted PEIrPSS layer ds 29.5 A taken as a reference to determine the MOC thickness from the total thickness increase. An ˚ is found, comaverage value of d MO C s 11 " 3 A ˚3 which is estiparing well with the 11 = 11 = 7 A mated from molecular modelling. From the molecular dimensions determined by bulk crystallography ŽFiggis et al., 1983; Baker and Goodwin, 1985; Bhula and Weatherburn, 1991., a mean intermolecular Žs inter-metal . distance of ap˚ and hence, a molecular proximately as 11 A, ˚3 can be assumed, volume of approximately 1700 A

The X-ray fluorescence under grazing angles was recorded simultaneously with the reflectivity measurements at the D4 station. A fast, energy dispersive silicon p-i-n detector ŽRontek, Berlin, ¨ Germany. of 0.1 ␮s deadtime and 150 eV energy resolution and a standard multi-channel data aquisition system was used ŽFast GmbH, Oberhaching, Germany.. The detector was placed at 90⬚ opposite the sample surface at a distance of a few centimeters. In this way, elastic scattering is suppressed by polarization and the fluorescence yield is maximized by a large solid angle. The fluorescence yield is then measured as a function of ␣ i .

3. Supramolecular complexes in self-assembled films The MOC samples were characterized by X-ray reflectivity within a few days after deposition, and at subsequent times. For polyelectrolyte layers, small, but observable changes in the structural parameters were found with time, in particular, a slow swelling of the layers at ambient conditions Žrelative humidity. ŽPlech et al., 2000. in agreement with previous findings reported in the literature ŽSchmitt et al., 1993.. However, in no case were the films destroyed or damaged. Contrarily, in most cases, the interfaces and layers tended to anneal with time, as evidenced by stronger oscillations in the reflectivity curve. In Fig. 2, representative X-ray reflectivity curves are shown for four different w1 = 1x MOC layers

Table 1 Simulation parameter corresponding to the w1 = 1x MOC systems shown in Fig. 2 ŽAn, 1999.: all density values ␳ are given in grcm3 ,

˚ A constant and fixed density of ␳ S i s 2.23 was assumed for the substrate all thickness values given in A. Probe

PEIrPSS PEIrPSSrw1 = 1x Hg PEIrPSSrw1 = 1x Cd PEIrPSSrw1 = 1x Co PEIrPSSrw1 = 1x Zn

Density

Roughness

Layer thickness

␳1

␳2

␳3

␴0

␴1

␴2

␴3

d1

d2

d3

D

1.73 1.82 1.67 1.51 1.53

1.89 1.88 1.7 1.77 1.48

᎐ 1.07 0.94 0.8 1.32

5.5 6.52 7.13 5.41 4

2 4.76 4.15 7 2

4.1 5.65 4.62 3.04 2

᎐ 2.3 2 4.47 3.2

14.5 14 17.9 14.35 13.5

15 14.7 14.5 10 14

᎐ 11 10.8 13.96 12

29.5 39.7 43.2 38.31 39.5

T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

Fig. 2. X-Ray reflectivity of four different w1 = 1x MOC layers deposited adsorbed onto a PSSrPEI polyelectrolyte cushion on silicon, along with the corresponding fits: Žfrom top to bottom: w1 = 1x Hg, w1 = 1x Cd, w1 = 1x Co, w1 = 1x Zn.. The curves have been shifted vertically for clarity. The thickness ŽKiessig. oscillations directly evidence the total film thickness and the successful adsorption of the MOC layers onto the oppositely charged polyelectrolyte ŽPSS.. The parameters of the simulation are shown in Table 1 ŽAn, 1999..

yielding a mass density of the adsorbed metal complexes of approximately ␳ 3 s 1.2 grcm3. This value is somewhat higher than the average of the fitted values Ždespite the large error bars in the latter quantities .. Fig. 3 shows two w2 = 2x Cd samples of the same composition GlasrPEIrPSS s w2 = 2x Cd along with the respective least-square fits. The thickness of the MOC layer is now determined to be d MO C ˚ ŽAn, 1999., corresponding to the s Ž20.3" 1.3. A ˚ . and the molecular length of the ligand Ž18.1 A size as determined from crystallography Žfor w2 =

61

˚3 C; Hanan et al., 2x Co 20.6 = 18.2 = 18.2 A 1997a,b.. After the adsorption of a MOC layer, the selfassembly was blocked, and no further polyanion layer could be adsorbed from aqueous solution. In other words, the necessary charge reversal did obviously not take place. Only upon addition of acetone at approximately 20 vol.%, could subsequent layers be absorbed. This finding can be explained by the fact that the PF6 counter ions balance the excess positive charge of the MOC and are insoluble in water. Only upon the addition of acetone to the aqueous PSS solution can they be released and replaced by polyanions ŽAn, 1999; Vix et al, 2001.. Note that PF6 metal complexes are typically soluble in acetone. Furthermore, the quality of multi-layers with alternating layers of MOC and PSS was significantly inferior Žin particular, regarding the roughness. than samples with an extra annealing layer of the form MrPSSrPEIrPSSrM, where M denotes any w1 = 1x MOC layer. Fig. 4 shows the reflectivity curves of three multi-layered samples composed of QuartzrPEIrPSSrwMrPSSrPEIrPSSx 3 , with M s w1 = 1x Co, w1 = 1x Zn, w1 = 1x Co, w1 = 1x Ru, w1 = 1x Fe, and w1 = 1x Zn, shifted vertically for clarity, along with the least-square fits ŽPerzl, 2000.. The lower curve of a sample with layers of different MOC illustrates the variety of combinations which are possible. The total thickness de˚ rived from the fits is D s 170, 151 and 204 A from top to bottom, respectively. The top rms˚ respectively, is roughness ␴ s 34, 34 and 47 A, considerably higher than the typical values of simple polyelectrolyte films. A finite interface width ␴ can originate in two effects: Ža. transverse fluctuations of an atomically sharp interface; and Žb. molecular interdiffusion or entanglement of the polyelectrolyte chains. The two effects can be distinguished from a combined study of specular and non-specular scattering. In the present case, the high ratios of specular to non-specular scattering, e.g. as observed in a series of rocking scans, indicate that the values of ␴i derived from the reflectivity fits, which are of the order of the layer thicknesses, are largely dominated by interdiffusion. Gener-

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T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

Fig. 3. Reflectivity of two w2 = 2x Cd samples of the same composition GlasrPEIrPSSrw2 = 2x Cd along with the respective ˚ ŽAn, 1999.. least-square fits. The thickness of the MOC layer is now determined to be d MO C s Ž20.3" 1.3. A

ally, a high amount of intermixing at the interfaces has been reported previously for poly-electrolyte self-assembled films ŽDecher, 1997. and seems to be inherent to this kind of growth process

by charge reversal. Diffuse X-ray scattering can be employed to investigate the lateral structural parameters of the samples, such as layer inhomogeneity, interface roughness, and at higher values

Fig. 4. The reflectivity curves of three multi-layered samples composed of QuartzrPEIrPSSrwMrPSSrPEIrPSSx 3 , with M s w1 = 1x Co, w1 = 1x Zn, w1 = 1x Co, w1 = 1x Ru, w1 = 1x Fe and w1 = 1x Zn, shifted vertically for clarity, along with the least-square fits. The lower curve of a sample with layers of different MOC illustrates the variety of combinations which are possible ŽPerzl, 2000..

T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

of parallel momentum transfer, q I , eventually also short range correlations and conformational properties of the macromolecules. This technique has recently been used for the first time in the characterization of thin self-assembled polyelectrolyte films Žmodel systems without metal complexes., illustrating only a weak increase in the power spectrum density of the polyelectrolyte film over the bare substrate. On longer length scales, the film conformally copies the substrate waviness ŽPlech et al., 2000.. Another important issue is the packing of the organo᎐metal complexes in the plane of the film. For these systems, two-dimensional crystalline Ž2D. ordering has been found previously at increasing 2D pressure along the isotherm of a monomolecular layer at the liquidrair interface ŽWeisbuch et al., 1998., as well as in thin MOC films adsorbed on mica. Hence, lateral ordering could also be expected in the present case of the complexes adsorbed onto the polyelectrolyte cushion, in particular, for the larger tetranuclear w2 = 2x complexes. In Fig. 5, the diffuse scattering

63

of a w2 = 2x Cd complex adsorbed on PSSrPEIrSi is displayed as a function of lateral momentum transfer along the momentum transfer q y parallel to the sample surface ŽSalditt et al., 1999.. The scattering geometry is sketched in the inset. The data has been taken at ␣ i s 0.5⬚ and ␣ f s 0.15⬚, where the diffuse intensity is enhanced by the Fresnel transmission function, the so-called Yoneda peak of diffuse scattering ŽDosch, 1992.. While the incidence and exit angles are kept constant, the angle 2␪ is varied out-of-the plane of incidence. Note that the data are purely diffuse, e.g. with no specular contribution at the peak. The instrument function has been determined from a 2␪ scan around the specular beam. Note the absence of any scattering peaks that would occur in the case of long-range lateral order in the packing of the complexes. The strong decay of diffuse scattering at small q I can be attributed to interfacial roughness, where the measured intensity SŽ q . is the Hankel transform of the height᎐height correlation function cŽ r . ŽSinha, 1988; Holy ´ et al., 1999; Tolan, 1999..

Fig. 5. The diffuse scattering distribution as measured in the out-of-plane scattering geometry Žsee inset.. At constant ␣ i s 0.5⬚ and ␣ f s ␣ c s 0.15⬚, the detector is moved along 2␪. Note that the central peak is purely diffuse since the specular beam ŽSB. is not ˚ most probably due to captured by the detector and can be attributed to film roughness on long length scales larger than 800 A, conformal substrate roughness. Note the absence of any peaks at higher q y which would be observed in the case of a 2D crystalline arrangement of complexes ŽSalditt et al., 1999..

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T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

There is no indication of additional contributions to the diffuse scattering from density fluctuations in the film ŽRauscher et al., 1995.. We can immediately draw the following conclusions: Ža. the organo᎐metal complexes did not self-assemble into a long-range ordered array. Žb. The ratio of diffuse to specular intensity is relatively low, excluding strong lateral density inhomogeneities or interface fluctuations. Žc. The low amount of diffuse scattering is contrasted with relatively high values of ␴ determined from the decay of the specular intensity, suggesting a high degree of intermixing at the interfaces, i.e. the interfaces are not locally sharp. Simultaneous to the specular reflectivity, the X-ray fluorescence of the metal ions in the complexes adsorbed on the surface has been measured. X-Ray fluorescence under grazing incidence has evolved into a powerful tool of surface chemical analysis with a sensitivity threshold orders of magnitude below monolayer coverages. By means of the characteristic fluorescence energies Ž K ␣ and K ␤ lines for Co, Cu, Zn, L␣ for Hg. excited by a monochromatic X-ray beam of 12.5 keV, this provides the most direct proof of the

complex adsorption ŽSalditt et al., 1999.. Moreover, the variation of the fluorescence yield with ␣ i contains quantitative information on the position and width of the metal distribution with respect to the substrate. A typical curve for the case of w1 = 1x Hg complexes is shown in Fig. 6, with the setup schematic shown in the inset. Close to the critical angle of reflection, the fluorescence yield, integrated around the Hg L␣ line at 10.1" 0.2 keV exhibits a maximum. This enhancement is a direct manifestation of the fluorescence source being located on the surface ŽDe Boer, 1991.. A quantitative analysis has been carried out in ŽPerzl, 2000. Žfitted, solid line in Fig. 6., providing a measure of the z position of the metal ion. However, the precision achievable depends crucially on the film thickness, background and sample flatness, as discussed in Perzl Ž2000.. After a calibration of the intensity, the data can also be used for a quantification of the adsorbed molar quantities, which can then be compared to the results of the reflectivity discussed above. In the case of different MOC layers within one sample, the observation of the fluorescence lines serves as unambiguous proof that the complexes of differ-

Fig. 6. X-Ray fluorescence yield of the Hg L␣ emission line measured simultaneously with the reflectivity curves as a function of ␣ i Žsetup see inset, Salditt et al., 1999.. The fluorescence source, i.e. the Hg atoms in the w1 = 1x Hg ŽII. complexes, must be located in a well defined layer near the surface to account for the fluorescence maximum near ␣ c , as confirmed by a quantitative analysis Žsolid line, Perzl, 2000..

T. Salditt, U.S. Schubert r Re¨ iews in Molecular Biotechnology 90 (2002) 55᎐70

Fig. 7. X-Ray fluorescence lines characteristic for Zn, Fe, and Ni, as observed for multi-layered MOC films. The subsequent sample built-up can be easily monitored by the observation of additional peaks.

ent kinds have indeed adsorbed, see Fig. 7 ŽPerzl, 2000..

4. Nucleic acids in self-assembled films The interaction of DNA with cationic polymers

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is currently an active field of research. As demonstrated from binding assays at varied ionic strength, the interaction is, in most cases, purely electrostatic. However, different scenarios of interaction and structure can be expected, depending on the linear charge density, the ionic strength, and on whether the poly-cations are linear, branched or dendrimeric. Apart from fundamental interest in supramolecular self-assembly, quantitative experiments and theoretic models will also be beneficial for the development of tailored complexes, e.g. in drug delivery applications. PEIrDNA complexes have attracted particular interest as vectors for gene therapy applications ŽPollard et al., 1998.. Similar to the case of DNA᎐cationic lipid complexes ŽRadler et al., ¨ 1997; Koltover et al., 1998a,b; Salditt et al., 1997., an understanding of the structural and colloidal properties of such complexes is needed to flank ongoing biomedical and clinical studies. As shown for the first time a few years ago, DNA can be deposited by the layer-by-layer selfassembly technique ŽLvov et al., 1993; Sukhorukov et al., 1996.. Studies of such planar films of DNA and synthetic polycations may also shed

Fig. 8. Reflectivity profiles of a PAHrDNA multi-layered film with layer sequence GlasrPEIrPSSrPAHrwDNArPAHx = 2. The inset illustrates the scattering length profile Žproportional to the electronic density. corresponding to the fit Žsolid line. based on a ˚ is observed. Note that the PEIrPSSrPAH part of the two-layer model and the Parratt algorithm. A total film thickness of D s 59 A film is significantly lower than the DNArPAH double layers.

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Fig. 9. Reflectivity profiles of PEIrDNA multi-layered films. The inset illustrates the thickness increase upon adsorption of ˚ wDNArPEIx layers indicating a mean thickness of Ds 27.7 A per double layer.

some light on their respective structure and interaction. Fig. 8 illustrates, how the density profile of a PAHrDNA film is obtained from a least-square fit of the measured X-ray reflectivity. Fig. 9 shows similar results for PEIrDNA. To our knowledge, it is the first study on PEIrDNA polymeric multi-layers. The basic unit PEIrDNA is found ˚ in the case of to have a thickness of D s 27.7 A salt-free deposition ŽFig. 9, inset.. However, our attempts to investigate the lateral short range ordering of the chains in these films by grazing incidence diffraction have so far not been successful. At low scattering volumes and contrast, the obviously relatively low degree of positional order could not be detected.

5. Membrane proteins in self-assembled films

When discussing proteins embedded in thin films, at least two different aspects come to mind. Firstly, a soft fixation of the proteins may allow for the application of spectroscopic and structural techniques which are not possible in bulk solutions or suspensions Žthough the opposite may also be true in many cases.. Secondly, functional

properties of proteins may be exploited if these proteins are stabilized and positionally or orientationally fixed in thin films. Both aspects require the development of suitable techniques to deposit stable thin films with high protein content. The self-assembly method has been demonstrated to be capable to incorporate functional globular proteins in the hydrophilic environment of polyelectrolyte cushions. In contrast to water soluble Žglobular. proteins, the case of membrane proteins deposited in thin films shall be briefly discussed here. To this end, the integral membrane-protein bacteriorhodopsin ŽBR. found in the purple membrane ŽPM. of the archeon Halobacterium salinarum may serve as an example. BR with its retinal chromophore works as a light-driven proton pump to provide the energy needed for cellular metabolism ŽOesterhelt and Stoeckenius, 1971.. Membrane proteins have since long been deposited in multi-lamellar stacks of bilayers on thin films. In most cases, these stacks were used for structural studies ŽNMR, X-ray diffraction, neutron scattering .; in some cases, including notably PM, for biotechnology applications also ŽBirge, 1990; Hampp, 1992.. For an early review of solid-supported membranes with native proteins, see Blaurock Ž1982.. The deposition of various native Žnaturally occurring. membrane systems and the preparation on solid supports for structural analysis by diffraction has traditionally suffered from a very modest degree of orientational alignment with respect to the substrate. Only more recently, Koltover et al. have reported a study of highly oriented multilamellar stacks of giant fused purple membranes ŽPM. containing the trans-membrane protein bacteriorhodopsin ŽBR. ŽKoltover et al., 1998a,b.. For the first time, the application of powerful interface scattering techniques like X-ray reflectivity, diffuse scattering, and truncation rod scattering to a membrane-protein system became possible. The diffuse scattering indicated a highly conformal buckling of the membranes on length ˚ The analysis of the scales of a few hundred A. truncation rods allowed the authors to determine inter-membrane correlations of the proteins at different hydration levels of the multi-layer. Novel

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and surprising results on the phase diagram of BR in PM including a detailed structural study of the melting transition was presented by Koltover et al. Ž1999.. Subsequently, Muller et al. demonstrated that ¨ not only fused, but also untreated PM patches can be deposited in form of highly oriented stacks, if the total film thickness is kept below approximately a micrometer ŽMuller et al., 2000.. Impor¨ tantly, the synchrotron data of such samples give a very clean account of intra-molecular interference signals Žprotein form factors, helix scattering., even when the lateral ordering in the membrane Žprotein structure factor. is very low Žsuch as in the high temperature phases of BR.. Furthermore, phase transitions, fluctuation and elasticity properties, as well as protein folding or unfolding may be probed at in-situ conditions. To this end, Muller et al. have shown that the ther¨ mal denaturing of bacteriorhodopsin can be observed by measuring the intra-molecular scattering signal Žform factor., independently of the lateral ordering of the proteins Žstructure factor. ŽMuller et al., 2000.. ¨ While the above method to deposit highly oriented films by slow sedimentation is appropriate

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for structural studies, this may not be the case for biomaterials and biotechnological applications. Note that BR films are used already commercially as holographic storage materials. In this context, stability is a major concern. Furthermore, in some applications, the stacks should be stable under conditions of excess water. Thermal stability is another issue to be addressed. For the case of BR, the protein has been shown to be quite stable against thermal denaturing ŽShen et al., 1993; Muller et al., 2000.. ¨ One solution for stable films is to incorporate native membranes into polyelectrolyte multilayers. The situation is sketched in Fig. 10, in Ža. real and Žb. reciprocal space. We have used the self-assembly method to adsorb PM patches onto a thin PEI layer on silicon or quartz substrates ŽSalditt et al., to be published.. The built-up of two bilayers of BRrPEI on the first PEI cushion could be evidenced by X-ray reflectivity, see Fig. 11. At the same time the well-known optical absorption peak could be observed at approximately 570 nm, indicating a functional state of BR. The total film thickness for the sample of composition SirPEIrwBRrPEIx = 2 is surpris˚ taking into account a memingly small, D s 92 A,

Fig. 10. Sketch of the PEIrPM film with integral membrane protein bacteriorhodopsin ŽBR., both in Ža. real and in Žb. reciprocal ˚ thickness. By the space. Instead of a thin water layer in-between the membranes there is a thin PEI cushion of a few A polyelectrolyte complexation Žacting as a electrostatic glue. the film is attached, and the highly charged membrane patches become immobilized, which is otherwise only possible at high osmotic pressure. In Žb. the different features in reciprocal space are sketched, including the multi-lamellar reflections, the scattering pattern of the transmembrane helices, and the hexagonal lattice reflections. The intensity distribution can be probed with high resolution by interface sensitive scattering techniques.

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Fig. 11. The reflectivity of a SirPEIrwBRrPEIx = 2 film along with fit Žsolid line. and the corresponding density profile ˚ is inferred for the film. Žinset.. A total thickness of D s 92 A

˚ This finding brane thickness of , ␦ m s 47 A. indicates that the PEI interdiffuses closely with the disordered loops of BR and essentially adsorbs in a flat two-dimensional layer on top of the lipid bilayer. Thus, we have shown that not only globular proteins can be adsorbed and complexed with synthetic polyelectrolytes in thin films, but also native, membrane patches with integral membrane proteins in the functional state. In future, the approach to integrating native membrane systems with high protein content in thin polyelectrolyte films by the self-assembly method may lead to a range of interesting devices. However, control should be achieved on the uprdown orientation of the membranes. 6. Summary We have presented different examples of supramolecular and biomolecular complexes deposited by the layer-by-layer self-assembly technique. Supramolecular metal᎐organic complexes ŽMOC. belonging to a family of w1 = 1x and w2 = 2x coordination arrays of fused terpyridine ligands could be adsorbed on polyelectrolyte layers, despite the fact that the charge of the complex is as small as two elementary charges Žw1 = 1x arrays.. In light of current theories of adsorption, it is very surprising that uniform coverage can be

achieved rather than a Poisson᎐Boltzmann-like distribution of the cationic complexes near the interface ŽNetz and Joanny, 1999; Joanny, 1999.. Even more surprisingly, the surface charge is reversed by the complexes, since polycations again adsorb on the MOC layers, as long as the counterions are soluble in the respective solution. Various combinations of MOC layers could thus be achieved in a multi-layered architecture. In essence, we are, therefore, facing a novel thin film material with a hierarchy of three structural levels: The primary structure is determined by the controlled self-assembly of the metallosupramolecular complexes, i.e. the metal coordination arrays with their functionalized organic ligands. The secondary structure is determined by the packing of the complexes in a two-dimensional sheet of essentially one molecular layer, while the ternary structure is finally governed by the sequence of layers imposed by the deposition process. Partial control of the films on all three levels can be achieved. The relationship between the hierarchical nanoscopic structure and the macroscopic properties of functionalized films consisting of the supramolecular units remains to be explored. Applications using optical, magnetic or catalytic properties of the supramolecular moieties may be envisioned. Beyond synthetic and supramolecular complexes, biomolecules can serve as interesting functional components in polymeric films. Polymeric thin film technology based on layer-bylayer self-assembly may become important in various biotechnology applications. Importantly, the method is not restricted to planar interfaces. To this end, Mohwald and co-workers have used ¨ spherical templates leading to hollow polymeric capsules, opening up new strategies and options for drug delivery as well as various other applications ŽMohwald et al., 2001; Caruso, 2001.. ¨ Here, we have shown that layer-by-layer selfassembly is compatible not only with the incorporation of single proteins, but also with native membrane patches with integral membrane proteins. Unlike multi-lamellar stacks ŽVogel et al., 2000. such films are stable in water. To investigate the film structure, interface-sensitive X-ray scattering provides an excellent analytical tool

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with high resolution in all three spatial dimensions, compatible with in-situ deposition conditions. However, low contrast and low positional ordering pose tremendous challenges in particular for the determination of the structural properties parallel to the film.

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