Letter pubs.acs.org/Langmuir
Layer-by-Layer Assembly of a Streptavidin−Fibronectin Multilayer on Biotinylated TiOX Michael Lehnert,†,‡ Christopher Rosin,† Wolfgang Knoll,§ and Michael Veith*,† †
Laboratory of Biophysics, Westphalian University of Applied Sciences, August-Schmidt-Ring 10, D-45665 Recklinghausen, Germany Department of Biology, Johannes Gutenberg University Mainz, Saarstr. 21, D-55099 Mainz, Germany § Austrian Institute of Technology (AIT), Donau-City-Str. 1, Vienna 1220, Austria ‡
ABSTRACT: The biomodification of surfaces, especially titanium, is an important issue in current biomedical research. Regarding titanium, it is also important to ensure a specific protein modification of its surface because here protein binding that is too random can be observed. Specific nanoscale architectures can be applied to overcome this problem. As recently shown, streptavidin can be used as a coupling agent to immobilize biotinylated fibronectin (bFn) on a TiOX surface. Because of the conformation of adsorbed biotinylated fibronectin on a streptavidin monolayer, it is possible to adsorb more streptavidin and biotinylated fibronectin layers. On this basis, an alternating protein multilayer can be built up. In contrast to common layer-bylayer technology, in this procedure the mechanism of layer adsorption is very specific because of the interaction of biotin and streptavidin. In addition, we showed that the assembly of this multilayer system and its stability are dependent on the degree of labeling of biotinylated fibronectin. Hence we conclude that it is possible to build up well-defined nanoscale protein architectures by varying the degree of labeling of biotinylated fibronectin.
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INTRODUCTION
As an alternative to covalent coupling to an antiadhesive polymer coating, proteins can also be immobilized directly on metallic or ceramic surfaces using the biotin−streptavidin system.14−17 Streptavidin is a 60 kDa protein from Streptomyces avidinii. It has four binding sites for biotin (also known as vitamin B7 or vitamin H), which streptavidin itself binds to with the strongest noncovalent bond known in nature.18,19 Because of the tetrameric arrangement of these binding sites, streptavidin is also used to immobilize biotinylated molecules on biotin-modified surfaces. The parameter for successful selfassembly of a streptavidin monolayer and the later adsorption of biotinylated molecules had already been studied in detail.20,21 In addition to gold, a streptavidin monolayer had also been built up on other surfaces including ceramics such as SiO2 and TiO2.22,23 Furthermore, an adsorption model for streptavidin was developed in which a certain biotin density is mandatory to achieve optimum streptavidin coverage of the surface, which is
The immobilization of proteins on surfaces is an important part of biosensing, surface functionalization, and biomedicine. To immobilize biomolecules, different methods can be applied depending on the type of protein and the purpose of the immobilization. The biofunctionalization of TiOX surfaces is hereby of great interest because titanium is the current implant standard and therefore TiOX is used as a model surface for studying protein adsorption.1−6 Because of the nonspecific random protein adsorption on TiOX surfaces, it is necessary to modify these surfaces with an antiadhesive coating to study the effects of specific protein immobilization.7−9 Such coatings are commonly called “blank” coatings because they resist protein adsorption. These coatings frequently consist of ethylene glycol and derived molecules or polymers as a result of their protein-repellent and thus celladhesion-suppression properties. The coupling of proteins to surfaces, modified with an antiadhesive blank coating, produces a highly specific response of the environment to the immobilized biomolecule.10−13 © 2013 American Chemical Society
Received: September 18, 2012 Revised: January 4, 2013 Published: January 12, 2013 1732
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Figure 1. SPR results for the assembly of the alternating multilayer system. (a) SPR kinetics of the sequential formation of the multilayer system. The formation of each protein layer is shown by a different color: Strep1 (cyan), bFn1 (red), Strep2 (blue), and bFn2 (green). As can be seen, there is always an increase in layer thickness and no decrease after rinsing, indicating specific protein adsorption. (b) SPF spectra of two layer systems with different DOLs of bFn (top: DOL = 44, bottom: DOL = 9). After the adsorption of each new monolayer, a resonance shift can clearly be observed. Furthermore, the immobilization of fluorescence-labeled streptavidin results in an increase in the fluorescence signal. Comparing both spectra, we can see that the formation of the second layer of streptavidin, at a higher DOL, results in a larger shift of the SPR signal as well as a greater increase in the fluorescence signal. (c) Calculated layer thicknesses of the different layers using Fresnel fitting. Multilayer constructed by biotinylated fibronectin with DOL = 9 having no filling; multilayers constructed with DOL = 44 are filled with a gray color.
up on TiOX surfaces for medical implants.29−31 Because of those qualities, they are a promising alternative for the specific functionalization of surfaces. For specific bioactivation, biotinylated fibronectin (bFn) can be immobilized onto this monolayer by means of molecular self-assembly. Fibronectin is a 440 kDa glycol protein with versatile functions and isoforms. It has binding sites for different proteins (e.g., collagen), which is a major component of the extracellular matrix (ECM).
necessary for the functional immobilization of biotinylated proteins and molecules.24,25 Such molecular architectures are very often used to analyze protein interactions by means of surface plasmon resonance (SPR) spectroscopy.24 It should be noted that biotin−streptavidin technology has also been used in other fields of bionanotechology.26−28 As we have recently shown, streptavidin monolayers also have protein-repellent properties and can be successfully built 1733
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Because of the observed derivation of layer thickness for the first layer of bFn, no significant difference can be assumed. As already shown, the DOL of bFn has no influence on the layer thickness,29−31 so similar conditions for the adsorption of the second layer of streptavidin are achieved. We can conclude that the only parameter influencing this adsorption should be the DOL of bFn. In contrast to Anamelechi et al.’s results, a correlation between the DOL of adsorbed bFn and the layer thickness of the second layer of adsorbed streptavidin can be clearly observed.39 The results of the SPR studies show that the second layer of streptavidin is approximately 2 times thicker when adsorbed onto a layer of bFn with a DOL of 44, compared to a layer of bFn with a DOL of 9. This result is supported by the fluorescence enhancement (Table 1).
Another motif binds integrins, which are located inside cellular membranes. Via these two binding sites, fibronectin mediates the adhesion of cells (e.g., osteoblasts) to the ECM.32−35 After the adsorption of bFn on a streptavidin -modified TiOX surface, a strong increase in osteoblast adhesion and activity can be monitored. Furthermore, this effect is stronger on TiOX surfaces modified with specifically adsorbed bFn in comparison to surfaces modified with randomly adsorbed native fibronectin.29−31 As we have recently shown, bFn adopts an extended conformation with exposed binding sites that differs from the compact conformation of native fibronectin, where these binding sites lie submerged within the protein and are therefore inaccessible.31 In this article, we show how bFn and streptavidin can be used to build up well-defined protein nanoscale architectures combining the self-assembly of biotin molecules and layer-bylayer technology.36 As mentioned above, when adsorbed onto a streptavidin layer, biotinylated fibronectin adopts a linear, extended conformation.31 In this conformation, not all biotins along the fibronectin axis will be connected to the streptavidin monolayer. Because of that, a second layer of streptavidin can be adsorbed on top of the first layer of biotinylated fibronectin. As a result, by sequential incubation of a biotinylated surface with streptavidin and bFn, an alternating protein multilayer can be built up (Figure 1). The multilayer system may also enhance the stability of the biomimetic surface coating. If it does, then this would simplify the handling of modified implants because the current stability of a single layer of bFn amounts to only a few hours. In contrast to commonly used layer-by-layer technology, molecular assembly as explained here results from highly specific molecular recognition.19,37,38 The assembly of the nanoscale molecular architecture was monitored in real time using SPR spectroscopy. Moreover, in this letter the degree of labeling (DOL) of biotins per fibronectin is analyzed as a parameter for the architectural buildup. Because Anamelechi et al. did not find any influence due to the DOL of randomly adsorbed bFn regarding the adsorption of streptavidin, during our experimental work we added the use of surface plasmon fieldenhanced fluorescence spectroscopy (SPFS).39 SPFS combines fluorescence and SPR spectroscopy and is known for its very high sensitivity.12,40,41
Table 1. Adsorption of Fluorescently Labeled Streptavidin Leads to a Fluorescence Enhancement Dependent on the DOL of bFn Used for the Multilayer Assemblya layer Strep1 Strep2 Strep1 Strep2
fluorescence signal increase
DOL of bFn 9 9 44 44
17.94 ± 0.04 58 ± 11
The relative fluorescence signal increase is caused by the adsorption of a second layer of fluorescently labeled streptavidin, compared to the signal of the first layer of fluorescently labeled streptavidin. a
As can be seen, at a DOL of 44 the fluorescence enhancement is about 58 ± 11% after the adsorption of the second layer of streptavidin. At a DOL of 9, the fluorescence signal showed an enhancement of only about 17.94 ± 0.04% after the adsorption of the second streptavidin layer. The difference between the results in this article and Anamelechi et al.’s results may be caused by the blank surface missing from Anamelechi’s work;39 we achieved it in our experiments by the streptavidin monolayer. In them, we first built up a monolayer of streptavidin on the metal surface that has protein-resistant properties. Because of this build up, there was no nonspecific streptavidin adsorption, and the results for the second streptavidin layer were unambiguous. In the last step of the multilayer assembly, a second layer of biotinylated fibronectin was adsorbed on top of the second layer of streptavidin. Here, a slightly higher layer thickness can be observed for the second layer of bFn at a DOL of 44 compared to a DOL of 9 (Figure 1c). These differences in layer thickness may be regarded as an effect of the higher density of the second layer of streptavidin. Because of the high deviation, these differences in layer thickness are only barely significant. However, at a DOL of 44, the second layer of bFn contains a layer thickness within the same range as the first layer of bFn. In the work described here, the formation of the alternating layer system was stopped after two iterations. Because the layer thickness of the second layer of bFn is within the same range as the thickness of the first layer of bFn, it should be possible to adsorb several more layers of streptavidin and bFn. As can be seen in Figure 2, the DOL also seems to play an important role in the stability of the multilayer system. At a DOL of 9, an immediately decrease in layer thickness can be observed. To describe the desorption of different monolayers of our multilayer system, a model was applied in which the last layer will desorb first because of the fact that the upper layer
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RESULTS AND DISCUSSION Two molar excesses, namely, 100:1 and 300:1 (biotin/ fibronectin), used in the biotinylation reaction delivered two different DOLs: 9 and 44. Biotinylated fibronectin (bFn) was thus used for protein multilayer assembly. As can be seen in the increasing reflectivity signal of the SPR kinetic diagram, by alternating streptavidin and biotinylated fibronectin incubation a sequential protein multilayer can be built up on biotinmodified TiOX surfaces (Figure 1a). After the injection of each protein, an increase in layer thickness can be observed. The DOL of bFn used in this experiment was 44. As can be seen, each protein layer was stable after rinsing, indicating no nonspecific adsorption. The thin black lines indicate the moment when an SPFS scan was taken. The resulting scan diagram is shown in Figure 1b in which a resonance shift after the adsorption of each new protein layer can be clearly observed. From those shifts, the resulting layer thicknesses were calculated. The results are presented in Figure 1c, together with the results, where bFn with a DOL of 9 was used to build up the alternating protein multilayer system. 1734
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MATERIALS AND METHODS
To analyze the assembly of an alternating streptavidin and bFn multilayer system, SPFS was used. For SPFS, LaSFN9 glass slides were prepared as previously described.31 Before preparation, all surfaces were cleaned in acetone, 2% Helmanex solution, distilled water, and absolute ethanol for 15 min in a supersonic cleaner. Substrates for SPFS were coated with 2 nm Cr and 48 nm Au to enable surface plasmon resonance. Cr and Au were thermally evaporated using an Edwards FL 400 AUTO 306. Titanium (Ti) was DC sputtered on all substrates using an Edwards AUTO 500 under an O2 atmosphere (30 sccm, 0.05 nm s−1). Afterwards, all substrates were stored under an argon atmosphere until use. The layer thickness was controlled in real time by a quartz crystal microbalance and later confirmed with SPR spectroscopy. For SPR spectroscopy, a thin TiOX layer of 5 nm was prepared because TiOX is a highly refractive material and a thick layer would make it impossible to observe surface plasmon resonance. Methanol (purity >99.9%) and ethanol (purity 99.5%) were purchased from Merck. N-(6-Aminohexyl)aminopropyl-trimetoxysilane (purity 95%) for silanization was obtained from ABCR and diluted in methanol. Sulfo-NHS-LC-biotin (Pierce) was dissolved in ethanol. Fluorescently labeled streptavidin (Atto-633-streptavidin/ Atto-Tec) and fibronectin (Millipore) were dissolved in PBS buffer (Sigma-Aldrich). For biotinylation, fibronectin was diluted to a final concentration of 0.5 μM and mixed with sulfo-NHS-LC-biotin. A stock solution of fibronectin (1 mg/mL, Millipore) was diluted in PBS to a final concentration of 0.5 μM and mixed with 25 μM sulfo-NHSLC-biotin dissolved in ethanol. To achieve well-defined conditions, the reaction was conducted in a thermomixer at 25 °C for 60 min and 500 rpm. Biotinylated fibronectin was separated from excess biotin reagent three times by size-exclusion chromatography (Zeba Spin desalting columns, 0.5 mL, Thermo Fisher Scientific Inc.). As we described in a previous article, changing the molar ratio between both molecules affects the degree of labeling (DOL) of fibronectin.31 In this article, two different molar ratios were applied in the reaction to achieve different DOLs for bFn, namely, 100:1 and 300:1 biotin/fibronectin. To verify the number of linked biotins to fibronectin, a fluorogenic biotin assay based on the displacement of 2-(4′-hydroxyazobenzene) benzoic acid (HABA, Sigma-Aldrich) from the biotin binding sites of fluorescently dye-labeled streptavidin (streptavidin DyLight488 conjugated, Thermo Fisher Scientific Inc.) was used. For biotin modification of the LaSFN9 glass substrates, the TiO2 surface was subsequently incubated with N-(6-aminohexyl)aminopropyl-trimetoxysilane (0.5 mM, 60 min; MeOH solvent) and sulfo-NHS-LC-biotin (0.5 mM, 120 min, EtOH solvent) as we described in a previous article.31 After each modification step, the surface was rinsed intensively with the solvent to remove physisorbed molecules. Protein adsorption was monitored with an SPF spectrometer in real time at a fixed angle close to the plasmon resonance angle. PBS buffer was used as the solvent. Fluorescently labeled streptavidin was adsorbed at a concentration of 0.5 μM for 60 min, and bFn was adsorbed at a concentration of 25 nM for 100 min. All experiments were carried out in double estimation. After the adsorption of each new layer, the surface was rinsed and an SPFS scan was taken. For that, a HeNe laser with a wavelength of 632.8 nm (JDS Uniphase 1125p) was used. This wavelength is within the same range as the absorption of the fluorescently labeled streptavidin. The angular scans were described by Fresnel equations using the following refractive indexes:
Figure 2. Long-term stability of the different layers of the alternating streptavidin and biotinylated fibronectin multilayer system.
may sterically hinder the desorption of the lower layer. Furthermore, after approximately 72 h an angle of surface plasmon resonance was monitored that was identical to that monitored after the adsorption of the second monolayer of streptavidin. On the basis of these results, the total desorption of the second monolayer of bFn can be assumed. In contrast to this, at a DOL of 44, there was almost no shift in the resonance angle within the first three days! Only on day four did a decrease in layer thickness occur. This enhanced stability is most probably due to an increased number of biotin−streptavidin bonds, which can be attributed to the larger number of biotin units along the fibronectin axis. However, by applying an alternating multilayer system of streptavidin and bFn at both DOLs, the stability of the first layer of bFn could be significantly increased from only a few hours to several days. In this study, the best stability of the multilayer system was found using highly biotinylated fibronectin. At the same time, a higher DOL leads to an enhanced adsorption of streptavidin. Hereby additional binding sites are also provided for the integration of additional biotinylated molecules or proteins. Therefore, we concluded that bFn with a high DOL is most suitable for the assembly of a biomimetic multilayer system.
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CONCLUSIONS In this article, we have presented a method to build up a multilayer protein system of alternating layers of streptavidin and bFn. To do so, self-assembly molecular techniques were applied together with layer-by-layer techniques. We demonstrated how the DOL of bFn used in these experiments influences the assembly of the protein multilayer. When the DOL is changed, streptavidin layers can be built up in a welldefined manner, with a resolution of 0.5 nm layer thickness. In addition, the DOL also plays an important role in the stability of the multilayer thickness. In this study, bFn was used because it is easy to modify and plays an important role in cellular processes. For future work where a multilayer system has been built up successfully, it may be interesting to add further biotinylated molecules. In doing so, biomimetic surface coatings with distinct functions and characteristics can be assembled easily by means of a self-assembly molecular process.
nTiOX = 2.32, nsilanes = 1.45, nbiotin derivatives = 1.50, nstreptavidin = 1.59,
and
n fibronectin = 1.40
Calculation and visualization were done using Fresnel fit software.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 1735
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a grant (FKZ1775X05) from the BMBF program in the context of this cooperative project. We thank Gabi Hermann at the Max Planck-Institute for Polymer Research in Mainz, Germany for her helpful technical support.
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