NANO LETTERS
Tuning Surface Energies with Nanopatterned Substrates
2006 Vol. 6, No. 2 267-270
Christine Selhuber, Jacques Blu2 mmel, Fabian Czerwinski, and Joachim P. Spatz* Max-Planck-Institute for Metals Research, Department New Materials and Biosystems, Heisenbergstr. 3, D - 70 569 Stuttgart, Germany, and UniVersity of Heidelberg, Institute for Physical Chemistry, Department of Biophysical Chemistry, Heidelberg, Germany Received November 15, 2005
ABSTRACT A novel approach to varying the surface energy of biofunctional substrates has been developed, where surface energies are controlled by utilizing tunable nanopatterned substrates. In this study we functionalized the nanopattern with streptavidin, providing an adhesive interface for biotinylated probes. To obtain the surface energies, we applied the Johnson−Kendall−Roberts model to the adhesion-induced deformation of elastic beads. The results reveal a linear relationship between surface energy and ligand density, demonstrating the capability of this technique to adjust surface energy.
Controlled adhesion and surface energies are highly relevant in many research areas from biosensors to microfluidics, in the wetting and dewetting of interfaces as well as in cell biology. In recent years, biofunctional nanopatterned substrates have attracted substantial attention to serve this purpose.1,2 Particularly in adhesion-mediated functions of proteins and cells where a molecular definition of binding points and multivalent interactions are crucial, nanopatterns are a first choice.4 In cell adhesion studies, where the assembly of proteins to hierarchical assemblies plays a key role, there is a big demand for surfaces where adhesion ligands can be positioned in individual patterns in order to study their effects on biological functions.5-7 For all of these studies, a nanopatterning method should ideally be easy to handle, applicable to a wide range of solid substrates and robust enough to sustain protein coupling chemistry. In this letter we demonstrate how a flexible nanopatterning method can be employed to adjust the strength of receptormediated adhesion. The adhesive strength is characterized by the detection of surface energy, which is defined by the free energy change that accompanies the adhesion of two solid surfaces. While this concept is often used to characterize unspecific interactions, it can also be extended to specific adhesion events without any restriction. For studying surface energies, we have chosen the well-characterized binding partners biotin and streptavidin as the ligand-receptor pair. In the experiment biotin was provided on an elastic biotinylated agarose bead, serving as one surface, whereas the streptavidin represented the adhesion ligand on a nanopat* Corresponding author. E-mail:
[email protected]. 10.1021/nl052256e CCC: $33.50 Published on Web 01/21/2006
© 2006 American Chemical Society
terned substrate. Because of the biotin-streptavidin interaction, the elastic bead adheres quasi-irreversibly to the substrate. During the adhesion, the elastic bead deforms and enlarges its contact area with the substrate. This phenomenon is well-described by the Johnson-Kendall-Roberts (JKR) model, which relates the deformation of adhering elastic particles to the surface energy, also called the interfacial energy, between the particles and the surface.12 The surface energy in this study depends on the surface density of the adhesion points, which is controlled by the nanopattern. The same approach of utilizing elastic beads for measuring surface energies was already used to study the biotinstreptavidin interaction as a function of pH and ionic strength.13 Our results for the surface energy between streptavidin-functionalized nanopatterns and biotinylated beads show that we have a method available to adjust the surface energy of bioadhesive samples over a wide range. To nanopattern substrates, an efficient and easy-to-use method has been developed in recent years.8-10 This approach is based on the self-assembly of diblock-copolymer micelles and allows one to deposit an array of nanometer-sized gold dots (5-7 nm in diameter) onto a solid support, where each gold dot can be used as anchor point for individual ligands. With this device the interligand distance can be controlled over a wide length scale from 30 up to 250 nm with a precision of several nanometers.11 The nanostructured substrates are prepared by dip-coating piranha cleaned glass slides with diblock copolymer micelles (polystyrene-block-poly(2-vinylpyridine)) containing a nanometer-sized cluster of gold hydrochloric acid in their core. Upon dip-coating onto hydrophilic glass slides, the micelles
Figure 1. Left: sketch of the experimental system. An elastic, biotin-functionalized agarose bead rests on top of a nanopatterned functionalized substrate (not to scale). Top right: scanning electron micrograph of a nanopatterned substrate. The bright spots show individual gold nanoparticles of approximately 6 nm in diameter. Bottom right: RICM image of an adhering bead. The adhesion area appears dark and is marked with a circle. The graininess of the interference pattern is evident even for the adhesion area and is a result of the roughness of the agarose bead.
self-assemble in a hexagonally close-packed arrangement so that a regular pattern of metal gold dots is left on the substrate after removing the micellar polymer coat by hydrogen plasma treatment. The interdot spacing of the hexagonal pattern is defined by the size of the micelle forming diblock copolymers.8,9 To prevent adhesion in the region between the gold dots, it is covered with a thin protein resistant layer of the poly(ethylene glycol) CH3-(O-CH2-CH2)43-NH-CONH-CH2-CH2-CH2-Si(OEt)3.11 Once the substrates are passivated, streptavidin is covalently coupled to the gold nanopattern. For this, mercaptoundecanoic acid is bound to the gold, leaving the carboxylic acid groups free for covalent binding of streptavidin (from streptomyces avidinii) through standard carbodiimide coupling chemistry.24 To verify successful functionalization, we applied fluorescent Atto532-Biotin (Attotec, Germany) to nanopatterned substrates and studied the fluorescence with a standard fluorescence microscope. The image analysis clearly showed enhanced fluorescence for streptavidin-functionalized nanopatterned substrates compared to nanopattern-free substrates that had been treated in the same way (data not shown here). This indicates that the poly(ethylene glycol) layer inhibits streptavidin adhesion and that the streptavidin remains in a functional state after its coupling to the gold dots. To characterize the interaction between beads and substrate we employed an interferometric technique, called reflection interference contrast microscopy (RICM). This technique enabled us to detect the contact area formed by the beads and the nanopatterned surface with high precision.14,15 The RICM is set up on an inverted microscope (Axiovert 200, Zeiss, Germany) equipped with a mercury lamp (HBO 103, Osram, Germany) and an antiflex objective (Plan Neofluar, 63x /1.25 Oil, Zeiss).16 Because of the reflection of the light 268
at two planes, at the glass surface and the bead, the resulting interferogram encodes information about the height of the bead’s contour. The interference pattern is in a first approximation described by I(b) r ) I1 + I2 + 2xI1I2 cos
(4πnλ h(b)r + ∆)
(1)
where I1 and I2 are the intensities of the light reflected at the bead (I1) and the glass (I2), respectively. n is the refractive index of the buffer solution, and λ is the wavelength of the irradiating light. h(x) is the height of the bead contour above the glass slide. For an object with nobject> nbuffer , the phase difference, ∆, becomes π, leading to destructive interference for small h, thus also for the adhesion area (see Figure 1). The interferograms were recorded with a Peltier-cooled 12 bit CCD camera (ORCA-ER, Hamamatsu, Japan), stored with SimplePCI (Compix Imaging Systems, PA) and further analyzed using ImageJ. The size of the adhesion area derived from RICM implies results on the elastic deformation of the bead that accompanies adhesion. Therefore, the elasticity of the beads is a crucial parameter for further analysis. To obtain the Young’s modulus of the agarose beads, we analyzed numerous force-indentation curves that were taken with an atomic force microscope (Nanowizard, JPK Instruments, Germany).17 With this device, a vertical force was applied to the center of the beads using tipless rectangular cantilevers (Mikromasch, Estonia). Prior to the experiments, the cantilevers were calibrated by thermal fluctuation analysis.18,19 The Young’s modulus, E, was then derived from the Hertz model,20 which relates the applied force, F, to the indentation, δ, of a bead of radius R Nano Lett., Vol. 6, No. 2, 2006
F)
4ExR 3/2 δ 3(1 - σ2)
σ is Poisson’s ratio and equals 0.5, assuming agarose is a rubber-like material. This yields a Young’s modulus of E ) 185.4 ( 2.8 kPa. The measurements were performed on a variety of bead sizes and did not vary from one to the other by more than 5%. During the experiments, we added biotin-linked agarose beads (Sigma, Germany) into a 1x PBS filled fluid chamber and left them to equilibrate on the streptavidin nanopatterns for approximately 1 h. Figure 2 shows data from a typical experiment, where the radius and contact area of a large number of beads were measured with bright-field microscopy and RICM, respectively. To obtain the surface energy, we applied the Johnson-Kendall-Roberts (JKR) model.12 For a bead that is pressed to a flat solid surface with force F, the JKR model yields its contact radius, a
Figure 2. Logarithmic plot of the contact area radii versus the bead radii for a streptavidin nanopattern. A linear fit of these data yields a slope of 0.68 ( 0.07, showing the validity of eq 2.
R a3 ) (F + 3πRW + x6πRWF + (3πRW)2) K where the elastic modulus K ) 4E/(3(1 - σ2)), R is the bead radius, and W is the surface energy. In this study the only external force, F, gravity, is negligible. Therefore, the expression simplifies to a3 )
6πR2W K
(2)
This approximation is justified because the slope of the linear fit in the logarithmic plot (see Figure 2) does not deviate significantly from 2/3, in agreement with eq 2. The surface energy, Wtot, is obtained with a least-squares fit of the data in Origin (OriginLab, MA) using eq 2. In the total surface energy, Wtot, a component of nonspecific interaction, Wnonspec, between the bead and the surface is included. To account for this, Wnonspec is measured on the nanopattern-free half of the substrates and then subtracted from Wtot for each data set, giving Wcorr as a first correction of surface energy. Figure 3 shows the surface energies, Wcorr , for different streptavidin densities on the substrates, revealing a linear relationship between the surface energy and ligand density. However, although the surface energy values had already been corrected for nonspecific interactions, the linear fit still shows a residue value, W0, for zero ligand density. This finding suggests that not all nonspecific interactions are taken into account with Wnonspec. We suppose that an interaction of the beads with the gold dots is responsible for this behavior. Gold has an enormously high surface energy21 of about 1 J/m2, and because of an incomplete coverage of either a single dot or several uncovered dots, a contribution to surface energy can arise. To check the influence of this interaction, we performed experiments with nanopatterned substrates where the gold dots were not functionalized with streptavidin but directly interacting with the beads. On these samples, we found surface energy values that were almost 1 Nano Lett., Vol. 6, No. 2, 2006
Figure 3. Linear dependence of corrected surface energy on ligand density. The comparatively large errors arise from the sensitivity of the method to bead structure and surface preparation.
order of magnitude larger than the ones obtained for functionalized nanopatterns. For a dot density of F ) 3.04 × 10-4/nm2 we measured a surface energy of Wgold ) 48.73 ( 2.82 µJ/m2 compared to Wcorr ) 8.92 ( 1.51 µJ/m2 for an identical, but functionalized nanopattern. A comparison of the residue value of W0 with Wgold gives an average gold dot coverage of 89.2%. To account for the gold-induced interaction, we corrected Wcorr further by subtracting W0 from the data, yielding values for the specific surface energy, Wspec, of the streptavidin-biotin interaction (Table 1). From affinity experiments, a binding energy contribution of about 35 kT for each streptavidin-biotin bond is estimated.22 In contrast to the situation in those experiments, where the ligand-receptor pair was in solution, the binding partners in our experiments are coupled to surfaces. As shown by Merkel et al. the free enthalpy of binding is strongly decreased when proteins are anchored to surfaces.23 Therefore, it is not surprising that our values of surface energy are 1 order of magnitude smaller than expected. Another contribution that is accountable for a reduced surface 269
Table 1. Surface Energies, Wspec, for the Streptavidin-Biotin Interaction after Correction for All Nonspecific Interactionsa ligand density, F (10-4/nm2)
Wspec (µJ/m2)
0.91 ( 0.53 3.04 ( 0.38 3.51 ( 0.64 4.64 ( 0.18 6.45 ( 0.30
1.36 ( 0.66 3.64 ( 1.57 3.99 ( 0.76 5.07 ( 1.08 7.30 ( 1.39
a With different ligand densities, we could vary surface energies by a factor of 5.3.
functionalized, tunable gold nanostructures. Recently, it was shown that our nanopatterning technique can even be used to establish substrates with micrometer-sized spots of different ligand densities.9,10 This applicability of nanostructures for imprinting surface energies on substrates or certain substrate regions opens new avenues for investigating the in situ adhesion of biomimetic systems such as giant vesicles or cells, especially because for these studies substrates with definable adhesive strengths are strongly desirable. Acknowledgment. We thank U. Schwarz, J. Curtis, and R. Richter for helpful discussions. C.S. was supported by the Boehringer Ingelheim Fonds (B.I.F.). Financial support from the Max-Planck-Society is acknowledged. The work has also been part of a STREP Program of the European Community (Nanocues). References
Figure 4. Surface reconstruction of a bead’s adhesion area obtained by applying eq 1 to an RICM image of a bead’s contact area. Assuming that the biotin-streptavidin interaction is restricted to a regime 15 nm in height, the RICM reconstruction shows that not more than about 10% of the bead’s surface can bind to the nanopatterned surface. This is another reason for the rather low measured surface energy values.
energy in our experiments is the surface roughness of the agarose beads, which prevents their full contact with the surface. In Figure 4, we show a reconstruction of the surface profile of a bead’s contact area from RICM calculated using eq 1. Despite the roughness of the beads on the 100 nanometer scale, the JKR model was still applicable but the effective surface energy is decreased because only about 1/10 of the bead surface can be linked to a streptavidin molecule on the surface. To summarize, we have measured a linear relationship between surface energy and ligand density for interacting biotin-streptavidin pairs. The ligand density variation, and thus surface energy adjustment, was achieved by utilizing
270
(1) Liu, G.-Y.; Ambro, N. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5165. (2) Moll, D.; Huber, C.; Schlegel, B.; Pum, D.; Sleytr, U.; Sara, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14646. (3) Briones, C.; Mateo-Marti, E.; Go´mez-Navarro, C.; Parro, V.; Roma´n, E.; Martı´n-Gago, J. A. Phys. ReV. Lett. 2004, 93, 208103. (4) Svedhem, S.; Pfeiffer, I.; Larsson, S.; Wingren, C.; Borrebaeck, C.; Ho¨o¨k, F. ChemBioChem 2003, 4, 393. (5) Bray, D.; Levin, M.; Morton-Firth, C. Nature 1998, 393, 85. (6) Geiger, B.; Bershadsky, A. Cell 2002, 110, 129. (7) Germain, R. N. Curr. Biol. 1997, 7, R640. (8) Glass, R.; Arnold, M.; Blu¨mmel, J.; Ku¨ller, A.; Mo¨ller, M.; Spatz, J. P. AdV. Funct. Mater. 2003, 13, 569. (9) Glass, R.; Mo¨ller, M.; Spatz, J. P. Nanotechnology 2003, 14, 1153. (10) Spatz, J.; Mo¨ssmer, S.; Hartmann, C.; Mo¨ller, M.; Herzog, T.; Krieger, M.; Boyen, H.; Ziemann, P.; Kabius, B. Langmuir 2000, 16, 407. (11) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blu¨mmel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. ChemPhysChem 2004, 5, 383. (12) Johnson, K.; Kendall, K.; Roberts, A. Proc. R. Soc. London, Ser. A. 1971, 324, 301. (13) Moy, V.; Jiao, Y.; Hillman, T.; Lehmann, H.; Sano, T. Biophys. J. 1999, 76, 1632. (14) Gingell, D.; Todd, I. Biophys. J. 1979, 26, 507. (15) Ra¨dler, J.; Sackmann, E. Langmuir 1992, 8, 848. (16) Ploem, J. Blackwell Scientific Publications: Oxford, 1975. (17) Binnig, G.; Quate, C.; Gerber, C. Phys. ReV. Lett. 1986, 56, 930. (18) Cleveland, J.; Manne, S.; Bocek, D.; Hansma, P. ReV. Sci. Instrum. 1993, 64, 403. (19) Hutter, J. L.; Bechhofer, J. ReV. Sci. Instrum. 1994, 64, 1868. (20) Hertz, H. J. Reine Angew. Mathematik 1882, 92, 156. (21) Metzger, R.; Xu, T.; Peterson, I. J. Phys. Chem. B 2001, 105, 7280. (22) Green, N. AVidin and StreptaVidin, AVidin-Biotin Technology, Methods in Enzymology; Academic Press Inc., San Diego, CA, 1990. (23) Nguyen-Duong, M.; Koch, K.; Merkel, R. Europhys. Lett. 2003, 61, 845. (24) Sehgal, D.; Vijay, I. K. Anal. Biochem. 1994, 218, 87. .
NL052256E
Nano Lett., Vol. 6, No. 2, 2006
