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Fabrication of Gradient Mesostructures by Langmuir-Blodgett Rotating Transfer Xiaodong Chen, Michael Hirtz, Harald Fuchs, and Lifeng Chi* Physikalisches Institut and Center for Nanotechnology (CeNTech), Westfa¨lische Wilhelms-UniVersita¨t, 48149 Mu¨nster, Germany ReceiVed October 6, 2006. In Final Form: December 21, 2006 In this letter, we present a simple yet novel method, Langmuir-Blodgett (LB) rotating transfer, to achieve a continuous gradient mesostructure in a well-ordered fashion over large areas. A mixed monolayer of phospholipid and dye is chosen as a model system to test the feasibility of LB rotating transfer to fabricate continuous gradient structures, which is confirmed by the simulation and experimental results. The technique presented here to obtain gradient structures is low-cost and high-throughput and can be extended to other systems of LB patterning.
Introduction Substrate-bound molecular gradients (chemical gradients) obtained in a well-controlled fashion offer an in vitro model to study the biological phenomena that occur in vivo, for instance, axon guidance, cell signaling, and proliferation.1 It has been demonstrated that chemical gradients on surfaces can influence the function and development of cells, biological recognition, and interaction.2,3 Different approaches to produce chemical gradients on surfaces have been reported, for instance, microfludic systems,4-6 controlled diffusion of reactive substances,7-9 and microcontact printing.10-12 Similarly, generating continuousgradient micro-/nanostructures on surfaces (topographical or pattern gradients) would be interesting for the study of cell motility and adhesion, cell mechanotransduction,13 and micro/nano analysis systems.14 However, the fabrication of structure gradients on surfaces has been much less frequently addressed than that of concentration gradients. The reason is that it is difficult and expensive to fabricate continuous-gradient micro/nanostructures on surfaces over the distances required for biological studies (at least a few hundred micrometers) on the basis of only top-down techniques, for instance, scanning probe lithography15 or optical lithography.14 This inspires the search for bottom-up techniques based on self-assembly because of their simplicity, high yield, and ease of implementation over large areas.16,17 * Corresponding author. E-mail:
[email protected]. (1) Baier, H.; Bonhoeffer, F. Science 1992, 255, 472-475. (2) Dertinger, S. K. W.; Jiang, X. Y.; Li, Z. Y.; Murthy, V. N.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12542-12547. (3) Ruardy, T. G.; Schakenraad, J. M.; van der Mei, H. C.; Busscher, H. J. Surf. Sci. Rep. 1997, 29, 3-30. (4) Fosser, K. A.; Nuzzo, R. G. Anal. Chem. 2003, 75, 5775-5782. (5) Jeon, N. L.; Dertinger, S. K. W.; Chiu, D. T.; Choi, I. S.; Stroock, A. D.; Whitesides, G. M. Langmuir 2000, 16, 8311-8316. (6) Jiang, X. Y.; Xu, Q. B.; Dertinger, S. K. W.; Stroock, A. D.; Fu, T. M.; Whitesides, G. M. Anal. Chem. 2005, 77, 2338-2347. (7) Chaudhury, M. K.; Whitesides, G. M. Science 1992, 256, 1539-1541. (8) Liedberg, B.; Tengvall, P. Langmuir 1995, 11, 3821-3827. (9) Riepl, M.; Ostblom, M.; Lundstrom, I.; Svensson, S. C. T.; van der Gon, A. W. D.; Schaferling, M.; Liedberg, B. Langmuir 2005, 21, 1042-1050. (10) Choi, S. H.; Newby, B. M. Z. Langmuir 2003, 19, 7427-7435. (11) Bhangale, S. M.; Tjong, V.; Wu, L.; Yakovlev, N.; Moran, P. M. AdV. Mater. 2005, 17, 809-813. (12) von Philipsborn, A. C.; Lang, S.; Loeschinger, J.; Bernard, A.; David, C.; Lehnert, D.; Bonhoeffer, F.; Bastmeyer, M. DeVelopment 2006, 133, 24872495. (13) Dalby, M. J. Med. Eng. Phys. 2005, 27, 730-742. (14) Cao, H.; Tegenfeldt, J. O.; Austin, R. H.; Chou, S. Y. Appl. Phys. Lett. 2002, 81, 3058-3060. (15) Fuierer, R. R.; Carroll, R. L.; Feldheim, D. L.; Gorman, C. B. AdV. Mater. 2002, 14, 154-157. (16) Boncheva, M.; Whitesides, G. M. MRS Bull. 2005, 30, 736-742.
Here, we report a simple yet novel method based on the Langmuir-Blodgett (LB) technique to achieve a continuous gradient mesostructure in a well-ordered fashion over large areas. The key improvement over the standard LB transfer technique is that a floating monolayer is transferred onto a solid substrate by rotating the substrate rather than vertically pulling the substrate. As a result, LB patterns with different dimensions and orientations that depend on the transfer velocity18,19 can be generated simultaneously. Experimental Section Materials. L-R-Dipalmitoyl-phosphatidylcholine (DPPC) was obtained as a powder from Fluka and used without further purification (chemical purity >99%). 2-(12-(7-Nitrobenz-2-oxa-1,3-diazol-4yl)-amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD) was obtained from Molecular Probes (Leiden, The Netherlands). These lipids were dissolved in chloroform (HPLC grade) purchased from AppliChem (Darmstadt, Germany). Gradient Stripe Pattern Formation. The mixed monolayers were obtained by spreading the lipid chloroform solutions on a water surface (Millipore, resistance 18.2 MΩ‚cm) using a microsyringe, and the transfer of the mixed monolayer was carried out using a computer-controlled commercial Langmuir-Blodgett (LB) film balance system (KSV 3000, Finland) combined with our LB rotator (our own design). Following solvent evaporation for 15 min, the monolayers were symmetrically compressed at a constant speed of 10 cm2/min. The monolayers at the mica surface were prepared as follows. First, the freshly cleaved mica substrate (purchased from Plano, Germany) was immersed in the pure water, and then the lipid chloroform solution was spread on the subphase. After the monolayers were compressed to the predefined target pressure and stabilized for 30 min, they were deposited onto the mica surface by LB rotating transfer at a constant surface pressure and a constant angular velocity. The temperature of the subphase was controlled by a thermostat (22.0 ( 0.3 °C), and the humidity of the laboratory was 50-70%. Software Interface to Control the LB Rotator. The software interface was developed with Visual Basic in Microsoft Visual Studio 2005. In addition to automated rotating transfer with defined angular velocity, direct access to the dipper position (“up” - sample parallel, “down” - sample perpendicular to the air-water surface) and an arbitrary angular position are possible. The software gives information about the rotation speed at different radii and can measure the actual motor speed for debugging and calibrating. (17) Geissler, M.; Xia, Y. N. AdV. Mater. 2004, 16, 1249-1269. (18) Chen, X. D.; Lu, N.; Zhang, H.; Hirtz, M.; Wu, L. X.; Fuchs, H.; Chi, L. F. J. Phys. Chem. B 2006, 110, 8039-8046. (19) Lenhert, S.; Gleiche, M.; Fuchs, H.; Chi, L. F. ChemPhysChem 2005, 6, 2495-2498.
10.1021/la062938x CCC: $37.00 © 2007 American Chemical Society Published on Web 02/03/2007
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Figure 1. (a) Geometrical view of a conventional LB dipper. (b) Scheme of LB vertical transfer in side view. (c) Schematic illustration of LB rotating transfer in front view. (d) Image of the LB rotating dipper connected with a commercial LB trough. Fluorescence Microscopy. An epi-fluorescence microscopy (Olympus BX41), equipped with 50× and 20× objectives and standard fluorescence filter sets, was used to record fluorescence images.
Results and Discussion For the conventional LB transfer (i.e., vertical transfer), the dipper is able to move the substrate only up or down along the Z axis (parallel to the normal of the water surface), as shown in Figure 1a,b, and the linear velocity for all points on the substrate is the same. From the geometric point of view, however, the transfer of the floating monolayer onto the substrate from the air-water interface can be realized by moving the substrate along the Z axis (vertical transfer) or rotating the substrate along the X axis (Figure 1a). From this consideration, we designed a new dipper to transfer the monolayer onto the substrate by rotating the substrate around X axis through the air-water interface (Figure 1c), as shown in Figure 1d. We term this kind of transfer LB rotating transfer. One characteristic point of LB rotating transfer that differs from the LB vertical transfer is that the linear velocity at different points on the substrate depending on the distance to the axis of rotation is different. First, we make a general analysis of the linear velocity at the three-phase contact line. Figure 2a shows a schematic illustration for LB rotating transfer in a frontal view. During the rotating transfer, the velocity controlled by the program is the angular velocity and not the linear velocity. However, we can converse the angular velocity to the linear velocity depending on the radius. Also, we can divide the transfer linear velocity (Vt) (parallel to the tangent to the radius) for the points at the threephase contact line into two velocities: the linear velocity perpendicular to the three-phase contact line (Vv) and the linear velocity parallel to the three-phase contact line (Vp)
Vt ) 2πωr Vv ) Vt cos R where r is the radius (i.e., the distance away from the axis of rotation), ω is the angular velocity, R is the angle between the direction of Vv and Vt, and θ is the angle between line a and the three-phase contact line (i.e., line b).
Figure 2. (a) Geometrical parameters that describe the linear velocity at the three-phase contact line. (b) Simulated distribution of the linear velocity perpendicular to the three-phase contact line (Vv) and orientation (denoted by white lines) of the stripes. d ) 23 mm, ω ) 0.07 rpm, l ) 60 mm, and w ) 20 mm.
Figure 3. Fluorescence microscope images (30 × 30 µm2) for the pattern along line a. The number in each image is the radius (r, mm), and the dotted line is parallel to the three-phase contact line shown in Figure 2a.
For transferring densely packed monolayers, such as a liquid condensed (LC) phase or a solid phase, rotating transfer should make no difference in monolayer structures and morphologies compared with vertical transfer. However, the LB transfer process itself can be used to induce phase transitions and stripe pattern formation from a homogeneous liquid expanded (LE) monolayer20 where the linear velocity can be used quantitatively to control the shape and size of a variety of patterns.18,19 In this case, rotating transfer is an efficient way to generate a pattern with different dimensions and orientations on the same substrate simultaneously. We chose a mixed monolayer of L-R-dipalmitoyl-phosphatidylcholine (DPPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4(20) Gleiche, M.; Chi, L. F.; Fuchs, H. Nature 2000, 403, 173-175.
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Figure 4. (a) Dependence of θ on the radius, which could be fitted well (red line, r2 ) 0.98) by the equation (inset) expected from the geometrical consideration. (b) Dependence of the lateral width of the luminescent stripe and dark stripe and periodicity of the gradient pattern on the radius and (c) linear velocity perpendicular to the three-phase contact line. (d) Area coverage of the luminescent stripe and NBD concentration as a function of the radius. All data in b-d are fitted by a monoexponential decay.
yl)-amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD) (2 mol %) as a model system to test the feasibility of LB rotating transfer. It has been previously demonstrated that linear luminescent stripe patterns with submicrometer widths can be obtained by means of LB vertical transfer of a DPPC/ NBD mixed monolayer onto a mica surface.21 The stripes are perpendicular to the transfer direction, and the width and periodicity of the luminescent stripes strongly depend on the transfer velocity. Because the linear velocity depends on the distance to the axis of rotation for LB rotating transfer, we expect a gradient structure on the surface. To get an overview of the patterning over the whole substrate, we develop a program to calculate θ and Vv with respect to d and ω for all points on the sample. The software produces color maps of the distributions of Vv and θ (denoted by white lines) (Figure 2b), which gives a good impression of the overall distribution and shape of the pattern as well as the associated gradient on the substrate. As an example, the mixed monolayer was transferred onto mica at a surface pressure of 2 mN/m with an angular velocity (ω) of 0.07 rpm. For this angular velocity, the linear velocity perpendicular to the three-phase contact line (Vv) can be tuned to a range of 0-30 mm/min depending on the radius (r). The obtained pattern dimensions were measured by fluorescence microscopy. However, it is difficult to measure the pattern in one image because of its length of several centimeters along the substrate and the small features of the pattern. Thus, we select two typical scenarios. One consists of the points along middle line a of the substrate (Figure 2a). For this case, θ (the angle between line a and the three-phase contact line, Figure 2a) is different for different points along line a. Another approach is to measure the points along the three-phase contact line, where θ is kept constant. Figure 3 shows the fluorescence microscopy images of gradient stripe patterns along line a in Figure 2a. The number in each image is the radius (r, mm) (i.e., the distance from a measured (21) Chen, X. D.; Hirtz, M.; Fuchs, H.; Chi, L. F. AdV. Mater. 2005, 17, 2881-2885.
Figure 5. Fluorescence microscopy images (30 × 30 µm2) for the pattern along line b (θ ) 35°). The number in each image is the radius.
point to the axis of rotation). From each image, we can obtain the value of θ. The angle decreases with increasing radius from the axis of rotation, as shown in Figure 4a, which could be fitted well by the expectation based on the transfer geometry (equation shown in the inset of Figure 4a). This confirms that our device works well. Moreover, one observes that the lateral width of the luminescent stripe and the periodicity strongly depends on the radius, which is monoexponentially decreasing as the radius increases, as shown in Figure 4b. In other words, the pattern is gradient rather than repetitive on the substrate.21 Because of the relationship between the linear velocity and radius (inset of Figure 4c), we can also obtain plots showing the lateral width and periodicity of luminescent stripes as a function of Vv, which are similar to the previous results for the velocity dependence.21 Moreover, the structure obtained here also shows a concentration gradient. First, we assume that the density of DPPC in the luminescent stripes is the same as that at the air-water interface (LE phase) and the molecular sizes of NBD and DPPC are the same. After we obtain the area coverage of luminescent stripes from fluorescence images, we can calculate the NBD concentra-
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Conclusions
Figure 6. Dependence of the lateral width of the luminescent stripe and dark stripe and periodicity of the pattern (line b) on the radius (θ ) 35°).
tion in the luminescent stripe, which shows a concentration gradient as shown in Figure 4d. Therefore, the method we developed here can realize the chemical gradient and structural gradient simultaneously. For the points along the three-phase contact line (line b) with a constant value of θ, for instance, 35°, the lateral width and periodicity of the luminescent stripes strongly depend on the radius and also behave in a gradient fashion, as shown in Figures 5 and 6. For this case, R is different for the points along line b, but the dependence of the lateral width on the radius is complicated compared with the case of line a. The lateral width decreases first but then increase with the radius. The result is consistent with we previously observed by vertical transfer.18,19
In summary, we have developed a simple yet novel method, LB rotating transfer, to produce a gradient mesostructure in a well-ordered fashion on a surface. One may expect that the combining rotating transfer with variations in the surface pressure18,19,22 during transfer as well as other LB-transfer-induced patterns would enrich the parameter space, allowing the formation of even more complex patterns. It is easy to extrapolate the LB rotating transfer presented here to other systems, such as nanoparticles,23,24 to obtain complex nanoparticles arrays. Moreover, this kind of transfer could be easily used to test the experimental conditions for exploring the pattern formation of other systems (i.e., high-throughput studies). With the help of LB lithography and nanoimprinting,25,26 the present gradient mesostructure would serve as a platform to study cell motility and adhesion, neuron guidance, and other processes involved in biological pattern formation. Acknowledgment. Dr. S. Lenhert is acknowledged for helpful discussions. This work was supported by the German-Israeli Foundation and SFB 424. LA062938X (22) Purrucker, O.; Fortig, A.; Ludtke, K.; Jordan, R.; Tanaka, M. J. Am. Chem. Soc. 2005, 127, 1258-1264. (23) Huang, J. X.; Kim, F.; Tao, A. R.; Connor, S.; Yang, P. D. Nat. Mater. 2005, 4, 896-900. (24) Huang, J. X.; Tao, A. R.; Connor, S.; He, R. R.; Yang, P. D. Nano Lett. 2006, 6, 524-529. (25) Lenhert, S.; Zhang, L.; Mueller, J.; Wiesmann, H. P.; Erker, G.; Fuchs, H.; Chi, L. F. AdV. Mater. 2004, 16, 619-624. (26) Lenhert, S.; Meier, M. B.; Meyer, U.; Chi, L. F.; Wiesmann, H. P. Biomaterials 2005, 26, 563-570.
