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Patterning Polymerized Lipid Vesicles with Soft Lithography Nidhi Mahajan,† Ruibo Lu,‡ Shin-Tson Wu,‡ and Jiyu Fang*,† Advanced Materials Processing and Analysis Center, Department of Mechanical, Materials, and Aerospace Engineering, and College of Optics and Photonics, University of Central Florida, Orlando, Florida 32816 Received November 2, 2004. In Final Form: December 4, 2004 The applications of soft lithography in patterning polymerized lipid vesicles of 1,2-bis(tricosa-10,12diynoyl)-sn-glycero-3-phosphocholine on glass substrates are reported. We demonstrate that the polymerized vesicles can be used as a high molecular weight ink to be transferred from a PDMS stamp onto a glass substrate to form two-dimensional stripes with a controlled separation. By combining channel flow with dewetting within microfluidic networks, we assemble the polymerized vesicle into three-dimensional stripes and one-dimension lines on glass substrates. Atomic force microscopy shows that these patterned vesicle structures are stable on glass substrates. The simple, stable, and precise immobilization of lipid vesicles on solid substrates will open up the possibility of integrating them in biosensors and microelectronic devices.
1. Introduction Self-assembled structures are gaining a lot of interest because of their thermodynamic stability and well-defined geometries.1,2 Lipid vesicles are supramolecular structures formed by the self-assembly of lipid molecules in an aqueous medium. They have been successfully used as nanocapsules for drug delivery.3 Functionalized lipid vesicles have emerged as promising nanoreactors for charge storage,4 signal amplification,5 energy transport,6,7 and mixing chemical compounds.8 It is well-known that the adsorption of lipid vesicles on solid substrates can lead to the formation of planar, extended bilayers through the fusion and rupture processes.9 Recently, a number of techniques have been developed to immobilize lipid vesicles on solid substrates such as oligonucleotide recognition,10 layer-by-layer assembly,11 biotin-streptavidin coupling,12 steric entrapment hydrophobic interaction,13 chemical binding,4,14 and attractive surface-polarizable binding.15 To address the challenge of patterning vesicles onto solid substrates, a novel strategy based on the combination of biomolecular * Corresponding author. E-mail:
[email protected]. † Advanced Materials Processing and Analysis Center. ‡ College of Optics and Photonics. (1) Lehn, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4763. (2) Whitesides, G. M.; Grzybowski, B. Science 2002, 295, 2418. (3) Ringsdorf, H.; Berhard, S.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1998, 27, 113. (4) Stanish, I.; Lowy, D. A.; Lee, Y.; Fang, J. Y.; Wong, E.; Ray, R. I.; Sing, A. J. Phys. Chem. B 2004, 108, 127. Stanish, I.; Lowy, D. A.; Tender, L. M.; Sing, A. J. Phys. Chem. B 2002, 106, 3503. (5) Bolinger, P. Y.; Stamou, D.; Vogel, H. J. Am. Chem. Soc. 2004, 126, 8594. (6) Alfonta, L.; Singh, A. K.; Willner, I. Anal. Chem. 2001, 73, 91. (7) Khairutdinov, R. F.; Hurst, J. K. Nature 1999, 402, 509, (8) Gust, D.; Moore, T. A.; Moore, L. Acc. Chem. Rev. 2001, 34, 40. (9) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (10) Yoshina-Ishii, C.; Boxer, S. G. J. Am. Chem. Soc. 2003, 125, 3696. (11) Katagiri, K.; Hamasaki, R.,; Ariga, K.; Kikuchi, J. J. Am. Chem. Soc. 2002, 124, 7892. (12) Jung, L. S.; Schumaker-Parry, J. S.; Campbell, C. T.; Yee, S. S.; Gelb. M. H. J. Am. Chem. Soc. 2000, 122, 4177. (13) Yang, Q.; Wallsten, M.; Ludahl, P. Biochim. Biophys. Acta 1988, 938, 243. (14) Kim, J. M.; Ji, E. K.; Woo, S. M.; Lee, H. W.; Ahn, D. J. Adv. Mater. 2003, 15, 1118. (15) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397.
recognition with micropatterned surfaces has been developed to control the location of immobilized vesicles on substrates.10,16,17 But these methods involve multistep chemical and/or biological reaction processes. In this paper, we demonstrate that soft lithography techniques18 such as microcontact printing (µCP) and microfluid network (µFN) can be used as a simple and direct method for the patterning of polymerized lipid vesicles on glass substrates. Atomic force microscopy (AFM) studies show that the patterned vesicle structures are stable on glass substrates. 2. Materials and Methods 1,2-Bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) was obtained from Avanti Lipids, Alabaster, AL. Its chemical structure is shown in Figure 1a. Vesicles of the DC8,9PC were prepared according to the standard procedure.19 Briefly, dried DC8,9PC was hydrated in water at a concentration of 2 mg/mL. The suspension was incubated at 50 °C and sporadically vortexed for 3 h and then extruded 10 times at 50 °C through a 0.1 µm Nucleopore membrane using a Lipex extruder. The resulting vesicles were polymerized with UV light. An oxygen plasma treated poly(dimethylsiloxane) (PDMS) stamp having a defined network of hydrophilic channels was used in µCP and µFN approaches. The hydrophiphilic channels are about 700 nm high and 2 µm wide. Glass slides were used as substrates in our experiments. They were cleaned in 1:1 methanol-hydrochloric acid for 30 min, followed by boiling in distilled water at 100 °C for 15 min and washing with water. The cleaned glass substrates are hydrophilic. Optical images of the patterned vesicle structures on glass substrates were obtained with an Olympus BX51 microscope. AFM (Dimension 3100, Digital Instruments) was used for the study of structures and stability of the patterned vesicles on glass substrates. Silicon nitride cantilevers (Nanosensors) with normal spring constants of about 30 N/m and resonant frequencies between 250 and 330 kHz were used. The cantilever was excited just below its resonant frequency. All AFM measurements were performed in the tapping mode in air under ambient conditions. (16) Stamou, D.; Duschl, C.; Delamarche, E.; Vogel, H. Angew. Chem., Int. Ed. Engl. 2003, 42, 5580. (17) Svedhem, S.; Pfeiffer, I.; Larsson, C.; Wingren, C.; Borrebaeck, C.; Ho¨o¨k, F. ChemBioChem. 2003, 4, 339. (18) Xia, Y. N.; Whitesides. G. M. Angew. Chem., Int. Ed. Engl. 1998, 37, 550. (19) Peek, B. M.; Callahan, J. H.; Namboodiri, K.; Singh, A.; Gaber, B. P. Macromolecules 1994, 27, 292.
10.1021/la0473153 CCC: $30.25 © 2005 American Chemical Society Published on Web 02/19/2005
Patterning Polymerized Lipid Vesicles
Figure 1. (a) Chemical structure of DC8,9PC lipid. Tapping mode AFM images of polymerized (b) and unpolymerized (c) DC8,9PC vesicles dried on glass substrates.
3. Results and Discussion Figure 1b shows an AFM image of the polymerized lipid vesicles adsorbed on a glass substrate. We find that they remain intact after drying in air for 2 days. Here, the apparent widths of the polymerized lipid vesicles in the AFM image are broadened by the tip convolution. The measured height of the polymerized vesicles is unaffected by the tip size, and we find that it is about 100 ( 30 nm, which agrees with the diameter of the vesicles measured by light scattering in solutions.19 The unpolymerized lipid vesicles are not stable on glass substrates after drying in air. They are found to collapse and form planar bilayer domains with a smooth surface (Figure 1c). It is clear that the polymerization in the bilayer wall prevents the collapse and fusion of the lipid vesicles on glass substrates. In the µCP approach, the polymerized vesicles were used as ink for contact printing. The PDMS stamp was immersed into a vesicle suspension for about 10 s and then placed on the surface of a cleaned glass substrate to form a conformal contact. After ∼5 min of contact, the stamp was carefully removed from the surface. Figure 2a is a low-resolution AFM image of a printed glass surface. Parallel vesicle stripes with a width of about 2 µm are observed over a large area on the glass substrate. This periodicity of the vesicle stripes corresponds well with the structure features in the PDMS stamp. The height profiles show that the parallel vesicle stripes are no more than 150 nm high. This means that only a single layer of the polymerized vesicles is deposited during the printing. From the high-resolution AFM image shown in Figure 2b, we find that the printed vesicles are not highly condensed. Isolated vesicles are clearly visible within the stripes. The vesicle stripes show sharp edges. There is no obvious diffusion observed when the sample is placed in an aqueous solution (pH 7.4-8.2) for 2 days. The strong immobilization of the printed polymerized vesicles might be a result of the electrostatic interaction between the negatively charged surface of the glass substrate and the
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positively charged headgroups of the DC8,9PC. The electrostatic interaction between silica colloidal particles with the tubes of the DC8,9PC has been used to from hollow silica cylinders.20 In the µFN approach, a drop of vesicle solution was placed along one of the open-ended channels of the PDMS mold. The polymerized lipid vesicles were filled into the network of the channels by capillary action. The vesicle solution-filled channels were allowed to dry in air at room temperature. After removal of the PDMS mold from the glass substrate, the patterned vesicles were imaged in air at room temperature. Figure 3a is an optical image of a patterned glass substrate with the polymerized lipid vesicles. The parallel vesicle stripes (lighter regions) are separated by 2 and 5 µm, respectively. The width of the vesicle stripes corresponds to the width of the channels recessed in the PDMS mold, suggesting that all polymerized vesicles were confined within the channels and did not diffuse across the glass surface following removal of the PDMS mold. Once the first layer of the parallel vesicle stripes were formed on the glass substrate, the second layer of the parallel vesicle stripes was added on it by repeating the process at 90° to form a crossbar array (Figure 3b). The AFM image in Figure 3c shows threedimensional (3-D) aggregates of the lipid vesicles within the stripes. The average thickness of the stripes measured by the AFM is about 380 nm, corresponding to three to four vesicle layers. In the case of using a very dilute vesicle solution, we find that the edges of the channels can act as pinning sites for the immobilization of the polymerized lipid vesicles. In our experiments, the solution-filled microchannels were dried in a vacuum chamber. When solvent evaporates rapidly, the residual solution is expected to recede into two corners of the hydrophilic channel (Figure 4a). As a result, the vesicles are transferred to the edges of the channels via the capillary force and then are immobilized through the electrostatic interaction with the glass substrate. Figure 4b is an AFM image of parallel vesicle lines which are formed by the dewetting and adsorption within the hydrophilic microchannels. Crosssection measurements show that these parallel lines are separated by 2 µm, which agrees with the width of the microchannels. This confirms that the polymerized vesicles are indeed transferred to the edges of the channels by the dewetting-induced capillary force before they are immobilized on the glass substrate. The above results also suggest the possibility of downsizing the printed vesicle features with respect to the size of the channels. Other groups have used the dewetting occurring within microchannel networks to align nanowires21,22 and position block-copolymer micelles.23 We note that the patterned vesicle lines are stable in air, but they can be selectively removed from the glass substrate with a lift-up method when the sample is placed under water. In the lift-up process, a PDMS mold was brought to contact with the vesicle lines on a glass substrate under water at a large pressure. The direction of the channels in the PDMS stamp was arranged to be perpendicular to the direction of the vesicle lines. After 5 min of contact under water, the PDMS mold was carefully peeled away. The sample was then taken out of the water and imaged by the atomic force microscope in air. We find (20) Baral, S.; Schoen, P. Chem. Mater. 1993, 5, 145. (21) Messer, B., Song, J. H., Yang, P. D. J. Am. Chem. Soc. 2000, 122, 10232-10233. (22) Chen, J.; Weimer, W. A. J. Am. Chem. Soc. 2002, 124, 785. (23) Levi, S. A.; Mourran, A.; Spatz, J. P.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Mo¨ller, M. Chem. Eur. J. 2002, 8, 3808.
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Figure 2. Low-resolution (A) and high-resolution (B) tapping mode AFM images of polymerized vesicle stripes printed on glass substrates with microcontact printing (µCP) approach.
Figure 4. (a) Schematic illustration of receding of vesicle solution within a channel during dewetting. The polymerized vesicles are confined at the two edges of the channel. (b) AFM image of patterned vesicle lines on a glass substrate. All vesicles align along the edges of the channels. Figure 3. Optical microscopy images of parallel (a) and cross (b) arrays of polymerized vesicle stripes formed on the glass substrate with a microfluid network (µFN) approach. (c) AFM image of parallel arrays of vesicle stripes. The capillary force is the main principle behind the patterning by this approach. All vesicles were confined within the fluid channels.
that the patterned vesicle lines in contact with the PDMS mold are selectively removed from the glass substrate (Figure 5). It is likely that the lipid vesicles, which are in contact with the stamp, are destroyed by physical contact with the stamp at large pressures and dissolve in water. The remaining vesicle lines show a two-dimensional “smectic” structure on the glass substrate. The same approach has been used to blot the supported lipid bilayers from the glass substrates.24 (24) Hovis, J. S., Boxer, S. G. Langmuir 2000, 16, 894.
Figure 5. AFM image of a patterned “smectic” vesicle structure formed by selectively removing the vesicles from parallel lines with a lift-up process.
Patterning Polymerized Lipid Vesicles
4. Conclusions We report the applications of soft lithography in patterning polymerized lipid vesicles on glass substrates. In the µCP approach, we demonstrate that the polymerized vesicles can be used as a high molecular weight ink to be transferred from the PDMS stamp onto glass substrates to form 2-D vesicle stripes with a controlled separation. Using the µFN approach, we are able to form 3-D parallel and crossbar arrays of the vesicles on glass substrates. 1-D vesicle lines can be formed on glass substrates by simply controlling the vesicle concentration and the dewetting occurring within the microchannels. In this case,
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the effective downscaling of printed vesicle features with respect to the channel size can be achieved. These printed vesicle structures are stable on glass substrates. The high precision assembly and stable immobilization of polymerized lipid vesicles on solid substrates will open up the possibility of integrating them in biosensors and microelectronic devices. Acknowledgment. This work was supported by University of Central Florida’s nano-initiative. LA0473153
