Langmuir 1999, 15, 8133-8139
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DNA Adsorption and Cationic Bilayer Deposition on Self-Assembled Monolayers Stefan Schouten,† Pieter Stroeve,* and Marjorie L. Longo* Center on Polymer Interfaces and Macromolecular Assemblies, Department of Chemical Engineering and Materials Science, University of California at Davis, Davis, California 95616 Received February 16, 1999. In Final Form: July 6, 1999 A cationic bilayer adsorbed to a self-assembled monolayer (SAM) of alkylthiols could be a useful substrate for DNA immobilization and patterning. Therefore, we studied the layer formation of a self-assembled system consisting of a base layer of a negatively charged SAM chemisorbed on gold, a middle layer of an electrostatically adsorbed cationic bilayer, and a top layer of double-stranded DNA that electrostatically adsorbs to the cationic bilayer. The formation of DNA, lipid, and alkylthiol layers was monitored by surface plasmon spectroscopy. Cationic lipids readily formed layers with thickness between 32 and 33 Å on selfassembled alkylthiols possessing terminal carboxylic acid groups within 24 h and at pH > 2. Fluorescence bleaching experiments indicated that these layers were homogeneous and relatively immobile. For comparison, we found that cationic lipids do not form layers on alkylthiols possessing a terminal alcohol group, while zwitterionic lipids formed bilayers on these surfaces and on the carboxylated surfaces with a thickness of approximately 38-44 Å. The use of self-assembled alkylthiols with diethylene glycol groups prohibited the formation of both cationic and zwitterionic lipid layers and also prevented DNA adsorption. Finally, DNA was adsorbed to cationic lipid bilayers which were electrostatically attached to negatively charged SAMs. The results indicate that DNA forms a layer of 8 Å calculated thickness, which is consistent with a monolayer possessing average interhelical distances of 50 Å, in agreement with other studies using different techniques. Hence this surface is useful for immobilizing DNA. No differences were observed in kinetics of deposition or the thickness of the DNA monolayer when different cationic lipids were used.
Introduction Recently, it has been demonstrated that doublestranded DNA adsorbs to cationic lipid bilayers deposited on mica.1,2 It was shown using atomic force microscopy that the DNA strands form a single monolayer with an interhelical spacing of approximately 5 nm. These substrate-supported, cationic lipid bilayers may, in the future, serve as a useful method of immobilizing3 and patterning DNA.4,5 However, mica (the substrate previously used to immobilize cationic bilayers1,2) is inherently fragile because of the weakly bound cleavage planes and is therefore a less-than-ideal candidate for patterning of molecules. An alternative is self-assembled monolayers (SAMs) on gold surfaces (Figure 1). SAMs can be readily functionalized, and detection of adsorbed substances is possible by surface plasmon spectroscopy6 (SPS) or surface plasmon microscopy7,8 (for positional detection in array formats). In addition, immobilization of DNA in any future array format that uses cationic lipids will require patterning into array elements coated with lipid bilayers, separated by regions in which the bilayer is absent. Here, we present * Authors to whom correspondence should be addressed: e-mail,
[email protected],
[email protected]; telephone, (530) 7546348; fax, (530) 752-1031. † Present address: Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB Den Burg, Texel, The Netherlands. (1) Fang, Y.; Yang, J. J. Phys. Chem. B 1997, 101, 441-449. (2) Mou, J.; Czajkowsky, D. M.; Zhang, Y.; Shao, Z. FEBS Lett. 1995, 371, 279-282. (3) Higashi, N.; Takahashi, M.; Niwa, M. Langmuir 1999, 15, 111116. (4) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Prog. 1998, 14, 356-363. (5) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (6) Aust, E. F.; Sawodny, M.; Ito, S.; Knoll, W. Scanning 1994, 16, 353-361. (7) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939-4947. (8) Frutos, A. G.; Corn, R. M. Anal. Chem. 1998, 70, 449A-455A.
Figure 1. Schematic representation of investigated system. A lipid bilayer is attached to an alkylthiol monolayer possessing a functional group with the opposite charge of the lipid headgroup.
SPS and fluorescence results in which we have tested several surface functionalizations of SAMs in order to identify functional groups on which cationic bilayers from vesicles (and, subsequently, DNA) readily deposit and functional groups which repel the deposition of cationic bilayers and DNA. In the past, attachment of lipid bilayers to SAMs has received some attention and therefore served as a backdrop for this work. Brink et al.9 deposited a weakly adsorbed (9) Brink, G.; Schmitt, L.; Tampe´, R.; Sackmann, E. Biochim. Biophys. Acta 1994, 1196, 227-230.
10.1021/la990162c CCC: $18.00 © 1999 American Chemical Society Published on Web 09/10/1999
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zwitterionic lipid, dimyristoylglycerophosphocholine, on gold surfaces coated with positively charged cysteamine. Steinem et al.10 found (using impedance spectroscopy) that bilayer formation of the negatively charged dimyristoylphosphatidylglycerol (DMPG) on a SAM of cysteamine was complete in 24 h. In addition, they (studied using impedance) and Stelzle et al.11 (studied using SPS) formed bilayers by adsorption of cationic lipid vesicles of dimethyldioctadecylammoniumbromide (DODAB) to gold surfaces coated with 3-mercaptopropionic acid and 11mercaptoundecanoic acid, respectively. Both groups also adsorbed negatively charged lipids (phosphatidylglycerols) to the same surfaces using Ca2+ ions. Liley et al.12 deposited a zwitterionic lipid, palmitoyloleoylphosphatidylcholine, with 3 mol % promastigote surface protease on gold surfaces coated with a SAM of 11-mercaptoundecanol at pH 7.5. Bilayer formation was successful and the binding of antibiodies could be detected by thickness increases. These previous studies prompted us to investigate the electrostatic attachment of cationic lipid bilayers to solid substrates and their utility for immobilizing DNA. Saturated cationic lipids with different headgroups, as well as an unsaturated cationic and zwitterionic lipid, were adsorbed from vesicles to negatively charged 11-mercaptoundecanoic acid functionalized surfaces. In addition, for the cationic bilayer deposition we obtained information on the pH sensitivity of vesicle deposition and lateral mobility of the lipid bilayers. In an attempt to identify additional anchors for cationic lipids, we examined the adsorption of cationic (and zwitterionic) lipids to alkylthiols with terminal alcohol groups, 11-mercaptoundecanol and 6-mercaptohexanol. To repel cationic bilayer formation and DNA layer formation for future micropatterning purposes, we used an alkylthiol with a diethylene glycol group because they are known to prohibit the adsorption of cells and proteins.4,13-15 Finally, we studied the interaction of λ-phage DNA with the prepared cationic lipid bilayers. In all of these studies (with the exception of fluorescence microscopy for the mobility studies), SPS was utilized to monitor adsorption and film thickness. Our results indicate the potential for using electrostatic attachment of DNA to cationic lipid bilayers prepared as described above as an alternative and convenient way to immobilize and pattern DNA on solid surfaces. Experimental Section Materials. All solvents and most chemicals were of analytical grade. 11-Mercaptoundecanoic acid (MUA), 6-mercaptohexanol (MHO), and 11-mercaptoundecanol (MUO) were purchased from Aldrich (Milwaukee, WI). The alkylthiol 11-mercaptoundecanediethylene glycol (MUDEG) was synthesized as described by Pale-Grosdemange et al.13 The lipid dihexadecyldimethylammonium bromide (DHDAB) was obtained from Aldrich, while 1,2-dipalmitoyl-3-trimethylammoniumpropane (DPTAP), 1,2dioleoyl-3-trimethylammoniumpropane (DOTAP), and 1-palmitoyl-2-oleoylglycero-3-phosphocholine (POPC) were purchased from Avanti Polar Lipids (Alabaster, AL). The cationic fluorescent probe used for fluorescent microscopy was 4-(4-(didecyl(10) Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J. Biochim. Biophys. Acta 1996, 1279, 169-180. (11) Stelzle, M.; Weismu¨ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981. (12) Liley, M.; Bouvier, J.; Vogel, H. J. Colloid Interface Sci. 1997, 194, 53-58. (13) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20. (14) Herbert, C. B.; McLernon, T. L.; Hypolite, C. L.; Adams, D. N.; Pikus, L.; Huang, C.-C.; Fields, G. B.; Letourneau, P. C.; Distefano, M. D.; Hu, W.-S. Chem. Biol. 1997, 4, 731-737. (15) Dubrovsky, T. B.; Hou, Z.; Stroeve, P.; Abbott, N. L. Anal. Chem. 1999, 71, 327-332.
Schouten et al. amino)styryl)-N-methylpyridinium iodide (4-Di-10-ASP) from Molecular Probes (Eugene, OR). The buffers tris(hydroxymethyl)aminomethane (tris) and 4-morpholineethanesulfonic acid (MES) were purchased from Aldrich. Lambda phage DNA isolated from Escherichia coli was purchased from Sigma (St. Louis, MO). Preparation of Gold Slides. Gold slides used for SPS experiments were prepared by vacuum evaporation ( 0.95), and the observed rate constant was similar with every lipid (Table 1). This suggests that the mechanism responsible for the adsorption of DNA to a cationic lipid bilayer is not dependent on the chemical structure of the lipid. Since lambda phage DNA has a large molecular weight (approximately 48 kilobase pairs), its diffusion coefficient in the aqueous solution is expected to be small. The results probably reflect the fact that the process is diffusioncontrolled and, therefore, the results are similar. Conclusions A supported lipid membrane system was investigated in which cationic lipid bilayers were firmly attached by electrostatic forces to a negatively charged alkylthiol monolayer, which itself was attached to a gold surface. The bilayer can be deposited within 24 h and at pH > 2. The lipid bilayers are not very mobile, as suggested by qualitative bleaching experiments using fluorescence microscopy. However, although mobility may be desired in some systems (e.g., diffusion of transmembrane proteins), our studies indicate that mobility is not necessary for adsorption of DNA to cationic lipid bilayers. Additionally, immobile layers may remain patterned longer than (29) Dan, N. Biophys. J. 1996, 71, 1267-1272. (30) Kinoshita, K.; Furuike, S.; Yamazaki, M. Biophys. Chem. 1998, 74, 237-249.
DNA Adsorption and Cationic Bilayer Deposition
diffusive layers. Using alkylthiols containing an alcohol group or a diethyleneglycol group, cationic lipid vesicles are effectively prohibited from adsorbing on the surface. Zwitterionic lipids do not adsorb on surfaces of diethyleneglycol groups, but do form bilayers on surfaces coated with alcohol groups and negatively charged carboxylic acid groups. DNA has been deposited on cationic lipid bilayers, and the results suggest that a monolayer is formed with average interhelical spacings of approximately 5 nm. DNA does not deposit onto a zwitterionic bilayer or diethyleneglycol groups. The DNA remains firmly attached to the cationic lipid bilayers even after rinsing with buffer. Thus, this may be a good method to immobilize and pattern DNA. For example, our results suggest that DNA will adsorb to cationic bilayers in carboxylated regions but will be excluded from regions functionalized with ethyleneglycol groups, where cationic lipids are not adsorbed.
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Acknowledgment. This work was supported in part by the MRSEC Program of the National Science Foundation under Award Number DMR-9808677. S.S. acknowledges the Center of Polymer Interfaces and Macromolecular Assemblies (CPIMA) for a Young Investigator Fellowship. M.L. acknowledges funding from NSF through the CAREER program (BES-9733764). P.S. acknowledges financial support from the Clorox Corporation. Prof. Dr. W. Knoll (Max-Planck-Institute for Polymer Research, Mainz, Germany) and Prof. C. W. Frank (Stanford University) are thanked for useful discussions. We thank L. Jong for providing MUDEG and Z. Hou for providing some of the gold-coated slides. We also thank Dr. C. Berger and B. Argo for their help with the SPS and Dr. A. Mc Kiernan for her assistance with fluorescence microscopy. LA990162C
