NANO LETTERS
DNA Multilayer Films on Planar and Colloidal Supports: Sequential Assembly of Like-Charged Polyelectrolytes
2005 Vol. 5, No. 5 953-956
Angus P. R. Johnston, Elizabeth S. Read, and Frank Caruso* Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The UniVersity of Melbourne, Victoria 3010, Australia Received March 30, 2005
ABSTRACT Multilayer films comprising solely negatively charged polyelectrolytes were sequentially assembled based on DNA hybridization. Films prepared from alternating layers of two-block homopolymeric nucleotides (polyA20G20/polyT20C20) grew linearly with increasing layer number, as verified by quartz crystal microgravimetry, UV−vis spectrophotometry and optical microscopy. Urea treatment of the films induced morphological changes, while exposure to low ionic strength solutions resulted in film disassembly. DNA multilayer films were also formed on silica particles, and DNA hollow capsules were obtained following dissolution of the template core.
Films containing DNA are of great interest because they have applications in sensing,1 diagnostics,2 electronics,3 and gene delivery.4 Additionally, for in vivo applications, DNA can be degraded into harmless waste products. To date, DNA films have been limited to a monolayer,1,2 such as in DNA chips, or part of a multicomponent system electrostatically deposited with polycations.5 Herein, we describe the layer-by-layer (LbL) assembly of multilayer films and hollow capsules consisting solely of DNA. LbL films are predominantly assembled by the alternate deposition of positively and negatively charged polymers,6 with film buildup facilitated by electrostatic interactions. However, in this study the DNA layers are held together by hydrogen bonding of the base pairs, and control of the morphology of the film can be achieved by manipulating the extent of hydrogen bonding and the repulsion between the negatively charged phosphate backbone. Although hydrogen bonding and hydrophobic interactions have been used previously to facilitate LbL assembly of uncharged polymers,7 to our knowledge this is the first example of LbL assembly of polyelectrolytes with the same charge. The DNA LbL films were assembled using DNA oligomers with blocks of repeating nucleotides (Scheme 1). Two DNA systems were investigated: poly-adenosine (polyA30) and poly-thymidine (polyT30) and a two-block oligomer system, polyA20G20 and polyT20C20 (see Supporting Information for experimental details). Block polymers of DNA were employed because homopolymeric regions of DNA exhibit * Corresponding author. E-mail:
[email protected]. 10.1021/nl050608b CCC: $30.25 Published on Web 04/20/2005
© 2005 American Chemical Society
Scheme 1. LbL Assembly of a polyAG and polyTC DNA Film, with PEI as the First Adsorbed Layer and polyT as the Electrostatically Adsorbed DNA Layer
a phenomenon known as slippage, where lateral movement of the two DNA strands can occur across the hompolymeric region. Such slippage may assist in the formation and stabilization of the DNA multilayers. The pairing of A with G in one oligomer and T with C in the other was chosen because G and T in the same oligonucleotide can hydrogen bond and could impair the assembly of the film. To follow the assembly of the multilayer DNA films, a quartz crystal microbalance (QCM) was used.8 Figure 1 shows frequency changes for negatively charged polyT30 adsorbing to a positively charged poly(ethyleneimine) (PEI) layer, and the hybridization of polyA20G20 to the polyT30 layer. The electrostatic interaction between polyT30 and PEI
Figure 1. Change in QCM frequency with the adsorption of polyT30 to a PEI layer and the subsequent hybridization of polyA20G20 to a polyT30 layer.
occurs over a short time scale (seconds) and is consistent with the response seen with the buildup of oppositely charged polyelectrolyte layers.9 In contrast, hydrogen bonding between the polyA20G20 and polyT30 is much weaker and the hybridization occurs over a longer time scale (minutes). The logarithmic adsorption curve is consistent with DNA hybridization.10 The addition of an oligonucleotide with a mismatched sequence to the PEI/polyT30/polyA20G20 film did not give a significant change in the QCM frequency (data not shown), indicating the hybridization is base pair specific. The buildup of multilayers can be seen in Figure 2a, which shows the frequency change and mass of the films as a function of layer number. The film made from alternate layers of polyT30 and polyA30 shows a decaying increase in the film mass as a function of layer number. There are two extreme modes of adsorption of DNA onto a surface, with the DNA either lying “flat” on the surface or adsorbing “endon” (see Supporting Information). The adsorption of a “flat” monolayer results in a thinner (and thus lighter) film than the “end-on” monolayer. For electrostatic interactions, “flat” adsorption on the surface would be favored, as the DNA strands can spread across the surface and electrostatically bind to a number of positively charged PEI sites. Using the Sauerbrey equation,11 the mass of polyT30 adsorbed in the first step (120 ng) is consistent with single-stranded DNA (ssDNA) adsorbed as a monolayer flat on the surface. PolyA30 hybridizes to polyT30 with approximately the same molar ratio (105 ng), indicating almost complete hybridization of the polyT sites, effectively forming a monolayer of double-stranded DNA (dsDNA) (see Supporting Information for details). Subsequent addition of polyT30 to the film had a lower level of adsorption (52 ng, approximately 60% of monolayer coverage), as there are few unhybridized polyA sites to bind to, and the hybridization can only occur through competitive hybridization to ds-polyA in the film. Consequently, growth beyond the second layer is restricted. To assist the buildup of the DNA multilayer assembly, a two block system of polyA20G20 and polyT20C20 was employed so that after each adsorption step half of the adsorbed layer should be free to hybridize with a subsequent layer. When the polyA20G20 oligomer hybridized to the polyT 954
Figure 2. (a) Frequency and mass change as a function of layer number and (b) absorbance at 260 nm as a function of layer number. For all samples PEI was the first layer. For polyAG/polyTC, layer 2 was polyT; layers 4, 6 and 8 were polyTC; and layers 3, 5 and 7 were polyAG. For polyT/polyA, even layers were polyT and odd layers were polyA. For the PSS/PAH film even layers were PSS and odd layers were PAH.
surface, a mass increase of 303 ng was observed, which is considerably greater than the mass of a single, “flat” monolayer. When the polyA20G20 hybridizes to the surface, the polyA region will hybridize to the bound polyT, but as there is little or no driving force for the polyG region to hybridize, these strands are likely to extend away from the surface, allowing higher coverage of the surface with polyA (as depicted in Scheme 1). The mass hybridized (303 ng) was equivalent to approximately 47% of the mass expected for “end-on” adsorption of the DNA and is 330% of the mass for “flat” monolayer adsorption. This trend continued regularly for subsequent layers of polyA20G20 and polyT20C20. In contrast to the polyA30/polyT30 system, the polyA20G20/ polyT20C20 film shows a linear increase in film mass with increasing layer number. The total mass of the film was approximately equivalent to the mass of a PSS/PAH film adsorbed under similar conditions with the equivalent layer numbers (Figure 2a). These data clearly demonstrate that DNA hybridization can be used to assemble multilayer films of similarly charged polyelectrolytes. UV-vis spectrophotometry was also employed to follow the buildup of the film by exploiting the absorbance of DNA at 260 nm. As with the QCM results, the multilayer buildup increased linearly with increasing layer number, indicating the formation of DNA multilayer films. Exposure of the films Nano Lett., Vol. 5, No. 5, 2005
Figure 3. AFM images (500 nm × 500 nm) of the polyA20G20/ polyT20C20 film (a) before urea treatment and (b) after 6 M urea treatment.
to PicoGreen, a fluorescent dye specific for dsDNA,12 resulted in highly fluorescent films (data not shown), confirming hybridization of the DNA strands. The ability to control the disassembly of a film has important implications for delivery and sensing applications. The polyA20G20/polyT20C20 film was stable in high ionic strength solutions, as the negative charge of the phosphate backbone is shielded. If the shielding was removed (by treating the film with water), repulsion between the strands caused the film to partially disassemble and 60% of the film mass was lost in 5 min. Some of the lower layers may remain adsorbed to the surface, and it is anticipated that an increase in the number of layers would lead to an increase in the percentage lost from the film. To disrupt the hydrogen bonding, the film was treated with 6 M urea. Urea treatment was performed in a high ionic strength buffer to ensure the negatively charged phosphate groups remained shielded. The treatment resulted in a small loss in mass from the film (7%). Further, AFM imaging of the films showed a distinct change in morphology after urea treatment. Before urea exposure the film had a smooth surface (rms ) 0.41) (Figure 3), but after urea treatment the film exhibited a more undulating surface with morphology consistent with that reported for LbL systems containing DNA alternately deposited with a positive polyelectrolyte (rms ) 0.59).13 We propose that the treatment of the film with a high concentration of urea in the presence of a high ionic strength buffer denatures the DNA; however, the extensive hydrogen bonding in the film, combined with entanglement of the oligomers prevents complete disassembly. Additionally, due to the use of homopolymeric blocks of nucleotide, slippage may also play a role in the stabilization of the film. When the urea is removed, the DNA renatures, but in a more “crosslinked” manner, with hybridization of the oligomers occurring across multiple layers of the film. This reorganization of the film will play an important role in controlling the morphology and release properties of the film. The assembly of an 8-layer polyA20G20/polyT20C20 film on 3 µm silica particles was also investigated. To confirm successful multilayer assembly, the DNA-coated particles were treated with PicoGreen and examined by optical microscopy. The resulting particles (Figure 4a,b) exhibited a highly fluorescent ring on the surface of the particles, indicating a film of dsDNA was present on the surface. Subsequent treatment of the particles with a hydrofluoric acid Nano Lett., Vol. 5, No. 5, 2005
Figure 4. Bright-field (a,c) and fluorescence (b,d) images of silica particles coated with 8 layers of polyA20G20/polyT20C20 (a,b) and the corresponding polyA20G20/polyT20C20 hollow capsules (c,d). The scale bar corresponds to all the images shown.
(HF)/ammonium fluoride (NH4F) buffer (pH 5) dissolved the silica core,14 yielding DNA hollow capsules. Using bright-field illumination the capsules were not visible (Figure 4c), however, when the sample was treated with PicoGreen the dsDNA shell of the capsule was clearly seen (Figure 4d). This confirms the formation of hollow capsules. We have demonstrated that it is possible to build LbL DNA thin films using polymers with the same charge on both planar and colloidal supports. Controlled disassembly of the films was achieved by changing the ionic strength of the solution and morphological changes in the film were achieved by treating the film with urea. Hollow capsules of DNA were formed by assembling the DNA film on silica particles and dissolving the silica core using HF. We are currently incorporating multiple blocks of repeating DNA into the films to further control the morphology of the films, and are studying the film properties with respect to temperature and pH stability, and permeability. These films and capsules are likely to find application in delivery and sensing devices where switchable film morphology is required. Acknowledgment. We thank Elvira Tjipto for AFM imaging and the ARC for funding under the Federation Fellowship and Discovery Project schemes. Supporting Information Available: Experimental details, orientation of adsorbed DNA and the expected monolayer mass coverage. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Boon, E. M.; Ceres, D. M.; Drummond T. G.; Hill M. G.; Barton J. K. Nat. Biotechnol. 2000, 18, 1096. (2) Schouten, S.; Stroeve, P.; Longo, M. L. Langmuir 1999, 15, 8133. 955
(3) Hartwich, G.; Caruana, D. J.; de Lumley-Woodyear, T.; Wu, Y. B.; Campbell, C. N.; Heller, A. J. Am. Chem. Soc. 1999, 121, 10803. (4) Zhang, J.; Chua, L. S.; Lynn, D. M. Langmuir 2004, 20, 8015. (5) Lvov, Y.; Decher, G.; Sukhorukov, G. Macromolecules 1993, 26, 5396. (6) (a) Decher, G.; Hong, J. D. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1430. (b) Decher, G. Science 1997, 277, 1232. (7) (a) Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2717. (b) Serizawa, T.; Kamimura, S.; Kawanishi, N.; Akashi, M. Langmuir 2002, 18, 8381. (8) Qsense D300 device with a flow cell (Va¨stra Fro¨lunda, Sweden). The 5 MHz AT-cut crystal was excited at its 5th overtone.
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(9) Picart, C.; Lavalle, Ph.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J.-C. Langmuir 2001, 17, 7414. (10) Ho¨o¨k, F.; Ray, A.; Norde´n, B.; Kasemo, B. Langmuir 2001, 17, 8305. (11) Sauerbrey, G. Z. Phys. 1959, 155, 206. (12) Chadwick, R. B.; Conrad, M. P.; McGinnis, M. D.; Johnston-Dow, L.; Spurgeon, S. L.; Kronick, M. N. Biotechniques 1996, 20, 676. (13) Shi, X.; Sanedrin, R. J.; Zhou F. J. Phys. Chem. B 2002, 106, 1173. (14) Yu, A.; Wang, Y.; Barlow, B.; Caruso, F. AdV. Mater. 2005, in press.
NL050608B
Nano Lett., Vol. 5, No. 5, 2005
