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Binary DNA Arrays on Heterogeneous Patterned Surfaces Gang Zhang, Xin Yan, Xueliang Hou, Guang Lu, Bai Yang,* Lixin Wu,* and Jiacong Shen Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, 119 Jiefang Road, Changchun 130023, P. R. China Received June 19, 2003. In Final Form: August 8, 2003 Studies of the deoxyribonucleic acid (DNA) molecule at surfaces are driven by both the inherent interest in understanding different aspects of this molecule and its importance in biomimetic materials science and molecular electronics. In this paper, we fabricate binary DNA/surfactant-modified DNA arrays on a heterogeneous patterned surface through twice controlled condensation and dewetting processes. Through the µCP technique, we created a hydrophobic/hydrophilic pattern, using a condensed microdroplet array as a template to fabricate a porous film of polystyrene (PS). With similarity to the “membrane-based patterning” method, this PS porous film was used as a mask, exposed hydrophilic regions inducing DNA to form as an array. After the PS porous films are peeled off, blocked surface sites emerged for the subsequent adsorption with other species. Through another condensation process, where a DNA aqueous solution droplet array could be used as a template, surfactant-modified DNA was sited in the ordered hydrophobic regions through a second dewetting process and hydrophobic interaction, and we fabricated binary arrays on the same surface. At the same time, the double-helical structure and function of DNA still remained. This provides a simple, flexible, and efficient patterning technique for biomolecules.
Introduction Biomacromolecules immobilized on solid surfaces are relevant to many fields of science and technology.1 Studies of the DNA molecules at surfaces are driven by both the inherent interest in understanding different aspects of these molecules and their potential applications in biomimetic materials science and molecular electronics.2 For this purpose, templating has been actively exploited as an effective route to direct and control the processes of assembling DNA. Methods to create DNA arrays on surfaces fall into two broad categories: base-by-base attachment to in situ building up different DNA strands at different sites in the array; the attachment of different complete strands at individual array sites.3 The second method has been used to create arrays, while the attachment chemistry of the DNA strands to the surface is vital. It is the delivery system, such as microcontact printing (µCP),4 microfluidic networks (µFNs),5 pin tool,6 ink jet,7 and photolithographic techniques or the combination of them, that controls the placement of biomolecules onto the array element. In addition, a direct-write “dip-pen” * To whom correspondence should be addressed. E-mail:
[email protected] (B.Y.);
[email protected] (L.W.). Tel: +86-4318924107 (B.Y.). Fax: +86-431-8923907 (B.Y.). (1) Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand Techniques; Academic: San Diego, CA, 1992. (2) (a) Fink, H. W.; Schoonenberger, C. Nature 1999, 398, 407. (b) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775. (c) Kasemo, B. Surf. Sci. 2002, 500, 656. (3) Gillmor, S. D.; Thiel, A. J.; Strother, T. C.; Smith, L. M.; Lagally, M. G. Langmuir 2000, 16, 7223. (4) (a) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1999, 37, 550. (b) Bernard, A.; Renault, J. P.; Michel, B.; Bosshard, H. R.; Delamarche, E. Adv. Mater. 2000, 12, 1067. (c) Hovis, J. S.; Boxer, S. G. Langmuir 2000, 16, 894. (d) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055. (5) (a) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. A. Science 1997, 276, 779. (b) Delamarche, E.; Bernard, A.; Bietsch, A.; Schmid, H.; Michel, B.; Biebuyck, H. A. J. Am. Chem. Soc. 1998, 120, 500. (6) Shalon, D. Genome Methods 1996, 6, 639. (7) Donnell-Maloney, M. J.; Little, D. P. Genet. Anal. Biomol. Eng. 1996, 13, 151.
nanolithography (DPN)8 has been successfully developed to deliver collections of molecules in a positive printing mode by using capillary transport of molecules from the atomic force microscope (AFM) tip to the solid substrate. On the other hand, the surfactant-modified DNA films as new materials with advanced functions attract attention for many practical applications in fields such as nonlinear optical materials, electroluminescent devices, and solidstate dye lasers.9 The most important aspect of this case is that only one molecule can have a seat at one site in the double-stranded structure. The ordered array of surfactant-modified DNA affords a heterogeneous functioned surface; it will extend the application of surfactantmodified DNA, despite receiving little attention until recently. Binary or multiple microarrays on the same surface10,11 are very important for testing the effects on DNA, cells of compounds, and other samples relevant to problems in environmental chemistry, pharmacology, and medicinal chemistry. The fabrication of binary or multiple microarrays is not easy, generally, as low-resolution patterns and “overlay-print” often occur or multi-tips/jets or “many times” operations are required by using the above systems, respectively. Using the “membrane-based patterning” (MEMPAT) method, Whitesides et al. patterned the extracellular matrix protein fibronectin (FN) to the surface and coated the remainder of the surface with a protein that resisted the attachment of cells, so cells were adhered selectively to the former FN pattern.12 It provided a feasible procedure to adhere cells selectively on a heterogeneous surface. (8) (a) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661. (b) Demers, L. M.; Ginger, D. S.; Park, S. J.; Li, Z.; Chung, S. W.; Mirkin C. A. Science 2002, 296, 1836. (c) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702. (9) (a) Kawabe, Y.; Wang, L.; Horinouchi, S.; Ogata, N. Adv. Mater. 2000, 12, 1281. (b) Peterson, O. G.; Snavery, B. B. Appl. Phys. Lett. 1968, 12, 238. (10) Demers, L. M.; Ginger, D. S.; Park, S. J.; Li, Z.; Chung, S. W.; Mirkin, C. A. Science 2002 296, 1836. (11) Chen, C. C.; Yet, C. P.; Wang, H. N.; Chao, C. Y. Langmuir 1999, 15, 6845.
10.1021/la035084e CCC: $25.00 © 2003 American Chemical Society Published on Web 10/07/2003
DNA Arrays on Heterogeneous Patterned Surfaces
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Figure 1. Schematic outline of the procedure to prepare binary arrays of DNA and surfactant DDAB-modified DNA.
In this paper, we have fabricated the binary DNA arrays on a heterogeneous patterned surface through twice controlled condensation of water vapor and controlled dewetting processes. Using this method, various types of molecules with different properties can be selectively placed at specific sites of a pattern within a particular type of micro- and nanostructure by different templates, concentrations, and processes. It provides a simple and efficient patterning technique for biomolecules. In addition, the ordered surfactant-modified DNA should be useful for the tailoring of fluorescent film. Experimental Section Deoxyribonucleic acid (DNA) sodium salts from fish testes and didodecyl dimethylammonium bromine (DDAB) were obtained from Amresco and Aldrich, respectively. Acridine orange (AO), fluorescein isothiocyanate (FITC), and FITC-labeled bovine serum albumin (BSA) were purchased from Sigma. Hexadecanthiol (HDT), 16-mercaptohexadecanoic acid (MHA), and polystyrene (PS) (Mr ) 50 000)were obtained from Aldrich. Silicone elastomer kit (Sylgard 184) was purchased from Dow Corning (Midland, MI). A thin film of gold (∼100 nm thick) was prepared using thermal evaporation onto the glass covers whose surfaces had been primed with thin layer (∼10 nm thick) of chromium. (12) (a) Ostuni, E.; Kane, R.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2000, 16, 7811. (b) Duffy, D. C.; Jackman, R. J.; Vaeth, K. M.; Jensen, K. F.; Whitesides, G. M. Adv. Mater. 1999, 11, 546.
Preparation. The stamp used in the microcontact printing (µCP) method was fabricated by casting poly(dimethyl siloxane) (PDMS) on a photolithographically prepared glass master, which was previously patterned with photoresist. The features of the photoresist pattern were replicated on the PDMS stamp surface after curing, and the PDMS stamp was peeled away carefully and was rinsed with ethanol and heptane for use. Preparation of surfactant DDAB-modified DNA was followed as described in the literature.13 Figure 1 depicts the procedure for fabricating ordered binary arrays of DNA/surfactant-modified DNA on a gold surface. Using the microcontact printing (µCP) method to prepare hydrophilic/ hydrophobic patterns was followed as in previous references.4 After this patterned surface was cooled below the dew point, regularly distributed water droplets were condensed on it. An ordered PS porous film was fabricated by dipping this water droplet-patterned surface in a PS solution of chloroform and immediately withdrawing followed by complete evaporation of chloroform and water. Then it was placed in a DNA aqueous solution vertically and evaporating water at 40 °C. After the PS porous film was peeled off from substrate with adhesive tape, DNA patterns were left on the surface. Then the DNA-patterned surface was cooled below the dew point again, and water droplets condensed on the DNA region and formed a DNA solution droplet array. As a template, this DNA solution droplet-patterned surface was dipped in a DDAB-modified DNA solution of chloroform and immediately withdrawn. After chloroform and water evaporated completely at room temperature, an ordered binary DNA/DDAB(13) (a) Tanaka, K.; Okahata, Y. J. Am. Chem. Soc. 1996, 118, 10679. (b) Ijiro, K.; Okahata, Y. J. Chem. Soc., Chem. Commun. 1992, 1339.
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Figure 2. Height image (left) and friction image (right) in LFM of typical patterned SAMs on gold surface. modified DNA array was left on the gold surface. Fluorophore ethidium bromide, acridine orange, and fluorescein isothiocyanate (FITC) were used to label DNA and DDAB-modified DNA for fluorescence imaging. Characterization. The optical images were recorded on a XSP-BM optical microscope (in reflection mode) and were captured with a Panasonic color charge-coupled device (CCD) and digitized with a frame grabber. Atomic force microscopy (AFM) observations of the sample surfaces were carried out with the commercial instrument (Digital Instrument, Nanoscope IIIa, Multimode), under ambient conditions at room temperature. AFM height images and lateral force microscopy (LFM) friction images were simultaneously recorded on contact mode AFM. Triangular Si3N4 cantilevers with pyramidal tips purchased from a Nanosensor were used. All tapping mode images were measured at room temperature in air with the microfabricated rectangle crystal silicon cantilevers (Nanosensor). Topography images were obtained using a resonance frequency of approximate 365 kHz for the probe oscillation. Confocal microscopic Raman spectra were obtained with a Renishaw Raman imaging microscope system 1000 spectrometer equipped with a Ge detector. The spectral resolution was 4 cm-1, and the laser power at the sample point was kept below 50 mW for the films. A Bio-Rad Radiance 2100 confocal laser scanning microscope (CLSM, England) was used to examine the labeled DNA and DDAB-modified DNA on the patterned surface. Specimens were exited at 488 nm.
Results and Discussion Fabrication of the Ordered Porous Polymer Films. By use of microcontact printing (µCP) and self-assembly techniques, SAMs of alkanethiolates terminated with COOH and CH3 were patterned on gold surfaces on the micrometer scale.14 From the contact mode AFM (LFM) friction image, we can measure different geomorphologies and properties between microdomains of the surface. Figure 2 displays AFM height and LFM friction images of typical patterned SAMs on a gold surface used in our experiment. Different from the smoothness in height image (both MHA and HDT are SAMs), a pattern can be seen in the friction image. The contrast in the friction image can be owed to the fact that the uncoated hydrophilic Si3N4 tip may generate a stronger friction force at the hydrophilic area than that at the hydrophobic area.15 The domains of ordered brighter circles and darker background represented the hydrophilic HOOC-terminated SAM and the hydrophobic CH3-terminated SAM, respectively. After this patterned surface was cooled below the dew point, regularly distributed water droplets were condensed on it. Figure 3 (left) shows an optical photograph of a (14) (a) Zhao, X. M.; Wilbur, J. L.; Whitesides, G. M. Langmuir 1996, 12, 3257. (b) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498. (15) (a) Takano, H.; Kenseth, J. R.; Wong, S. S.; O’Brien, J. C.; Porter, M. D. Chem. Rev. 1999, 99, 2845. (b) Green, J. B. D.; McDermott, M. T.; Porter, M. D.; Siperko, L. M. J. Phys. Chem. 1995, 99, 10960. (c) Kendall, K. Nature 1986, 319, 203.
Figure 3. Optical microscopy (OM) images of a water droplet array (left) and AFM height image (tapping mode) of porous PS film (right).
regular array of condensed water droplets formed on the patterned gold surface due to the preferential nucleation and growth of water droplets within the hydrophilic areas during the condensation process.16 The patterned condensation figure reflects the lateral pattern of varying wettability on the gold surface just like the friction image of LFM.17 Subsequently, a liquid film of polymer solution was deposited on the water-patterned surface by dipping it in a dilute chloroform solution of PS and withdrawing immediately. After chloroform and water evaporated completely at room temperature, an ordered porous PS film was left on the gold surface.18 When PS solutions with concentration from 2 to 20 mg/mL were used, 2D ordered porous polymer films were formed on the surface. Figure 3 (right) shows an AFM image of the sample that was prepared from a 10 mg/mL PS solution. It reveals that the film possessed a position-dependent thickness. Raised rims (about 380 nm in height) surrounding each hole appeared in the film, while film far from the holes was thinner (approximate 97 nm in thickness). It is clear that the chloroform solution preferentially dewet from water droplets according to the heterogeneous nucleation mechanism,19 exposing the regularly arrayed water droplets to air and giving rise to the raised rims surrounding them. When solutions with concentrations below 1 mg/ mL were used, a second dewetting occurred and 2D ordered polymer rings rather than porous films were formed on the surface.20 On the basis of the above results, it is clear that the size, shape, area, and uniformity of the ordered porous films are stable when the concentration of PS solution is above 2 mg/mL, and a high concentration of PS solution can increase the thickness of PS porous films. For the 2D pattern size, the condensation process and the size of condensed water droplets are crucial. DNA Array on the Patterned Surface. The above ordered porous film was dipped in a 2.6 mg/mL DNA solution with vertical placement, and after evaporation of water at 40 °C, the DNA molecules were deposited on the HD-patterned gold substrate. During the deposition process, the strong capillary forces at a meniscus between the substrate and the DNA solution would induce hy(16) (a) Gau, H.; Herminghaus, S.; Lenz, P.; Lipowsky, R. Science 1999, 283, 46. (b) Herminghaus, S.; Gau, H.; Mo¨nch, W. Adv. Mater. 1999, 11, 1393. (c) Kumar, A.; Whitesides, G. M. Science 1994, 263, 60. (17) Lo´pez, G. P.; Biebuyck, H. A.; Frisbie, C. D.; Whitesides, G. M. Science 1993, 260, 647. (18) Braun, H. G.; Meyer, E. Thin Solid Film 1999, 345, 222. (19) Lu, G.; Li, W.; Yao, J. M.; Zhang, G.; Yang, B.; Shen, J. C. Adv. Mater. 2002, 14, 1049. (20) (a) Karapanagiotis, I.; Evans, D. F.; Gerberich, W. W. Langmuir 2001, 17, 3266. (b) Thiele, U.; Mertig, M.; Pompe, W. Phys. Rev. Lett. 1998, 80, 2869.
DNA Arrays on Heterogeneous Patterned Surfaces
Figure 4. AFM height image (tapping mode) of DNA-patterned surface (left) and confocal microscopic Raman spectra (right) of (a) pure DNA powder, (b) DNA-patterned surface, and (c) SAM on the Au surface.
Figure 5. Confocal fluorescence images of the acridine orange labeled DNA-patterned surface (left) and the FITC-labeled BSApatterned surface (right).
drophilic DNA to assemble into the bottom of pores.21 After the porous PS film and excess DNA on it were peeled off from substrate, a DNA array was fabricated on the surface. Figure 4 (left) shows the AFM (tapping mode) of ordered arrays of DNA on the patterned surface. From the image, it can be seen that there is clear pattern on the surface; the size of the DNA pattern cell is about 4 µm in diameter and 150 nm in height. The same sample was measured by confocal microscopic Raman spectroscopy; the confocal laser can be focused on the dispersed domains and the continuous region for collect Raman spectra, respectively. From the Raman spectra (Figure 4, right), we can see that, in comparison to the Raman spectrum of pure DNA molecules (a), there are typical bands of DNA (such as 1683.6, 1571.0, and 1486.6 cm-1) occurring in the spectrum of dispersed domains (b), while there is no signal at the continuous region (c). It can be confirmed that DNA molecules were adsorbed on the dispersed circular domains and were not adsorbed on the other regions. For the observation of pattern sizes and shapes, fluorophore acridine orange and FITC were used as labels to visualize the patterns. In Figure 5 (left), the magnified confocal fluorescence image showed that the AO-labeled DNA spatially localized onto the patterned surface and were arrayed in a red fluorescence pattern. The size and shape of the pattern were corresponding to its AFM image. In addition, FITC-labeled BSA was employed to extend this method. In Figure 5 (right), the fluorescence image showed an ordered green FITC-BSA array. When the DNA solutions of concentrations from 0.1 to 3.0 mg/mL were used, different thicknesses from 5 to 180 nm on the patterned surface could be observed. Except for size and shape of patterns controlled by µCP technique, the concentration of the DNA solution is a very important (21) (a) Jiang, P.; Bertone, J. F.; Hwang, K. S.; Colvin, V. L. Chem. Mater. 1999, 11, 2132. (b) Vlasov, Y. A.; Bo, X. Z.; Sturm, J. C.; Norris, D. J. Nature 2001, 414, 289. (c) Gu, Z. Z.; Fujishima, A.; Sato, O. Chem. Mater. 2002, 14, 760.
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Figure 6. AFM height images and their cross section of binary DNA arrays fabricated from 12 mg/mL (left) and 6 mg/mL (right) DNA-DDAB solution.
factor for the figuration and microstructure of the pattern on the heterogeneous surface. So it was applied in wider fields than that for a monolayer DNA pattern created by the simple µCP technique using the same stamp. We can fabricate various DNA arrays with different patterns and structures by use of different templates with suitable concentrations of DNA solution. So it gives a chance for the further application of DNA in both basic and biotechnological science. Binary DNA Arrays on the Patterned Surface. The surfactant-modified DNA still retained the double-helical structure and function of DNA though it is immiscible with aqueous solutions.13 Since surfactant-modified DNA is soluble in most organic media such as chloroform, it is possible to array it on the hydrophobic regions of a patterned surface. Just like the fabrication of the ordered porous polymer films, when a hydrophilic/hydrophobic patterned surface was cooled below the dew point, water droplets condensed on the hydrophilic domains and formed a water droplet array. This water droplet-patterned surface was dipped in a DDAB-modified DNA solution of chloroform and immediately withdrawn. After chloroform and water evaporated completely at room temperature, an ordered DDAB-modified DNA array was left on the gold surface. For the previous DNA-patterned surface remaining with the original hydrophilic/hydrophobic pattern, it is possible to subsequently array hydrophobic DDAB-modified DNA on the hydrophobic regions. The DNA-patterned surface was cooled below the dew point again, and water droplets condensed on the DNA region and formed a DNA solution droplet array. As a template, this water droplet-patterned surface was dipped in a 12 mg/mL DDAB-modified DNA solution of chloroform and immediately withdrawn. After chloroform and water evaporated completely at room temperature, ordered binary DNA/DDAB-modified DNA arrays were left on the gold surface. Figure 6 (left) shows the AFM height image and its cross section of the resulting binary surface. It is very clear that surrounding the dispersed DNA domains a continuous film of DNA-DDAB complex with dispersed raised rims was formed. Just as for PS, the resulting patterned surface structure could be affected by the concentration of DDAB-modified DNA solution. Thinner DNA-DDAB complex film (about 135 nm thinner than DNA domains) corresponds to the lower concentration (6 mg/mL) of DNA-DDAB solution (Figure 6, right). Confocal microscopic Raman spectroscopy was used to measure the above binary DNA arrays. The confocal laser can be focused on the dispersed domain and continuous
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Figure 8. AFM height image and phase image of a binary DNA surface in its condensation process, lacking condensation, and/or the evaporation after condensation occurred.
Figure 7. Confocal microscopic Raman spectra (top) of (a) dispersed domain and (b) continuous region of binary DNA arrays and the fluorescence image of DNA-acridine orange/ DDAB-modified DNA-FITC binary arrays (bottom).
region for collect Raman spectra, respectively. From the Raman spectra of the dispersed domain and continuous region (Figure 7 top), we can see that there were typical bands of DNA and DDAB-modified DNA occurring in the spectra of the dispersed domain and continuous region, respectively. These results confirm that the binary arrays were made from DDAB-modified DNA at continuous regions and DNA in the dispersed areas, and the dispersed DNA domains were not covered by DDAB-modified DNA. For the direct observation of binary patterns, acridine orange and fluorescein isothiocyanate (FITC) were used for labeled DNA and DDAB-modified DNA, respectively, to visualize the binary fluorescence pattern. From Figure 7 (bottom), a confocal fluorescence image, we can see that the red-dispersed domains were surrounded with green continuous regions. Compare with the red pattern of DNA, the green pattern just fills the continuous regions between the dispersed DNA domains exactly. So this confirms the binary pattern we prepared. At the same time, bright rings could be seen at boundaries between two regions due to stronger fluorescence intensity of raised rims and a few diffuse fluorophores. In addition, since the surfactant prefers to set to the interface between hydrophobic and hydrophilic phases, it is possible that trace-free surfactant may induce local miscibility at the interface of two phases when the modified DNA phase was added, thereby blurring the edge resolution. The binary structure of DNA was influenced by condensation and dip coating processes significantly, since the porous structure was a replica of water droplets condensed on the hydrophilic DNA domains. Lacking condensation or the evaporation of water after condensation could lead to the decrease in the diameter of the dispersed domains, and furthermore, they could be covered by a continuous region. Figure 8 shows the AFM image
of the binary structure of DNA; a homogeneous surface with ordered higher domains could be seen from its phase image. It is clear that the entire surface of dispersed DNA domains was covered by DDAB-modified DNA. We have patterned not only DNA/surfactant-modified DNA binary arrays but also bovine serum albumin/ surfactant-modified DNA binary arrays on a patterned surface using this method. This could be easily extended to fabricate ordered binary arrays made of other biomolecules. The only requirement seems to be that one must be dissolved in water and the other one dissolved in a volatile organic solvent. Conclusion In summary, we have fabricated DNA arrays with different patterns and structures by use of different templates with suitable concentrations of DNA solution. It was applied in wider fields than that monolayer DNA pattern created by the simple µCP technique using the same stamp. Subsequently, we modified another hydrophilic DNA, transferred it into organic solution, and arrayed it on the hydrophobic regions of patterned surface to fabricate the binary DNA/surfactant-modified DNA pattern on the same surface; at the same time, the doublehelical structure and function of DNA still remained. This twice controlled condensation and dewetting process extends the applications under different conditions and environments of the species. It is a simple and inexpensive binary patterning technique for biomolecules, which should be useful for the arrays of DNA, proteins, cells, and other biomaterials, testing the effect on DNA, cells of compounds, and other samples relevant to problems in environmental chemistry, pharmacology, and medicinal chemistry. The surfactant-modified DNA patterned films as new materials with advanced functions may be applied in nonlinear optical materials, electroluminescent devices, and solid-state dye lasers fields. Acknowledgment. This work was supported by the NSFC (29925412, 50073007 and 20204003) and the Major State Basic Research Development Program (G2000078102). We thank Prof. Changyou Gao and Dr. Jie Feng for the help with the confocal laser scanning microscope (CLSM). LA035084E
