Honeycomb-Patterned Films Fabricated by Self-Organization of DNA

Aug 13, 2009 - In this paper, DNA-based honeycomb films were successfully ... Investigation of the effects of substrate, concentration, and solvent on...
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Honeycomb-Patterned Films Fabricated by Self-Organization of DNA-Surfactant Complexes Hang Sun, Wen Li, and Lixin Wu* State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, P. R. China

Langmuir 2009.25:10466-10472. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/17/19. For personal use only.

Received January 26, 2009. Revised Manuscript Received July 17, 2009 In this paper, DNA-based honeycomb films were successfully constructed from a simple solution casting process at high relative humidity, through the encapsulation of DNA with a cationic surfactant ditetradecyldimethylammonium (DTDA). Cationic surfactants electrostatically attach to phosphate anions of DNA in a molar ratio of 1:1 in the DNA-DTDA complex. Investigation of the effects of substrate, concentration, and solvent on the morphology of the microporous films demonstrated the wide generality and high reproducibility of the formation of DNA-DTDA selforganized microporous films. The morphology of the microporous structures can be adjusted by slightly changing the concentration of the complex solution. DNA exists in the double helical B-form in the microporous film confirmed by circular dichroism spectrum. Dye molecule rhodamine B was loaded into the DNA-DTDA complex, presenting a fluorescent microporous film. DNA-DTDA complex as a host material provides a new method for tailoring fluorescent microporous films. The DNA-based ordered honeycomb films should be attractive for use in optical and optoelectronic devices, separation, ion transportation, and biosensors.

Introduction DNA is not only an important biological material, but is also a fascinating anionic polyelectrolyte with a unique double helical structure. However, film materials prepared from DNA are often water-sensitive and have insufficient mechanical strength for device applications. To incorporate DNA into various organized systems and perform an assortment of functions, cationic surfactants are usually applied to modify its surface properties by interacting with counterions present in the DNA molecule.1 The formed DNAsurfactant complexes are no longer soluble in water but are readily dissolved in organic solvents and will continue to maintain its double-stranded structure.1,2 DNA-surfactant complexes present a new type of material capable of demonstrating several advanced functions. These complexes have attracted a great deal of attention for practical applications in many fields such as nonlinear optical materials, electroluminescent devices, and solid state dye lasers.3,4 Highly ordered thin films of DNA-based assemblies have been previously prepared by a variety of methods including spin coating, simple casting, and the Langmuir-Blodgett technique.5-8 Furthermore, the hydrophobicity of DNA-surfactant complexes provides a possibility for the construction of heterogeneous functioned surfaces, although little attention to this topic has been received. By employing microcontact printing techniques and *To whom correspondence should be addressed. E-mail: [email protected]

(1) Ijiro, K.; Okahata, Y. J. Chem. Soc., Chem. Commun. 1992, 1339–1341. (2) Steckl, A. J.; Spaeth, H.; Singh, K.; Grote, J.; Naik, R. Apply. Phys. Lett. 2008, 93, 193903. (3) Steckl, A. J. Nat. Photonics 2007, 1, 3–5. (4) Kawabe, Y.; Wang, L.; Horinouchi, S.; Ogata, N. Adv. Mater. 2000, 12, 1281–1283. (5) Neumann, T.; Gajria, S.; Tirrell, M.; Jaeger, L. J. Am. Chem. Soc. 2009, 131, 3440–3441. (6) Smitthipong, W.; Neumann, T.; Gajria, S.; Li, Y.; Chworos, A.; Jaeger, L.; Tirrell, M. Biomacromolecules 2009, 10, 221–228. (7) Hou, X.; Sun, L.; Xu, M.; Wu, L.; Shen, J. Colloids Surf., B 2004, 33, 157– 163. (8) Tanaka, K.; Okahata, Y. J. Am. Chem. Soc. 1996, 118, 10679–10683. (9) Zhang, G.; Yan, X.; Hou, X.; Lu, G.; Yang, B.; Wu, L.; Shen, J. Langmuir 2003, 19, 9850–9854.

10466 DOI: 10.1021/la900322d

dewetting processes, we have fabricated binary surface patterns of DNA/DNA-surfactant complexes on solid substrates.9 Ordered microporous structures have attracted considerable attention recently due to their potential application in diverse areas such as membranes, photonic, or optoelectronic devices, and catalysis.10-14 The breath figure technique provides a selforganized method for the preparation of ordered microporous films with submicro- and nanometer scales. This is accomplished by providing humid conditions to the surface of polymer solution in a volatile solvent.15,16 In this technique, condensed water droplets form as a result of rapid cooling at the solution surface due to solvent evaporation. These droplets will then self-organize into a well-ordered, hexagonal array that acts as a template directing the formation of ordered microporous structures. By using this method, a variety of materials such as surface modified nanoparticles as well as linear, star-like, block, amphiphilic, and conjugated polymers, have been successfully employed for the fabrication of ordered microporous films.16 Of particular interest is the incorporation of biomaterials into microporous structures for both medical and nonmedical uses. However, biomolecules usually need to be modified by polymers through covalent linkage, and multistep reactions not only could cause the biomolecules to deactivate, but also seriously limit applications.17,18 The amphiphilic character of DNA-surfactant complexes as well as their large molecular weights, make DNA-surfactant complexes good candidates for the preparation of DNA-based ordered (10) Tanev, P. T.; Chibwe, M.; Pinnavaia, T. J. Nature 1994, 368, 321–323. (11) Jiang, P.; Hwang, K. S.; Mittleman, D. M.; Bertone, J. F.; Colvin, V. L. J. Am. Chem. Soc. 1999, 121, 11630–11637. (12) Wijnhoven, J. E. G. J.; Vos, W. L. Science 1998, 281, 802–804. (13) Imada, M.; Noda, S.; Chutinan, A.; Tokuda, T.; Murata, M.; Sasaki, G. Apply. Phys. Lett. 1999, 75, 316–318. (14) Hulteen, J. C.; Jirage, K. B.; Martin, C. R. J. Am. Chem. Soc. 1998, 120, 6603–6604. (15) Widawski, G.; Rawiso, M.; Francois, B. Nature 1994, 369, 387–389. (16) Bunz, U. H. F. Adv. Mater. 2006, 18, 973–989. (17) Stenzel, M. H. Aust. J. Chem. 2002, 55, 239–243. (18) Kadla, J. F.; Asfour, F. H.; Bar-Nir, B. Biomacromolecules 2007, 8, 161– 165.

Published on Web 08/13/2009

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microporous films. More interestingly, dyes could intercalate into DNA, giving rise to an increasingly multifunctional, self-assembling film.19,20 Based on previous studies of the surface patterning of organicinorganic hybrid complexes,21-23 we herein demonstrate the selforganization of DNA into honeycomb structures on solid substrates by a simple solution casting of DNA-ditetradecyldimethylammonium (DTDA) complex at high relative humidity. We have systematically investigated the effects of the substrate type, DNA-DTDA complex concentration, solvent type, and two different DNA-surfactant complexes on the morphology of the microporous films. Furthermore, we have loaded the dye molecule rhodamine B (RhB) into the DNA-DTDA complex and obtained a fluorescent honeycomb film. The present research draws a convenient route to fabricate DNA-based honeycomb films, and establishes the multicomponent self-assembly in honeycomb films to endow the DNA-surfactant complex with fluorescent properties, providing the films with increased functionality.

Experimental Section Materials. Fish testes DNA-sodium salt (DNA 3 Na) fragment was purchased from AMRESCO and used as received. Ditetradecyldimethylammonium bromide (DTDA 3 Br) and didodecyldimethylammonium bromide (DDDA 3 Br) were synthesized in the previous work.22 Rhodamine B (RhB) and poly(vinylphosphonic acid) (PVPA) were purchased from the British Drug Houses Ltd. and Sigma-Aldrich, respectively. The distilled water (Milli-Pore 18.2 MΩ/cm) and chloroform of analytical grade (water content: ca. 0.03 wt %) (Beijing Chemical Works) were used in the experiments. The calculation for DNA concentration in the experiment was based on the method used by Shoyab,24 by assuming each purine and pyrimidine base in DNA occurs in an equal ratio. Preparation of DNA-Surfactant Complexes. The DNAsurfactant complexes were prepared by replacing sodium counterions of DNA with cationic surfactants. DNA-DDDA complex was synthesized and characterized in the previous work.7 DNADTDA complex was prepared as described in the literature with a simple modification. Approximately 0.175 g (0.338 mmol) of DTDA 3 Br in 200 mL of distilled water was stirred at 80 °C for 2 h and cooled to room temperature, resulting in the complete dissolution of DTDA 3 Br in water. Next, 20 mL of aqueous solution containing 0.1 g (0.154 mmol/base pair) of DNA 3 Na was added to the DTDA 3 Br aqueous solution and stirred for 12 h at room temperature. The precipitate was collected by filtration, washed five times with distilled water, and then dried under vacuum at 25 °C for 48 h. IR analysis (KBr, cm-1) for DNADTDA: ν =3209, 2925, 2854, 1697, 1651, 1468, 1246, 1062, 722, 528. Anal. Calcd. for DNA-DTDA ((C5H5N5)0.25(C5H5N5O)0.25(C4H5N3O)0.25(C5H6N2O2)0.25C5O5PH7C30H64N,H76.25C39.75N4.75O6P, 747.78): C 63.85, H 10.28, N 8.90, by assuming each purine and pyrimidine base exists in DNA in an equivalent ratio.24 Found: C 63.38, H 9.80, N 8.45. PVPA-DTDA complex was prepared for the control experiment according to the similar procedure by initially neutralizing PVPA with aqueous NaOH solution. Then, 50 mg (0.097 mmol) of DTDA 3 Br in 60 mL of distilled water was stirred at 80 °C for 2 h and cooled to room temperature, resulting in the complete dissolution of DTDA 3 Br in water. Next, 10 mL of aqueous solution containing 5.2 mg (19) Okahata, Y.; Ijiro, K.; Matsuzaki, Y. Langmuir 1993, 9, 19–21. (20) Hou, X.; Xu, M.; Wu, L.; Shen, J. Colloids Surf., B 2005, 41, 181–187. (21) Bu, W.; Li, H.; Sun, H.; Yin, S.; Wu, L. J. Am. Chem. Soc. 2005, 127, 8016– 8017. (22) Sun, H.; Li, H.; Bu, W.; Xu, M.; Wu, L. J. Phys. Chem. B 2006, 110, 24847– 24854. (23) Sun, H.; Li, H.; Wu, L. Polymer 2009, 50, 2113–2122. (24) Shoyab, M. Biochem. Arch. Biophys. 1979, 196, 307–310.

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(0.048 mmol/repeating unit) of PVPA was treated with NaOH aqueous solution to adjust the pH of the solution to 13. Then, the PVPA solution was added to the aqueous DTDA 3 Br solution and was stirred for 12 h at room temperature. The precipitate was collected by filtration, washed five times with distilled water, and then dried under vacuum at 25 °C for 48 h. We have also tried to adjust the pH of the aqueous PVPA solution to 9 and 11 for complexing with DTDA, however, no evident precipitate formed after mixing.

Preparation of RhB-Loaded DNA-DTDA Complex. RhB-loaded DNA-DTDA complex was prepared according to the literature.20 A 10 mL solution of DNA-DTDA chloroform solution (0.75 mg/mL) and a 10 mL solution of RhB chloroform solution (11.75 μg/mL) were mixed together at room temperature (the molar ratio of DNA phosphate to dye, P:D =40:1). The mixed solution was allowed to stand overnight in a dark environment to ensure all of dye molecules bound to the DNA. RhBloaded DNA-DTDA complex was obtained by evaporating the chloroform to dryness. Then, the sample was further dried under vacuum at 25 °C until its weight remained constant. RhB-loaded PVPA-DTDA complex and RhB-loaded polymethylmethacrylate (PMMA) were prepared according to the same procedure for the control experiments. Preparation of Honeycomb-Patterned Films. Typically, the microporous thin films were prepared by direct casting 20 μL of DNA-DTDA complex or dye-loaded complex chloroform solution (0.75 mg/mL) onto the glass substrates under a moist airflow. The humid conditions were achieved by bubbling nitrogen gas through a water-filled conical flask. As the nitrogen gas percolates through the water, it becomes saturated with water vapor (temperature: 25 °C), and exits the flask through a glass nozzle and is vertically applied above the surface of the sample solution. The airflow was controlled at 200 mL/min, which was monitored by a flow meter to ensure reproducibility. The relative humidity of the moist airflow is 85% which was confirmed by a hygrometer. The internal diameter of the glass nozzle used to apply moist airflow is 0.6 cm, and the distance between the solution surface and the nozzle is ca. 1.2 cm. The white thin films covering an area of ca. 1 cm2 were left behind after the complete evaporation of the solvent and water within 30-60 s. The control experiments without humid airflow have been conducted under ambient atmosphere (relative humidity: 20-30%) and no microporous structures developed, leaving only unpatterned flat films as a result. Measurements. The optical photographs were taken with an Olympus BX-51 optical microscope (OM). Scanning electron microscopic (SEM) images were collected on a JEOL JSM6700F field emission scanning electron microscope. Atomic force microscopy (AFM) images were carried out on a commercial instrument (Digital Instrument, Nanoscope III, and Dimension 3000TM) in tapping mode at room temperature in air. Each image was confirmed by measurements with three films in at least five different well-separated 20  20 μm2 sites. Transmission electron microscopy (TEM) images were obtained with a JEOL-2010 electron microscope operating at 200 kV. Dynamic light scattering (DLS) was performed on a DAWN EOS enhanced optical system (Wyatt Technology Corporation). UV-vis spectra were carried out on a Shimadzu 3100PC spectrometer. Circular dichroism (CD) measurements were performed with a JASCO J-810 spectropolarimeter. Solution samples were measured using a 1 cm quartz cell at the same base concentration ([DNA]=300 μM/base pair), and background correction was performed using the quartz cell filled with the corresponding solvent. Films were prepared on the 1  3 cm quartz plates for measurement. The honeycomb-patterned film was measured at relative humidity >60% and the common casting film was measured under ambient atmosphere (relative humidity: 20-30%). The high humidity was achieved by putting a bottle of water in the sample chamber, and measuring the humidity by a hygrometer. As the relative humidity rose to >60%, we then took our measurements. DOI: 10.1021/la900322d

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Figure 1. (A) Chemical structure and (B) schematic drawing of DNA-DTDA complex. Background correction was performed using a clean quartz plate. Three measurements were performed and averaged for each sample. The signals of each sample have been normalized for the weight and concentration of DNA. Fourier transform infrared (FT-IR) spectral measurements were completed on a Bruker IFS66 V FT-IR spectrometer equipped with a DGTS detector (32 scans for solids in KBr pellets, and 128 scans for films on CaF2 slides), and the spectra were recorded with a resolution of 4 cm-1. Element analysis (C, H, N) was carried out on a Flash EA1112 analyzer from ThermoQuest Italia SPA 1H NMR (TMS) spectra were recorded on a Bruker UltraShieldTM 500 MHz spectrometer. X-ray photoelectron spectroscopy (XPS) measurements were carried out on an ES-CALAB Mark (VG Company, U.K.) photoelectron spectrometer using a monochromatic Al KR X-ray source. Luminescence measurements were performed on a HORIBA Jobin Yvon FL3-TCSPC fluorescence spectrophotometer. X-ray diffraction (XRD) was performed on a Rigaku X-ray diffractometer (D/max rA, using Cu KR radiation at a wavelength of 1.542 A˚), and the data were collected from 0.7° to 10°.

Results and Discussion

Figure 2. FT-IR spectra of (A) native DNA (a), DTDA 3 Br (b), and DNA-DTDA complex (c) in solid state (KBr), and (B) DNA-DTDA microporous film (a) and DNA-DTDA common casting film (b) on CaF2 slides.

Figure 3. 1H NMR spectra of DTDA 3 Br (top) and DNADTDA complex (bottom) in CDCl3 (TMS).

Characterization of DNA-DTDA Complex. DNADTDA complex was prepared by mixing two aqueous solutions of DNA 3 Na and DTDA 3 Br at room temperature (Figure 1). The synthetic procedure was described in detail in the Experimental Section. The as-prepared complex is no longer soluble in water but readily dissolved in organic media such as chloroform, dichloromethane, benzene, and so forth, giving transparent solutions. This feature implies that the hydrophilic surfaces of DNA have been successfully modified by the hydrophobic alkyl chains of surfactants. For DNA-DTDA complexes, the typical vibration absorptions of DNA and DTDA appear in the IR spectrum, indicating the formation of the complex (Figure 2).1 Furthermore, we focused on the CdO stretching modes in the 1800-1500 cm-1 region to examine the DNA structure in the complex. The band centered at 1690 cm-1 arises due to the C6dO6 stretch of base paired guanine plus C2dO2 stretch of uracil and thymine. The band centered at 1660 cm-1 arises mainly from the stretching vibrations of C6dO6 of free guanine, C2dO2 of free cytosine, and C4dO4 of free uracil or thymine. A double strand to single strand transition results in a decrease in the intensity of the band at approximately 1690 cm-1 with a concomitant increase of the band at approximately 1660 cm-1.25,26 DNA-DTDA complex exhibits a stronger absorption at 1697 cm-1 relative to the band at 1652 cm-1, implying the double-stranded state of DNA in the DNA-DTDA complex. It is noted that in our case, surfactantfree or “native” DNA shows a little stronger absorption at 1652 cm-1 relative to the band at 1697 cm-1, which is different from the literature results,25,26 implying that there are some native DNA molecules that have potentially become single-stranded. We think the pelletization process of the native DNA for IR measurement causes the native DNA to become single-stranded,

and in the case of DNA-DTDA complex, cationic surfactants protect the double strands of DNA to remain annealed to one another during the pelletization process. Ogata and co-workers reported that “cationic surfactants can protect the conformation of the DNA double strands from change in ethanol solution and in films” according to the CD spectra.27 In our manuscript, IR experiments demonstrate that it is easy for DNA to remain in its double-stranded structure in the DNA-DTDA complex in KBr pellets compared with the “native” DNA in KBr pellets. Moreover, 1H NMR spectra also confirm the supramolecular complexation of DTDA with DNA (Figure 3). Only the signals of the protons of DTDA are found clearly while the signal of DNA could not be observed at all due to the limited molecular motion. After the electrostatic complexation, the triplet peaks at 3.503.53 ppm representing -NCH2 and singlet peak at 3.41 ppm representing -NCH3 of DTDA shift to the higher field at 3.31 ppm, and become a broadened single peak due to the electrostatic interaction. Meanwhile, the proton peaks of other CH2 groups near the headgroup shift to high field comparing with that of DTDA 3 Br. These data indicate that the head groups of DTDA are bound to DNA through electrostatic interactions, and the tight complexation of the head groups to DNA influences the motion of the hydrocarbon chains of the complex in solution.7 XPS measurements did not detect the signals of Na(1s) ions from DNA or Br(3d) ions from DTDA, suggesting that in the DNADTDA complex, all phosphate anions interact with DTDA cations, and no residual Br- or Naþ exist within the DNADTDA complex (Supporting Information (SI) Figure S1). Furthermore, 1H NMR results also confirm that there are no residual DTDA molecules which have not been complexed with DNA in the production of the DNA-surfactant complex. Therefore, we believe that the cationic surfactants attach to phosphate

(25) Banyay, M.; Sarkar, M.; Gr€aslund, A. Biophys. Chem. 2003, 104, 477–488. (26) Cristofolini, L.; Berzina, T.; Erokhin, V.; Tenti, M.; Fontana, M. P.; Erokhina, S.; Konovalov, O. Colloids Surf., A 2008, 321, 158–162.

(27) Wang, L.; Yoshida, J.; Ogata, N.; Sasaki, S.; Kajiyama, T. Chem. Mater. 2001, 13, 1273–1281.

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Figure 5. Honeycomb-patterned film prepared from 0.75 mg/mL of DNA-DTDA complex: (A) SEM image, (B) high-magnified SEM image of (A) tilted 60°, and (C) AFM image.

Figure 4. CD spectra of (a) pure DNA in aqueous buffer solution (20 mM NaCl, 10 mM Tris, pH 7.8), (b) DNA-DTDA complex in chloroform, and (c) DNA-DTDA microporous film.

anions of DNA in a strict 1:1 ratio. The result of elemental analysis also supports the fact that cationic surfactants and phosphate anions form a 1:1 complex in the DNA-DTDA complex, by assuming each purine and pyrimidine base exists in DNA in an equal ratio.24 To further validate that DNA-surfactant complexes retain its double helical structure in chloroform, UV-vis spectra were used to confirm that DNA-DTDA complexes exhibit a nearly identical absorption coefficient in chloroform when compared to native DNA in aqueous solution (SI Figure S2).27 To gain insight into the change in the secondary structure of DNA, CD spectra of DNA in aqueous solution and DNA-DTDA complex in chloroform were conducted as presented in Figure 4. For DNA in aqueous buffer solution (20 mM NaCl, 10 mM Tris, pH 7.8), there is a positive Cotton effect at 279 nm and a negative Cotton effect at 246 nm, which indicates the B-form of the DNA double helix. In the case of DNA-DTDA complex in chloroform (water content: ca. 0.03 wt %), the positive Cotton effect at 279 nm shifts to 290 nm with the decrease in θ value compared with that of native DNA in aqueous solution, implying the C-form of the DNA double helix.7,8 Recently, Steckl and co-workers reported that DNA-cetyltrimethylammonium (CTMA) complex in butanol retains most of the spectral features of the DNA in aqueous solution, except a 7-8 nm shift to longer wavelengths.2 It is noted that the conformation of DNA in the complex solution is greatly affected by the water content in organic media, while the surfactants employed in the complexes show little effect on it.7,27 Thus, the different DNA conformations between DNADTDA-chloroform solution in our case, and the reported DNA-CTMA-butanol solution maybe a result of different water content found in both chloroform and butanol solutions. However, Steckl and co-workers did not mention the water content in butanol,2 so we found it difficult to do a more careful comparison. To further illustrate physicochemical properties of the DNADTDA complex in chloroform, the solution was further characterized by DLS and TEM measurements. Approximately 5 mg of DNA-DTDA complex was dissolved in 10 mL of chloroform, and the solution was allowed to stand overnight at room temperature (25 °C) for DLS measurement. Meanwhile, the same solution was applied to a copper grid for TEM characterization after the complete evaporation of chloroform under ambient conditions (25 °C, relative humidity: 20-30%). The DLS measurement shows the presence of large aggregates with the hydrodynamic radius, Rh, of 126 nm (SI Figure S3). TEM imaging confirms that the aggregates form a highly compacted globular structure (SI Figure S4), and the radius of the aggregates ranges from 60 to 80 nm, which is smaller than observed by DLS. The condensation of the complexes during the evaporation of Langmuir 2009, 25(18), 10466–10472

chloroform leads to the shrinkage of the aggregates, thus providing a reasonable mechanism for the size of the aggregates observed by TEM to be smaller than what is observed using DLS. This compact globular structure has been reported for DNA-surfactant complexes in low-polarity organic solvents.28 The combination of thermodynamic solubility of the amphiphilic DNA-surfactant complexes, as well as their complete electric neutrality should contribute to the formation of the compact globular structure observed in low-polarity organic solvents. Importantly, globular aggregation of DNA-DTDA complexes in chloroform is favorable for the formation of ordered microporous structures.16 Honeycomb-Patterned Films of DNA-DTDA Complex. The microporous films of DNA-DTDA are prepared by casting chloroform solutions of these complexes onto glass substrates under a moist airflow (relative humidity: 85% as determined by a hygrometer) applied to the solution surface. These films exhibit bright iridescent colors when viewed with reflected light, indicating a periodic refractive index of variation throughout the thickness of the film. Figure 5 shows representative SEM and AFM images of the microporous film cast from 0.75 mg/mL of DNA-DTDA complex in chloroform. The top view of the SEM image reveals that the double-layered honeycomb pattern exists at an angle with respect to the top and bottom layers with high regularity. The structure of the double-layered walls can be clearly seen from the high-magnified SEM image observed at an angle of 60 degrees. The two hexagonal lattices are connected quasihorizontally by pillars at the vertex of the hexagons. A more in depth look through AFM further confirms the double-layered structure observed by SEM. A protuberance is found on each corner of the hexagons on both top and bottom layers. According to the section analysis, the bottom layer of the wall possesses a height of ca. 60 nm and the upper layer a height of ca. 80 nm; the upper surface of the walls are arc-shaped and the peaks are higher than the valleys by ca. 80 nm. The double-layered honeycomb pattern is interesting and several groups reported relevant structures by using rod-coil block copolymer, hyperbranched polymer, polyimide, and Ptcomplex, respectively.29-33 Condensation and intermolecular attraction of neighboring molecules during solvent evaporation, as well as the influence of the surface tension of the solution are reported to afford a unique, spatial architecture, rather than giving the conventional, continuous microporous film. Here, by employing this DNA-DTDA complex, we obtained similar double-layered structure, and the shrinkage of the aggregates of (28) Sergeyev, V. G.; Pyshkina, O. A.; Lezov, A. V.; Mel’nikov, A. B.; Ryumtsev, E. I.; Zezin, A. B.; Kabanov, V. A. Langmuir 1999, 15, 4434–4440. (29) Cheng, C.; Tian, Y.; Shi, Y.; Tang, R.; Xi, F. Langmuir 2005, 21, 6576–6581. (30) Chang-Soo, L.; Kimizuka, N. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 4922– 4926. (31) Cheng, C.; Tian, Y.; Shi, Y.; Tang, R.; Xi, F. Macromol. Rapid Commun. 2005, 26, 1266–1272. (32) Yabu, H.; Tanaka, M.; Ijiro, K.; Shimomura, M. Langmuir 2003, 19, 6297– 6300. (33) Liu, C.; Gao, C.; Yan, D. Angew. Chem., Int. Ed. 2007, 46, 4128–4131.

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DNA-DTDA complexes on the evaporation of chloroform seems to play an important role. The possible mechanism for the formation of double-layered honeycomb pattern of DNADTDA complex could be described as follows: (1) When the moist airflow passes across a volatile solution of DNA-DTDA complex, the rapid evaporation of the solvent induces a cooling of the solution surface followed by condensation of water vapor into droplets on the solution surface; (2) The water droplets grow with time by molecular condensation until they reach a self-limiting, narrow size distribution. Ultimately, they begin to organize into a hexagonally arranged, close-packed lattice, with high surface coverage being driven by principle of the lowest free energy.16,34 Coalescence of the water droplets is inhibited by the complexes adsorbed at the interface between the water droplets and the solution; (3) Upon evaporation of chloroform, the organized film initially forms a template fashioned by the arrangement of water droplets; (4) Finally, the slow evaporation of the residual water droplets and the remaining chloroform contribute to the further regulation and fixation of the film into the structures we have observed here. Concomitantly, the condensation of the aggregates (As mentioned above, the DNA-DTDA complexes take a globular aggregate in chloroform.) leads to the shrinkage and incline of the walls, thereby forming a double-layered morphology with the top and bottom layers being angled with respect to one another in the finished film structure. Ordered microporous films were only achieved with the relative humidity higher than 70%. When films were prepared under ambient conditions, with relative humidity below 30%, no honeycomb morphology can be observed. This further confirms that the microporous structure is templated by the water droplets condensed on the surface of the evaporating solution. It should be noted that the variation in humidity does not affect the microporous structure of the film once assembled. Both the DNA-DTDA microporous film and its common casting film exhibit a stronger absorption at 1697 cm-1 relative to the band at 1652 cm-1, implying the double-stranded structure of DNA in both of these films remains intact (Figure 2).25,26 The IR spectra were obtained under vacuum, and the vacuum condition does not destroy the double-stranded structure of DNA in the films. Furthermore, from the magnification of the IR spectra in the DNA region 1600-1700 cm-1 (SI Figure S5), we can see that the peak at 1652 cm-1 is a little stronger in the common casting film than that in the microporous film when the spectra were normalized based on the peak at 1697 cm-1, and we think the different conformations of DNA in the two films should contribute to the different spectral features, which was confirmed by the following CD measurements. To gain insight into the secondary structure of DNA in the films, CD spectra were employed, as presented in Figure 4. The microporous film (relative humidity >60%) shows a similar Cotton effect with that of native DNA in aqueous solution (Figure 4c), indicating that DNA retains the B-form of the double helix in the microporous film.7 In the common casting film prepared under ambient atmosphere (relative humidity: 20-30%), DNA exists in the C-form of the double helix, which is similar with the case of DNA-DTDA complex in chloroform (SI Figure S6). Similar to our work, Ogata and co-workers reported that the DNA conformation was gradually changed from B type to A, Z, and Z types in the films of three kinds of DNA-aliphatic amine of N-alkyl-substitution complexes, as the relative humidity decreased from 50 to 20%.27 It is reported that DNA keeps its double-stranded (34) Karthaus, O.; Maruyama, N.; Cieren, X.; Shimomura, M.; Hasegawa, H.; Hashimoto, T. Langmuir 2000, 16, 6071.

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structure in films containing DNA-aliphatic amine of N-alkylsubstitution complex, and DNA is melted into a single stranded structure in the DNA-aliphatic amine film.26,35-37 Cristofolini and co-workers attribute DNA melting in the DNA-aliphatic amine film to variations in pH caused by the protonation of the amino headgroups.26 Shabarchina and co-workers attribute DNA melting in the DNA-aliphatic amine film to the replacement of the intramolecular hydrogen bonding in the A-T and G-C pairs by intermolecular base-amine hydrogen bonding.36 Additionally, they also pointed out melting within the film containing the DNA-aliphatic amine of N-alkyl-substitution complex under dry conditions. Recently, Neumann and co-workers reported that DNA-DDDA films undergo a reversible structural transition from double- to single-stranded DNA, as well as a change in surfactant organization, from a bilayered to a monolayered structure, as the water content in the film decreases.5 Furthermore, the melting of DNA was ascribed to the hydrophobic interactions between the surfactant tails and DNA bases. Thus, water molecules are important in retaining the B-form of the DNA helix in the film state, since DNA conformation was changed and melted into a single-stranded structure as the relative humidity decreased.5,8,27,36 Furthermore, we have performed XRD measurement to examine the structure of the DNA-DTDA microporous film. As seen in SI Figure S7, the DNA-DTDA microporous film shows single diffraction peak at 2.32°, corresponding to the spacing of 3.8 nm. In order to simulate the film structure, we first analyzed the structure of the alkyl chains in the film according to the IR result. It is well-known that antisymmetric and symmetric stretching vibrations of the methylene group are strong indicators of the chain conformation: low wavenumbers (2915-2918 and 2846-2850 cm-1) of the bands are characteristic of highly ordered alkyl chains, while their upward shifts (29242928 and 2854-2856 cm-1) are indicative of the increase in gauche conformers, implying that the alkyl chain conformation tends toward disorder.38 The IR result in Figure 2 shows that the methylene antisymmetric and symmetric stretching bands of the DNA-DTDA film appear at 2925 and 2854 cm-1, respectively, implying that the alkyl chains are disordered in the film. The similar intensity of antisymmetric and symmetric stretching vibrations of the methylene group indicates an interdigitated DTDA bilayer in the film.5 Thus, DNA should be surrounded by disordered and interdigitated alkyl chains in the DNADTDA film. The interdigitated DTDA bilayer is ca. 2.6 nm by using the reported interdigitated DDDA bilayer of 2.4 nm plus the length (0.25 nm) of two methylene groups.5 It is noted that the methylene antisymmetric and symmetric stretching bands moved to 2925 and 2854 cm-1 in DNA-DTDA film, relative to 2923 and 2852 cm-1 in DNA-DDDA film.5 Thus, the alkyl chains in DNA-DTDA film are more disordered than those in DNADDDA film, and the actual length of DTDA bilayer should be much smaller than 2.6 nm. Furthermore, in our previous work we found that DODA (dioctadecyldimethylammonium cation) could form an almost fully interdigitated bilayer with a distance of 2.4 nm in the DODA-polyoxometalate film.39 Based on this (35) Sukhorukov, G. B.; Feigin, L. A.; Montrel, M. M.; Sukhorukov, B. I. Thin Solid Films 1995, 259, 79–84. (36) Shabarchina, L. I.; Montrel, M. M.; Sukhorukov, G. B.; Sukhorukov, B. I. Thin Solid Films 2003, 440, 217–222. (37) Shabarchina, L. I.; Montrel, M. M.; Savintsev, I. V.; Sukhorukov, B. I. Russ. J. Phys. Chem. 2003, 77, 1862–1867. (38) MacPhail, R. A.; Strauss, H. L.; Snyder, R. G.; Elliger, C. A. J. Phys. Chem. B 1984, 88, 334–341. (39) Bu, W.; Li, H.; Li, W.; Wu, L.; Zhai, C.; Wu, Y. J. Phys. Chem. B 2004, 108, 12776–12782.

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Article Table 1. Vapor Pressure and Molecular Weight of the Used Solvents (20 °C)

vapor pressure (kPa) molecular weight

Figure 6. Optical micrographs of the honeycomb-patterned films prepared by casting 0.75 mg/mL of DNA-DTDA complex onto (a) mica, (b) gold, and (c) silicon substrates, respectively.

Figure 7. Honeycomb-patterned films of DNA-DTDA complex prepared at different concentrations: SEM images of (A) 0.5 mg/mL, (B) 1 mg/mL, and AFM images of (C) 0.5 mg/mL, (D) 1 mg/mL, and (E) the local magnification of (D).

spacing, we mimicked the length of DTDA almost fully interdigitated bilayer as ca. 1.9 nm by subtracting four methylene lengths, 0.51 nm, from 2.4 nm. Thus, both the disorder and the deep interdigitattion of the alkyl chains contribute to the small length of DTDA bilayers in DNA-DTDA film. And, it is reasonable that the spacing of 3.8 nm corresponds to the dimension of a model structure of a double-stranded DNA (ca. 2 nm in diameter) and a deep interdigitated and disordered DTDA bilayer (1.8 nm as expected), and not a single-stranded DNA and DTDA monolalyer. We also investigated the possible effects of the substrate type, DNA-DTDA concentration, solvent type, and DNA complexed with different surfactants on the morphology of the microporous films. Similar structured surfaces were observed on mica, gold, and silicon substrates (Figure 6). Noticeably, the structure of the microporous films can be varied by slightly adjusting the concentration of the complex (Figure 7). At the concentration of 0.5 mg/mL, ordered honeycomb-patterned films occur, however, the bottom layer of the wall becomes very thin, which could not be seen clearly by SEM imaging. When the concentration increases to 1.0 mg/mL, the film morphology develops into a “web-like” structure, with highly ordered hexagonal structures outlined by large-sized, randomly distributed structures which form the “weblike” structure observed (Figures 7B, D). More interestingly, the “web-like” structure conforms to the pores quite well, namely, the pores do not destroy the order of the web at all. A close AFM examination of the microporous films prepared at three different concentrations (0.5, 0.75, and 1.0 mg/mL) shows that the films all assume a double-layered structure. Films prepared from dichloromethane and benzene solutions also show porous structures, but the holes are less regular than those formed from chloroform (SI Figure S8). The primary reason for the different regularity of the microporous films is the different evaporation rates of chloroform, dichloromethane, and benzene. Low evaporation rate allows water droplets to have more time to coalesce and grow larger on the surface of the solution, consequently forming larger pores. However, slow evaporation of solvent may lead to the unavoidable coalescence, which decreases the order of the Langmuir 2009, 25(18), 10466–10472

dichloromethane

chloroform

benzene

46.50 84.93

21.28 119.38

10.67 78.11

microporous films. On the other hand, rapid evaporation of solvent leads to the instability of the solution surface, and the microporous film may be solidified well before the arrangement of the condensed water droplets. As a result, the proper evaporation rate of the solvent is essential for the formation of ordered microporous films. Evaporation rates of the solvent depend mostly on vapor pressure above the solvent as well as the molecular weight of the solvent (Table 1).40,41 Typically, higher vapor pressure and lower molecular weight lead to faster evaporation rates. Here, as the evaporation rate decreases from dichloromethane to chloroform, and to benzene, the average sizes of the holes in the microporous films increase, continuously. And only the film casting from chloroform solution with proper evaporation rate shows an ordered microporous structure. All of the investigations in the paper demonstrate the wide generality and high reproducibility for the DNA-DTDA selforganized microporous films. We also tried more hydrophilic DNA-DDDA complex to fabricate microporous structure. However, only a disordered morphology was obtained at concentrations ranging from 0.5 to 2.0 mg/mL (SI Figure S9). Thus, DNA enwrapped with more hydrophilic surfactant is unfavorable for the construction of ordered microporous films, which is consistent with our previous studies on microporous films formed from surfactant-encapsulated polyoxometalate complexes.22 Furthermore, we have tried to prepare DNA complexes with the surfactants possessing longer alkyl chains, such as DNADHDA (DHDA: dihexadecyldimethylammonium cation) complex and DNA-DODA (DODA: dioctadecyldimethylammonium cation) complex, for pattern formation. However, we failed to obtain the two complexes with cationic surfactants and phosphate anions in a strict ratio of 1:1 due to the decreased solubility of DHDA and DODA in water. Thus, we could not give an example of microporous films fabricated from DNA complexes with more hydrophobic surfactants to establish the trend of the formation of ordered microporous films. Luminescent Honeycomb-Patterned Film of RhB-Loaded DNA-DTDA Complex. The patterned film structure could be suitably decorated, and we loaded RhB into DNA-DTDA complex to endow the microporous film with fluorescence. In the solution of RhB-loaded DNA-DTDA complex, no proton signals of RhB were observed at P:D (the molar ratio of DNA phosphate to dye) of 40:1 except those signals of DTDA, implying that RhB was in a confined state through binding to DNA, but is not replacing DTDA or within DTDA bilayers (SI Figure S10). And, DNA-DTDA complex retains the intact structure after loading RhB. From the emission band at 576 nm, we know that the dye molecules exist in their monomer state within the complex in chloroform. As shown in Figure 8, the honeycomb-patterned film of RhB-loaded DNA-DTDA complex shows homogeneous red fluorescence. The strong dye emission at 590 nm indicates the weak aggregation of dye molecules in the solid film formed during the evaporation of the solvent.42 The microporous film of (40) Peng, J.; Han, Y.; Yang, Y.; Li, B. Polymer 2004, 45, 447–452. (41) Tian, Y.; Ding, H.; Jiao, Q.; Shi, Y. Macromol. Chem. Phys. 2006, 207, 545– 553. (42) Machida, S.; Morisada, M.; Horie, K.; Okahata, Y. Polym. J 1999, 31, 1179–1184.

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matrix. Therefore, we believe that it is DNA in the complex which accommodates the dye molecules and makes them well dispersed in the patterned film, a process which could not be demonstrated by the conventional polymers.

Conclusions

Figure 8. (A) Optical fluorescence microscope image of the microporous film prepared from RhB-loaded DNA-DTDA complex (P/D, 40:1) in chloroform, and (B) the emission spectra of (a) RhBloaded DNA-DTDA microporous film, (b) RhB-loaded PVPADTDA microporous film, and (c) RhB-loaded PMMA microporous film at room temperature with the same casting amount (excitated at 260 nm).

RhB-loaded DNA-DTDA complex shows a stronger absorption at 1697 cm-1 relative to the band at 1652 cm-1, similar to that of DNA-DTDA microporous film, implying the double-stranded structure of DNA remains in the RhB-loaded DNA-DTDA microporous film (SI Figure S11).25,26 Furthermore, XRD of RhB-loaded DNA-DTDA microporous film shows similar single diffraction peak to that of DNA-DTDA microporous film at 2.32° (SI Figure S7), suggesting that RhB does not deform the DNA helix when it binds in the DNA-DTDA film. To understand the advantages of using DNA-DTDA complex as a host material for the preparation of luminescent microporous film over conventional polymers, PVPA and PMMA were applied for the control experiments (SI Figure S12). For this purpose, we have prepared the PVPA-DTDA complex as described in the Experimental Section. In the RhBloaded PVPA-DTDA microporous film, large disordered holes were observed throughout the film, and the corresponding emission of RhB shifts to 600 nm with a concomitant intensity decrease, implying the aggregation of the dyes in PVPA-DTDA microporous film is stronger than that in the DNA-DTDA microporous film. In the case of RhB-loaded PMMA film, not only does the microporous structure become more disordered, but also the corresponding emission of RhB shifts to a longer wavelength, 615 nm, with a much heavier intensity decrease, indicating a more serious aggregation of the dyes in the PMMA

10472 DOI: 10.1021/la900322d

A supramolecular, self-organized strategy for the facile fabrication of DNA-based honeycomb structures was demonstrated with wide generality and high reproducibility through the encapsulation of a cationic surfactant DTDA to DNA. DNA exists in the double helical B-form in the microporous film. The morphology of the microporous structures can be adjusted by slightly changing the concentration of the complex solution. RhB dye molecules were loaded into the DNA-DTDA complex, presenting a fluorescent microporous film. DNA-DTDA complex as a host material provides a new method for tailoring fluorescent microporous films. The DNA-based ordered honeycomb films should present attractive applications in optical and optoelectronic devices,2 separation, ion transportation, and biosensors.43 Acknowledgment. We acknowledge the financial support from National Basic Research Program (2007CB808003), National Natural Science Foundation of China (20731160002, 20703019), PCSIRT of Ministry of Education of China (IRT0422), Lance Wollenberg at West Virginia University for help with manuscript preparation and Open Project of State Key Laboratory of Polymer Physics and Chemistry of CAS. Supporting Information Available: XPS, UV-vis, DLS and TEM of DNA-DTDA complex. SEM image of the patterned surface of DNA-DDDA complex. 1H NMR spectrum of RhB-loaded DNA-DTDA complex. XRD of DNA-DTDA microporous film and RhB-loaded DNADTDA microporous film. CD of DNA-DTDA common casting film. Optical fluorescence microscope images of the microporous films prepared from RhB-loaded PVPADTDA complex and RhB-loaded PMMA. This material is available free of charge via Internet at http://pubs.acs.org. (43) Jiang, S.; Liu, M. Chem. Mater. 2004, 16, 3985–3987.

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