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In Situ Measurements of the Dynamics of Single Giant DNA Molecules at the Toluene-Trioctylamine/Water Interface by Total Internal Reflection Fluorescence Microscopy Satoshi Tsukahara,* Michinori Suehara, and Terufumi Fujiwara Department of Chemistry, Graduate School of Science, Hiroshima UniVersity, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan ReceiVed September 8, 2007. In Final Form: January 4, 2008 The dynamics of single giant deoxyribonucleic acid (T4GT7DNA, 165 600 base pairs) molecules was examined near and at the toluene/water, toluene-trioctylamine mixtures/water, and trioctylamine/water interfaces by total internal reflection fluorescence microscopy. The results were considerably affected by the trioctylamine content. With pure toluene or mixtures of lower trioctylamine volume contents (%VA), the randomly coiled DNA molecules diffused to near the interfaces. With mixtures of higher %VA (9 and 50%), the DNA molecules were stretched and adsorbed at the interfaces. There are a large number of anionic phosphate groups ((-O)2PO2-) in the DNA molecule that have an electrostatic affinity to protonated trioctylamine existing at the interface. In the case of pure trioctylamine, globular DNA molecules were adsorbed at the interface and also existed in the aqueous phase.
Introduction There is no doubt that total internal reflection spectroscopy is a significant method for research into surfaces and interfaces. The technique is widely applied to fixed solid/liquid and gas/ solid interfaces.1 Its application to the liquid/liquid interface is confined2 because the interface is easily disturbed and is hard to fix. The combination of this technique and fluorescence microscopy is rarely used in studies on the liquid/liquid interface. We have already developed a thin-layer two-phase microcell3 and have shown that it is very useful in microscopic measurements of reactions and phenomena occurring at liquid/liquid interfaces.3-6 Deoxyribonucleic acid (DNA) is an essential macromolecule in biology. Fluorescence microscopy has clarified that single DNA molecules show several kinds of interesting motion in aqueous solutions, such as a conformational change (random coil-globule transition), intramolecular motion, and 3D Brownian motion.7-9 Furthermore, it is known that the conformational change of DNA molecules is deeply related to their hydration. So far, the dynamics of single DNA molecules has been examined in homogeneous aqueous solutions or at solid surfaces but it has been scarcely studied at movable liquid/liquid interfaces, where the physical properties of solvents and solvation change drastically. The liquid/liquid interface is sometimes treated as a model of biological membranes. Furthermore, it was recently found that polyanionic DNA molecules could be extracted by * Corresponding author. E-mail:
[email protected]. Phone: +81 82 424 7425. Fax: +81 82 424 7424. (1) Kang, S. H.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2001, 73, 10911099. Jeong, S.; Park, S.-K.; Chang, J. K.; Kang, S. H. Bull. Korean Chem. Soc. 2005, 26, 979-982. (2) Ishizaka, S.; Kim, H.-B.; Kitamura, N. Anal. Chem. 2001, 73, 2421-2428. Tsukahara, S. Anal. Chim. Acta 2006, 556, 16-25, and references therein. Tsukahara, S. Anal. Chim. Acta 2006, 556, 112-120. (3) Hashimoto, F.; Tsukahara, S.; Watarai, H. Anal. Sci. 2001, 17(Suppl.), i81-i83. Hashimoto, F.; Tsukahara, S.; Watarai, H. Langmuir 2003, 19, 41974204. (4) Tsukahara, S.; Kitaguchi, H.; Watarai, H. Chem. Lett. 2007, 36, 148-149. (5) Kamiya, Y.; Tsukahara, S.; Fujiwara, T. Chem. Lett. 2007, 36, 344-345. (6) Tsukahara, S.; Suehara, M.; Fujiwara, T. Anal. Sci. 2007, 23, 375-378. (7) Yoshikawa, K.; Matsuzawa, Y. Physica D 1995, 84, 220-227. (8) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Matsumoto, M.; Doi, M. FEBS Lett. 1991, 295, 67-69. (9) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 2401-2408. Dias, R. S.; Innerlohinger, J.; Glatter, O.; Miguel, M. G.; Lindman, B. J. Phys. Chem. B 2005, 109, 10458-10463.
cationic surfactants into organic solvents.10 The neutralization or association of DNA molecules with them would occur near the interface, but the process has not been observed directly at a single-molecule level. From these, the dynamics of single DNA molecules at the liquid/liquid interfaces is very interesting, but there are no studies on it except our first report on the dodecane/ water interface.6 The present study focuses on the dynamics of single giant DNA molecules near and at liquid/liquid interfaces. Experimental Section A giant double-stranded DNA (T4GT7DNA, 165 600 base pairs) was purchased from Nippon Gene, which was labeled with a fluorescent dye, YO-PRO-1 (Molecular Probe). Water was purified with a Milli-Q system (Milli-Q SP. TOC., Millipore). Two membrane filters (0.2 and 0.02 µm in pore size, Advantec and Whatman, respectively) were used to remove a few tens of nanometers of dust particles in solvents and DNA-free aqueous solutions.6 The DNA aqueous solution contained 3.3 × 10-13 M (1 M ) 1 mol dm-3) or 2.0 × 10-12 M DNA, 3.0 × 10-7 M YO-PRO-1, 1.0 × 10-2 M tris(hydroxymethyl)aminoethane, 1.0 × 10-3 M EDTA, and 4% 2-mercaptoethanol by volume. The last was added to reduce the photobleach of fluorescence.6,7 The pH of the DNA solution was adjusted to 8.0. Toluene (purity g99.7%, Kanto Chemical) was the main organic-phase solvent. Dimethylditetradecylammonium bromide (purity g97%, Tokyo Chemical Industry), dioctadecylamine (purity g99%, Fluka), and trioctylamine (purity g98%, Kanto) were examined as additives to toluene. Figure 1 shows an illustration of the instrument system that includes an inverted microscope (IX-51, Olympus) with a water-immersion objective (UPlanApo, 60×, NA 1.2, Olympus) and a CCD camera (WAT-100N, Watec). The lateral resolution is expressed by the diffraction limit of light (d); it was estimated to be 0.22 µm at 520 nm with d ) λ/2NA (λ, the wavelength of light). The thin-layer two-phase microcell was the same one fabricated in the previous report.6 The interface was irradiated by 473 nm light from a DPSS CW laser (power 20 mW; Suwtech) after it passed through ND filters and was focused by a lens (12 mm in focal length). For laser irradiation, a rectanglar prism (BK-7, 1 × 1 × 1 cm) was placed on the microcell through index-matching oil.4 The angle of incidence was set to 68°, which was larger than the critical angle of the toluene/ (10) Goto, M.; Ono, T.; Horiuchi, A.; Furusaki, S. J. Chem. Eng. Jpn. 1999, 32, 123-125. Goto, M.; Momota, A.; Ono, T. J. Chem. Eng. Jpn. 2004, 37, 662-668.
10.1021/la7027719 CCC: $40.75 © 2008 American Chemical Society Published on Web 02/01/2008
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Figure 1. Illustration of the total internal reflection fluorescence microscopy for the liquid/liquid interfaces prepared in the thin-layer two-phase microcell.
Figure 2. Illustration of irradiation by laser light under the total internal reflection condition. (a) Part of a DNA molecule existing in the evanescent-wave region and (b) a whole DNA molecule adsorbed at the interface were selectively excited. water interface (63°). For the observation of DNA molecules in the aqueous phase, common epi illumination with a Hg lamp (100 W) and a mirror unit (U-MWIB2, Olympus; emission at 460-490 nm, observation at 510-900 nm) was used for the excitation. The microscopic pictures were obtained by the CCD camera at the video rate (33 ms/frame) and recorded to a video tape. The pictures were transferred to a personal computer as digital pictures to be analyzed. All of the experiments were carried out at 20 ( 2 °C.
Results and Discussion Under the total internal reflection condition, the penetration depth of the evanescent wave (de) in the aqueous phase can be written as11
de )
λ 4πxno sin2 θ - na2
(1)
2
where no and na are the refractive indices of the organic and aqueous phases, respectively, and θ is the angle of incidence; the de value was estimated to be 96 nm. In the present study, the DNA molecules were not extracted into the organic phases; they existed in the aqueous phase or at the interface. Therefore, the DNA molecules in the evanescent-wave region or at the interface can be selectively detected under the total internal reflection condition, as shown in Figure 2. Diffusion of Single DNA Molecules near the Toluene/Water Interface. Figure 3 shows a typical example of microscopic fluorescence pictures at the toluene/water interface. The wide white area is the one irradiated by the laser light, where YO-PRO-1 emitted weak fluorescence. The brighter white images in the black circles correspond to single DNA molecules. The DNA molecule labeled with 1 appeared in Figure 3b first, stayed and slightly moved in Figure 3c,d, and disappeared in Figure 3e. (11) Hansen, W. N. In AdVances in Electrochemistry and Electrochemical Engineering; Delahey, P., Tobias, C. W., Eds.; John Wiley and Sons: New York, 1973; Vol. 9, pp 1-60.
This means that the DNA molecule continuously existed in the evanescent-wave region for three frames (100 ms). However, the DNA molecule labeled with 2 existed there for only one frame (33 ms) in Figure 3d. Similarly, the existence of DNA molecules in every video frame was examined for 43 DNA molecules. For each DNA molecule observed, the number of frames in which the DNA molecule continuously existed was counted, and the histogram in Figure 4 was made. Clearly, half of them (51%) were observed in only one frame, and almost all of them were continuously observed for less than 133 ms. This histogram can be compared with the Poisson distribution, whose probability (P(k)) is expressed as P(k) ) e-ββk/k!, where k is the frame number and β is the expected number of occurrence. The frequency (F(k)) can be written as F(k) ) nP(k), where n is the total number. The Poisson distribution treats an on-off event; it corresponds to a DNA molecule existing in the evanescent-wave region (on) and it being just under the region (off) in the present case (Figure 2). The optimal n and β values were determined to be 60 and 1.15 by the least-squares method, respectively, and the calculated F(k) values were superimposed in Figure 4. They agreed well with the observed counts at k g 1, meaning that almost all of the DNA molecules near the toluene/ water interface behaved in the same manner. The calculated F(0) corresponds to the frequency at which a DNA molecule exists just under the evanescent-wave region and does not enter there. The count at k ) 0 that should be compared with the F(0) value could not be obtained experimentally because it could not be distinguished as to whether a DNA molecule existed just under the evanescent-wave region or did not exist there. The β value corresponds to the average time (tav) during which a DNA molecule stays in the evanescent-wave region; its value is 38 ms () 1.15 × 33 ms). This short stay implies that the DNA molecules were not adsorbed at the interface. To confirm the diffusion of the DNA molecules, the diffusion coefficient (D) of the DNA molecules near the toluene/water interface in the depth direction was roughly estimated to be 2 × 10-13 m2/s with D ≈ de2/tav. If the DNA molecule was assumed to be a sphere, then its hydrodynamic radius (rh) was evaluated with the Einstein-Stokes equation D ) kT/(6πηrh), where k is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the medium. With η of water (1.0 mPa s at 20 °C), rh was estimated to be 0.9 µm. The apparent size of the DNA molecule in Figure 3c is 1.2 µm, which is comparable to rh. However, the intrinsic diameter and length of the chainlike DNA molecule are 2 nm and 57 µm, respectively,6 and thus its volume is 1.8 × 10-4 µm3. If the DNA were a solid sphere, then its radius (rs) would be 0.035 µm. It is natural that rh is much larger than rs because of the hydration and the intramolecular motion of the DNA molecules. Clearly, the depth of the evanescent wave (de) is smaller than the rh or the apparent size of the DNA molecule, indicating that only a part of the DNA molecule was observed in Figure 3b-d. The diffusion of a substance is commonly evaluated from the motions of its center of gravity, but its diffusion coefficient can be obtained from the motions of a part of the substance in principle. These indicate that the rh value is valid and thus the DNA molecules collided with the toluene/water interface and rebounded from there by diffusion. In aqueous solution, the DNA molecules show fast intramolecular motions at room temperature, and they are round like balls of wool. When the DNA molecule is fully hydrated, that is, in the random-coil state, its observed size is 2-4 µm by optical microscopy.7 Figure 3f shows a fluorescence image of the DNA molecule in the aqueous phase; the image size is 2 to 3 µm, which agrees well with the reported value.7 These facts
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Figure 3. (a-e) Continuous microscopic fluorescence pictures of single DNA molecules near the toluene/water interface under the total internal reflection condition. The white area was laser irradiated. The brighter white images in the circles in b-d correspond to single DNA molecules. Laser power: 20 mW. (f) Microscopic fluorescence picture of a single DNA molecule in the aqueous phase excited by common epi illumination. The contrast of all of the pictures was adjusted for clarity.
Figure 4. Distribution of counts as a function of frame number in which each DNA molecule was continuously observed in the evanescent-wave region at the toluene/water interface. The circles (O) express the frequency of the Poisson distribution.
mean that the DNA molecules in the aqueous phase are in the random-coil state. The previous study suggested that the DNA molecules were also not adsorbed at the dodecane/water interface.6 A DNA molecule has organic bases, but it has a large number of hydrophilic and anionic phosphate groups ((-O)2PO2-) at neutral and basic pH values. Hence, the DNA molecules could not be adsorbed at these common interfaces. Adsorption of Single Stretched DNA Molecules at TolueneTrioctylamine/Water Interfaces. Goto et al. extracted DNA molecules with cationic alkylammonium ions into organic phases.10 The DNA molecules were transported across the liquid/ liquid interfaces when their phosphate groups were neutralized and they became hydrophobic. Therefore, for the sake of a strong interaction with DNA molecules at the interface, several surfaceactive organic cations were essential, and the above-mentioned additives were examined. Unfortunately, toluene containing dimethylditetradecylammonium bromide (1.0 × 10-2 M) could not be used because the DNA aqueous solution burst when the organic solution was placed on it. As for dioctadecylamine, the acid-dissociation constant (pKa) of its conjugated acid is above
10, and thus it can be protonated at the interface at pH 8 and the protonated form can work as a surfactant. However, the behavior of the DNA molecules near the interface with dioctadecylamine at a concentration of 1.0 × 10-7-1.0 × 10-3 M was similar to that near the bare toluene/water interface. Dioctadecylamine is a solid and is slightly soluble in toluene. However, obvious effects were observed for trioctylamine at higher volume contents (%VA). The pKa of its conjugated acid is about 10. It is a liquid at room temperature and freely miscible with toluene. Furthermore, the refractive index of it is close to that of toluene, and thus total internal reflection occurs for toluene-trioctylamine mixtures and water interfaces under the same optical conditions. When %VA was not lower than 0.5%, some adsorption of the DNA molecules was observed at the interfaces. Figure 5 shows a series of pictures of a stretched DNA molecule at the interface at %VA ) 9%. The DNA moved somewhat and its form slowly changed at the interface. It continuously stayed at the interface at least for 467 ms, which is much longer than the staying time of the DNA molecules near the bare toluene/ water interface (38 ms). The DNA molecules other than that in Figure 5 also stayed at the interface for longer than 500 ms. All of them were not released from the interface and finally showed a photocut at the interface as mentioned in the following text. Furthermore, the form was quite different from that at the toluene/ water interface. Therefore, the stretched DNA was adsorbed at the toluene-trioctylamine/water interface. The whole length of the stretched DNA image in Figure 5a is 23 µm, which is about 0.4 times as long as the full length. Therefore, its thickness should be about 5 nm (2.5 times the intrinsic diameter), but the image is much thicker owing to the diffraction limit of light. The radius of gyration (Rg) or end-to-end distance (Ree) of single DNA molecules was sometimes employed to characterize the conformation and flexibility of DNA molecules quantitatively.8,9,12 The Rg value of T4GT7DNA in aqueous solutions was reported to be 1.2 µm.8 The Rg and Ree values of the DNA (12) Makita, N.; Ullner, M.; Yoshikawa, K. Macromolecules 2006, 39, 62006206.
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Figure 5. Series of microscopic fluorescence pictures of a stretched single DNA molecule at the toluene-trioctylamine mixture (91:9) and water interface. The cut positions are indicated by white arrows. The contrast was adjusted for clarity.
Figure 6. Microscopic fluorescence pictures (a-c) at the interfaces under the total internal reflection condition and those (d-f) in the aqueous phases excited by common epi illumination. Organic phase: (a, d) toluene-trioctylamine (91:9); (b, e) toluene-trioctylamine (50:50); (c, f) trioctylamine. DNA concentration, 3.3 × 10-13 M; laser power, 2.6 mW. The contrast was adjusted for clarity.
molecule shown in Figure 5a were estimated to be 5 and 12 µm, respectively, and these values changed slightly with the short elapse of time (Figure 5a-d). The other interfacial DNA molecules showed various forms and degrees of stretching, and their Rg values are about 3 to 4 times as large as that in the aqueous solutions. Rg should be estimated by a statistical calculation with many images of a single DNA molecule for a long time. Rg values of single DNA molecules should be compared with each other for the sake of homogeneity of adsorbed DNA molecules. Furthermore, deep discussions on the physical meanings of Rg at 2D interfaces and on comparison of it with Rg in 3D solutions are necessary. These microscopic measurements, quantitative analyses, and discussions are not achieved at this stage but will be undertaken in the near future.
After 200 ms, the DNA was cut in Figure 5e,f; the cut positions were indicated by white arrows. Their number was four, thus the DNA molecule was divided into five fragments. Each fragment diffused away at the interface independently. Some researchers reported that the excitation by high-power light caused the fragmentation of DNA molecules,13 and therefore irradiation by focused laser light led to the fragmentation of the interfacial DNA molecules in the present study. These stretched DNA molecules were also observed at the interface at %VA ) 50%, as shown in Figure 6b. The Rg value of the DNA molecule in Figure 6b was estimated to be 2.5 µm, (13) Lyon, W. A.; Fang, M. M.; Haskins, W. E.; Nie, S. Anal. Chem. 1998, 70, 1743-1748. Yoshikawa, Y.; Suzuki, M.; Yamada, N.; Yoshikawa, K. FEBS Lett. 2004, 556, 39-42.
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Figure 7. Illustration of DNA molecules in the (a) toluene/water, (b) toluene-trioctylamine/water, and (c) trioctylamine/water systems.
which was also larger than that in aqueous solutions. At these interfaces, protonated trioctylamine should exist so that the fluorescence of YO-PRO-1 is relatively weaker dissimilar to that in Figure 3. As mentioned above, the randomly coiled DNA molecules were not adsorbed at the bare toluene/water interface, as shown in Figure 7a. However, the stretched DNA molecules were adsorbed at the toluene-trioctylamine/water interfaces, as shown in Figure 7b, because there are a large number of anionic phosphate groups ((-O)2PO2-) in the DNA molecule that have an electrostatic affinity to protonated trioctylamine existing at the interface. This makes the interfacial DNA molecules more stable electrostatically. The homogeneity of adsorbed trioctylammonium ions is not examined by common macroscopic methods, but such in situ microscopic measurements would investigate the homogeneity. Stretched DNA molecules were observed on fixed solid surfaces,1,14 but the present study found for the first time that such stretched DNA molecules were adsorbed at movable liquid/liquid interfaces. Figure 6d,e shows that randomly coiled DNA molecules existed in the aqueous phases, meaning that trioctylamine had no effect on the conformation of the DNA molecules in the phases in these cases. The form of the DNA molecules is easily changed near the interface because they are in the flexible random-coil state in the aqueous phase. Their stretching process should occur near the interface, but it could not be observed for 0.2 s; after that time, they showed the photobleach and photocut by excitation. Adsorption of Single Globular DNA Molecules at the Trioctylamine/Water Interface. With pure trioctylamine, smaller images of DNA molecules were observed as shown in Figure 6c. They vibrated somewhat at the interface, and their form was not changed. The release of DNA molecules from this interface was not observed; they stayed at the interface continuously for at least 20 s and finally showed photobleach and disappeared. These findings indicate that the DNA molecules (14) Zheng, H.-Z.; Pang, D.-W.; Lu, Z.-X.; Zhang, Z.-L.; Xie, Z.-X. Biophys. Chem. 2004, 112, 27-33. Wang, H.; Grimes, S.; Anderson, D. L.; Serwer, P. J. Microsc. 2004, 213, 172-179.
were strongly adsorbed at the interface, which is dissimilar to the bare toluene/water interface. They did not show the photocut. Globular DNA molecules existed and showed the diffusion in the aqueous phase as in Figure 6f. Therefore, each small image in Figure 6c corresponds not to a part of a randomly coiled DNA molecule but to a globular DNA molecule adsorbed at the interface. It was reported that an addition of cations to a DNA aqueous solution caused a globule transition of DNA as a result of its neutralization.8,9 In the present case, protonated trioctylamine existing at the interface and in the aqueous phase at higher concentrations could induce the transition of DNA molecules to the globule state, as shown in Figure 7c.
Conclusions The present study developed a new methodology that enabled one to observe single DNA molecules existing in the evanescentwave region (96 nm) in the aqueous phase or at the liquid/liquid interfaces. Also, it proposed a method for evaluating the diffusion coefficient of the DNA molecules in the evanescent-wave region. Furthermore, it was found that the electrostatic affinity of the DNA molecules to trioctylammonium ions at the interfaces was the dominant factor in the adsorption of DNA molecules. The dynamics as well as the conformation of DNA molecules at movable interfaces is an important subject to be clarified from the viewpoints of interfacial chemistry, extraction chemistry, and biological chemistry. The present approach is a basic study of research on the interaction of DNA molecules with biological membranes. A higher-sensitivity detection system is now being constructed, and detailed dynamics of DNA molecules at interfaces will be measured and analyzed. Acknowledgment. This work was supported by a Grantin-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture, Japan (nos. 16350046 and 19350040). LA7027719
