Measuring the Length Distribution of Self-Assembled Lipid Nanotubes

Jan 26, 2009 - Health Technology Research Center, National Institute of Advanced ... Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi...
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Anal. Chem. 2009, 81, 1459–1464

Measuring the Length Distribution of Self-Assembled Lipid Nanotubes by Orientation Control with a High-Frequency Alternating Current Electric Field in Aqueous Solutions Ken Hirano,*,† Masaru Aoyagi,‡ Tomomi Ishido,† Toshihiko Ooie,† Hiroshi Frusawa,§ Masumi Asakawa,‡ Toshimi Shimizu,‡ and Mitsuru Ishikawa† Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan, Nanotube Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, 305-8565, Japan, and Department of Environmental Systems Engineering, Graduate School of Engineering, Kochi University of Technology, Tosayamada-cho, Kochi, 782-8502, Japan The present work addresses the length distribution of selfassembled lipid nanotubes (LNTs) by controlling the orientation of the LNTs using an alternating current (ac) electric field in aqueous solutions. The effect of the ac field on the orientation and rotation of individual LNTs was examined to evaluate the optimum orientation frequency by visualizing the individual LNTs in real time. By using the high-frequency ac field, we have successfully measured the length distribution for two different types of LNTs and have quantitatively analyzed the maximum occurrences of the length distribution as well as the extension of the longer length region. Self-assembled lipid nanotubes (LNTs) with a hollow cylindrical architecture have attracted much interest due to their resemblance to carbon nanotubes in size, structure (unique hollow cylinders with high aspect ratio), and potential chemical and physical properties.1 Three research groups reported first on the formation of LNTs from bilayer-forming amphiphiles independently and almost simultaneously.2-4 Once self-assembly starts, lipid molecules spontaneously self-assemble into a well-defined tubular morphology without special instruments and without consuming energy. LNTs are composed of multiple lipid bilayer membranes self-assembled from amphiphiles that have both hydrophilic and hydrophobic groups. The outer and inner surfaces of LNTs are * To whom correspondence should be addressed. Ken Hirano, Ph.D., Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST). E-mail: [email protected]. Phone and fax: +81-87869-3569. † Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST). ‡ Nanotube Research Center, National Institute of Advanced Industrial Science and Technology (AIST). § Kochi University of Technology. (1) Shimizu, T.; Masuda, M.; Minamikawa, H. Chem. Rev. 2005, 105, 1401– 1443. (2) Yager, P.; Schoen, P. E. Mol. Cryst. Liq. Cryst. 1984, 106, 371–381. (3) Yamada, K.; Ihara, H.; Ide, T.; Fukumoto, T.; Hirayama, C. Chem. Lett. 1984, 10, 1713–1716. (4) Nakashima, N.; Asakuma, S.; Kunitake, T. J. Am. Chem. Soc. 1985, 107, 509. 10.1021/ac8022795 CCC: $40.75  2009 American Chemical Society Published on Web 01/26/2009

hydrophilic;5,6 thus, LNTs are easily dispersed in water. Furthermore, functionalized LNTs act as cytomimetic tubules, capsules for biomolecules and nanoparticles,1,7-9 containers for nanoscale chemical reactions,10-12 channels for nanoscale separation,1,13 and carriers for drug delivery.14-17 Recently, a limited number of LNTs have become available at the 100 g scale.18 This achievement highly motivates us to push LNTs into practical use. To understand the properties of LNTs in detail, we must explore techniques for manipulating individual LNTs. However, few well-developed techniques exist compared with the number available for manipulating carbon nanotubes. The microinjection technique19 and optical manipulation with laser trapping20,21 have been demonstrated to align LNTs on a glass surface and analyze their physical properties. Manipulation techniques using an ac (5) Masuda, M.; Shimizu, T. Langmuir 2004, 20, 5969–5977. (6) Kamiya, S.; Minamikawa, H.; Jung, J. H.; Yang, B.; Masuda, M.; Shimizu, T. Langmuir 2005, 21, 743–750. (7) Kameta, N.; Masuda, M.; Minamikawa, H.; Mishima, Y.; Yamashita, I.; Shimizu, T. Chem. Mater. 2007, 19, 3553–3560. (8) Shimizu, T. J. Polym. Sci. Part A: Polym. Chem. 2008, 46, 2601–2611. (9) Kameta, N.; Masuda, M.; Mizuno, G.; Morii, N.; Shimizu, T. Small 2008, 4, 561–564. (10) Yang, B.; Kamiya, S.; Yoshida, K.; Shimizu, T. Chem. Commun. 2004, 500, 501. (11) Yang, B.; Kamiya, S.; Shimizu, Y.; Kashizaki, N.; Shimizu, T. Chem. Mater. 2004, 16, 2826–2831. (12) Yui, H.; Guo, Y.; Koyama, K.; Sawada, T.; John, G.; Yang, B.; Masuda, M.; Shimizu, T. Langmuir 2005, 21, 721–727. (13) Sott, K.; Lobovkina, T.; Lizana, L.; Tokarz, M.; Bauer, B.; Konkoli, Z.; Orwar, O. Nano Lett. 2006, 6, 209–214. (14) Schnur, J. M.; Price, R.; Rudolph, A. S. J. Controlled Release 1994, 28, 3– 13. (15) Spargo, B. J.; Cliff, R. O.; Rollwagen, F. M.; Rudolph, A. S. J. Microencapsulation 1995, 12, 247–254. (16) Meilander, N. J.; Pasumarthy, M. K.; Kowalczyk, T. H.; Cooper, M. J.; Bellamkonda, R. V. J. Controlled Release 2003, 88, 321–331. (17) Kameta, N.; Minamikawa, H.; Masuda, M.; Mizuno, G.; Morii, N.; Shimizu, T. Soft Matter 2008, 4, 1681–1687. (18) Shimizu, T.; Asakawa, M. Latest Researches in AIST. http://www.aist.go.jp/ aist_e/latest_research/2006/20060807/07.html, 2006. (19) Frusawa, H.; Fukagawa, A.; Ikeda, Y.; Araki, J. A.; Ito, K.; John, G.; Shimizu, T. Angew. Chem., Int. Ed. 2003, 42, 72–74. (20) Fujima, T.; Frusawa, H.; Minamikawa, H.; Ito, K.; Shimizu, T. J. Phys.: Condens. Matter 2006, 18, 3089–3096. (21) Arai, F.; Endo, T.; Yamauchi, R.; Fukuda, T.; Shimizu, T.; Kamiya, S. J. Rob. Mechatron. 2007, 19, 198–204.

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electric field should also be useful for manipulating LNTs as well as other micrometer-sized rods or spheroids, such as bacteria cells22,23 and carbon nanotubes;24,25 however, we have not exploited electrical manipulation of LNTs, with its advantages of synchronicity and simultaneity, in controlling the orientation of individual LNTs. The length of LNTs is a basic dimension for evaluating the characteristics of self-assembled LNTs, such as their length distribution. To accurately measure the length of individual LNTs in aqueous solutions, which are a favorable environment for them, we must not only suppress their rotational and translational Brownian motion but also acquire an ensemble of measurements for evaluating the length distribution. Indeed, it is indispensable for length measurements that we control the orientation of individual LNTs synchronously and simultaneously. To meet this requirement, a measurement method based on ac electric field orientation is expected to be effective. In the present work, we investigated a technique based on an ac electric field for controlling the orientation of LNTs and measuring their length in aqueous solutions. First, we examined the orientation of individual LNTs under an ac and a direct current (dc) electric field to determine which is suitable for our purpose. We found that the use of an ac field is suitable. To find the minimum rotation time and the frequency for stable orientation, in which individual LNTs stay and rotate without displacement from their positions, we also measured the rotation time of a single LNT driven by ac frequencies. To obtain basic information about how a rotating object was affected by the electric orientation force, we also roughly estimated the torque generated during rotation from the relationship between length and angular velocity of individual LNTs. Using our technique, we successfully measured the length distribution of two types of LNT and quantitatively analyzed the distribution’s maximum as well as its extension to the longer nanotube region. EXPERIMENTAL SECTION Preparation of LNT. The LNTs were made of renewableresource-based synthetic glycolipid, N-(9-octadecanoyl)-β-D-glucopyranolsilamide (GL-AIST) (Figure 1a), and had a hollow cylindrical architecture composed of multiple lipid bilayer membranes (Figure 1b). The LNTs were prepared by a modification of the literature method.6 The GL-AIST (40.0 g) was dissolved in methanol (500 mL) at 50 °C. Activated charcoal was added to the solution. After 2 h of stirring at the same temperature, selfassembled LNT formed and precipitated. The precipitated LNT was corrected, and the activated charcoal was removed by filtration. The extracted LNT was dried under reduced pressure. It appeared as hard white lumps and was ground into powder. We used two different types of LNT in powdered condition from the same extracted LNT before dissolving it in an aqueous solution. We refer to fine-powdered LNTs prepared with a mortar as GLNT-1 and slightly rough-powdered LNTs prepared with a (22) Washizu, M.; Kurahashi, Y.; Iochi, K.; Kurosawa, O.; Aizawa, S.; Kudo, S.; Magariyama, Y.; Hotani, H. IEEE Trans. Ind. Appl. 1993, 29, 286–294. (23) Nishioka, M.; Katsura, S.; Hirano, K.; Mizuno, A. IEEE Trans. Ind. Appl. 1997, 33, 1381–1388. (24) Krupke, R.; Hennrich, F.; Lohneysen, H. V.; Kappes, M. M. Science 2003, 301, 344–347. (25) Guo, Z. H.; Wood, J. A; Huszarik, K. L.; Yan, X. H.; Docoslis, A. J. Nanosci. Nanotechnol. 2007, 7, 4322–4332.

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Figure 1. (a) Structure of glycolipid GL-AIST used for self-assembled LNTs. (b) Schematic of the wall structure of an LNT formed from GLAIST. Spheres and cylinders represent the hydrophilic sugar headgroup and the hydrophobic tails of the glycolipid molecules, respectively. (c) FE-SEM and (d) TEM images of LNTs of GLNT-1. The scale bars are 2 µm and 500 nm. (e) Optical micrograph of the unstained LNTs of GLNT-1 immobilized on the electrodes, represented by black areas. Scale bar is 5 µm; the electrode gap is 10 µm.

Figure 2. Controlling the orientation of individual LNTs on the electrode system using an ac electric field. (a) Geometry of the orientation electrodes, which are ∼100 µm in width and have a gap of ∼200 µm between facing electrodes (1-1′ or 2-2′). (b) LNTs in Brownian motion without application of an ac electric field. (c) LNTs oriented by applying an ac electric field to the 1-1′ pair of electrodes. (d) LNTs oriented by applying an ac electric field to the 2-2′ pair of electrodes.

mechanical mill as GLNT-2. We used mainly GLNT-1; however, GLNT-2 was used only as a reference in the analysis of the length distribution (Figure 8). Parts c and d of Figure 1 show the images of a field emission scanning electron microscope (FE-SEM) and transmission electron microscope (TEM), respectively, for individual GLNT-1s. The observed LNTs were completely isolated and

Figure 3. Fluorescence images of two individual LNTs in Brownian motion in an aqueous solution. The time interval between images is 0.66 s. The scale bar is 5 µm.

formed no bundles. Averaged i.d. and o.d. of the LNTs were 70 nm (SD, 15.7 nm) and 192 nm (SD, 32.5 nm), respectively, for GLNT-1 and 90 nm (SD, 19.8 nm) and 297 nm (SD, 65.4 nm), respectively, for GLNT-2 from analysis of the TEM images. Figure 1e is an optical micrograph of the individual LNTs of GLNT-1, which is a comparison image against fluorescent-labeled LNTs. To observe this micrograph clearly with an ordinary optical microscope, individual LNTs were immobilized on electrodes by dielectrophoretic assembly18 in the presence of an ac electric field with a frequency of 100 kHz and a strength of 5 × 105 Vp/m. The gap between each pair of electrodes was 10 µm. Orientation Control of LNT. Figure 2a shows the geometry of aluminum electrodes on a coverslip for controlling the orientation of LNTs. Four electrodes (∼100 µm wide), 1-1′ and 2-2′ in Figure 2a, were arranged in two facing pairs with a gap of ∼200 µm between each pair and were placed at right angles to each other. A 30 µL aliquot of an LNT solution was dropped on the electrodes, which were fabricated on a coverslip and sandwiched with another small coverslip to seal them with glue to prevent the LNT solution from evaporating. Individual LNTs in water are randomly moved by Brownian motion in three dimensions (Figure 2b). To control their orientation, we applied an ac electric field and switched alternatively and orthogonally between the pairs of electrodes 1-1′ and 2-2′ (Figure 2a). Thus, individual LNTs are oriented synchronously in one direction (Figure 2c) and then in the other (Figure 2d) by switching the ac electric field. The strength of the dc and ac fields was constant for all experiments in the present study: 2 × 104 V/m and 2 × 104 Vp/m, respectively. The electrodes, without the electric field applied, were floated without a connection to ground to avoid distortion of the electric field that interferes with controlling the LNT orientation. The voltages were generated with a function generator (Agilent, dc to 15 MHz) and amplified with a high-speed power amplifier (NF Electronic Instruments, HSA4101). Visualization of the LNT. To visualize individual LNTs in sharp contrast in an aqueous solution, we stained LNTs with fluorescence dye 4′,6-diamidino-2-phenylindole (DAPI). The mixture of LNTs and DAPI was composed of 0.3 mg/mL LNT and 0.3 µM DAPI in ultrapure water. The mixture was incubated for 1 min at room temperature (∼25 °C) before use. Fluorescence images of individual LNTs were observed with an inverted fluorescence microscope (Nikon TE-2000) equipped with a highly sensitive EB-CCD camera (Hamamatsu Photonics). Fluorescence images of LNTs were recorded on digital videotape in real time and analyzed with an ARGUS-20 image processor (Hamamatsu

Figure 4. Comparison between direction of an individual LNT under (A) a dc electric field at 2 × 104 V/m and (B) an ac electric field at 2 × 104 Vp/m. (a-c) Fluorescence images of an oriented individual LNT using the 1-1′ electrodes in Figure 2a. A positive electrode was placed on the left side of each image in part A. The time interval of each image is 1 s. The scale bar is 10 µm. (d) Change in displacement ∆x of the center of gravity of an individual LNT along the x axis with time.

Photonics) to evaluate orientation and rotation dynamics and also the length distribution. RESULTS AND DISCUSSION As the averaged o.d. of LNTs made of GLNT1 [192 nm (SD, 32.5 nm)] was near or smaller than the diffraction-limited resolution of optical microscopes, it is difficult to obtain sharp contrast and distinguish a single LNT with optical microscopy (Figure 1e). To visualize individual LNTs in sharp contrast in an aqueous solution without immobilization, we stained LNTs with fluorescence dye DAPI. Figure 3 shows images of individual LNTs observed with a fluorescence microscope. The labeled LNTs were clearly observed in real-time without aggregation. The LNTs in an aqueous solution were diffused three-dimensionally by Brownian motion and easily went out of the focus of the microscope (Figure 3a-f). Thus, it is difficult to identify the length and orientation of each LNT. First, we evaluated the influence of dc and ac electric fields on the orientation dynamics of individual LNTs to determine which electric field is suitable for controlling individual LNTs in an aqueous solution. Figure 4 compares the direction of an individual LNT under dc and ac electric fields. The principal axis of an individual LNT was aligned with the direction of a dc (Figure 4A,a-c) and an ac (Figure 4B,a-c) electric field. Under the dc field, an individual LNT was electrophoretically moved to the positive electrode because LNTs are charged negatively (Figure 4A,d and Movie S-1 in the Supporting Information). In contrast, an individual LNT was oriented without displacement under an ac electric field at a frequency higher than 1 MHz in Figure 4B,d. The orientation under an ac electric field occurred due to interaction of the external electric field and the induced dipole moment in the LNT because the dipole moment is oriented parallel to the eternal field lines.22,23 At frequencies lower than 1 Analytical Chemistry, Vol. 81, No. 4, February 15, 2009

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Figure 5. Rotation and orientation of an individual LNT using an ac field. (a) Orientation using the 1-1′ electrodes in Figure 2a. (b) Rotation by changing the electrodes from 1-1′ to 2-2′. (c) Orientation using the 2-2′ electrodes after 90° rotation. (d) Change in the rotation angle with time. The origin is the time when the ac electric field was switched from the 1-1′ to the 2-2′ electrodes. The scale bar is 5 µm.

Figure 6. Dependence of the angular velocity ω on the frequency of the ac electric field.

MHz, an individual LNT was slowly moved along the electrodes and immobilized on an electrode by dielectrophoretic assembling (Figure 1e). This steady orientation without displacement under a high-frequency ac electric field in an aqueous solution provides a technique suitable for in situ length measurement of individual LNTs. In addition, applying a dc electric field generated heat, followed by a turbulence flow and bubbles due to electrolysis (Movie S-1 in the Supporting Information). Thus, we used only a high-frequency ac electric field to avoid turbulence flow and bubbles. To characterize the rotation and orientation of LNTs under a high-frequency ac field, we evaluated rotation time from video images of an individual LNT. Figure 5a-c shows the manipulation of a single LNT from horizontal to vertical orientation. Figure 5a shows the image of an individual LNT rotated with a pair of horizontal electrodes (Figure 2c). Switching the ac electric field with a pair of vertical electrodes (Figure 2d) changed the orientation of the LNT (Figure 5b) and then aligned it vertically (Figure 5c). This orientation control of an individual LNT is shown in Movie S-2 in the Supporting Information. To numerically evaluate a change in the orientation of a single LNT, we use an angular velocity ω, defined as the rotation angle of π/2 (in radians) over the rotation time (s). Figure 5d shows a change in the angle of an individual LNT with time, from the slope of which ω is evaluated. 1462

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Figure 7. Dependence of the angular velocity ω (b) and rotation time 1/ω (2) on the length of the LNT L. Estimated torque T is indicated by solid lines.

Figure 6 shows the dependence of angular velocity ω on the frequency of the ac electric field from 1 to 15 MHz. The amplitude of the electric field was kept at 2 × 104 Vp/m. The frequency dependence of ω showed a maximum at 4 MHz. This maximum frequency also provided stable orientation without displacement for LNTs; thus, 4 MHz is suitable for quickly rotating individual LNTs using an ac electric field. Note that the use of frequencies lower than 1 MHz made it difficult to rotate a single LNT in the present experimental setup due to disturbance from bubbling by electrolysis and turbulence flow resulting from Joule’s heating. Furthermore, at frequencies higher than 15 MHz, individual LNTs did not rotate because insufficient force was generated. The orientation force under an ac electric field is generated due to interaction of the external electric field and the induced dipole moment in the LNT. The generation of the dipole moment requires a certain time which is given by the relaxation time of the processes leading to polarization.22,23 In the ac electric field, the dipole strength is dependent on the frequency. With increasing frequency, the dipole moment decreases because the generated dipole is delayed with respect to the external ac electric field. Figure 7 shows the dependence of ω and rotation time 1/ω on the length L of individual LNTs from 2.98 to 32.6 µm at 4 MHz. The rotation time 1/ω increased with increasing length of individual LNTs. Approximation of a single LNT to a prolate spheroid, not to a cylinder, makes it possible to evaluate torque T given by22 T ) 8πηa3Kω

(1)

where η is the viscosity of water and a is the length of the major axis of a prolate spheroid. The torque rotates a prolate spheroid at angular velocity ω around its minor axis. The parameter K )

2 3

2 - β2 1 1 + β2 1+β - + log β 1-β 2β3

(2)

Figure 8. Histograms of the length L for two types of LNTs, (a) GLNT-1 and (b) GLNT-2. Each histogram was fitted to a Gaussian curve (solid line). (inset) Each histogram of 7 µm bin width was fitted to an exponential curve (solid line) for evaluation of the relaxation length λ.

includes the eccentricity β of a prolate spheroid

β)

1 - ( ab )

2

(3)

where b is the length of the minor axis of a prolate spheroid. Both the length and the o.d. of an individual LNT were substituted for parameters a and b in the approximation. To estimate torque T, we used a diameter of 190 nm and the viscosity η of water as 0.890 mPa s at 25 °C. The torque T was estimated at ∼8 pN µm, which is indicated by a solid line in Figure 7. Two fitting curves for 4 and 16 pN µm are also included in Figure 7 for reference. The fitting curve for ∼8 pN µm means that the torque generated by the ac field is approximately constant in the present range of ω and L. For LNTs longer than 15 µm, ω seems to deviate outward from the ∼8 pN µm curve, for reasons unknown at present. It may result from the distribution of the LNT’s diameter. The torque of 8 pN µm was approximately 4 times larger than that of 2.2 pN µm for the biological motor of an Escherichia coli flagellum.26 The larger torque suggested that rotation of individual LNTs by an ac field might be applied to analysis and control of the rotation of biological motors, for instance, ATP synthesis by rotating ATPase instead of the use of Actin filaments and magnetic beads.27-29 (26) Inoue, Y.; Lo, C.-J.; Fukuoka, H.; Takahashi, H.; Sowa, Y.; Pizota, T.; Wadhams, G. H.; Homma, M.; Berry, R. M.; Ishijima, A. J. Mol. Biol. 2008, 376, 1251–1259. (27) Noji, H.; Yasuda, R.; Yoshida, M.; Kinosita, K., Jr. Nature 1997, 386, 299– 302.

Using an ac field, we analyzed the length distribution of the LNTs. Parts a and b of Figure 8 show histograms illustrating the length distribution of GLNT-1 and GLNT-2, respectively. The length of individual LNTs was measured from fluorescence images by rotating each LNT at 4 MHz. The apparent size in the fluorescence image of individual LNT is larger than the actual size by approximately 0.3 µm due to the light blurring effect.30 In addition, a quantization error of the digitized fluorescence images is ±0.11 µm because one dimension length of a fluorescence image is divided 100 µm by 480 pixels, i.e., one pixel is 0.21 µm square. A measured principal axis length of an individual LNT is theoretically 0.6 ± 0.21 µm larger. In fact, the length measurement was limited for LNTs longer than 2 µm by difficulty in dividing particle LNTs from cylinder LNTs shorter than 2 µm due to the limited spatial resolution of the present measurement system. To obtain adequate statistics, each histogram includes ∼160 occurrences from individual LNTs. The peak of each distribution depends on the preparation process of the sample. Peaks occur in the histograms at 5.52 and 2.66 µm, both evaluated from Gaussian fitting in Figure 8a,b. Note that GLNT-1 was longer than GLNT-2 by a factor of 2. The distribution is characterized by one maximum and a trailing distribution to the longer-length region, both of which depend on the preparation process of the sample. To evaluate the extent of the longer-length region, each histogram was reproduced by resizing the bin width from 1 to 7 µm and fitted with a single exponential curve (insets in Figure 8a,b). The fitting exponential function was k0 + k1e-L/λ, where k0, k1, and λ are fitting parameters, and L is the length of an LNT. The λ evaluated from the curve fitting can be named the relaxation length. Thus, relaxation length λ is useful as a measure of the extent of the longer LNT region. The value of λ was estimated at 7.95 and 3.62 µm for GLNT-1 and GLNT-2, respectively. The relaxation length of GLNT-1 is longer than that of GLNT-2 by a factor of 2. Indeed, the length of GLNT-1 was extended to ∼50 µm in Figure 8a, although GLNT-2 was distributed in the region shorter than 20 µm. Thus, these results clearly demonstrate that length measurement based on rotation by a high-frequency ac electric field is a useful tool for analyses of the length distribution such as the maximum length and the extent of the longer-length region, both of which depend on the preparation process of the sample. CONCLUSIONS In conclusion, we have quantitatively evaluated the length distribution for LNTs prepared by different processes by controlling the orientation of individual LNTs using a highfrequency ac electric field. The maxima of the length distribution and the extension of the longer-length region depend on the preparation process. These results strongly suggest that the length measurement method in the present study will be a useful tool for exploring the length distribution of LNTs made (28) Adachi, K.; Oiwa, K.; Nishizaka, T.; Furuike, S.; Noji, H.; Itoh, H.; Yoshida, M.; Kinosita, K., Jr. Cell 2007, 130, 309–321. (29) Rondelez, Y.; Tresset, G.; Nakashima, T.; Kato-Yamada, Y.; Fujita, H.; Takeuchi, S.; Noji, H. Nature 2005, 433, 773–777. (30) Yoshikawa, K.; Yoshikawa, Y.; Koyama, Y.; Kanbe, T. J. Am. Chem. Soc. 1997, 119, 6473–6477.

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of different materials. We also observed rotation and orientation of individual LNTs in real time under a dc and an ac electric field by fluorescence video microscopy. The results show that a frequency of 4 MHz and a torque of ∼8 pN µm were suitable for stable orientation without displacement or undesirable immobilization on the surface of electrodes. This technique for stable orientation in an aqueous solution seems to have the potential to manipulate an individual LNT for length measurement, analysis of single biomolecules, such as biological motors, and in situ measurement of the length of an LNT before and after manipulation by laser trapping. Furthermore, the frequency and the torque properties of LNTs might be useful for detection and classification of LNTs encapsulating various materials, because the orientation force depends on the reflective index, or the permittivity, of the object oriented.31 (31) Pohl, H. A. Dielectrophoresis; Cambridge University Press: Cambridge, U.K., 1978.

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Our present work will support important progress in method for analyzing not only LNTs but also other nanotubes.

ACKNOWLEDGMENT This work was supported by the PRESTO program of the Japan Science and Technology Agency (JST) and a Grant from the Industrial Technology Research Program from NEDO, Japan. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review October 30, 2008. Accepted January 6, 2009. AC8022795