Anal. Chem. 2003, 75, 4347-4350
Accelerated Articles
DNA Manipulation and Retrieval from an Aqueous Solution with Micromachined Nanotweezers Gen Hashiguchi,*,† Takushi Goda,† Maho Hosogi,† Ken Hirano,‡ Noritada Kaji,‡ Yoshinobu Baba,‡ Kuniyuki Kakushima,§ and Hiroyuki Fujita§
Department of Intelligent Mechanical Systems, Faculty of Engineering, Kagawa University, 2217-20 Hayashi-cho, Takamatsu 761-0396, Japan, Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan, and Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
We have demonstrated DNA handling with micromachined nanotweezers that consist of a pair of opposing nanoprobes and integrated thermal expansion microactuators for changing the probe gap. The probe tips coated with a thin Al layer were dipped into a droplet of a solution containing λ-DNA molecules labeled with fluorescence dye, and then an ac electric field was applied between probes for several seconds. DNA molecules were then captured between the probe tips and retrieved from the solution to the air. The DNA capture between the probe tips could be performed more successfully on the droplet surface than in the underwater region. We also conducted an observation of the retrieved DNA molecules by transmission electron microscope and found that the thickness of the retrieved DNA molecules under the condition of this experiment was ∼21 nm when the time of the applied ac power (1 MHz, 20 Vpp) was 20 s.
Micromachining techniques based on IC-compatible fabrication technology have been applied to a great variety of microelectromechanical systems through the integration of moving mechanisms, sensors, and electronics in one chip. However, recent exploding interests in nanotechnology expect to realize a new class of function by means of nanometer-size mechanical parts, i.e., nanoelectromechanical systems (NEMS). One of the promising applications in NEMS is a handling device for nanoparticles such as DNA molecules. Washizu et al. first demonstrated alignment * Corresponding author. E-mail:
[email protected]. † Kagawa University. ‡ The University of Tokushima. § The University of Tokyo. 10.1021/ac034501p CCC: $25.00 Published on Web 07/30/2003
© 2003 American Chemical Society
and immobilization of DNA molecules between fixed electrodes1 by dielectrophoresis means. Porath et al. also reported a single DNA trapping between 8-nm gaps Pt electrodes.2 Nakao et al. fabricated a hook-shaped nanoprobe by electron beam deposition and extracted DNA fibers from a pretreated rice cell nucleus.3 Furthermore, tweezerlike nanoprobe devices (nanotweezers) were recently developed for manipulation of nanoparticles. Kim and Lieber demonstrated handling and electrical measurements of nanoclusters using carbon nanotube-based nanotweezers.4 Akita and co-workers also reported carbon nanotube nanotweezers similar to that of Kim and Lieber.5 Furthermore, Watanabe et al. presented electrical measurements of a DNA molecule using a triple-probe atomic force microscope consisted of carbon nanotube probes.6 Although these reports demonstrate successful manipulation of nanoparticles, the operation of nanotweezers in an aqueous solution has not been reported so far. From the viewpoint of recent interest in molecular physics, the direct manipulation of biomolecules dispersed in an aqueous solution with nanotweezers is to be noted as one of the important techniques for its analytical utility. For this purpose, we have recently developed a new type of micromachined nanotweezer having two opposed sharp nano(1) Yamamoto, T.; Kurosawa, O.; Kabata, H.; Shimamoto, N.; Washizu, M. IEEE Trans. IA 2001, 37, 1625-1633. (2) Porath, D.; Bezryadin, A.; de Vries, S.; Dekker, C. Nature 2000, 403, 635637. (3) Ooi, T.; Matsumoto, K.; Nakao, M.; Otsubo, M.; Shirakata, S.; Tanaka, S.; Hatamura, Y. Proceedings of the 13th Annual International Conference on Micro Electro Mechanical Systems; Miyazaki, Japan, January 23-27, 2000; pp 580-583. (4) Philip, K.; Lieber, C. M. Science 1999, 286, 2148-2150. (5) Akita, S.; Nakamura, Y.; Mizooka, S.; Takano, Y.; Okawa, T.; Miyatake, Y.; Yamanaka, S.; Tsuji, M.; Nosaka, T. Appl. Phys. Lett. 2001, 79, 1691-1693. (6) Watanabe, H.; Manabe, C.; Shigematsu, T.; Shimotani, K.; Shimizu, M. Appl. Phys. Lett. 2001, 79, 2462-2464.
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Figure 2. Experimental setup of retrieval of DNA molecules. The nanotweezer was attached to a 3D positioning system to approach a water droplet containing DNA molecules. DNA molecules were stretched and attracted to the probe tips by applying ac power of 1 MHz, 20 Vpp. Figure 1. SEM photograph of the micromachined nanotweezers. Inset: magnified view at the probe tip taken by a TEM. The radius of the tip was estimated to be 4 nm.
probes.7 The nanotweezer is fabricated only by IC-compatible microfabrication techniques so that mass production is possible. Here, in this paper, we present a successful λ-DNA manipulation with the nanotweezers as well as a brief description of the developed nanotweezers. Although Chiu and Zare have reported successful manipulation of λ-DNA molecules by optical means,8 we will show the methods using micromachined nanotweezers to retrieve stranded λ-DNA molecules from an aqueous solution to the air; this is a distinctive feature in comparison with conventional techniques such as laser trapping or dielectrophoresis9,10 in which DNA molecules are only trapped and moved in an aqueous solution. EXPERIMENTAL SECTION To retrieve DNA molecules from an aqueous solution, we have designed and developed a new type of nanotweezer having the following characteristics; (1) Two nanoprobes are facing one another having a gap adjusting to DNA molecules we intend to retrieve, (2) The rigidity of the probe arms is strong enough not to be bent by surface tension or viscosity of aqueous solutions in order to avoid unintentional change of the gap during retrieval experiments, (3) The gap of the two probe tips can be changed by monolithically fabricating microactuators. The third feature is optional but might be useful to investigate mechanical or electromechanical properties of DNA molecules since that is a function to apply mechanical stress to retrieved DNA molecules. Figure 1 shows a scanning electron microscope (SEM) photograph of the developed nanotweezers. The device was made of silicon on insulating (100) substrate and fabricated only by silicon micromachining techniques. Two sharp silicon probe tips having a radius of less than 10 nm were facing in a straight line at the end of the probe arms. This structure can be obtained by a combination process of anisotropic wet (7) Hashiguchi, G.; Fujita, H. Proceedings of the IEEE Sensors 2002; June 1114, 2002; No. 48. (8) Chiu, D. T.; Zare, R. N. J. Am. Chem. Soc. 1996, 118, 6512-6513. (9) Hughes, M. P. Nanoelectromechanics in Engineering and Biology; CRC Press: New York, 2003; Chapter 5. (10) Hirano, K.; Baba, Y.; Matsuzawa, Y.; Mizuno, A. Appl. Phys. Lett. 2002, 80, 515-517.
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etching of silicon and local oxidation of silicon techniques. Although the initial gap between the probe tips was designed to be 16 µm, which corresponds to the length of a λ-DNA molecule we used in this study, we also prepared nanotweezers having the gap of 18 µm for comparison. Thermal expansion actuators, which dimensions are 500 µm in length, 50 µm in width, and 25 µm in thickness, were integrated together with probes. The hinge structure magnifies the displacement of the thermal expansion actuator by 5 times at the end of probe arms (1 mm long); therefore, the probe moves 1 µm toward the opposite one when 0.2 W is applied to the thermal expansion actuator. The inset micrograph in Figure 1 is a magnified view of the probe taken by a transmission electron microscope (TEM). We confirmed that the radius of the probe tip was ∼4 nm, which was obtained by dry oxidation and successive oxide etching with HF solution. After sharpening the probe tips, 50-nm-thick Al film was deposited on the area of the probe tips, arms, and electrode pads. The Al layer acts as an anchor material to DNA molecules and is also used as an electrical conductor from electrical pads to probe tips. The details of the entire device fabrication and performance of the integrated actuators were described elsewhere.7 Having described the nanotweezers, we move to set up of a DNA retrieval experiment. It has been known that a DNA molecule adheres to metals such as Al or Pt1,11 at its ends. When an alternating (ac) electric field is applied to a solution containing DNA molecules, the DNA molecules are uncoiled and aligned along the electric field; eventually each end of the DNA molecules is attracted to the opposed electrodes until stripes are formed.9 Thus, the DNA molecule could be trapped between the electrodes.2 We intended to retrieve DNA molecules by replacing the fixed electrodes with gap-controlled nanotweezers. Figure 2 shows an illustration of the experimental setup of DNA molecule retrieval. The fabricated nanotweezers were mounted on a three-dimensional positioning system that was attached to a fluorescent microscope with a photomultiplier system. Only a small droplet, which contains λ-DNA (48.5 kbp, 16 µm long) in a concentration of 10 µg/mL stained by YOYO1 in a concentration of 1 µg/mL, was put on a glass slide, and then the nanotweezers approach the droplet. We examined two methods according to whether the probe head is dipped into the (11) Ueda, M.; Baba, Y.; Iwasaki, H.; Kurosawa, O.; Washizu, M. Jpn. J. Appl. Phys. 1999, 38, 6568-6569.
Figure 3. Series of fluorescent photographs taken in the DNA retrieval experiment in case A using 16-µm-gap nanotweezers (underwater region). (a) A scene when ac power (1 MHz, 20 Vpp) was applied to the nanotweezers. (b) Observation by an optical microscope in the air after taking up the nanotweezers from the solution. (c) Observation of the retrieved material (DNA molecules) by a florescent microscope.
Figure 4. Fluorescent microscope photographs at the gap of probe tips in case B (surface region) taken (a) during ac power (1 MHz, 20 Vpp) application and (b) after retrieval to the air.
DNA solution (case A) or put on the boundary between the solution and the air, that is, surface tension area (case B). After the nanotweezers were positioned, ac voltage (1 MHz, 20 Vpp) was applied between the probe tips for dozens of seconds. The probe head was then carefully taken up from the solution using a microactuator of the three-dimensional positioning system. The motion of DNA molecules was recorded as a fluorescent image during this operation. TEM observation was also carried out to determine the thickness of the retrieved DNA molecules. EXPERIMENTAL RESULTS Figure 3 shows a series of photographs taken during the DNA retrieval experiment in case A using 16-µm-gap nanotweezers. While ac voltage was applied, DNA molecules were attracted to high electric field regions; consequently, DNA flow was induced toward the probe gap. We could observe many DNA molecules attracted to both sides of the probe tips as shown in Figure 3a by bright fluorescence. After a 20-s application of the ac voltage, we took up the nanotweezers from the water droplet and observed the probes by an optical microscope. We found that a stringlike structure was captured between the probe tips as shown in Figure 3b. To identify the captured material, we conducted fluorescence observation again in air and confirmed the bright fluorescence from the captured material as shown in Figure 3c; this strongly suggests that the material is related to DNA. In addition, note that the fluorescence could be only observed from the captured material; this indicates other DNA molecules attracted to the probe tips were dropped when the nanotweezers were taken from the DNA solution; in other words, only the stretched DNA molecules were fixed to the probe tips. As described above, we have demonstrated the successful DNA retrieval from a DNA solution in case A; however, sometimes the
DNA molecule was not captured. We suppose that this is caused by strong flow of DNA molecules around the probe tips, which tend to prevent DNA molecules from stretching between the probes. In case B, on the other hand, the motion of DNA molecules during application of ac power was quite different from that in case A as shown in Figure 4a. The bright fluorescent area was observed between the probe tips; this means a lot of DNA molecules converged at the gap of probe tips rather than flowing through the gap. After switching off the ac power and taking up the nanotweezers from the solution, we confirmed that the DNA molecules were retrieved between the probe tips as shown in Figure 4b. We repeated the experiment using 16-µm-gap nanotweezers and obtained the same successful results in all trials; we believe that DNA retrieval on the boundary between the solution and the air is more reliable than that in the underwater region. We then conducted the case B experiments using 18-µm-gap nanotweezers. Most of the trials to retrieve DNA molecules failed, but we have also experienced successful retrieval of DNA molecules a few times even for the 18-µm-gap nanotweezers. At present, we suppose the 18-µm-gap nanotweezers retrieved DNA molecules longer than 16 µm, which yielded in the solution owing to a coalescence feature at the ends of the λ-DNA molecules. As shown in Figure 3b, the retrieved DNA molecule looks very thin. However, we believe that a bundle of DNA molecules was retrieved rather than a single DNA molecule, since the diameter of a single DNA molecule is only 2 nm. Therefore, we conducted a TEM observation to measure the thickness of the retrieved DNA molecules correctly. The DNA sample, which was retrieved under the same conditions as case B with the exception that the time of the ac power was 20 s, was set in the TEM chamber together with the nanotweezers. Figure 5 shows TEM photographs of the retrieved DNA molecules. The scale bar was calibrated by a Analytical Chemistry, Vol. 75, No. 17, September 1, 2003
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Figure 5. TEM photograph of the retrieved DNA molecules. The scale bars were calibrated by a crystallographic image of a graphite sheet. The thickness of the retrieval DNA molecules was estimated to be 21 nm when 1 MHz, 20 Vpp ac power was applied during 20 s.
crystallographic image of a graphite sheet. Although one end of the connection between the DNA molecules and probe was off and the DNA wire was curled, we found that the thickness of the bundle of DNA molecules is ∼21 nm. If the cross section of the bundle is supposed to be a circle and a single DNA molecule occupies it densely, the number of the retrieved DNA molecules might be estimated to be 110 pieces by simple scaling. In fact, it can be considered that the number of retrieved DNA molecules depends on a lot of factors such as density of DNA molecules, shape of the probes, temperature of the solution, and so on. We hope to obtain detailed data concerning the number of retrieved DNA molecules. DISCUSSION The successful DNA retrieval strongly depends on the flow of DNA molecules at the gap of the probes. DNA molecules, which are negatively charged particles, can be induced to move dielectrophoretic forces in a solution. As shown in Figure 1, since the shape of our probes is tapered, the generating electric field around the probes is unsymmetrical with the line connecting the opposing tips. Therefore, in case A, DNA molecule flow through the probe gap from the tapered side to the bottom side of the probes is inevitably induced. On the other hand, in case B, where the surface tension acts dominantly on DNA molecules rather than buoyant force, the motion of the DNA molecules would be restricted by the two-dimensional surface region. From the observation of motion of the DNA molecules in case B, it looks as if the DNA molecules converge at the gap of the probe tips rather than flow. The successful retrieval in case A was apparently because it occurs when the probe tips are placed either near the bottom or near the surface of the droplet on the glass slide in (12) De Pablo, P. J.; Moreno-Herrero, F.; Colchero, J.; Herrero, J. G.; Herrero, P.; Baro´, A. M.; Ordejo´n, P.; Soler, J. M.; Artacho, E. Phys. Rev. Lett. 2000, 85, 4992-4995.
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which the flow of DNA molecules will be disturbed. In fact, we could observe a stagnant DNA flow when the probes were placed near the bottom. In comparison with the two methods, we could say case B is more reliable than case A for DNA retrieval. This will be attributed to the difference in the concentration of DNA molecules between the probes; that is, the concentration of DNA molecules was very high in case B but very low in case A. However, if we intend to retrieve a few or a single DNA molecule, case A might be profitable because we can observe the motion of even a single DNA by fluorescent microscope during the DNA retrieval experiment. Up to now, we have achieved a single DNA capture between the probe tips in the droplet of solution, but have not yet confirmed the single DNA retrieval from the solution. In addition to the function of the DNA retrieval described above, we should insist on the useful advantage of our nanotweezers. Since the probes of the nanotweezers also can be employed for electrodes of electrical measurements, the nanotweezers are immediately used for a “nanotester” of DNA molecules. The measurements will be expected to be very reliable because our probes are perfectly isolated from any substrates. We have conducted measurements of retrieved λ-DNA molecules several times, but even picoampere-order currents could not observed; this strongly assists the numerical study reported in ref 12. Furthermore, our nanotweezers can apply mechanical stress to DNA molecules. Therefore, electromechanical characteristics of DNA molecules such as piezoelectricity might be investigated in corporation with the feature of electrical probes. In the future, we intend to develop a mechanical-electrical measurement system for DNA molecules using the newly developed nanotweezers. CONCLUSION We have demonstrated DNA retrieval from a water droplet of a DNA solution by micromachined nanotweezers that we have recently developed. We confirmed the thickness of the retrieved DNA molecules by TEM observation and found that a bundle of DNA molecules with a diameter of 21 nm was retrieved when 1 MHz, 20 Vpp ac power was applied for 20 s. We have also conducted electrical measurements of retrieved DNA molecules using the nanotweezers as electrical probes. We expect this micromachined nanotweezer will offer a new class of research tools for bionanoscience. ACKNOWLEDGMENT The authors thank Y. Mihara and F. Oohira for their support to this work. We also acknowledge the VDEC staff of The University of Tokyo for providing photolithographic masks.
Received for review May 12, 2003. Accepted July 15, 2003. AC034501P
