Nanomanipulation

DOI: 10.1002/smll.200701320

Dielectrophoretic Trapping of DNA Origami**

Anton Kuzyk,* Bernard Yurke, J. Jussi Toppari, rma Veikko Linko, and Paivi To

Owing to its exceptional self-assembly properties, DNA could become a key player in bottom-up fabrication of nanoscale systems.[1,2] A striking example of a DNA self-assembly technique is DNA origami which involves folding long single-stranded DNA with the help of short oligonucleotides.[3] Each short oligonucleotide can serve as a pixel. Therefore, origami structures can be decorated with complex patterns with 6-nm resolution to form a nanobreadboard, that is, a planar template for attachment of various materials (proteins,[4] carbon nanotubes, metal nanoparticles). Controlled positioning of DNA origami structures on the chip is a crucial open challenge for the realization of the nanobreadboard idea. Here we present a fully developed dielectrophoresis-based method for trapping DNA origami structures. The method gives a high yield of single-structure trapping between nanoelectrodes and controlled positioning of origami structures on a chip. The method provides a means of bridging bottom-up and top-down fabrication approaches in nanotechnology. Dielectrophoresis (DEP) is a manipulation method based on the movement of a polarizable particle in a nonuniform electric eld.[5] The DEP force is proportional to the
[] A. Kuzyk, Dr. J. J. Toppari, V. Linko, Prof. P. Torma Nanoscience Center, Department of Physics University of Jyvaskyla P.O. Box 35, FIN-40014 (Finland) Fax: (358) 142-604-756 E-mail: ankuzyk@cc.jyu. Dr. B. Yurke Bell Laboratories Alcatel-Lucent Murray Hill NJ 07974 (USA) Prof. P. Torma Department of Engineering Physics Helsinki University of Technology P.O. Box 5100 FIN-02015 HUT (Finland) Dr. B. Yurke Present address: Materials Science and Engineering Department Boise State University Boise, ID 83725 (USA) [] The authors thank P. W. K. Rothemund, E. Winfree, R. Barish, and R. Hariadi for useful discussions and J. Ylanne for help with lab facilities. This work was supported by Academy of Finland (project numbers 118160, 115020, 213362), NSF grant CCF-0622046, and it was conducted as part of a EURYI scheme awards (see www.esf.org/euryi). A. K. thanks the National Graduate School in Nanoscience and Teknii a kan Edistamissatio. : Supporting Information is available on the WWW under http:// www.small-journal.com or from the author.
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polarizability of the particle and the eld gradient. On the micrometer scale, DEP has been widely used as a trapping technique for a variety of objects, such as cells, viruses, long DNA molecules, and nanotubes.[6,7] It has been also demonstrated that DEP can be used for manipulation and trapping of objects on the nanoscale, such as short DNA molecules,[811] proteins,[11,12] and gold nanoparticles.[13,14] Recently, DEP was used for the large-scale assembly of carbon nanotubes.[15] In this Communication, we report a fully developed AC-DEP technique for trapping of DNA origami structures. Arbitrary shapes can be fabricated with DNA origami by choosing a proper set of short oligonucleotides (so-called staple strands). The origami structures are typically of the size 100 nm 100 nm and nearly two-dimensional (2D). The new challenges related to trapping of such complex self-assembled structures are 1) how to combine the designed self-assembly with trapping, considering that different environments (buffers) are required for the two processes, 2) how to nd the optimal trapping parameters for these nanoscale objects of high aspect ratio, 3) how to preserve the shape and structure of the origami objects in the trapping process, and 4) how to enforce the orientation of these objects of nontrivial shapes. To respond to (1), we applied a ligation procedure that allows changing of buffers. To respond to (2) and (3), optimal parameters were found by carefully studying the dependence of the DEP process on frequency, voltage, and buffer properties. In general, trapping of origami structures was found to be more sensitive to the parameters than single DNA trapping, and somewhat higher frequencies and lower voltages are required for trapping origami structures than for trapping a single DNA strand. To respond to (4) the orientation was enforced by thiol modication of certain areas of the origami structures that then preferentially attached to the gold nanoelectrodes. To our knowledge, this is the rst demonstration of DEP manipulation of complex, designed selfassembled structures. For the trapping, we used ngertip-type gold electrodes, with widths of 2025 nm and gaps of 7090 nm, fabricated on a SiO2 substrate using standard electron beam lithography (see Figure 1a). Two types of DNA origami structures were used for trapping (see Figure 1b): a rectangular (71 nm 98 nm) structure and a disk-shaped (approximately 100 nm in diameter) structure with three holes (the so-called smiley). For details of origami fabrication see the Supporting Information. Two oligonucleotides modied with thiol groups (the red dots in Figure 1a) were incorporated in the middle of each side of the origami structure to allow their attachment to the gold electrodes through SAu bonding. In our previous studies we showed that without any linkers DNA molecules diffuse very fast from the DEP trapping region after the DEP voltage is switched off, that is, unspecic physisorbtion is not strong enough to keep the object in the trap.[9] The sample of origami structures was ligated using T4 DNA ligase. The ligation procedure is known to increase the thermal and mechanical stability of DNA structures.[16] In the nal step of preparation of the solution of origami structures, the annealing buffer was replaced by a Hepes-based buffer of smaller conductivity. Two types of buffers were used (see Experi-

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Figure 1. Trapping DNA origami structure with dielectrophoresis. a) Schematic view of the origami trapping experiments. b) AFM image of origami structures used for DEP trapping. The image is taken on a MICA surface using tapping mode AFM in liquid. c) AFM image of a single smiley. d) Rectangular origami trapped with the optimal DEP parameters (on SiO2 surface, tapping mode AFM in air). The scale bar is 100 nm.

mental Section): buffer 1 containing magnesium acetate (pH % 7.2, s % 300 mS cm1) and buffer 2 containing magnesium chloride (pH % 7.2, s % 590 mS cm1). Note that the buffer should have low enough conductivity (well below 1 mS cm1) for DEP trapping to work.[17] We found that the ligation procedure was necessary for the origami structures to be stable in our buffer of choice that is very weakly conductive (low salt concentration). Altogether, we have fabricated and observed more than 200 samples to obtain reliable statistics of the process. DEP experiments were performed by incubating a drop of origami solution onto the surface of the nanoelectrode sample while applying a sinusoidal AC voltage to the electrodes. The DEP frequency was varied from 1 to 15 MHz, and the voltage

Figure 2. Effect of the DEP frequency on the trapping of an origami structure. a) DEP frequency below optimal, origami structures are trapped along the electrodes but not in the gap. b) Optimal DEP frequency, one origami structure is trapped precisely in the gap and another one is trapped at the end of the electrode. The scale bar is 100 nm.

from 0.6 to 2.4 Vpp (peak-to-peak value). With careful tuning of the DEP parameters, it is possible to trap a single origami structure precisely between the electrodes (Figure 1c and d). In order to trap a single origami structure precisely in the middle of the electrodes, we found that high frequencies should be used. AC dielectrophoresis helps to avoid electrophoretic effects, that is, simple attraction of the charged DNA at any place on a charged electrode. This is one reason why higher frequencies are favorable. On the other hand, polarizability of DNA goes down with frequency[9,10] and, therefore, an optimum has to be determined. We found that, for DEP frequencies below 10 MHz, origami structures are mostly observed along the electrodes in the region around the gap, and the yield of origami structures trapped precisely in the gap is very low, below 1%. For optimal frequencies, somewhat above 10 MHz, the yield becomes as high as 10% and there are clearly fewer origami structures in other places that are not in the gap. An example is shown in Figure 2 with rectangular origami structures in buffer 1. The results agree qualitatively with our earlier experiments where double-stranded DNA was more localized in the trap under higher DEP frequencies.[8] However, we found that somewhat higher frequencies are optimal for origami structures than for individual double strands, possibly due to higher polarizability of the former. We also found that origami structures undergo positive DEP (attracted to eld maxima rather than minima) for the whole applied frequency range. The DEP voltage is another crucial parameter that should be carefully tuned so that only a single object is trapped. In our previous experiments of trapping single DNA we showed that, for each frequency, there is a certain threshold voltage below which no trapping is observed and above which the amount of trapped DNA grows rapidly with voltage;[9,10] this was also observed for DEP trapping of gold nanoparticles.[14] The effect occurs because the trapping potential caused by the voltage should overcome the Brownian motion, which determines the minimum voltage. Naively, one could then expect that higher voltages would lead to stronger trapping over a larger area. Indeed, with voltage even slightly above the optimal, the trapping of origami structures takes place not only in the gap but also along the electrodes close to the gap. The effect is demonstrated in Figure 3 with smileys in buffer 1. Another nding is that smaller voltages are required at higher frequencies for the successful trapping of origami structures, which is somewhat surprising considering that the polarizsmall 2008, 4, No. 4, 447450

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Figure 3. Effect of DEP voltage on the trapping of an origami structure. a) DEP voltage above optimal, smileys are trapped not only in the gap but also along the electrodes close to the gap. b) Optimal DEP voltage, a single origami structure is trapped precisely in the gap. The scale bar is 100 nm.

ability is expected to decrease with frequency. A plausible explanation is given by the liquid ow due to AC electroosmosis,[18] which reaches a maximum at a certain frequency. The optimal frequency for the trapping of an origami structure is rather high and may be located above this maximum. For frequencies above the maximum, the harmful liquid ow decreases with increasing frequency and therefore lower voltages are sufcient. The best yield for trapping one origami structure precisely between the electrodes was obtained using 12.5 MHz frequency, and voltages of 1 Vpp for magnesium chloride containing buffer 2 and 0.8 Vpp for magnesium acetate containing buffer 1. As expected, for the buffer of higher conductivity the trapping voltage should be higher. The yield for trapping a single origami structure depends on the buffer. For buffer 2 the yield for trapping a single origami structure was about 5% and for buffer 1 about 10% (percentage of successful trapping experiments; yields are the same for smiley and rectangular origami structures). The higher yield is most probably because of the reduced uid ows (electrothermal and AC-electro-osmotic) in the buffer of lower conductivity.[18] Here we should note that with proper DEP parameters (optimal DEP frequency and DEP voltage above optimal) trapping of multiple origami structures (Figure 3a) can be achieved with almost 100% yield. As can be seen from the images, the origami structures are often folded. We do not know presently whether the problem is technical (such as folding during the water wash) or fundamental (such as the DEP process favors folded shapes
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Figure 4. Height prole of DNA origami structures on SiO2 surface. a) AFM image of a sample. The scale bar is 100 nm. b) Height proles along the white lines in (a).

due to increased polarizability). The former could be solved by technical improvements; the latter may require the attachment of material on the origami structure that helps it to preserve its 2D shape. An important observation from the AFM images is that the origami structures tend to show a height of approximately 2 nm (Figure 4), whereas the typical height of a single double-stranded DNA on SiO2 is approximately 1 nm.[19] This observation suggests that DNA double strands inside origami structures are less deformed due to the interaction with the SiO2 surface than a single double-stranded DNA, which may be of high interest in connection to studies of DNA conductivity where the conformation of DNA is expected to play a major role.[8,19] In summary, we have developed a dielectrophoresis-based method for trapping DNA origami structures. The method has a high yield and provides a platform for realization of the origami-based nanobreadboard. We believe that this approach can also be used for trapping other DNA structures, such as DNA nanotubes[20] or DNA self-assembled lattices,[21] and can nd application in less-than-100-nm scale device fabrication. Our results constitute an example of successful combination of bottom-up and top-down approaches where electrodes are fabricated using lithography methods and the trapped object is made by designed self-assembly.

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Experimental Section
Nanoelectrode fabrication: Fingertip-type gold electrodes with widths of 2025 nm and gaps of 7090 nm were fabricated on a SiO2 substrate using standard electron beam lithography and evaporation of metal (1 nm of Ti followed by 1520 nm Au) in an ultrahigh vacuum (UHV) chamber. Origami solution preparation: Two types of DNA origami structures were used for trapping (see Figure 1b): a rectangular (71 nm T 98 nm) structure, and a disk-shaped (%100 nm diameter) structure with three holes (the so-called smiley). Detailed information on these kinds of origami structures can be found on pages 2628 and 3235 of Supplementary Note 1 of reference [3]. These structures were fabricated by thermal annealing (from 90 -C to 20 -C, at rate of 1 -C minS1, in 0.1 -C steps) of 50 mL solution of 10 nM of single-stranded viral DNA (from the M13mp18 virus) with 10T excess concentration of staple strands. Staple strands along the edges of origami structures were left out to prevent the aggregation of origami structures because of the stacking interaction. Two thiol-modied oligonucleotides were incorporated in the middle of each end of the origami structures to allow attachment to the gold electrodes through SAu bonding. After annealing, the origami sample was ligated using T4 DNA ligase. In the nal step of origami solution preparation, the 1T TAE MgRR buffer used for origami annealing (1X Tris-Acetate-EDTA (TAE) with 12.5 mM magnesium acetate, pH % 8.1, s % 3.5 mS cmS1) was changed to the Hepes-based buffer of smaller conductivity using spin ltering, which should also wash out T4 DNA ligase and glycerol from the ligation procedure. Two types of buffers were used: (buffer 1) 6.5 mM Hepes, 2 mM NaOH, 1 mM magnesium acetate (pH % 7.2, s % 300 mS cmS1) and (buffer 2) 6.5 mM Hepes, 2 mM NaOH, 1 mM magnesium chloride (pH % 7.2, s % 590 mS cmS1). Final concentration of the origami structures in the solution for the DEP experiments was about 1 nM. Origami structures are stable at least for a week in our buffers of choice, if kept at room temperature. For details of origami fabrication, see the Supporting Information.
[1] [2] [3] [4] [5] N. C. Seeman, Nature 2003, 421, 427431. T. H. LaBean, H. Li, Nano Today 2007, 2, 2635. P. W. K. Rothemund, Nature 2006, 440, 297302. R. Chhabra, J. Sharma, Y. Ke, Y. Liu, S. Rinker, S. Lindsay, H. Yan, J. Am. Chem. Soc. 2007, 129, 1030410305. H. A. Pohl, Dielectrophoresis: the Behavior of Neutral Matter in Nonuniform Electric Field, Cambridge University Press, Cambridge, UK 1978. P. J. Burke, in Encyclopedia of Nanoscience and Nanotechnology, Vol. 6(Ed.: H. S. Nalwa ), American Scientic Publishers, Los Angeles, CA 2004, pp. 623641. M. P. Hughes, Nanotechnology 2000, 11, 124132. S. Tuukkanen, A. Kuzyk, J. J. Toppari, V. P. Hytonen, T. Ihalainen, P. Torma, Appl. Phys. Lett. 2005, 87, 183102. S. Tuukkanen, A. Kuzyk, J. J. Toppari, H. Hakkinen, V. P. Hytonen, E. Niskanen, M. Rinkio, P. Torma, Nanotechnology 2007, 18, 295204. S. Tuukkanen, J. J. Toppari, A. Kuzyk, L. Hirviniemi, V. P. Hytonen, T. Ihalainen, P. Torma, Nano Lett. 2006, 6, 13391343. R. W. Clarke, J. D. Piper, L. Ying, D. Klenerman, Phys. Rev. Lett. 2007, 98, 198102. R. Holzel, N. Calander, Z. Chiragwandi, M. Willander, F. F. Bier, Phys. Rev. Lett. 2005, 95, 128102. L. Bernard, M. Calame, S. J. van der Molen, J. Liao, C. Schonenberger, Nanotechnology 2007, 18, 235202. R. J. Barsotti, M. D. Vahey, R. Wartena, Y. M. Chiang, J. Voldman, F. Stellacci, Small 2007, 3, 488499. A. Vijayaraghavan, S. Blatt, D. Weissenberger, M. Oron-Carl, F. Hennrich, D. Gerthsen, H. Hahn, R. Krupke, Nano Lett. 2007, 7, 15561560. P. ONeill, P. W. K. Rothemund, A. Kumar, D. K. Fygenson, Nano Lett. 2006, 6, 13791383. S. Suzuki, T. Yamanashi, S. Tazawa, O. Kurosawa, M. Washizu, IEEE Trans. Indust. Appl. 1998, 34, 7583. A. Castellanos, A. Ramos, A. Gonzalez, N. G. Green, H. Morgan, J. Phys. D: Appl. Phys. 2003, 36, 25842597. A. Yu. Kasumov, D. V. Klinov, P.-E. Roche, S. Gueron, H. Bouchiat, Appl. Phys. Lett. 2004, 84, 10071009. a) D. Liu, S. H.Park, J. H. Reif, T. H. LaBean, Proc. Natl. Acad. Sci. USA 2004, 101, 717722. b) J. C. Mitchell, J. R. Harris, J. Malo, J. Bath, A. J. Turbereld, J. Am. Chem. Soc. 2004, 126, 16342 16343. c) P. W. K. Rothemund, A. Ekani-Nkodo, N. Papadakis, A. Kumar, D. K. Fygenson, E. Winfree, J. Am. Chem. Soc. 2004, 126, 1634416352. E. Winfree, F. Liu, L. A. Wenzler, N. C. Seeman, Nature 1998, 394, 539544. Received: December 29, 2007 Published online: March 18, 2008

[6]

[7] [8] [9] [10] [11] [12] [13] [14] [15]

[16] [17] [18] [19] [20]

[21]

Keywords:

dielectrophoresis . DNA self-assembly . nanoelectrodes . nanomanipulation

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