Nanomanipulation

DOI: 10.1002/smll.200701320

Dielectrophoretic Trapping of DNA Origami**

Anton Kuzyk,* Bernard Yurke, J. Jussi Toppari,

Veikko Linko, and Paivi Torma

Owing to its exceptional self-assembly properties, DNA couldbecome a key player in bottom-up fabrication of nanoscalesystems.[1,2] A striking example of a DNA self-assemblytechnique is ‘‘DNA origami’’ which involves folding longsingle-stranded DNA with the help of short oligonucleo-tides.[3] Each short oligonucleotide can serve as a pixel.Therefore, origami structures can be decorated with complexpatterns with 6-nm resolution to form a ‘‘nanobreadboard’’,that is, a planar template for attachment of various materials(proteins,[4] carbon nanotubes, metal nanoparticles). Con-trolled positioning of DNA origami structures on the chip is acrucial open challenge for the realization of the ‘‘nanobread-board’’ idea. Here we present a fully developed dielectro-phoresis-based method for trapping DNA origami structures.The method gives a high yield of single-structure trappingbetween nanoelectrodes and controlled positioning of origamistructures on a chip. The method provides a means of bridgingbottom-up and top-down fabrication approaches in nano-technology.

Dielectrophoresis (DEP) is a manipulation method basedon the movement of a polarizable particle in a nonuniformelectric field.[5] The DEP force is proportional to the

polarizability of the particle and the field gradient. On themicrometer scale, DEP has been widely used as a trappingtechnique for a variety of objects, such as cells, viruses, longDNA molecules, and nanotubes.[6,7] It has been alsodemonstrated that DEP can be used for manipulation andtrapping of objects on the nanoscale, such as short DNAmolecules,[8–11] proteins,[11,12] and gold nanoparticles.[13,14]

Recently, DEPwas used for the large-scale assembly of carbonnanotubes.[15]

In this Communication, we report a fully developedAC-DEP technique for trapping of DNA origami structures.Arbitrary shapes can be fabricated with DNA origami bychoosing a proper set of short oligonucleotides (so-calledstaple strands). The origami structures are typically of the size100 nm! 100 nm and nearly two-dimensional (2D). The newchallenges related to trapping of such complex self-assembledstructures are 1) how to combine the designed self-assemblywith trapping, considering that different environments(buffers) are required for the two processes, 2) how to findthe optimal trapping parameters for these nanoscale objects ofhigh aspect ratio, 3) how to preserve the shape and structure ofthe origami objects in the trapping process, and 4) how toenforce the orientation of these objects of nontrivial shapes.To respond to (1), we applied a ligation procedure that allowschanging of buffers. To respond to (2) and (3), optimalparameters were found by carefully studying the dependenceof the DEP process on frequency, voltage, and bufferproperties. In general, trapping of origami structures wasfound to be more sensitive to the parameters than single DNAtrapping, and somewhat higher frequencies and lower voltagesare required for trapping origami structures than for trapping asingle DNA strand. To respond to (4) the orientation wasenforced by thiol modification of certain areas of the origamistructures that then preferentially attached to the goldnanoelectrodes. To our knowledge, this is the first demonstra-tion of DEP manipulation of complex, designed self-assembled structures.

For the trapping, we used fingertip-type gold electrodes,with widths of 20–25 nm and gaps of 70–90 nm, fabricated on aSiO2 substrate using standard electron beam lithography (seeFigure 1a). Two types of DNA origami structures were usedfor trapping (see Figure 1b): a rectangular (71 nm! 98 nm)structure and a disk-shaped (approximately 100 nm indiameter) structure with three holes (the so-called smiley).For details of origami fabrication see the SupportingInformation. Two oligonucleotides modified with thiol groups(the red dots in Figure 1a) were incorporated in the middle ofeach side of the origami structure to allow their attachment tothe gold electrodes through S–Au bonding. In our previousstudies we showed that without any linkers DNA moleculesdiffuse very fast from the DEP trapping region after the DEPvoltage is switched off, that is, unspecific physisorbtion is notstrong enough to keep the object in the trap.[9] The sample oforigami structures was ligated using T4 DNA ligase. Theligation procedure is known to increase the thermal andmechanical stability of DNA structures.[16] In the final step ofpreparation of the solution of origami structures, the annealingbuffer was replaced by a Hepes-based buffer of smallerconductivity. Two types of buffers were used (see Experi-

["] A. Kuzyk, Dr. J. J. Toppari, V. Linko, Prof. P. TormaNanoscience Center, Department of PhysicsUniversity of JyvaskylaP.O. Box 35, FIN-40014 (Finland)Fax: (#358) 142-604-756E-mail: ankuzyk@cc.jyu.fi

Dr. B. YurkeBell LaboratoriesAlcatel-LucentMurray HillNJ 07974 (USA)

Prof. P. TormaDepartment of Engineering PhysicsHelsinki University of TechnologyP.O. Box 5100FIN-02015 HUT (Finland)

Dr. B. YurkePresent address:Materials Science and Engineering DepartmentBoise State UniversityBoise, 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 numbers118160, 115020, 213362), NSF grant CCF-0622046, and it was con-ducted as part of a EURYI scheme awards (see www.esf.org/euryi). A.K. thanks the National Graduate School in Nanoscience and Teknii-kan Edistamissaatio.

: Supporting Information is available on the WWW under http://www.small-journal.com or from the author.

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mental Section): buffer 1 containing magnesium acetate(pH$ 7.2, s$ 300mS cm%1) and buffer 2 containing magne-sium chloride (pH$ 7.2, s$ 590mS cm%1). Note that thebuffer should have low enough conductivity (well below1mS cm%1) for DEP trapping to work.[17] We found that theligation procedure was necessary for the origami structures tobe stable in our buffer of choice that is very weakly conductive(low salt concentration). Altogether, we have fabricated andobserved more than 200 samples to obtain reliable statistics ofthe process.

DEP experiments were performed by incubating a drop oforigami solution onto the surface of the nanoelectrode samplewhile applying a sinusoidal AC voltage to the electrodes. TheDEP frequency was varied from 1 to 15MHz, and the voltage

from 0.6 to 2.4 Vpp (peak-to-peak value). With careful tuningof the DEP parameters, it is possible to trap a single origamistructure precisely between the electrodes (Figure 1c and d).

In order to trap a single origami structure precisely in themiddle of the electrodes, we found that high frequenciesshould be used. AC dielectrophoresis helps to avoid electro-phoretic effects, that is, simple attraction of the charged DNAat any place on a charged electrode. This is one reason whyhigher 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 10MHz, origami structures aremostly observed along the electrodes in the region around thegap, and the yield of origami structures trapped precisely in thegap is very low, below 1%. For optimal frequencies, somewhatabove 10MHz, the yield becomes as high as 10% and there areclearly fewer origami structures in other places that are not inthe gap. An example is shown in Figure 2 with rectangularorigami structures in buffer 1. The results agree qualitativelywith our earlier experiments where double-strandedDNAwasmore localized in the trap under higher DEP frequencies.[8]

However, we found that somewhat higher frequencies areoptimal for origami structures than for individual doublestrands, possibly due to higher polarizability of the former. Wealso found that origami structures undergo positive DEP(attracted to field maxima rather than minima) for the wholeapplied frequency range.

The DEP voltage is another crucial parameter that shouldbe carefully tuned so that only a single object is trapped. In ourprevious experiments of trapping single DNAwe showed that,for each frequency, there is a certain threshold voltage belowwhich no trapping is observed and above which the amount oftrapped DNA grows rapidly with voltage;[9,10] this was alsoobserved for DEP trapping of gold nanoparticles.[14] Theeffect occurs because the trapping potential caused by thevoltage should overcome the Brownian motion, whichdetermines the minimum voltage. Naively, one could thenexpect that higher voltages would lead to stronger trappingover a larger area. Indeed, with voltage even slightly above theoptimal, the trapping of origami structures takes place not onlyin the gap but also along the electrodes close to the gap. Theeffect is demonstrated in Figure 3 with smileys in buffer 1.Another finding is that smaller voltages are required at higherfrequencies for the successful trapping of origami structures,which is somewhat surprising considering that the polariz-

communications

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

Figure 2. Effect of the DEP frequency on the trapping of an origamistructure. a) DEP frequency below optimal, origami structures aretrapped along the electrodes but not in the gap. b) Optimal DEPfrequency, one origami structure is trapped precisely in the gap andanother one is trapped at the end of the electrode. The scale bar is100 nm.

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ability is expected to decrease with frequency. A plausibleexplanation is given by the liquid flow due to AC electro-osmosis,[18] which reaches a maximum at a certain frequency.The optimal frequency for the trapping of an origami structureis rather high and may be located above this maximum. Forfrequencies above the maximum, the harmful liquid flowdecreases with increasing frequency and therefore lowervoltages are sufficient.

The best yield for trapping one origami structure preciselybetween the electrodes was obtained using 12.5MHz fre-quency, and voltages of 1Vpp for magnesium chloridecontaining buffer 2 and 0.8Vpp for magnesium acetatecontaining buffer 1. As expected, for the buffer of higherconductivity the trapping voltage should be higher. The yieldfor trapping a single origami structure depends on the buffer.For buffer 2 the yield for trapping a single origami structurewas about 5% and for buffer 1 about 10% (percentage ofsuccessful trapping experiments; yields are the same for smileyand rectangular origami structures). The higher yield is mostprobably because of the reduced fluid flows (electrothermaland AC-electro-osmotic) in the buffer of lower conductiv-ity.[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 beachieved with almost 100% yield.

As can be seen from the images, the origami structures areoften folded. We do not know presently whether the problemis technical (such as folding during the water wash) orfundamental (such as the DEP process favors folded shapes

due to increased polarizability). The former could be solved bytechnical improvements; the latter may require the attachmentof material on the origami structure that helps it to preserve its2D shape. An important observation from the AFM images isthat the origami structures tend to show a height ofapproximately 2 nm (Figure 4), whereas the typical heightof a single double-stranded DNA on SiO2 is approximately1 nm.[19] This observation suggests that DNA double strandsinside origami structures are less deformed due to theinteraction with the SiO2 surface than a single double-strandedDNA, whichmay be of high interest in connection to studies ofDNA conductivity where the conformation of DNA isexpected to play a major role.[8,19]

In summary, we have developed a dielectrophoresis-basedmethod for trapping DNA origami structures. The method hasa high yield and provides a platform for realization of theorigami-based nanobreadboard.We believe that this approachcan also be used for trapping other DNA structures, such asDNA nanotubes[20] or DNA self-assembled lattices,[21] andcan find application in less-than-100-nm scale device fabrica-tion. Our results constitute an example of successfulcombination of bottom-up and top-down approaches whereelectrodes are fabricated using lithography methods and thetrapped object is made by designed self-assembly.

Figure 4. Height profile of DNA origami structures on SiO2 surface.a) AFM image of a sample. The scale bar is 100 nm. b) Heightprofiles along the white lines in (a).

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

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communications

Experimental Section

Nanoelectrode fabrication: Fingertip-type gold electrodes with

widths of 20–25 nm and gaps of 70–90 nm were fabricated on a

SiO2 substrate using standard electron beam lithography and

evaporation of metal (1 nm of Ti followed by 15–20 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 nmT 98 nm) structure, and a disk-shaped ($100 nm dia-

meter) structure with three holes (the so-called smiley). Detailed

information on these kinds of origami structures can be found on

pages 26–28 and 32–35 of Supplementary Note 1 of reference [3].

These structures were fabricated by thermal annealing (from 90 -Cto 20 -C, at rate of 1 -C minS1, in 0.1 -C steps) of 50mL 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-modified oligonucleotides were incorpo-

rated in the middle of each end of the origami structures to allow

attachment to the gold electrodes through S–Au bonding. After

annealing, the origami sample was ligated using T4 DNA ligase. In

the final step of origami solution preparation, the 1T TAE MgRRbuffer used for origami annealing (1X Tris-Acetate-EDTA (TAE) with

12.5mM magnesium acetate, pH$8.1, s$ 3.5mS cmS1) was

changed to the Hepes-based buffer of smaller conductivity using

spin filtering, which should also wash out T4 DNA ligase and

glycerol from the ligation procedure. Two types of buffers were

used: (buffer 1) 6.5mM Hepes, 2mM NaOH, 1mM magnesium

acetate (pH$7.2, s$300 mS cmS1) and (buffer 2) 6.5mM Hepes,

2mM NaOH, 1mM magnesium chloride (pH$7.2, s$590mS

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.

Keywords:dielectrophoresis . DNA self-assembly . nanoelectrodes .nanomanipulation

[1] N. C. Seeman, Nature 2003, 421, 427–431.[2] T. H. LaBean, H. Li, Nano Today 2007, 2, 26–35.[3] P. W. K. Rothemund, Nature 2006, 440, 297–302.[4] R. Chhabra, J. Sharma, Y. Ke, Y. Liu, S. Rinker, S. Lindsay, H. Yan, J.

Am. Chem. Soc. 2007, 129, 10304–10305.[5] H. A. Pohl, Dielectrophoresis: the Behavior of Neutral Matter in

Nonuniform Electric Field, Cambridge University Press, Cambridge,UK 1978.

[6] P. J. Burke, in Encyclopedia of Nanoscience and Nanotechnology,Vol. 6(Ed.: H. S. Nalwa ), American Scientific Publishers, LosAngeles, CA 2004, pp. 623–641.

[7] M. P. Hughes, Nanotechnology 2000, 11, 124–132.[8] S. Tuukkanen, A. Kuzyk, J. J. Toppari, V. P. Hytonen, T. Ihalainen, P.

Torma, Appl. Phys. Lett. 2005, 87, 183102.[9] S. Tuukkanen, A. Kuzyk, J. J. Toppari, H. Hakkinen, V. P. Hytonen, E.

Niskanen, M. Rinkio, P. Torma, Nanotechnology 2007, 18, 295204.[10] S. Tuukkanen, J. J. Toppari, A. Kuzyk, L. Hirviniemi, V. P. Hytonen, T.

Ihalainen, P. Torma, Nano Lett. 2006, 6, 1339–1343.[11] R. W. Clarke, J. D. Piper, L. Ying, D. Klenerman, Phys. Rev. Lett.

2007, 98, 198102.[12] R. Holzel, N. Calander, Z. Chiragwandi, M. Willander, F. F. Bier,

Phys. Rev. Lett. 2005, 95, 128102.[13] L. Bernard, M. Calame, S. J. van der Molen, J. Liao, C. Schonen-

berger, Nanotechnology 2007, 18, 235202.[14] R. J. Barsotti, M. D. Vahey, R. Wartena, Y. M. Chiang, J. Voldman, F.

Stellacci, Small 2007, 3, 488–499.[15] A. Vijayaraghavan, S. Blatt, D. Weissenberger, M. Oron-Carl, F.

Hennrich, D. Gerthsen, H. Hahn, R. Krupke, Nano Lett. 2007, 7,1556–1560.

[16] P. O’Neill, P. W. K. Rothemund, A. Kumar, D. K. Fygenson,Nano Lett.2006, 6, 1379–1383.

[17] S. Suzuki, T. Yamanashi, S. Tazawa, O. Kurosawa, M. Washizu,IEEE Trans. Indust. Appl. 1998, 34, 75–83.

[18] A. Castellanos, A. Ramos, A. Gonzalez, N. G. Green, H. Morgan, J.Phys. D: Appl. Phys. 2003, 36, 2584–2597.

[19] A. Yu. Kasumov, D. V. Klinov, P.-E. Roche, S. Gueron, H. Bouchiat,Appl. Phys. Lett. 2004, 84, 1007–1009.

[20] a) D. Liu, S. H.Park, J. H. Reif, T. H. LaBean, Proc. Natl. Acad. Sci.USA 2004, 101, 717–722. b) J. C. Mitchell, J. R. Harris, J. Malo, J.Bath, A. J. Turberfield, 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, 126,16344–16352.

[21] E. Winfree, F. Liu, L. A. Wenzler, N. C. Seeman, Nature 1998, 394,539–544.

Published online: March 18, 2008Received: December 29, 2007

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