Large-area spatially ordered arrays of goldnanoparticles directed by lithographically confinedDNA origamiAlbert M. Hung1,2, Christine M. Micheel2,3, Luisa D. Bozano2, Lucas W. Osterbur2,4, Greg M. Wallraff2

and Jennifer N. Cha1,2*

The development of nanoscale electronic and photonicdevices will require a combination of the high throughput oflithographic patterning and the high resolution and chemicalprecision afforded by self-assembly1–4. However, the incorpor-ation of nanomaterials with dimensions of less than 10 nminto functional devices has been hindered by the disparitybetween their size and the 100 nm feature sizes that can beroutinely generated by lithography. Biomolecules offer abridge between the two size regimes, with sub-10 nm dimen-sions, synthetic flexibility and a capability for self-recognition.Here, we report the directed assembly of 5-nm gold particlesinto large-area, spatially ordered, two-dimensional arraysthrough the site-selective deposition of mesoscopic DNAorigami5 onto lithographically patterned substrates6 and theprecise binding of gold nanocrystals to each DNA structure.We show organization with registry both within an individualDNA template and between components on neighbouringDNA origami, expanding the generality of this methodtowards many types of patterns and sizes.

As device features shrink towards a critical limit, extensiveresearch has focused on the development and implementation ofnew lithographic approaches to fabricate increasingly smallerfeature sizes with high yields and minimal defects in a cost-effectivemanner. However, there remain significant hurdles to addressingfeatures smaller than 20 nm. In light of this, there have beenefforts to use self-assembly or ‘bottom-up’ approaches for pattern-ing. Although DNA and proteins are particularly suited for nano-scale assembly, providing registry and spatial orientation betweennanoscale components means that the biomolecular systems them-selves must also be organized into ordered arrays. One major barrierto achieving this has been the disparity between the resolution easilyobtained by optical lithography and the length scales of typical bio-logical systems. In this work, the two length scales are bridged usingDNA origami5: a mesoscopic biological template large enough to bepatterned on lithographically generated features but with a chemicalcomposition that also enables directed assembly of sub-10-nmmaterials through molecular recognition.

Two-5,7–11 and three-dimensional12–15 DNA-based scaffolds havegarnered significant interest for directing the assembly of nanoscalematerials, because specific molecular binding sites can be incorpor-ated within the templates with a precision of 1–2 nm (refs 3,16,17).In the case of gold nanocrystals, patterns as complex as two-dimen-sional arrays of differently sized particles18,19, three-dimensional col-loidal crystals20,21 and spiral chains22 have been assembled. Despiterecent progress23,24, the ability to control accurately the placement

and spacing between individual nanocrystals to produce large-areaarrays of arbitrary design has yet to be achieved. This requiresboth a high level of organization of the DNA templates on surfacesas well as the defect-free assembly of nanoparticles on every DNAscaffold. For practical purposes, technologically relevant substrates,and the removal of buffer and salts, are also desirable. Towards thisend, three key processes are needed that must be made compatiblewith one another: (1) patterning or adsorption of the templates ontoareas defined by top-down lithography; (2) removal of solventwithout destroying or removing the template; and (3) obtainingmaximum binding yields between the nanoparticles and the DNAtemplates while maintaining positional accuracy (Fig. 1).

Recently, we demonstrated adhesion of individual DNA origamistructures in solution to clean, hydrophilic oxide surfaces6. Largenumbers of DNA structures adsorbed to oxide surfaces in highmagnesium concentrations (Fig. 2a) but not to surfaces renderedhydrophobic by treatment with hexamethyldisilazane (HMDS),providing a means to pattern the DNA structures on lithographi-cally defined features with high fidelity (Fig. 2b). However, to usethe adsorbed DNA templates to direct the assembly of nanoscalematerials, a benign and facile method was needed to remove themagnesium and buffer solutions while leaving dried and intactDNA origami adhered to the oxide surfaces. Alcohol solutionshave previously been used to dry plasmid and lambda DNA onmica, with no obvious deleterious effects25,26. Although moreMg2þ was needed to adsorb DNA to oxide than to mica, ethanoltreatment was successfully adapted for drying DNA structures inplace on both patterned and non-patterned oxide (Fig. 2d,e).When samples were washed with pure water instead of ethanol/water mixtures, the DNA origami completely desorbed from theoxide surface. Wicking away excess solvents or complete evapor-ation left behind salt and resulted in non-specific adsorption ofexcess DNA structures.

We postulate that the addition of ethanol to the DNA solutionsresults in rapid diffusion of water into the ethanol, thereby loweringthe solvation state of the magnesium-coordinated DNA strands andcausing the adsorbed DNA structures to associate more intimatelywith the oxide surface. The ethanol treatment also causes DNAstructures that are weakly or partially bound to the oxide surfaceto adsorb more strongly and occasionally overlap with othersurface-bound DNA origami (Fig. 2e and Supplementary Fig. S1).Although stacked DNA origami structures were never observed insolution atomic force microscopy (AFM) studies, probablybecause they were weakly bound and easily knocked off by the scan-ning probe, they were frequently observed after drying. Reducing the

1Department of Nanoengineering, 9500 Gilman Drive M/C 0448, University of California San Diego, La Jolla, California 92093-0448, USA, 2IBM AlmadenResearch Center, 650 Harry Road, San Jose, California 95120, USA, 3The National Academies, Washington, DC 20001, USA, 4Department of MaterialsScience and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA. *e-mail: jencha@ucsd.edu

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molar amounts of DNA origami in solution helped to mitigatethese defects.

Having established a way to pattern and dry the DNA origami onlithographically defined substrates, a compatible method was

needed to attach nanomaterials to the DNA structures withoutnecessarily incorporating the nanomaterials as active ingredientsof the origami synthesis. Of the binding functionalities availablefor this purpose, complementary base-pairing is among the easiest

5’ A30 3’

Au

-O-S

i(CH

3)3

-O-S

i(CH

3)3

-O-S

i(CH

3)3

-OH

-OH

-OH

-O-S

i(CH

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3)3

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37 °C

Patterned HMDS/SiO2

2−3 h

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and dry

Anneal

iii

iii

5’ HS-Tx 3’

Figure 1 | Process for assembling two-dimensional nanoparticle arrays. The schematic illustrates the three key steps: (i) high-yield origami and nanocrystal

binding, (ii) controlled DNA origami adsorption and (iii) drying and salt removal.

a b c

d e f

Figure 2 | Binding of DNA origami to a surface. a–c, Adsorbed triangular DNA origami are imaged by AFM in solution. a, The DNA origami can bind to

clean silicon oxide surfaces in large numbers (scale bar, 400 nm). b, By patterning triangular binding sites of clean oxide in a HMDS film with electron-beam

lithography, the DNA structures can adsorb at specific locations and with a preferred orientation; the lithographically patterned triangular sites have sides of

110 nm and alternate between columns pointed up and columns pointed down. c, Modification of staples to extend A30 strands from the corners of the

triangular structure allows the origami to bind 5-nm gold nanoparticles coated with thymine oligomers (T8 in this case) when the two are mixed in solution.

d–f, Ethanol treatment is used to dry the DNA structures to the substrate, allowing them to be imaged by AFM in air. A large number of origami can be

captured in this way with no visible damage to their structure (d; scale bar, 50 nm; inset, 1.5 mm across). The ethanol treatment can dry patterned origami,

preserving the placement and orientation of each structure (e). Some non-specifically bound and overlapping origami structures are also captured (e, inset,

200 nm across). The lithographically patterned triangular sites have sides of 90 nm, which may not control the rotation of the origami as effectively as the

larger sites in b. DNA scaffolds bound with 5 nm gold nanoparticles (coated with T30 strands in this case) can also be dried down by ethanol treatment, but

the nanoparticle concentration must be kept low to limit the non-specific deposition of nanoparticles (f). As a result, the yield of corners bound with

nanoparticles is lower. Images a,c–f are height contrast images; b is an inverted phase contrast image. Scale bars in b,c,e,f, 500 nm.

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to implement27, but its efficiency is dependent on multiple variables,including nanoparticle concentration, annealing temperature andDNA linker lengths. To obtain large-area arrays of 5-nm gold nano-crystals, optimal parameters must be determined to push bindingyields with the DNA origami towards 100%.

First, gold nanocrystals were coated with poly-thymine strandseither 8 (T8) or 30 (T30) bases long (Supplementary Fig. S2).Steric and electrostatic repulsion between DNA strands stabilizedthe gold nanocrystals in the high-ionic-strength buffer solutionsnecessary for DNA adsorption to oxide. To ensure high binding effi-ciency of the gold nanocrystals to the DNA scaffolds yet maintainpositional accuracy, four free 30-mer poly-adeninine strands (A30)were incorporated at specific sites �5 nm apart at each corner ofthe triangular origami (Supplementary Fig. S3). On annealingthe polythymine-coated nanocrystals with corner-modified A30DNA triangles in solution at 37 8C, both the T30- and T8-coated5-nm gold nanocrystals bound selectively to the triangle corners(Fig. 2c,f ). Through AFM imaging and counting of bound nano-crystals to the DNA origami corners, the overall binding efficienciesas a function of annealing temperature, thymine linker length andnanoparticle concentrations were determined (Fig. 3). In all cases,the T8-coated gold nanoparticles demonstrated a significantlyhigher binding percentage to the A30-modified DNA trianglesthan the T30-coated gold, exceeding 85% binding with 10 nM par-ticle concentrations. Surprisingly, the T30-conjugated nanoparticlesshowed more efficient binding to the DNA origami only at elevatedannealing temperatures and higher nanocrystal concentrations. Onehypothesis for the lower binding yields of T30-coated gold is thatexcess T30 strands were more difficult to remove by microcentrifugefiltration. Free T30 strands could compete for binding tothe A30-modified DNA triangles. Another possibility is that theT8-coated gold nanocrystals diffuse more rapidly than theT30-conjugated gold, thereby increasing the probability of nanocrys-tal–origami contact. The higher binding yields obtained by anneal-ing the DNA origami and T30-coated nanocrystals at 37 8C mightalso be attributed to an acceleration of diffusion kinetics athigher temperatures.

For certain applications it may be more advantageous to firstpattern the DNA templates on a substrate and introduce nanoscaleobjects in subsequent steps, so annealing of nanoparticles to surface-bound DNA origami was also studied. Despite concerns thatethanol treatment and drying might destabilize the DNA structuresat the molecular level, solution AFM images (Supplementary

Fig. S4) of previously dried origami detected no degradation ofthe DNA structure, and the polyadenine strands were clearly ableto hybridize to the thymine-coated nanoparticles. The bindingyields of nanocrystal attachment to already patterned DNA origamiincreased with higher nanoparticle concentrations (Fig. 3), butnever exceeded the values obtained by solution annealing. Pre-patterning the DNA origami on oxide may entrap some A30 strandsbeneath the dried DNA origami and thus hinder binding to thegold nanocrystals. It is also plausible that nanocrystal diffusion nearthe surface is slower28 and more nanocrystals in solution are neededto increase the probability of finding a binding site on the surface.

Subsequent patterning studies used pre-annealing of the goldnanocrystals to the DNA origami in solution because it affordedhigher nanoparticle binding yields, fewer process steps, and theability to work with low nanoparticle concentrations, which is criti-cal for minimizing non-specific adsorption of excess nanocrystals tothe substrate. Thus, A30 corner modified DNA triangles hybridizedto T8-conjugated 5-nm gold nanoparticles were adsorbed and driedon electron-beam patterned oxide substrates with controlled place-ment and orientation to create a two-dimensional array of bothDNA origami and gold nanocrystals (Fig. 4). The addition of thegold nanoparticles to the DNA origami neither hindered bindingnor caused reorientation of the DNA structures on patternedoxide surfaces. Obtaining clean, well-ordered DNA origami andgold nanocrystal arrays required some optimization of the DNAorigami and gold nanocrystal concentrations to reduce defects(Supplementary Figs S5,S6). To evaluate the degree of order of thetwo-dimensional 5-nm gold nanocrystal arrays, Fourier transformsof both the AFM images and schematic representations of ideal pat-terns were performed (Fig. 4). The positions of the spots in theFourier transforms are primarily determined by the lithographicpattern, which is represented by a rectangular unit cell with unitvectors a¼ 430 nm between columns of triangles of the same orien-tation and b¼ 200 nm between rows. However, there are specificspots, denoted by their Miller indices (l, m) that are characteristicof either the triangle or nanoparticle patterns alone. The trianglepattern gives intense spots at (0, +1), (+1, +1), (+2, +1) and(+2, 0), whereas the Fourier transform of the nanoparticle patternshows six spots at (0, +2) and (+3, +1) arising from a distortedhexagonal pattern, illustrated by the red lines in Fig. 4d. No spotswould be seen at odd values of l if there was no control over theorientation of the triangles, because a¼ 215 nm would then bethe relevant unit vector. The Fourier transform of the AFM image

100

408

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Solution annealed at 37 °C

Solution annealed at 37 °C

Solution annealed at 23 °CSurface annealed at 23 °C

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200Nanoparticle concentration (nM)

Per c

ent o

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bou

nd w

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0

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Figure 3 | Binding efficiencies of 5-nm gold nanoparticles to corners of A30-modified triangular DNA origami either in solution or adsorbed on oxide

substrates. The effect of nanoparticle concentration, annealing temperature and length of thymine linker coating the nanoparticles is also shown. Numbers

indicate the total number of corners counted.

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shows up as a convolution of these two spot patterns, and the visi-bility of the characteristic six nanoparticle spots, in particular,demonstrates the order and fidelity with which the 5-nm com-ponents could be arranged over a large area.

Ordered, geometrically packed arrays of multiple DNA origamiconjugated with gold nanocrystals could also be obtained by

confining several origami within larger domains of oxide patternedin HMDS. As shown in Fig. 5a, adsorbing unmodified DNA tri-angles to long, rectangular lithographic features resulted in close-packed chains of multiple triangles with alternating left and rightorientations. By depositing DNA templates pre-hybridized withgold nanoparticles on these patterns, linear arrays of gold

a b c

d e

f g

(2, 1)(2, 1)

(2, 0)

(2, 0)

(2, 0)

(0, 1)

(0, 1)

(3, 1)

(3, 1)

(3, 1)

(3, −1)

(0, 2)

(0, 2)

(5, 1)(0, 2)

ab

Figure 4 | Two-dimensional arrays of gold nanoparticles directed by lithographically confined DNA origami. a, AFM image of origami first bound with

T8-coated gold nanoparticles, adsorbed to substrates patterned by electron-beam lithography and then dried. The electron-beam pattern consists of columns

of equilateral triangles with sides of 100 nm, alternately oriented up and down. Scale bar, 500 nm. b–e, Schematic representations of idealized origami-only

(b) and nanoparticle-only (d) patterns and their respective Fourier transforms (c,e) allow for the identification of first-order peaks unique to each pattern.

Peaks are labelled by Miller indices (l, m) based on the unit cell vectors a and b, as shown in panel b. f,g, A Fourier transform of the real AFM image shows

clear spots that can be attributed to a significant degree of long-range order in the arrangement of both the DNA origami (green) and the 5-nm gold

nanoparticles (red).

a b c d

Figure 5 | Geometrically packed arrays of multiple DNA origami conjugated with gold nanocrystals. a, Solution AFM phase image (contrast inverted) of

DNA triangles alone adsorbed to electron-beam patterned 360 nm� 80 nm rectangles shows limited geometric tiling of the triangles. b–d, Tiling is also

observed in AFM height images of gold nanoparticle (T8) bound origami adsorbed and dried to electron-beam patterned rectangles of different dimensions:

270 nm� 60 nm (b), 405 nm� 90 nm (c), and 240 nm� 120 nm (d). Rectangles of the appropriate size are added for comparison. Scale bar, 500 nm and

applies to all AFM images.

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nanocrystals with relatively large periodicities were obtained.Nanoparticle hybridization may improve order by hybridizing totwo or more adjacent origami triangles, pinning them together atthe corners. Simple lithographic confinement offers significant flexi-bility in the variety of tiling arrangements possible on a single sub-strate, and one can imagine extending the rectangles to drawmacroscopically long lines and complex paths.

Ultimately, the presence of DNA may not be necessary or desir-able for many devices. Preliminary data suggest that either dryetching or DNA melting may be used to remove the DNAorigami from the surfaces and still keep the nanoparticle arraysintact, but more careful quantitative studies are necessary to optimizeand confirm the removal of DNA and residual salts. Compared toultraviolet/ozone treatment, desorbing the DNA structures in warmbuffer is less likely to leave behind inorganic salts (SupplementaryFig. S7) and keeps intact other organic functionalities such as theHMDS surface pattern and the DNA on the nanoparticles, whichcan then be used to assemble additional components.

This method of combining top-down lithography and bottom-up DNA self-assembly offers the possibility of dictating the arrange-ment of a wide variety of sub-10-nm components over macroscopicareas with characteristic length scales ranging from a few to severalhundreds of nanometres. Much of the power of this strategy lies inits versatility. Both the DNA template and the lithographic patterncan be rationally designed to create not just periodic arrays butcomplex networks. A wide range of biological and chemical function-alities may be used to bind different nanoscale components to singleor tiled arrays of DNA templates, opening the possibility of smartrelease materials as well. Thus, we have demonstrated a combinationof several compatible techniques that can be rearranged to suit manydesired applications in nanoscale electronics, photonics or biology.

MethodsTriangular DNA origami were assembled according to the published procedure5 in astandard thermal cycler (Applied Biosystems Geneamp PCR System 9700). DNAoligonucleotide staples were obtained from Integrated DNA Technologies(Coralville, IA), and single-stranded M13mp18 phage DNA (M13) was obtainedfrom New England Biosciences. All chemicals were obtained from Sigma Aldrich,unless otherwise stated. For the triangular origami, four staples at each corner weremodified by extending 30 (or 8) adenine nucleotides on the 30 end (seeSupplementary Information). These A8- or A30-modified DNA staples wereintroduced into the DNA scaffolds in place of the original DNA staples. Afterthermal cycling, excess staples were removed by filtering the origami solutionthrough a Nanosep 30 kDa molecular weight cutoff (MWCO) centrifuge filter (Pall)using a single-speed benchtop microcentrifuge (VWR Galaxy Mini) at 2,000g. In asingle filter, 100 ml of origami solution was washed three times with 500 ml of10� TAE (400 mM tris acetate, 10 mM EDTA) and 125 mM MgCl2, spinning downto �50 ml of sample each time. The solution was brought to a volume of 50 ml, anorigami concentration of �1.4 nM, and stored at 210 8C until use.

Gold nanoparticles with a diameter of 5 nm (British Biocell International,obtained through Ted Pella) were concentrated and coated withbis-( p-sulphonatophenyl)phenylphosphine dihydrate dipotassium salt(Strem Chemicals) according to an established procedure29. After concentration,the nanoparticles were mixed with a 200� molar excess of 50-thiolated thymine8-mer or 30-mer (T8 or T30) oligonucleotides and allowed to incubate for at least12 h. To remove excess DNA and salts, the particles were washed four times in a30 kDa MWCO centrifuge filter, using 400–500 ml of water (HPLC grade),spinning down to �25 ml each time. The nanoparticles were resuspended in200 ml of water at a final concentration of 3–4 mM and stored at 4 8C.

Patterned substrates were prepared from clean silicon wafers with 100 nmthermal oxide primed with hexamethyldisilazane (HMDS) in a vapour chamber.Features were patterned into poly(methyl methacrylate) resist by electron-beamlithography and the developed areas cleaned with O2 plasma for 7 s in a plasmaetcher (March Plasma Systems, PX-250). The resist was stripped by ultrasonicationin N-methylpyrrolidone (NMP) at 55 8C for 20 min. The substrates were brieflyrinsed with 2-propanol followed by water, then dried in a nitrogen stream andused immediately.

Adsorption of DNA origami to clean oxide or hydrophilic oxide patterns inHMDS-primed (hydrophobic) wafers was achieved as previously reported6. Briefly, a1–2 ml drop of origami suspension in 10� TAE/MgCl2 was placed on the substrateand allowed to incubate for 2–3 h in a loosely capped container ringed with a moistKimwipe to prevent excessive evaporation. After incubation, the adsorbed DNAorigami could be dried in place by treating the sample with ethanol solutions. The

sample with the drop of origami solution still on it was first dipped briefly (5–8 s) ina solution of 50% (v/v) ethanol in water then immersed for an hour in 90% (v/v)ethanol/water. The sample was removed from ethanol and allowed to dry underambient conditions before AFM imaging.

The effectiveness of binding DNA-conjugated gold nanoparticles to A30-modified DNA triangles in suspension was studied at a constant concentration ofDNA templates of �0.7 nM and varying nanoparticle concentrations of 10, 50 and200 nM. The solutions were mixed in 10� TAE/MgCl2 buffer in 10–20 ml aliquots,annealed for 20 min either at 37 8C in the thermal cycler or at room temperature,and imaged by solution AFM on mica. Binding to adsorbed origami was studied byadsorbing and ethanol drying origami on clean oxide substrates, exposing theorigami to 2 ml of 50 or 200 nM nanoparticles in 10� TAE/MgCl2 for 20 min, thenimaging the sample by solution AFM. For the adsorption of nanoparticle-decoratedorigami to electron-beam patterned substrates, a 10 ml solution of 1.4 nM origamiand 40 nM nanoparticles in 10� TAE/MgCl2 was annealed at 37 8C for 20 min. Themixture was diluted to half the original concentration and adsorbed to the patternedsubstrate as usual. Before ethanol treatment, the sample was dipped in clean10� TAE/MgCl2 to remove most of the free nanoparticles first. This additional stepwas not necessary and not conducted if there were no nanoparticles in solution.

UV-vis absorption measurements were performed with a Cary 400spectrophotometer (Varian). Tapping-mode AFM was carried out on an MFP-3DStand Alone AFM (Asylum Research) or a Veeco Instruments Dimension 3100 (dryimaging only). NP-S silicon nitride sharpened tips (tip C, 15 kHz in solution, VeecoProbes) were used for solution imaging. OTESPA silicon tips (300 kHz, VeccoProbes) were used for imaging in air. For imaging in solution, 35–40 ml of bufferwas added to 1–5 ml of sample solution or to the dried origami. On oxide,10� TAE/MgCl2 was used; 1� TAE and 12.5 mM MgCl2 was used for nanoparticlebinding studies on mica. The images presented in this paper were flattened andplane-fitted. Typical height ranges were 6210 nm.

Received 1 July 2009; accepted 16 November 2009;published online 20 December 2009

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AcknowledgementsThis work was financially supported by the Center on Polymer Interfaces andMacromolecular Assemblies (award no. NSF DMR 0213618), the Office of Naval Research(award no. N00014-09-01-0250) and UCSD startup funds. The authors thank B. Davis foroptical lithography, P.W.K. Rothemund for helpful discussions and comments on themanuscript, and S. Swanson, C. Rettner and M. Sanchez for helpful discussions.

Author contributionsA.M.H., C.M.M. and J.N.C. conceived and designed the experiments. A.M.H., C.M.M. andL.W.O. performed the experiments. L.D.B. contributed the lithographically patternedsubstrates. A.M.H. and J.N.C. analysed the data. A.M.H. and J.N.C. co-wrote the paper. Allauthors discussed the results and commented on the manuscript.

Additional informationThe authors declare no competing financial interests. Supplementary informationaccompanies this paper at www.nature.com/naturenanotechnology. Reprints andpermission information is available online at http://npg.nature.com/reprintsandpermissions/.Correspondence and requests for materials should be addressed to J.N.C.

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