Gold Nanolenses Self-Assembled by DNA Origami - ACS Photonics

Mar 30, 2017 - Department of Chemistry & SALSA, Humboldt Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany. ∥ Leibniz IPHT Jena ...
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Gold nanolenses self-assembled by DNA origami Christian Heck, Julia Prinz, André Dathe, Virginia Merk, Ondrej Stranik, Wolfgang Fritzsche, Janina Kneipp, and Ilko Bald ACS Photonics, Just Accepted Manuscript • DOI: 10.1021/acsphotonics.6b00946 • Publication Date (Web): 30 Mar 2017 Downloaded from http://pubs.acs.org on April 2, 2017

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Gold nanolenses self-assembled by DNA origami Christian Hecka,b,c, Julia Prinza, André Dathed, Virginia Merkc, Ondrej Stranikd, Wolfgang Fritzsched, Janina Kneippb,c, Ilko Bald*a,b a Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24–25, 14476 Potsdam, Germany. b BAM Federal Institute for Materials Research and Testing, Richard-Willstätter-Str. 11, 12489 Berlin, Germany. c Department of Chemistry & SALSA, Humboldt Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany. d IPHT Jena, Albert-Einstein-Straße 9, 07745 Jena, Germany. * Corresponding author: [email protected]

Abstract Nanolenses are self-similar chains of metal nanoparticles which theoretically can provide extremely high field enhancements. Yet, the complex structure renders their synthesis challenging and has hampered closer analyses so far. Here, DNA origami is used to self-assemble 10, 20 and 60 nm gold nanoparticles as plasmonic gold nanolenses (AuNLs) in solution and in billions of copies. Three different geometrical arrangements are assembled and for each of the three designs, surface-enhanced Raman scattering (SERS) capabilities of single AuNLs are assessed. For the design which shows best properties, SERS signals from the two different internal gaps are compared by selectively placing probe dyes. The highest Raman enhancement is found for the gap between the small and medium nanoparticle, which is indicative of a cascaded field enhancement.

TOC Graphic:

Key words: Plasmonics, DNA origami, SERS, nanolenses, gold nanoparticles

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Assemblies of plasmonic nanoparticles are promising building blocks in the development of nanoscopic optical devices: They can guide light1 and focus it2 at scales far below the diffraction limit. Upon irradiation with light of their resonance wavelength, localized surface plasmons are excited in plasmonic nanoparticles, which amplifies the electromagnetic field in their proximity. If two or more nanoparticles are in close proximity, the individual surface plasmons can couple and create spots of very high field intensity.3–6 Such high local fields can be exploited for the enhancement of signals in Raman,7–9 infrared10,11 and fluorescence

spectroscopy.12–14

Collinear

assemblies

of

self-similar

gold

nanoparticle chains (so-called gold nanolenses, AuNLs) are particularly interesting plasmonic objects.15,16 Due to their incremental architecture, a cascaded enhancement of the electromagnetic field was predicted, that can potentially outperform other plasmonic structures such as dimers or rods.17,18 In addition to the high field enhancements, nanolenses are predicted to be efficient generators of second harmonic local fields19 and further, to be capable of surface plasmon amplification by stimulated emission (spaser).20 In order to build such plasmonic nanostructures and to learn more about the basic principles of signal enhancement in different processes, molecules or materials, exquisite control over relative positioning and type of the constituent nanoparticles is needed.21,22 In the case of gold nanoparticles, highly versatile interactions with organic molecules can be exploited to control their positions and interaction. Thanks to the programmable nature of DNA-DNA interaction, DNA nanotechnology,23 and namely scaffolded DNA origami,24 have developed into key technologies for selfassembling nanocomponents of many kinds.25–27 Gold nanoparticles that have been coated with DNA strands28,29 can be bound at defined positions on such DNA origami scaffolds.30–33 With orthogonal coating strand sequences, different nanoparticles can be programmed to bind at selected positions. By using this self-assembly principle, we present a bottom-up technique to synthesize plasmonically active AuNLs in solution, in billions of copies, which is in contrast to typical top-down methods, where only few structures are produced.34,35 Several approaches that use DNA origami to assemble metal nanoparticle homomers for surface-enhanced Raman scattering (SERS) measurements have been reported,36–40 utilizing two to four nanoparticles of the same kind, and recently reaching single 2 ACS Paragon Plus Environment

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molecule sensitivity.41,42 AuNLs represent more complex aggregates, which are more challenging to assemble, yet promising in their plasmonic applications. As will be demonstrated here, DNA origami scaffolds can arrange individual gold nanoparticles of appropriate size-range (10 nm, 20 nm, and 60 nm) in strictly defined patterns and close enough so that they act as nanolenses. The results of the experiments and theoretical considerations reported here indicate that the gold nanoparticle assemblies enable efficient plasmon coupling, and the generation of high local optical fields. The three gold nanoparticles are assembled in three different spatial arrangements (Scheme 1). The collinearity and order of the assemblies are controlled by capture strands with specific recognition sequences on the DNA origami scaffold. For one of the nanolens designs discussed here, SERS signals from the two internal gaps are compared by selectively labelling gold nanoparticles with Raman probes. As predicted in the literature,18 the strongest enhancement is found located between the medium-sized and smallest nanoparticle.

Scheme 1 Scheme and representative AFM images of the three AuNL assemblies. Triangular DNA origami scaffolds43,44 with capture strands (colored) bind gold nanoparticles coated with complementary DNA sequences. In AuNL 20b-10-60, the 20 nm gold nanoparticle is positioned on the opposing face of the DNA origami scaffold. Scale bars: 100 nm.

Results & Discussion

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As base for our assemblies, the sharp triangle introduced by Rothemund24 is used, which has a side length of 127 nm and consists of a single layer of doublestranded DNA. It self-assembles when 208 short ssDNA strands, termed staple strands, and the long scaffold ssDNA are subjected to a temperature program. Extended staple strands are used at those positions on the DNA origami scaffold where gold nanoparticles are to be bound (extension typically by 28 bases). If the sequence of such an exposed capture strand is complementary to the DNA on a specific nanoparticle, both strands hybridize and the nanoparticle is immobilized at the respective position. The number of capture strands on the DNA origami scaffold is adjusted with respect to gold nanoparticle size: 60 nm particles are bound by four to five capture strands, 20 nm particles by four, and 10 nm particles by three. The capture strand positions used to realize the three different AuNL geometries are displayed in the SI (S8-S10). Gold nanoparticles are coated with 3’ thiol-modified DNA using a protocol modified from Zhang et al.45 The DNA coating of the nanoparticles serves two purposes: (i) the selective hybridization to complementary capture strands on the DNA origami scaffold, and (ii) stabilization against aggregation in the high ionic strength buffer that is required for DNA origami integrity. In practice, the particle stabilization turned out to be pivotal. DNA bases greatly differ in their affinity to gold surfaces. The resulting unspecific interaction then blocks the thiol-gold docking during the coating procedure. By that it decreases the final coating density and consequentially also the gold nanoparticle stability.46 This becomes crucial when working with larger nanoparticles (d >15 nm), which in general have a lower stability. With thymine having the lowest affinity to gold,47 particles preferentially are coated with oligo-T strands. For the assembly of complex structures such as AuNLs, orthogonal sequences are required, which makes the assembly more cumbersome, especially, if for plasmonic applications larger particles are needed. With the coating protocols displayed in the SI, sufficiently stable nanoparticles can be obtained. In order to enable the characterization of the three AuNL designs regarding their ability to enhance Raman scattering signals, the 10 nm gold nanoparticles carry the fluorescent dye tetramethylrhodamine (TAMRA). For these particles, a decreased stability is observed. TAMRA is attached at the solution-facing 5’-end of 4 ACS Paragon Plus Environment

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the DNA coating. This lack of interaction of the dye with the gold nanoparticle surface should lead to very small or negligible contributions of chemical enhancement effects, and a main contribution to potential SERS enhancement by electromagnetic enhancement. Coated gold nanoparticles are bound to the DNA origami scaffold and the resulting nanolenses are purified by agarose gel electrophoresis. For further characterization, the samples are deposited onto silicon wafers. During this process, AuNLs are exposed to high concentrations of Mg2+ (110 mM), which shields and bridges negative charges. With this and drying effects, the coated particles are assumed to touch, leading to a nominal distance between the gold spheres of ca. 3-4 nm. Areas around a scratch on the silicon wafer are mapped with a Raman microscope and then scanned by AFM. Both images are superimposed and the detailed structures of those AuNLs giving SERS signals are determined. Apart from the electromagnetic enhancement occurring due to the high local field, the SERS signal from an individual AuNL is related to the number of TAMRA molecules participating in the SERS process, and hence to the number of TAMRA-modified DNA strands on the surface of the labelled gold nanoparticles. In order to determine this number, a protocol from Hurst et al.48 is applied. The average number was 99 TAMRA molecules for the 10 nm particles in AuNLs 2010-60 and 20b-10-60, and 39 TAMRA molecules for the 10 nm particles in AuNLs 10-20-60. The coating density between the two batches varies since different DNA coating strand sequences are employed. For each design, a set of 20 single AuNLs was examined; the resulting average SERS spectra and respective SERS spectra with strongest signal are shown in Fig. 1 A-C. The average SERS spectra of the three AuNL designs exhibit several differences, with 20-10-60 showing the strongest SERS signal, followed by 20b10-60, whereas design 10-20-60 has the weakest one. The single AuNL analysis enables a close look at how differences in average SERS signals between designs are constituted: In the histograms of Fig. 1 D-F, each design yields a majority of spectra with relatively low signals just around the limit of detection. The main difference between the three designs lies in the number and intensity of the small amount of spectra of relatively high intensity (compare Fig. 1 D, 1 E, and 1 F). Whereas six AuNLs of design 20-10-60 yield a signal-to-noise ratio above six, for 5 ACS Paragon Plus Environment

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design 20b-10-60 only two individual AuNLs show SERS signals of similar intensity. For design 10-20-60, none of the 20 examined AuNL yield signals of this strength.

Fig. 1 (A-C) Average spectrum and most intense Raman spectrum obtained from each respective AuNL design: (A) 20-10-60, (B) 20b-10-60, (C) 10-20-60. Characteristic bands of TAMRA are highlighted in yellow. (D-F) Distribution of signal-to-noise in the individual SERS spectra: (D) 20-10-60, (E) 20b-10-60, (F) 10-20-60. The intensity of the most prominent TAMRA band of each respective spectrum is divided by the noise σ. The limit of detection is indicated by a dotted line. One AuNL of design 20-10-60 showed a particularly strong SERS signal (Fig. 1 A, red), relating to an enhancement factor (EF) of 1.4 * 106. This EF is underestimated, because it is determined based on the total number of TAMRA molecules. Only a fraction of them are in the region where the local field is very high, therefore, the number of molecules that contribute to the SERS signal is expected to be considerably smaller. Further effects such as the additional 6 ACS Paragon Plus Environment

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resonance of the TAMRA molecules at the wavelength of the excitation laser (532 nm) alter the electromagnetic enhancement of SERS. The corresponding AFM, SEM and localized surface plasmon resonance (LSPR) data for this wellperforming AuNL are displayed in Fig. 2. A complimentary data set for a AuNL with low enhancement is shown in Fig. S1 of the SI.

Fig. 2 (A) AFM, (B) SEM, and (C) LSPR data for the AuNL of design 20-10-60 with a particularly strong SERS signal (spectrum displayed in Fig. 1 A). The AFM image reveals the DNA origami structure beneath the AuNL in dark red. Scale bars: 100 nm.

The estimates of the enhancement are on the same order of magnitude as those found by Finite Difference Time Domain (FDTD) simulations (Fig. 3). The simulation results demonstrate how the electric field enhancement is affected when gold nanoparticle positions differ in the different nanolens designs. It should be noted that the actual geometry of an individual AuNL might deviate from the ideal, collinear arrangement, since in the experiments reported here (i) the AuNLs are dried on the surface, (ii) the nanoparticles are assembled on a planar DNA origami scaffold, and (iii) the nanoparticle-anchoring strands allow for some flexibility and imply variation between individual nanolenses. The SERS enhancement of the different AuNL designs strongly depends on how closely individual structures match ideal collinear geometries with smallest gaps (Fig. 3 A-C). The relatively small enhancement obtained with design 10-20-60 (see Fig. 1 C for SERS spectra) can be explained by the flexibility of the 10 nm gold nanoparticle: In AuNL 10-20-60, the TAMRA-labelled 10 nm particle is free to 7 ACS Paragon Plus Environment

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move as far the DNA tethers allow. The simulation shows that an additional 5 nm gap between 10 nm and 20 nm gold nanoparticle causes the SERS signal in design 10-20-60 to drop by two orders of magnitude (Fig. 3 C, F). This effect is considerably stronger than the variations expected for designs 20-10-60 and 20b10-60, where the 10 nm particle is wedged between the larger particles and not free to move. In these two designs, the centers of the spherical particles are expected to be not in a line when assembled on a perfectly flat surface (see schemes in Fig. 3 D, E). The simulations show this to decrease the EF by one order of magnitude in the least favorable case. The initial idea for design 20b-10-60 was to optimize the geometry in a way that the DNA origami scaffold would lift the 10 nm particle into the plane between the 20 and 60 nm particles. However, the poor SERS performance of the design indicates that when dried on a surface, the scaffold is not sturdy enough to do so. Furthermore, AuNLs of type 20b-10-60 suffer from the additional DNA origami spacer separating the 20 nm gold nanoparticle. The strong SERS enhancement for some structures of design 20-1060 indicates an optimal plasmonic coupling with a geometry closer to the one shown in Fig. 3 A than in 3 D. In these cases, the structures might be benefitting from the relative flexibility of the single-layered DNA origami scaffold.

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Fig. 3 (A-F) Electromagnetic SERS enhancement based on FDTD simulations of the local fields for the three designs at the excitation wavelength (532 nm) and at the wavelength corresponding to the TAMRA signal at 1650 cm-1 (583 nm). (A-C) in-line, no gap (D, E) out-of-line, (F) 5-nm-gap arrangement. The polarization direction of the incident light is along the longitudinal axis of the AuNLs. The red line in the schematics illustrates the plane of observation; the plane is chosen to intersect the position of greatest field intensity. The DNA origami scaffold separating the 20 nm gold nanoparticle in 20b-10-60 AuNLs (B, E) is indicated in grey. Zoom-ins of the areas around the 10 nm particles are shown in Fig. S3 of the SI.

Apart from misalignment, it was recently shown that the plasmonic response of the system can also be greatly influenced by the morphology of the individual gap.49 Both effects are expected to contribute to the large signal fluctuations observed for the AuNLs. LSPR spectra from the sets of AuNLs have been obtained by dark field spectroscopy. The wavelengths of the absorbance maxima λmax are in good accordance with scattering spectra simulated by the FDTD method (see SI).

Comparison of field enhancement in different gaps For AuNLs, a cascaded field enhancement is predicted, meaning that that the electromagnetic field, and thus the SERS signal, is higher in the gap between the small and the medium-sized particle, compared to the gap between the small and the large particle.15,18 In order to learn more about the distribution of the electromagnetic enhancement within AuNLs, experiments with 20-10-60 AuNLs were carried out, where either 20 nm (20*-10-60) or 60 nm (20-10-60*) gold nanoparticles were TAMRA-labeled, respectively (Fig. 4 B). In this way, the Raman signal is only enhanced in one of the two gaps and the signal intensities can directly be compared from the two experiments. SERS spectra from 17 single 20*10-60 AuNLs and 11 single 20-10-60* AuNLs have been measured and the intensity of the strongest TAMRA signal in each spectrum has been determined. For the comparison of signals from the two individual gaps, the different numbers of TAMRA molecules in the gaps are considered, since nanoparticles of different size support different densities of dye molecules. The SERS signals are normalized 9 ACS Paragon Plus Environment

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by the densities of TAMRA molecules on the gold nanoparticles, assuming that the SERS signal is proportional to the number of dye molecules in the area of highest enhancement, and that for both gaps these areas share similar size. The resulting average SERS signal IAuNL,d from 20*-10-60 is approximately 30% higher than the one from 20-10-60* (Fig. 4 C).

Fig. 4 (A) FDTD simulation result indicating the electromagnetic Raman enhancement of the two gaps in AuNL 20-10-60. Positions of gold nanoparticles are shown by the black wireframes. (B) Schematic of the selective placement of TAMRA molecules (bright green) for probing of the different inter-particle gaps. (C) SERS signals from AuNLs with TAMRA-labeled 20 nm or 60 nm gold nanoparticles. The SERS signal from single AuNLs (IAuNL) is corrected for TAMRAdensity (IAuNL,d) and for signal contribution from isolated, TAMRA-labeled gold nanoparticles (IAuNL,d,i).

When the incident light is polarized parallel to the longitudinal axis of the nanolens, the regions of high fields are located in the gaps between the particles. For light polarized perpendicular to the longitudinal axis, mainly transverse modes are excited that should provide field enhancements similar to singleparticle modes.50,51 Under the illumination used in our experiments, both, longitudinal and transverse modes are excited. Therefore, also the enhancement contributed by the whole nanoparticle surfaces, apart from the gaps, must be taken into account, and average SERS signals from TAMRA-coated, isolated 10 ACS Paragon Plus Environment

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nanoparticles of the respective size are subtracted from the signal obtained with the nanolenses. The difference in SERS intensity between the gaps increases considerably: the signal IAuNL,d,i from the gap between 20 nm and 10 nm gold nanoparticle then is approximately 6 times higher than that from the other gap (between the 10 nm and 60 nm gold nanoparticle). This matches well with the FDTD simulations, which predict a factor of 2 for a 3.4 nm gap of DNA coating between the particles (Fig. 4 A). With smaller gap sizes, larger factors were predicted. In spite of the signal fluctuations and a relatively low number of analyzed AuNLs, these results provide an experimental indication that the gap between medium and small nanoparticle in AuNLs provides stronger field enhancement than that between the large and the small nanoparticle.

Conclusion In summary, we assembled plasmonic AuNLs from 10, 20 and 60 nm gold nanoparticles in three different designs (20-10-60, 20b-10-60 and 10-20-60) and present a systematic investigation of these nanolenses. For sets of single AuNLs from each arrangement, AFM images, SEM images, LSPR spectra and SERS signals of a probe dye have been determined, which gives insight into the Raman enhancement of the different arrangements and into the distribution of SERS signals from individual structures. The experiments indicate that design 20-10-60 yields the most structures with strong SERS enhancement and also shows a narrow distribution of LSPR wavelengths. The localization of SERS enhancement in 20-10-60 AuNLs has been examined further by selectively labelling the 20 nm and 60 nm gold nanoparticles, respectively, with TAMRA. The highest enhancement is located in the gap between the 20 nm and 10 nm gold nanoparticle, in accordance with theoretical predictions and indicating the predicted cascaded field enhancement. In the present designs, the AuNLs do not yet match the sensitivity of published dimers.41 In order to reach the giant Raman enhancements predicted for AuNLs,17,18 it will be of importance to strengthen the cascading effect, i.e., to increase the number of coupled nanoparticles and to decrease the thickness of the DNA coating. The former will require additional sets of capture strands on the present DNA origami scaffold. The latter will be realized by hybrid coatings with 11 ACS Paragon Plus Environment

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separated

functionalities

for

binding

and

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stabilization.

The

use

of

a

complementary, three-dimensional scaffold that ensures a truly collinear assembly then will make it possible to compete with, if not outcompete, the field enhancements provided by simple homodimers. With applications of complex plasmonic structures in mind, and with the need for experimental systems to study plasmon-enhanced spectroscopic effects, it is desirable to control also the position of probe molecules. Using DNA origami technology, it is easy to incorporate binding functionalities that immobilize defined numbers of analyte molecules at defined positions on the DNA origami scaffold.52 The structures presented here are prepared by self-assembly in solution, in billions of copies. They can be useful for analytical applications, and besides, enable fundamental investigations of plasmon-related processes.

Methods DNA origami scaffolds DNA origami scaffolds are assembled in 1x TAE buffer with 11 mM MgCl2 as described earlier.39 Staple strand and capture strand sequences can be found in the SI (S8 – S10).

Gold nanoparticle coating pH3 procedure (modified from Zhang et al.45]): Citrate-stabilized gold nanoparticle solution (BBI Solutions, 1 nM (60 nm), 10 nM (20 nm), 5 nM (10 nm)) in 0.02 % sodium dodecyl sulfate (SDS) is mixed with DNA coating strands (metabion, 15 µM (60 nm), 22.5 µM (20 nm), 2.5 µM (10 nm), respectively) and incubated for 30 min. 0.5 M citrate buffer (pH 3) is added to a final concentration of 10 mM and the solution is incubated for another 45 min. 2.5 M NaCl is added to a final concentration of 300 mM and after shaking for 3 h, 2.5 M NaCl is added again to a final concentration of 600 mM. After overnight incubation, 400 µl 1x TAE with 11 mM MgCl2 and 0.02 % SDS are added. Gold nanoparticles are either sedimented by centrifugation (60, 20 nm particles) or separated by centrifugal filters (10 nm particles, Amicon Ultra 0.5 100 kDa, Millipore) and supernatant/flow through are removed. After four more cycles of buffer addition and centrifugation, particles are stored at 4 °C. The DNA coating should allow optimum plasmon coupling between the gold nanoparticles. Thus we have minimized the length 12 ACS Paragon Plus Environment

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of coating strands to 13 bases, with 9 base recognition sequences. For shorter lengths, a rapid loss in stabilization efficiency was reported.47 The coating strands have the sequence (XTT)3T4-SH, with X representing A, G, or T, respectively. For the experiments with TAMRA-labeled 60 nm gold nanoparticles, a 1:1 mix of 5’-TAMRA-labeled / unlabeled (TTT)3T4-SH strands is used for coating. The pH3-coating procedure does not yield stable 20 nm nanoparticles with TAMRA(GTT)3T4-SH DNA. Instead, a small-step salt-ageing protocol at pH 7 is applied: Citratestabilized 20 nm gold nanoparticles (BBI Solutions) are phosphinated as described earlier.39 A solution of 6 nM bis(p-sulfonatophenyl)phenylphosphine-coated 20 nm particles with 13.5 µM thiol-modified DNA (metabion, 1:1 mix TAMRA-(GTT)3T4-SH / (GTT)3T4-SH) in 0.02 % SDS, 50 mM NaCl and 0.5x TAE is incubated for 1 h, then NaCl concentration is increased to 100 mM and the solution is left shaking overnight. The next day, NaCl concentration is increased to 700 mM in 50 mM steps every 40 min. After overnight incubation, particles are purified by five cycles of buffer addition (400 µl 1 x TAE with 11 mM MgCl2 and 0.02 % SDS), centrifugation and supernatant removal. The surface coverage with TAMRA-strands is determined by the dithiothreitol replacement technique48: DNA-coated gold nanoparticles are mixed with 0.5 M dithiothreitol, which replaces DNA on the particle surface. After overnight incubation, gold nanoparticles are separated by centrifugation. The concentration of TAMRA-DNA in the supernatant is determined by fluorescence spectroscopy. The data for 60 nm and 20 nm TAMRA-coated gold nanoparticles is displayed in Figure S6 in the SI.

AuNL assembly Coated gold nanoparticles are bound to the DNA origami scaffold in several steps: For AuNLs 20-10-60 and 20b-10-60 (see Scheme 1 for schematic structures), the small 10 nm particle is first immobilized at the middle position, then 20 nm and 60 nm particles for the peripheral positions are added. AuNL 10-20-60 is assembled in three steps, in the order 10 nm, 20 nm, 60 nm. The first hybridization step is facilitated by a temperature ramp from 45 °C to 25 °C over 71 min. In order to prevent the detachment of nanoparticles, subsequent hybridization steps have been performed at room temperature (90 min). A ratio of 1:1 between DNA origami scaffold and the respective gold nanoparticles was found to show the best assembly yield. The stepwise 13 ACS Paragon Plus Environment

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hybridization enables process control by AFM: with binding yields of almost 100% in the first step, each AuNL includes one 10 nm gold nanoparticle. Additionally, the characteristic SERS signals of TAMRA from individual AuNLs serve as final proof for the presence of the 10 nm gold nanoparticle in the AuNLs. In AFM or SEM images, the 10 nm-sized particles are often covered by the larger particles. Unbound gold nanoparticles are typically removed by agarose gel electrophoresis. Bands in the gel containing the assembled AuNLs are isolated by electroelution into a sucrose-filled pocket54 and concentrated by centrifugation.

Si sample preparation 6 µl of the sample solution are placed onto a plasma-cleaned ~1 cm2 silicon wafer (CrysTec) and 30 µl 110 mM MgCl2 10x TAE buffer are added. The sample is incubated for 1 h, washed with 4 ml ethanol/water 1:1 and blow-dried with compressed air.

Raman measurements Raman measurements were carried out on a WITec alpha300 confocal Raman microscope with a 100x Olympus MPlanFL N objective (NA=0.9), 600 gr/mm grating and 532 nm excitation laser. Areas of 25*25 μm2 were scanned with 0.5 μm step size, 4 s integration time and 2.0 * 104 W/cm2 laser power. The diffraction-limited spot was estimated to be 1.3 μm wide.

Estimation of enhancement factors TAMRA’s fluorescence under 532 nm illumination prevents bulk Raman measurements of the non-enhanced dye. Since EFs for single 60 nm gold nanoparticles (EFAuNP) are known from literature, EFs for AuNLs (EFAuNL) can be determined indirectly when AuNLs and TAMRA-coated, single 60 nm gold nanoparticles are measured under the same conditions. EFAuNL then can be defined as:

 = (  /  )(  /  ) with IAuNL – SERS intensity of strongest TAMRA band from single AuNL, IAuNP – average SERS intensity from single, TAMRA-labeled 60 nm gold nanoparticles (13.9 cts, n=14), NAuNP – number of TAMRA molecules on single 60 nm gold nanoparticles, NAuNL – number of TAMRA molecules on single 10 nm gold nanoparticles, EFAuNP – enhancement 14 ACS Paragon Plus Environment

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factor of single 60 nm gold nanoparticles (7.5 * 103).55 An estimation of errors as well as SERS spectra from single TAMRA-labeled 60 nm gold nanoparticles can be found in the SI. The observation of the latter was enabled by the resonance Raman contribution of the dye molecules.53

FDTD simulations FDTD simulations for all AuNLs were carried out with Lumerical FDTD Solutions 8.6.3, using a mesh size of 0.25 nm in the plotted area. Thickness (1.7 nm) and refractive index (1.7) of the ssDNA coating are based on the findings of Thacker et al.40 Since in Raman scattering, exciting and emitted light are wavelength-shifted and thus experience different enhancement, the electric field intensity enhancements at 532 nm (laser) and 583 nm (λ of the usually most prominent TAMRA band, ≙1650 cm-1) are multiplied. For Rayleigh scattering simulations, substrate layers were modeled with thicknesses of 2 nm (DNA origami scaffold), 2 nm (SiO2 substrate) and an infinite layer of silicon below. Scattering cross sections from s and p polarized light were added incoherently to account for the unpolarised illumination used in the dark field setup. The following refractive indices were used: DNA origami scaffold: 2.1, gold: Johnson and Christy,56 silicon and SiO2: Palik,57 surrounding medium: 1.0. The used 3D models are shown in Figure S2 and S5 in the SI.

Comparison of SERS signals from different gaps Correction for different densities of TAMRA on the gold nanoparticles:

, =

 /

with n - number of TAMRA strands on the coated gold nanoparticles (2988 for 60 nm particles, 130 for 20 nm particles, see S5 in the SI for details), d – particle diameter. Correction for signal contribution of isolated TAMRA-coated gold nanoparticles:

,, = , −  , with IAuNP,d – density-corrected SERS intensity of strongest TAMRA band from isolated gold

nanoparticles

(53

cts/(TAMRA/nm2)

for

60

nm

particles

(n=14),

18 cts/(TAMRA/nm2) for 20 nm particles (n=11)). 15 ACS Paragon Plus Environment

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Acknowledgements This research was supported by the Deutsche Forschungsgemeinschaft (DFG), a Marie Curie FP7 Integration Grant within the 7th European Union Framework Programme, the European Regional Development Fund (EFRE), by the University of Potsdam and the Federal Institute of Materials Research (BAM). V.M. & J.K. acknowledge an ERC Grant (259432, MULTIBIOPHOT); C.H. & V.M. are grateful for funding through the DFG (GSC 1013, SALSA). We thank Franka Jahn (IPHT Jena) for SEM imaging.

Supporting Information Available: Experimental details for LSPR measurements. Error estimation for enhancement factors. Data set for a AuNL of design 20-10-60 with low Raman enhancement. 3D models used in FDTD simulations. Magnified FDTD simulation data. Simulated scattering cross sections for all AuNL designs. Fluorescence data used to determine Tamra coverages for 20 nm and 60 nm gold nanoparticles. SERS spectra from TAMRA-coated 60 nm gold particles. Maps of capture strand placement. AFM images used for comparison of AuNL designs. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsphotonics.XXXXXXX.

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