Single-Step Organization of Plasmonic Gold Metamaterials with Self-Assembled DNA Nanostructures

Self-assembled DNA nanostructures hold great promise as nanoscale templates for organizing nanoparticles (NPs) with near-atomistic resolution. However, large-scale organization of NPs with high yield is highly desirable for nanoelectronics and nanophotonic applications. Here, we design five-strand DNA tiles that can readily self-assemble into well-organized micrometer-scale DNA nanostructures. By organizing gold nanoparticles (AuNPs) on these self-assembled DNA nanostructures, we realize the fabrication of one- and two-dimensional Au nanostructures in single steps. We further demonstrate the one-pot synthesis of Au metamaterials for highly amplified surface-enhanced Raman Scattering (SERS). This single-step and high-yield strategy thus holds great potential for fabricating plasmonic metamaterials.

Structural DNA nanotechnology offers unprecedented opportunities for assembling Au nanostructures [11]. During the past two decades, numerous DNA nanostructures with various shapes in multiple dimensions were fabricated with one-pot assembly [23][24][25]. Particularly, the DNA origami techniques [26], which fold long single-stranded DNA into desired shapes, allow for creation of custom-designed and discrete DNA nanostructures. Because of the nanometerscale precision and full addressability, DNA nanostructures are employed as templates [16,27,28] or linkers to organize Au nanoparticles (NPs) into multidimensional discrete nanostructures, lines, lattices, and crystals [29][30][31]. The typical procedure to create these AuNPs nanostructures usually contains two steps: assembly of DNA templates and organization of AuNPs, which would take several hours and even days [2,9,10]. These studies have mostly focused on studies of versatile strategies to fabricate complex plasmonic architectures with multiple functions or tailored optical response. Despite the rapid progress, a robust design strategy of DNA nanostructures together with timesaving protocols for AuNPs organization remains highly desirable [32][33][34][35][36]. Here, we demonstrate the realization of plasmonic Au metamaterials through one-pot assembly of AuNPs with five DNA strands and then investigate their optical applications.

Results
To assemble plasmonic gold metamaterials, ribbon-like DNA origami nanostructures (RDNs) were used as templates, in which three shorter DNA strands (N 1 , N 2 , and N 3 ) not only fold two longer DNA strands (S 1 and S 2 ) into repeated rectangular units but connect neighbor units into long ribbons (Figure 1(a)). By extending N 3 with a capture sequence as a binding site, AuNPs functionalized with thiol-DNA (SH-DNA-AuNPs) are mixed together with five strands in one pot to generate one-dimensional (1D) AuNPs lines. This strategy is also applied to organize gold nanorods (AuNRs) which functionalized with thiol-DNA (SH-DNA-AuNRs) into 1D AuNRs lines (Figure 1(b)). To obtain two-dimensional (2D) DNA lattices, S 1 and S 2 are extended with complementary overhangs of 15 bases into S 3 and S 4 . Assisted by mica surface, five strands (N 2 , N 3 , N 4 , S 3 , and S 4 ) are directly assembled into 2D lattices (Figure 1(c)). Using these lattices which have two binding sites on N 3 and N 4 as templates, 2D AuNPs lattices can be directly assembled with five strands in one pot assisted by mica (Figure 1(d)).
With the optimization of sequences for N 1 , N 2 , N 3 , S 1 , and S 2 by SEQUIN [37], we surprisingly found that five DNA strands can self-assemble into RDNs in 20 minutes at room temperature in 1×TAE/Mg 2+ buffer. AFM measurements showed that the lengths of RDNs were ranging from hundreds of nanometers to several micrometers and the width of RDNs was ∼17 nm, which is in agreement with its theoretical value (Figures 2(a) and S1). Using one-pot assembly strategy, we generated AuNPs lines with a high To better understand the assembly process, we employed the two-step protocol to generate the AuNPs lines; that is, the RDNs templates were firstly assembled followed by anchoring of SH-DNA-AuNPs onto template surfaces. The coverage rate of thus assembled AuNPs lines was 45% ( Figure S2), which is significantly lower than the one obtained with the onepot assembly strategy. In the one-pot assembly process, SH-DNA-AuNPs might hybridize with strand N 3 that followed by folding with other strands into lines. While in the two-step assembly, the negative charged DNA nanoribbon may hinder the hybridization of SH-DNA-AuNPs to N 3 . In theory, the gap between neighboring N 3 on the template is ∼ 6 nm, which is big enough to anchor 5 nm-sized AuNPs. Different experimental conditions were systematically investigated. Micrometer-long AuNPs lines could be assembled with 99% coverage rate (Figures 2(c), 2(d), and S3), with the optimized 1:1 concentration ratio of SH-DNA-AuNPs to N 3 . Considering that the RDNs can be easily assembled at room temperature, different initial temperatures for one-pot assembly were studied. When the initial temperature was equal to or higher than 65 ∘ C, micrometer-long AuNPs lines could be formed (Figures 2(e) and S4). We also monitored the annealing time for one-pot assembly when the initial temperature was 65 ∘ C. After 2 h annealing, the percentage of micrometer-long AuNPs lines in the product was 36%, which was increased to 57% as prolonging the annealing time to 4 h (Figures 2(f) and S5). We also examined the thermal stability of the 5 nm-sized AuNPs lines by keeping the samples in water bath with different temperature for 20 min. The AFM imaging results indicated that the structures were stable when they were heated to 40 ∘ C ( Figure S6).
We further applied the one-pot assembly strategy to organize AuNPs of different sizes (Figure 3). TEM images validated the one-pot assembly of 10 nm-, 20 nm-, 30 nm-, 50 nm-, and 80 nm-sized AuNPs lines (Figures 3(c)-3(g) and S7), which were obtained by annealing five strands with SH-DNA-AuNPs in 1×TAE/Mg 2+ buffer from 65 ∘ C to 25 ∘ C in 4 h. To obtain high-quality 1D AuNPs lines, the concentration ratio of SH-DNA-AuNPs to N 3 was optimized for AuNPs of different sizes. For example, 4 strands of N 3 with other DNA strands form an 18 nm-long unit of nanoribbon, which are adapted to the size of 20 nm-sized AuNPs (Figure 3(i)), and the concentration ratio of 20 nm-sized AuNPs to N 3 was 1:4. Correspondingly, the concentration ratio of SH-DNA-AuNPs to N 3 was 1:2 and 1:6 for 10 nm-and 30 nm-sized AuNPs, 1:10 and 1:16 for 50 nm-and 80 nm-sized AuNPs, respectively.
With the increase of the size of AuNPs, the length of the AuNPs lines decreased from micrometers to hundreds of nanometers. These phenomena were adapted to the supposition we mentioned above; that is, SH-DNA-AuNPs may 12 nm~ 10 nm  firstly hybridize with N 3 ; therefore, it is more difficult for SH-DNA-AuNPs-N 3 of larger sizes to further hybridize with other strands to form lines. Using the same one-pot assembly strategy, AuNRs (20×70 nm) were organized to form AuNRs lines with the concentration ratio of SH-DNA-AuNRs to N 3 of 1:14, confirmed by TEM imaging results (Figures 3(h) and S7).
To widen the range of one-pot assembly strategy, we extended S 1 and S 2 with complementary 15-base overhangs. Five strands (N 2 , N 3 , N 4 , S 3 , and S 4 ) were mixed together in 1×TAE/Mg 2+ buffer and annealed from 65 ∘ C to 25 ∘ C for 12 h. Although there were some connections between RDNs, network-like DNA nanostructures other than 2D lattices were formed, demonstrated by AFM imaging results ( Figure  S8). When mica was annealed together with five strands, the Mg 2+ in buffer helped DNA strands to absorb onto mica surface and 2D DNA lattices were generated, which is validated by AFM images (Figures 4(a) and 4(b)). The growth processes of 2D lattices were proposed to be assisted by mica; that is, earlier formed RDNs could adsorb on negatively charged mica surface as nuclei and then single strands or smaller RDNs diffuse onto the surface for 2D crystallization. Using the one-pot assembly strategy, SH-DNA-AuNPs could be assembled into 2D AuNPs lattices assisted by mica (Figure 4(c)). AFM and SEM images demonstrated the formation of different-sized AuNPs 2D lattices (Figures 4(d)-4(g), S9, and S10).
Because metal nanostructures can excite collectively coupled plasmons when in interacting with light, [15,16] AuNPs metamaterials impelled us to consider their potential plasmonic properties [27,38]. Previous studies showed that proper distances between individual AuNP could generate hot spots that induce stronger SERS signals. As most of the distances between the AuNPs in our 1D AuNPs lines and 2D AuNPs lattices were less than 10 nm, their coupling of the plasmons was detected by UV-vis absorption ( Figure S11). We used 4-MBA as a Raman-active molecule which could covalently attach to AuNPs via Au-S bond. The frequencies of 1580 cm −1 and 1075 cm −1 in the SERS spectra were attributed to 4-MBA. SERS signals were hardly observed from 5 nm-and 10 nm-sized AuNPs lines or lattices, because of the weak coupling of the larger gaps between small-sized AuNPs. However, large-sized AuNPs lines and lattices both induced enhanced SERS signals. Take the peak at 1075 cm −1 for an example; an enhanced signal generated by 80 nm-sized AuNPs lattices was twenty times higher than that of 20 nm-sized AuNPs lines (16833.93 to 804.98 a.u. at 1075 cm −1 , Figure 5). Based on Maxwell's curl equations, the electrical field in different-sized AuNPs line upon illumination (Figure 6) was simulated by the comprehensive finite difference time domain (FDTD) method. On this basis, the enhancement could be naturally interpreted by the increasing of the hotspots created in the differentsized AuNPs lines. The same enhanced phenomenon was observed when comparing 20 nm-and 10 nm-sized AuNPs lattices.

Discussion
We designed five-strand DNA tiles for the fabrication of DNA nanostructures in micrometer scale, which facilitate the realization of 1D and 2D plasmonic gold metamaterials. Using one-pot assembly strategy, AuNPs of different sizes are organized for high-quality 1D lines and 2D lattices, which could generate enhanced Raman scattering. This strategy is of great importance for single-step, rapid, and high-yield assembly of AuNPs into plasmonic metamaterials. As semiconductor and information technologies approach their physical limits, it is a great challenge to combine the functionality of biological systems and semiconductors. Our strategy would provide a potential tool to deal with interfacing biological materials with conventional semiconductors for practical applications.

TEM Characterization.
For TEM imaging, 10 L of the sample was dropped on a carbon-coated grid (400 mesh; Ted Pella, Inc.). After 15 min deposition, the excess amounts of solution drop on the grid were wicked by a drop of water and then adsorbed into a filter paper. TEM images were obtained by a Tecnai G2-20S TWIN system, which was operated at 100 kV in a bright-field mode.
4.6. SEM Characterization. 2D AuNPs lattices on the mica were washed by pure water and dried under nitrogen. The samples were rinsed with positively charged nanogold solution for 15 s to increase the conductivity. Then the mica was stuck to the sample table by conductive tape. SEM imaging was performed using a HITACHI S4800 at 80 kV in dark field mode.

SERS Characterization.
Samples of AuNPs lines were incubated in 1 mM 4-MBA ethanolic solution for 2-3 h. 25 mL of the sample was deposited on a silicon substrate and performed in solution for SERS characterization. 10 L 10 mM 4-MBA ethanolic solution was deposited onto the mica surfaces with 2D AuNPs lattices for over 3 h, followed by a thorough rinse with pure ethanol. The SERS characterization was performed on mica surface. For all measurements, the confocal Raman microscope (Invia, Renishaw, England) was employed with a 633 nm He-Ne laser at the power of 0.08 mW.
Other experimental parameters were as follows: objective 20×, acquisition time 10 s, slit of 100 m, resolution grating of 1800 grooves, and spectra ranged from 1000 to 1800 cm −1 .

Data Availability
All data is available in the main text or the Supplementary Materials.