Results - simulation

More detailed information(such as specific information about the reagents) will be published separately


  • comparison between tetrahedron and octahedron

  Metamolecules, which constitue metafluid, have to has symmetry stucture and high magnetic response. So, we choose the final candidates of projects; tetrahedron and octahedron which are the symmetric geometry with lowest complexity (which means relatively easy to fabricate compared with higher symmetry structures. To compare the magnetic response between two clusters we performed numerical simulation.
Tetra octa compare biomod.png
Fig 1.(a) and (b) represent electric response(blue line) and magnetic response(red line). (c),(d) represent magnetic field distribution (color) of tetrahedron and octahedron at their magnetic resonance wavelength(668[nm] and 720[nm], respectively).

  At graph (a),(b) in figure 1, electric means radiated power of sum of electric dipole and quadrupole, magnetic means radiated power of magnetic dipole. The methods of calculating electric and magnetic response are shown in 'Materials & Methods - simulation'.
  In both bottom pictures in Figure 1 the magnetic responses under impinging light were mapped with rainbow color. Red color indicate strong magnetic response whereas blue color indicate weak magnetic response (same scale bar in both figure). The strong magnetic response can be identified in the center of the both structure. However, the octahedral structure shows stronger magnetic response in relatively large area than tetarhedron stucture. It indicate that there is high induced magnetic response. The result of comparison in top two figures(fig 1(a), (b)) also show that octahedron structure can induce more stronger magnetic response under incoming light which means octahedron structure is more appropriate to induce negative refraction in liquid phase. Thus, we chose an octahedron structure to construct metafluid.



Results - experiment

More detailed information(such as specific information about the reagents) will be published separately

  • DNA origami folding and purification
  Designed barrel like DNA origami structure was folded and purified by following protocols in methods. Gel purification data shows that more than 70% yield of the structures are well folded(fig 1.). In addition, the handle modification on the DNA origami structure was preliminary confirmed by slower gel band.
  Gel purification was further confirmed by TEM imaging. Purified origami was sampled on TEM gird and stained with Uranly formate solution. As one can identify below(fig 2.), desired origami strictures are well assembled and purified.


BIOMOD result gel purification.png
Fig 1. 1.85% agarose gel purification of designed DNA origami structure. The depicted lanes contain the following samples: 1kb DNA Ladder, M13mp18 scaffold strand, barrel-like DNA origami structure(w/o handle modification), barrel-like DNA origami structure with handles modification which act as binding sites. Orange rectangle indicate gel band cutting location.



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Fig 2. TEM imaging of the purified DNA origami structure.



  • Seed AuNP synthesis
  Seed AuNPs should be spherical and homogeneous as possible because the optical properties and further grown AuNPs quality are highly dependent on the quality of seed AuNPs. For this, our team synthesized ~18nm AuNPs by citrate capping methods. Because spherical AuNPs show clear reddish scattered light, we can preliminary identify the quality of synthesized AuNPs by varying synthesis condition(fig 3.). Based on this, our team narrowing down reaction condition to synthesize spherical and homogeneous AuNPs. These processes were further supported by TEM imaging process(fig 4.). As a result, our optimum condition(2.02mM SC with new beaker) produces spherical AuNPs with narrow size distribution as one can identify in below figure(fig 4.).


BIOMOD LABBOK July 1st sodium citrate.png
Fig 3. Seed AuNPs synthesis results. Experiment was conducted by varying sodium citrate(SC) concentration to achieve desired quality of AuNPs. 2.52mM SC was set as a reference condition. Old beaker indicate originally used one for other purpose which treated with aqua regia. New beaker was also treated with aqua regia before use.

BIOMOD LABBOK July 1st sodium citrate TEM.png
Fig 4. Seed AuNPs synthesis results with TEM analysis. Size distribution was further analyzed by TEM image process (using Image J). Average size of 2.02mM old beaker(bottom, left) is 18.76nm and 2.02mM new beaker - 1(bottom, right) is 16.64nm.

  • DNA functionalization of seed AuNP
  To attach AuNPs on the DNA origami structure, AuNPs should be functionalized with DNA strands, For this, our team used thiolated DNA strands which form covalent bond with Au atoms. To lead the high binding yield between DNA functionalized AuNPs and origami structures, thiolated DNA strands should be densely grafted on the AuNPs surface. For this, proper amount of monovalent cation should be injected because the existence of monovalent cation reduce the electrostatic repulsion between DNA strands which hinder the dense functionalization of DNA strands on the AuNP surface. However, when AuNPs were exposed at high salt concentration, low stability of AuNPs can lead to aggregation of particles. Thus, the optimal condition should be set so that DNA functionalization density on seed AuNPs should be maximized while minimizing aggregation between AuNPs in buffer solution.
  Thus, we varied two variables: final salt concentration, and oligonucleotide modification(monothiol vs. dithiol). To compare thiolated DNA coating density and stability of particles, our team conducted gel purification of functionalized AuNPs(figure 5, 6, 7, 8)



BIOMOD LABBOK July 4th salt aging.png
Fig 5. DNA functionalization process snap shot. Written concentrations indicate the final concentration of monovalent cation. (Top) Functionalization of AuNPs using monothiolated DNA strands. At relatively high salt concentration, color of AuNPs turned to darkish color which is the evidence of low stability. (Middle) After 1 day incubation of above samples. During incubation, as thiolated DNA strands bind to AuNPs, the reddish color recovered. (Bottom) Functionalization of AuNPs with dithiolated using dithiolated DNA strands. Clear reddish color have maintained at all salt concentration.

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Fig 6. Gel purification of DNA functionalized AuNPs with monothiolated DNA strands (salt injection interval = 20min).



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Fig 7. Gel purification of DNA functionalized AuNPs with dithiolated DNA strands. (salt injection interval = 20min)



Biomod gel graph.png

Fig 8. Gel intensity analysis. Relative intensity indicates the intensity ratio of gel pocket against the each lane.

  In the all experimental condition, as final salt concentration increases, leading bands migrate slowly, indicating higher DNA density on AuNPs. However, the relative amount of AuNPs in the pocket increas at high concentrations which show direct evidence of low stability of particles in buffer condition. These tendencies are clearly identified in figure 9. At this point we can set the optimum salt condition because above certain salt concentration, the leading band become saturated. Thus, by functionalizing DNA strands at these saturation conditions, one can maximize DNA strands density on AuNPs while minimizing instability problems of particles
As an attempt to further increase the AuNPs stability and homogenous functionalization of DNA strands, we changed oligonucleotide modification from monothiol(fig 6) to dithiol(fig 7). As one can clearly figure it out by comparing the gel data, tailing of the bands are significantly decreased while AuNPs in the pockets are tremendously reduced(fig 8) which indicate that AuNPs stability and homogenous functionalization of DNA strands on the particles are significantly increased.
Thus, our team set the optimum final salt concentration(800mM for dithiolated DNA strands) and oligonucleotide modification(dithiol modification) for DNA functionalization of AuNPs.



  • Assembling seed AuNPs into octahedron
  To realize metafluid by assembling AuNPs onto DNA origami structures, the binding yield between DNA functionalized AuNPs and DNA origami should be maximized. Even though previous researches already reported high yield arrangement of AuNPs on DNA origami templates[1], specific condition should be changed as origami structures or size of AuNPs changes. Thus, our team set the condition of binding between AuNPs and origami structure with high yield.
Biomod obrick gel purification.png

Fig 9. 1.25% agarose gel purification of assembled AuNPs cluster. The depicted lanes contain the following samples: 1kb DNA Ladder, M13mp18 scaffold strands, barrel-like DNA origami structures, assembled AuNPs cluster by barrel-like DNA origami and unbounded AuNPs. In the lane 4, the faint dark band in the middle is assembled cluster whereas the leading dark band is unbounded AuNPs. The cluster band was extracted and purified for TEM analysis

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ig 10. TEM images of the assembled AuNP clusters (octahedron).


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Fig 11. Quantitative analysis of the assembled clusters for yield calculation. Tri: trimer, Tetra: tetramer, Penta: pentamer, Hexa: hexamer(octahedron). Totally, 127 clusters are counted for analysis

  To clearly identify assembled structures, the clusters were purified by agarose gel electrophoresis(fig 9). The faint dark band was extracted and purified by Freeze N Squeeze extraction spin column for TEM analysis. TEM images show that the assembled clusters are well formed as expected geometry (fig 10). Quantitative analysis from TEM images confirms that the assembled clusters are not only well formed but also fabricated in relatively high yield(fig 11). Even though the considerable clusters are still malformed, further purification methods such as density gradient centrifugation can be used to selectively extract target structure. Thus, our team believe that ~50% yield of assmbled clusters is enough to show the feasiblity of our strategy for realizing metafluid.


  • AuNPs further growth on DNA origami surface
  As a next step, our team confirmed that assembled AuNPs can be further grown in buffer condition without aggregation between particles in spherical geometry.

Result further growth.png Result TEM data.png Result TEM data2.png
Fig 12. (a)Assembled AuNPs further growth process and (b)results. a) During growth process, AuNPs does not aggregated in buffer condition even after washed by centrifugation. b)TEM image of the assembled AuNPs after further growth steps. To verify grown state more clearly, AuNP clusters in tetramer geometry (nano-ring consist of four particles) were used. c) Further growth result of non-preassembled AuNPs which does not show emerging between AuNPs

  As seen in figure 12(a), our team successfully set the AuNP further growth condition with high stability in salt and buffer condition using surfactant capping method. The grown clusters were confirmed by TEM imaging(fig 12(b)).
Due to the surfactant during reaction, DNA origami structures cannot be identified in TEM images. But, the geometrical arrangement of AuNPs(fig. 12(b)) indicate the existence of origami inbetween of AuNPs. Otherwise, the AuNPs would be closely packed without any void. Also, in some of our structures the AuNPs are merged by growth reaction. This result shows clear evidence that the structures are the growth result of pre-assembled seed AuNPs by barrel-like origami structure not by randomly assembled cluster from droplet drying.
Even though the sphericity ratio of the particles are not so high and some clusters are merged, we confirmed that assembled AuNP clusters can be successfully grown in salt and buffer condition. We expect that by further optimizing growth or surfactant capping condition, particles sphericity could be improved while minimizing merging problem between particles



  • Reference
[1] Toward Self-Assembled Plasmonic Devices: High-Yield Arrangement of Gold Nanoparticles on DNA Origami Templates


Conclusion and Outlook


  By utilizing DNA nanotechnology, our team successfully arrange seed AuNPs in specific structure and further growth it to achieve target size of AuNP clusters. Our team chose octahedron structure which shows strong magnetic response against the impinging light using the Finite Element Method(FEM) simulation. We assembled octahedron clusters with ~50% yield and verified the feasibility of seeding growth method using clusters with 3 to 4 AuNPs. Even though the sphericity of the assembled particles are not so high and some clusters are merged, we confirmed that assembled AuNP clusters can be successfully grown in salt and buffer condition.


  Eventhough our works show the possibility that our approaches can be used to realize negative refraction in liquid phase, there are many issues must be solved for realization. First of all, octahedral AuNP clusters formation yield should be further increased. Even though additional purification such as density gradient centrifugation can be to selectively disperse octahedral clusters in suspension with ~50% of formation yield, this approach unavoidable accompanies loss of the assembled cluster. Thus, high yield is essential for practical application. Second, further growth processes should be optimized further. In our research, due to the low stability of AuNPs in salt condition, growth conditions are highly restricted which lead to polygonal AuNPs geomerty. Becasue the high sphericity is essential for homogeneous optical properties, AuNPs growth process should be further optimized.


  Despite the mentioned issues, our results reveal that seeding growth method utilizing DNA nanotechnology can pave the to negative refractive index in liquid phase which provide a practical platform for the versatile translation of metamaterials into various real-world applications.