Biomod/2011/TeamJapan/Sendai/Results/Atomic Force Microscope: Difference between revisions

Revision as of 06:30, 1 November 2011

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 <td>

<ul id="verticalmenu" class="glossymenu"> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai">Home</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Strategy">Strategy</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Design">Design</a></li> <li><a href="#">Experiments</a>

   <ul>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis">Electrophoresis</a> 
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Atomic_Force_Microscope">AFM</a> 
   </ul>

</li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Computational_design/Simulation" >Simulation</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Notes">Notes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Team">Team</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Resources">Resources</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Sitemap">Sitemap</a></li>

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High-speed AFM

We used a high-speed AFM(RIBM, Nano Live Vision：NLV) for observation. This high-speed AFM enables us to capture moving molecules in solution clearly without blurring.
So we can confirm not just the molecular robot, but also observe at video-rate the molecular robot moving over the DNA origami field by using this.
In the following flowchart it is drawn the different project stages that were verified by the AFM.

Observation of 2D and 3D structures

Triangular prism 2D structure

Figure 2. caDNAno design: 2D view of origami unfolded body

First,we checked 2D nanostructures.The shape of 3D nanostructures resemble that of aggregation.On the other hand,the shape of 2D nanostructures are rectangle and differ from aggregation clearly. That is why 2D nanostructures are easier to observe than 3D ones.Therefore, before doing the annealing for getting the triangular prism we verified the 2D structure. The size of 2D structure in AFM is 67 nm × 23 nm(Figure 1).
As compared to our design(60 nm × 20 nm),the maximum error is 15percent.From electoric rebounding between double helix, the size of 2D nanostrucutre fall within the error range. We can affirm 2D nanostructures since our original design (Figure 2) depicts similar dimensions (60 nm × 20 nm)

There are surplus scafold(M13) from the middle of 2D nanostructure(Figure 3). We experimented to cut surplus M13 by PCR because we were afraid that surplus M13 inhibit folding and moving of structure. After we cut surplus M13,we also observed 2D structure made from cut M13(Figure 1). It proved that our experiment by PCR succeeded and we got 2D nanostructure using cut M13.

Sample conditions:

・Concentration M13:staples (1:10)
・Mg₂⁺ concentration 12.5 mM
・Annealing time: 3 hours from 90°C to 25°C
・Buffer: Tris-HCl
・Only cut M13

We confirmed the size of 2D structures in this experiment.
Next experiment, we reffered to the size of 2D structures and checked 3D structures.

Triangular prism 3D structure

Figure 3 is our design of 3D structure and Figure 4 shows the 3D structure of triangular prism in AFM. 3D structure consists of the three DNA sheets (Figure 3).

As compared to the figure of 2D structures, there are some differences. We can see the folded structure in figure 4.The folded structure was not observed in Figure 2. This structure was formed by self-assemby using three edging staples. There were two sticky ends in both ends of the 2D structures.

It is found that the shape of the structure seemed to be like triangle because all DNA sheets were adsorbed on mica (Figure 4). The reason why all DNA sheets were adsorbed on mica is that the 3D structure on mica was distorted and M13 linked to DNA sheets were cut by AFM tip (Figure 5). It seems that the 3D structures were not stable in the presence of the external force from AFM.

The length of the side is almost equal to the design. We think that 3D structures are not distorted in the solution if there was no external force from AFM.
Therefore, we were able to make 3D structure.

Condition

・M13:staples= 1:10(ratio of concentration)
・Mg²⁺ concentration 12.5 mM
・anealing time:3 hours
・Buffer:Tris-HCl
・cutting M13

Detail:Observation 2D and 3D structures

Observation Field　

Figure 6 is design of the Field. Figure 7 is AFM image of the Field.
According to this image, redundant M13 was also observed.
The size of Field in AFM picture is similar to that of design.
Therefore we succeeded in observing the Field.

Condition

・M13:staples= 1:10(ratio of concentration)
・Mg2+ concentration 12.5 mM
・anealing time:3 hours
・Buffer:Tris-HCl

We got clear pictures of Field.
Before this experiment,the 2D and 3D structure were also observed using the high-speed AFM.
In other words,we accomplished observing all the elements necessary for our race by using high-speed AFM.
Next,we move on to combine the 3D structure with Field.

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Observation structure on the Field

Triangular prism 2D structure on the Field　

　 Luckily we obtained the image of the 2D structure attached to the field using high-speed AFM.
In this sample, there ought to be only robots (2D structures which couldn't be folded and 3D structures ) but several fields were observed in this sample.
We supposed that the leftover fields on cantilever adsorbed on mica.
We scanned this field with 2D structure about 10 times but the structure didn't go away. Next, we raised the scanning speed then the structure was gone.
The structure was detached from the field because cantilever scratched the structure.

It seems that robots can be attached to the field.

Condition

・M13:staples= 1:10(ratio of concentration)
・Mg²⁺ concentration 12.5 mM
・Annealing time:3 hours
・Buffer:Tris-HCl

Condition for the robots to attach the fields

In order to optimize the condition for reproducible observation

Please click for more information!!

Detail:Observation structure on the Field

Flow chart of experiments to bind the robots and fields
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 <td>

　Observation diary　

We wrote the observation diary for AFM. If you want to check it, please click Observation diary.

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+<FONT SIZE=4 COLOR=#FF0000>'''Please click for more information!!'''</FONT><br/>
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 <p style="text-align">
 *[http://openwetware.org/index.php?title=Biomod/2011/TeamJapan/Sendai/Result_Atomic_Force_Microscope/detail_Observation_struture%2Bfield '''Detail:Observation structure on the Field''']

Biomod/2011/TeamJapan/Sendai/Results/Atomic Force Microscope: Difference between revisions

Revision as of 06:30, 1 November 2011

Contents

High-speed AFM

Observation of 2D and 3D structures

Triangular prism 2D structure

Triangular prism 3D structure

Observation Field

Observation structure on the Field

Triangular prism 2D structure on the Field

Condition for the robots to attach the fields

In order to optimize the condition for reproducible observation

Observation diary

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