Biomod/2012/Titech/Nano-Jugglers/Results
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} </style> </head> <BODY> <div id="biomodlink"> <<a href="http://openwetware.org/wiki/Biomod">BIOMOD</a>|<a href="http://openwetware.org/wiki/Biomod/2012">2012</a>|Titech Nano-Jugglers </div> <div id="header"> <div id="navigation"> <div id="menu"> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers"><br>Home<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Team/Students"><br>Team<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Project"><br>Project<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Results">Results<br>&<br>Methods</a></font></li> <li class="ach"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Achievements"><br>Achievements<br><br></a> <li class="sup"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Protocols"><br>Suppl. Info.<br><br></a></li> <li class="none"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Acknowledgement"><br>Acknowledgements<br><br></a></li> </ul> </div> </div> </div> </BODY> </html>
Results
<html><body><td align="center" width="300px"><A href=#0._Construction_of_Biomolecular_Rocket title="Body"><img src="http://openwetware.org/images/b/b5/BM.jpg" border=0 width=310 height=240></a></td></body></html> |
Construction of Biomolecular Rocket
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<html><body><A href=#1._Power_supply_for_the_rail-free_movement_of_Biomolecular_Rocket title="Rail-free"><img src="http://openwetware.org/images/7/7f/Rail-free%E3%80%80movement_kinesin.jpg" border=0 width=310 height=220></a></body></html> Power supply for the rail-free movement of Biomolecular Rocket |
<html><body><A href=#2._Realization_of_high-speed_movement_of_Biomolecular_Rocket title="High-speed"><img src="http://openwetware.org/images/8/89/High-speed_movement.jpg" border=0 width=310 height=220></a></body></html> Realization of high-speed movement of Biomolecular Rocket |
<html><body><A href=#3._Introduction_of_a_photo-switchable_DNA_system_for_the_directional_control title="Control"><img src="http://openwetware.org/images/d/dd/Control_image.jpg" border=0 width=310 height=220></a></body></html> Introduction of a photo-switchable DNA system for the directional control |
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0. Construction of Biomolecular Rocket
<html><body><font size="5">We constructed Biomolecular Rocket with a microbead, catalysts, and designed DNAs.</font></body></html> Biomolecular Rocket is composed of a micrometer-sized body and many catalytic engines. The body consists of a microbead with a diameter of 10 μm, and catalytic engines consist of platinum nanoparticles or catalase molecules. The catalytic engines are conjugated to the body using a DNA-based linker in a spatially selective manner.
>>see more methods
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<html><body><td align="center"><img src="http://openwetware.org/images/4/4d/Charts.jpg" border=0 width=200 height=440></a></td></body></html> |
0.1. Selective coating of the body
<html><body><font size="5">We succeeded in selective coating of a micrometer-sized bead by vapor deposition of Au and Cr.</font></body></html>- >>see more methods
<html><body><td align="center"><img src="http://openwetware.org/images/a/a5/Vapor_deposition.jpg" border=0 width=330 height=110></a></td></body></html> - >>see more methods
- Figure 0.1a, b and c are microscope images of 40 μm microbeads. Figure 0.1a shows microbeads before vapor deposition of metals. Figure 0.1b shows microbeads after vapor deposition of Au on the microbeads. Figure 0.1c shows microbeads after additional vapor deposition of Cr on the Au-deposited microbeads. The microbeads had three types of surface areas because the angular alignment of the beads was changed when Cr was deposited on the Au-deposited microbeads.
- Similarly, Figure 0.1e, f and g are microscope images of 10 μm microbeads. Figure 0.1e shows microbeads before vapor deposition of metals. Figure 0.1f shows microbeads after vapor deposition of Au on the microbeads. Figure 0.1g shows microbeads after additional vapor deposition of Cr on the Au-deposited microbeads. The 10 μm microbeads probably had three types of surface areas. From these result, we conclude that selective coating of microbeads for Biomolecular Rocket was achieved.
0.2. Design of linker DNA strands
- <html><body><font size="5">We designed linker DNA strands that can stably hybridize.</font></body></html>
- >>see more methods
We designed 2 kinds of DNA duplex that had different stability. We called the longer one as DNA sequence L, and the shorter one as DNA sequence S. We called their complementary strands as DNA sequence L* and DNA sequence S*. |
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- In both Figure 0.2a and 0.2b, Tm is far from Rt. So, we achieved DNA design that can hybridize stably.
0.3. DNA conjugation
0.3.1. Selective conjugation of DNA to polystyrene surface area
<html><body><td align="center"><img src="http://openwetware.org/images/f/f5/Polystyrene_EDAC.jpg" border=0 width=300 height=240></a></td></body></html> <html><body><font size="5">We succeeded in DNA conjugation to polystyrene surface area by using EDAC.</font></body></html>
- >>see more methods
For visualizing the results of DNA conjugation to polystyrene surface area, we hybridized fluorescent complementary DNA and observed them under blue light by microscope.
- >>see more methods
- Figure 0.3.1a, b, and c are images of 10 μm selective coated beads under visible light. Figure 0.3.1a', b', and c' are images of 10 μm selective coated beads under blue light. Figure 0.3.1a anda' shows selective coated beads after conjugated DNA to polystyrene area and hybridize fluorescent cDNA. Figure 0.3.1b and b' shows selective coated beads after conjugated DNA to polystyrene area. Figure 0.3.1c and c' shows selective coated beads after mixing fluorescent DNA. Selective coated beads exhibit fluorescence because fluorescent cDNA was excited by blue light. From these results, we conclude that selective conjugation of DNA to polystyrene surface area was achieved.
0.3.2. Selective conjugation of DNA to metal surface area
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/8/87/Metal_SAM.jpg" border=0 width=300 height=240></a></td></body></html> <html><body><font size="5">We succeeded in DNA conjugation onto Au surface area by the reaction of SAM.</font></body></html>
- >>see more methods
For visualizing the results of DNA conjugation to metal surface area, we hybridized linker DNA to Au plate surface.
- >>see more methods
- Figure 0.3.2a is a images of Au plate that linker DNA conjugated partially. Figure 0.3.2b is a images after soaking up DNA buffer of Au plate. Figure 0.3.2a is a images after washing away of partially DNA conjugated Au plate. Final concentration of phosphate and NaCl buffer are the same in these spot. But 1, 2 and 3 are added NaCl concentration immediately after 24 hours of incubation, the other hand 4, 5, and 6 are added NaCl as salt aging process. Au plate reveals partially hydrophilic, this is because physical property of Au surface was assimilated to physical property of DNA. From these result, we conclude that selective conjugation of DNA to metal surface area was achieved.
0.4. Catalyst attachment with DNA hybridization
<html><body><td align="center"><img src="http://openwetware.org/images/1/19/Conjugation_catalyst.jpg" border=0 width=300 height=230></a></td></body></html> <html><body><font size="5">DNA hybridization will allow to attach catalyst engine to the body of Biomolecular Rocket before long.</font></body></html>
- >>see more methods
- >>see more methods
- We will attach platinum particles to the body of Biomolecular Rocket by taking advantage of DNA hybridization. DNA strand and its complementary DNA transit to take thermodynamically stable forms. To visualize catalyst attachment, we will attach Pt particles to Au plate (instead of Au deposited beads) , but there is a few difference between DNA conjugated materials and natural materials. So, We must prove the technology of attaching catalyst. For example, buffer concentration and temperature influence DNA hybridization. In addition, We also design DNA strands of DNA sequence L and DNA sequence S that have the first 15 bases from 5' end as a linker. This DNA was also designed not to make unexpected structures. Probably, this linker part will allow much leeway for attaching the body, in that decrease the effect of steric hindrance.
1. Power supply for the rail-free movement of Biomolecular Rocket
<html><body><font size="5">We achieved the power supply for the rail-free movement.</font></body></html>
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<html><body><td align="center"><img src="http://openwetware.org/images/b/b3/WikiRFBM.jpg" border=0 width=240 height=240></a></td></body></html> |
1.1. DNA hybridization in solution of H2O2
- <html><body><font size="5">We succeeded in DNA hybridization in 1%-5% H<sub>2</sub>O<sub>2</sub> solution stably.</font></body></html>
- To visualize the stability of DNA duplex in 1%-5% H2O2 solution, we use PAGE electrophoresis. It shows the difference of molecular weight of nucleic acid that comes from denaturetion or hybridization in the form of bands.
- >>see more methods
- Figure 1.1 is a image of DNA hybridization after immersing in solution of H2O2. Lane 1, 2, 3, and 8 shows dsDNA, that is hybridized after immersing in solution of 0%-5% H2O2 for 90 minutes. Lane 4, 5, 6, and 7 shows ssDNA, after immersing in solution of 0%-5% H2O2 for 90 minutes. Influence of H2O2 solution within 90 minutes was few for DNA hybridizing, because the line of dsDNA appear in the same positions. Also influence of H2O2 solution within 90 minutes was few for ssDNA in that the line of ssDNA appear in the same positions. From this results, we achieved DNA hybridization in solution of H2O2.
1.2. Power supply for rail-free movement by using catalase catalytic engine
- <html><body><font size="5">We scceeded in rail-free movement by taking advantage of catalase catalytic ability.</font></body></html>
- Catalase has Catalytic ability of decomposing H2O2, like platinum. We conjugated catalase to the polystyrene hemisperical area, so this catalase hemispherical bead moves by emission of O2 bubbles without rails.
- >>see more methods
- Figure 1.2 reveals the movement of 10 μm catalase hemisphere movement in solution of 3% H2O2. We can easily distinguish catalase conjugated bead or natural bead in that their movement in solution of 3% H2O2 is very different. Natural beads didn't move at all, on the other hand catalase hemisphere emitted bubbles and moved quickly. From this results, we realized power supply for rail-free movement by using catalase catalytic engine.
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<html><body><img src="http://openwetware.org/images/1/1b/Crf.jpg" border=0 width=320 height=240></body></html> |
1.3. Power supply for rail-free movement by using platinum catalytic engine
- <html><body><font size="5">We achieved bubble emissions with platinum micro-particles in solution of H<sub>2</sub>O<sub>2</sub>.</font></body></html>
- We provide 1 μm platinum particles, and Cr coating to create platinum hemisphere. Then added 3% H2O2 solution and observed their movement.
- >>see more methods
- Movie 1.3 reveals the catalytic reaction of 3% H2O2 and 1 μm beads that have platinum hemispherical area. We can recognize that bubbles are emitted in H2O2 solution. This bubbles are emitted from 1 μm beads that have platinum hemispherical area by decomposing H2O2. From this results, we conducted that Power supply for rail-free movement by using platinum catalytic engine.
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<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/8/89/Simple_beads.jpg" border=0 width=240 height=180></a></td></body></html> |
2. Realization of high-speed movement of Biomolecular Rocket
<html><body><font size="5">We achieved high-speed movement of Biomorecular Rocket by bubble emission.</font></body></html>
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<html><body><img src="http://openwetware.org/images/2/26/Hst.jpg" width=300 height=230></body></html> |
2.1. Analyses of the speed of platinum in solution of H2O2 by High-speed camera
- <html><body><font size="5">We succeeded in analyses of the speed of plutinum in solution of H<sub>2</sub>O<sub>2</sub> by High-speed camera</font></body></html>
- >>see more methods
a
<html><iframe width="440" height="330" src="http://www.youtube.com/embed/7pBw4FWEt3I" frameborder="0" allowfullscreen></iframe></html>
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<html><iframe width="440" height="330" src="http://www.youtube.com/embed/E1rtI0mS5Zs" frameborder="0" allowfullscreen></iframe></html>
Movie. 2.1 Analyses of the speed of platinum in solution of H2O2.
a Platinum movement in solution of H2O2 b Analises of the speed of plutinum by High-speed camera
- Movie 2.1b disclosed the values of Acceleration, Velocity, Coordinate x, and Coordinate y of platinum movement. Not only that, by observation of these values, we could determine relationships between bubble radius growth and the speed of platinum, so we were able to simulate of movement of our rocket.
2-2.Simulation for speeding-up of Biomolecular Rocket movement
- <html><body><font size="5">From our numerical simurations of high-speed, We carried out numerical simulations of the high-speed movement of Biomolecular Rockets.</font></body></html>
- <html><body><td align="center" width="150px"><img src="http://openwetware.org/images/f/f0/Simulation_speed.jpg" border=0 width=240 height=180></a></td></body></html>
3. Introduction of a photo-switchable DNA system for the directional control
<html><body><font size="5">We developed a photo-switchable DNA system for directional control with photoresponsive DNA.</font></body></html>
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<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/8/8a/DC.jpg" border=0 width=300 height=230></a></td></body></html> |
3.1. Design of photoresponsive DNA
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/4/45/Photo_design.jpg" border=0 width=210 height=210></a></td></body></html> <html><body><font size="5">We designed photoresponsive DNA strands for achieving photo-switchable DNA system.</font></body></html>
>>see more methods
We designed photoresponsive DNA that can hybridize stably with its cDNA. To visualize the conformation of photoresponsive DNA, we analyze Abs of photoresponsive DNA. Numerical values of Abs depends on the concentration of material corresponding to the absorption wavelength.
- We called DNA as A:Photoresponsive DNA sequence S, and B:Photoresponsive DNA sequence S*. Figure 3.1 reveals that Abs of A+B around 260nm was below those of A and B, and also Abs of A+B around 330 nm was less than that of A. This is because the concentration of azobenzene decreased by hybridization of A with B.
- Calculation an average absorbance of A and B Abs around 260 nm, theorical value of A+B was 0.168. Compared to this, measured value of absorbance is 22.6% lower. We thought this difference comes from that DNA formed duplex and interactions between base pairs, so decrease the UV absorbance relative to single strands.
- In this point, we believe that the complementary photo-responsive DNAs can form duplex. So, we concluded that these results mean A and B hybridized successfully.
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Fig.3.1 Abs of photoresponsive DNA results, wave length around 260 nm reveals the concentration of DNA. Around 330 nm reveals the concentration of trans-formed azobenzene. |
3.2. Directional control with photo-switchable DNA system
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/f/f3/Azobenzene_image_dissociation.jpg" border=0 width=240 height=180></a></td></body></html> <html><body><font size="5">We have scceeded in a photo-switchable DNA system.</font></body></html>
- >>see more methods
Photo-switchable DNA duplex can easily dissociate its duplex by irradiating UV-light. We put this switching system in the rocket in order to control our rocket. To visualize a photo-switchable DNA system, we checked Abs.
- >>see more methods
- For investigating the relationship between the strength of UV light and the time of dissociation to determine the valid time, we examine 2 type of UV light. Figure 3-2.1 represents spctrum of Abs in condition of UV-light(30 mW/cm2) irradiation. Abs of A+B around 260 nm was increasing gradually from 0 minutes to 5 minutes. This result means dsDNA was completely dissociated after irradiating UV-light for 5 minutes. Moreover, Abs of A+B around 330 nm was decreasing from 0 minutes to 5 minutes. This means trans-formed azobenzene changed its form to cis-formation. Therefore, we achieved photoresponsive DNA which was designed by us would be dissociated by irradiating UV-light for 5 minutes.
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Fig. 3.2.1 Spectrum analysis of photoresponsive DNA duplex(A+B) in condition of UV-light(30 mW/cm2) irradiation |
- Then, we tested the dissociation of photoresponsive DNA under the condition of different strength of UV-light(180 mW/cm2). First, from Figure 3.2.2b, we could find that dsDNA was dissociated gradually from 0 seconds to 50 seconds because maximum Abs around 260 nm was increasing. Second, Figure 3.2.2c shows that trans-formed azobenzene decreased because Abs around 330 nm was decreasing from irradiation for 0 seconds to 50 seconds. Finally, Figure 3.2.2 shows cis-formed azobenzene increased. As we did experiences for many times, we noticed that there was maximum wave length around 480 nm. By researching, we reached the fact that Abs around 480 nm shows the existence of cis-formed azobenzene. So, we can say that cis-formed azobenzene increased because Abs around 480 nm was increasing from irradiation for 0 seconds to 50 seconds. To sum up, we concluded that photo-seichable DNA system achieved after 50 seconds irradiation of UV-light(180 mW/cm2). In brief, we can conclude that photoresponsive dsDNA was dissociated completely after the irradiation of UV-light (180 mW/cm2) for 50 seconds.
- From these results, we conducted that Dissociation of photoresponsive DNA by UV-light irradiation.
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Fig. 3.2.2 Spectrum analysis of photoresponsive DNA duplex(A+B) in condition of UV-light(180 mW/cm2) irradiation |
3-3.Simulation for directional control of Biomolecular Rocket
- sss
- If you want to see all of our methods, click here