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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

Construction of Biomolecular Rocket

We constructed Biomolecular Rocket composed of a micrometer-sized body and many catalytic engines. The catalytic engines were conjugated to the body using DNA-bead linkers in a spatially selective manner.

Shown in detail below

<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

We realized rail-free movement by power generation with catalytic reactions of platinum and catalase.

Shown in detail below

We realized the high-speed movement by power generation with catalytic reactions of platinum and catalase, and analyzed the speed.
We carried out numerical simulations of the high-speed movement of Biomolecular Rockets.

Shown in detail below

We developed photo-switchable DNA for the control of Bimolecular Rocket using UV light irradiation.
We investigated directional control of Biomolecular Rocket in the simulation.

Shown in detail below

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.
We constructed the Biomolecular Rocket through the following four steps.

1. The microbead body was selectively coated by vapor deposition of metals (Au and Cr).
2. We designed DNA sequences for spatially-selective hybridization of catalytic engines.
3. The DNA molecules were conjugated to a designated metal surface of the microbead body.
4. Catalyst engines were attached to the microbead body with selective hybridization of DNAs
we designed.

>>see more methods

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

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.

40 μm silica beads	a	b	c
10 μm polystyrene beads	d	e	f
Image of micro-beads	<html><body><img src="http://openwetware.org/images/d/d5/Polystyrene.jpg" border=0 width=240 height=180></body></html>	<html><body><img src="http://openwetware.org/images/4/41/After_first_deposition.jpg" border=0 width=240 height=180></body></html>	<html><body><img src="http://openwetware.org/images/3/30/After_second_deposition.jpg" border=0 width=240 height=180></body></html>
Fig. 0.1 Selective coating of micro-beads by vapor deposition.

0.2. Design of linker DNA strands

<html><body><font size="5">We designed DNA sequences for the linkers to attach catalytic engines to the microbead body.</font></body></html>

>>see more methods

We designed two kinds of DNA sequences with different stability. One is DNA sequence L. This sequence forms a very stable duplex with its complementary sequence L^* at a room temperature. Another is DNA sequence S. The duplex of DNA sequence S and S^* has less stability than DNA sequence L and L^* but is stable at a room temperature.

DNA sequence L 5’-CGTCTATTGCTTGTCACTTCCCC-3'	DNA sequence S 5’-AATACCCAGCC-3’
DNA sequence L^* 5'-GGGGAAGTGACAAGCAATAGACG-3'	DNA sequence S^* 5’-GGCTGGGTATT-3’

Figure 0.2a and 0.2b show calculated melting profiles of duplexes L-L^* and S-S^*, respectively. Calculated melting temperatures of these duplexes were higher than a room temperature.

a	b
Fig. 0.2. (a) The melting profile of duplex L-L^. (b) The melting profile of duplex S-S^. RT and T_m indicate a room temperature (24°C) and a melting temperature, respectively. We calculated the melting profiles using NUPACK software.

0.3. DNA conjugation

0.3.1. Selective conjugation of DNA to polystyrene surface area

<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.

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.

Under visible light	a	b	c
Under blue light	a'	b'	c'
conditions	<html><body><img src="http://openwetware.org/images/d/d3/EDACBEADS1.JPG" border=0 width=280 height=150></a></body></html>	<html><body><img src="http://openwetware.org/images/3/38/EDACBEADS2.JPG" border=0 width=280 height=150></a></body></html>	<html><body><img src="http://openwetware.org/images/2/23/EDACBEADS3.JPG" border=0 width=280 height=150></a></body></html>
Fig. 0.3.1 Images of DNA conjugation to polystyrene surface area by fluorescence complementary DNA strand.

0.3.2. Selective conjugation of DNA to metal surface area

<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.

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, on 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.

a	b	c
d<html><body><img src="http://openwetware.org/images/1/1a/Auplate1.jpg" border=0 width=300 height=160></a></body></html>	e<html><body><img src="http://openwetware.org/images/1/12/Auplate2.jpg" border=0 width=300 height=160></a></body></html>	f<html><body><img src="http://openwetware.org/images/f/fd/Auplate3.jpg" border=0 width=300 height=160></a></body></html>
g	Fig. 0.3.2 Confirmation of DNA conjugation to metal area (a) Incubated for 48 hours at a gold plate (b) Soaked up the water after incubation (c) Washed away gold plate surface by 3 × SSC after soaking up the water (d) Image of spot 1, 4 (e) Image of spot 2, 5 (f) Image of spot 3, 6 (g) Condition of gold plate

0.4. Catalyst attachment with DNA hybridization

<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

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.

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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>

    Biomolecular Rocket can move straightforward by emitting bubbles. Platinum and catalase catalysts decompose H₂O₂ and emit H₂O and O₂ bubbles. When the O₂ bubbles are emitted from the surface of Biomolecular Rocket, the momentum changes induces driving force, and rail-free movement is accomplished.
    We achieved power supply for the rail-free movement through the following points.

1. DNA hybridization was not affected by the solution of H₂O₂
2. Platinum catalyst could emit sufficient amount of bubbles for rail-free movement
3. Catalase catalyst could emit sufficient amount of bubbles for rail-free movement

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 used 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 show dsDNA, that is hybridized after immersing in solution of 0%-5% H₂O₂ for 90 minutes. Lane 4, 5, 6, and 7 show ssDNA, after immersing in solution of 0%-5% H₂O₂ for 90 minutes. Influence of H₂O₂ solution within 90 minutes was a bit for DNA hybridizing, because the lines of dsDNA appear in the same positions. Also influence of H₂O₂ solution within 90 minutes was a bit for ssDNA in that the lines of ssDNA appear in the same positions. From this results, we achieved DNA hybridization in solution of H₂O₂.

Fig. 1.1 Results of DNA hybridization after immersing in solution of H₂O₂

dsDNA bands show in lane 1, 2, 3 & 8. In lane 4, 5, 6 & 7 show ssDNA bands. Lane 1 & 8 are the same, lane 2 & 3 are immersed in H₂O₂ solution (1% and 5%) for 90 minutes. In lane 5 & 7 show the results of ssDNA in solution of H₂O₂. In lane 4 & 6 are the control bands of ssDNA.

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 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 result, we realized power supply for rail-free movement by using catalase catalytic engine.

Fig. 1.2 The image of the movement of 10 μm beads that had catalase hemispherical area in the solution of 3% H₂O₂

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 provided 1 μm platinum particles, and Cr coating to create platinum hemispherical area. 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. These bubbles are emitted from 1 μm beads that have platinum hemispherical area by decomposing H₂O₂. From this result, we conducted that Power supply for rail-free movement by using platinum catalytic engine was achieved.

Movie. 1.3 Image of the catalytic reaction of 3% H₂O₂ and 1 μm beads that have platinum hemispherical area

↑Page Top

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>

    Catalytic engines emit large amount of bubbles, and this reaction supplies powerful driving force to BiomolecularRocket. By observation and simulation of the behavior of platinum movement behavior in detail, we found that the speed of Biomolcular Rocket is faster than the speed of kinesin.
    We realized high-speed movement of Biomolecular Rocket through the following points.

1. Platinum catalyst could emit sufficient amount of bubbles for high-speed
movement of Biomolecular Rocket
2. Compared the speed of kinesin, platinum hemispherical beads and Biomolecular
Rocket to dictate which is the fastest one.

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>	b <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 Biomolecular Rocket.

2.2. Comparison of the velocity among Biomolecular rocket and kinesin

<html><body><font size="5">Biomolecular Rocket moved at ten times faster than kinesin moves, twice faster than usual platinum motor without DNA in simulation, .</font></body></html>

↑Page Top

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>
    Photoresponsive DNA structure is changed by UV light irradiation, then dissociation of DNA duplex will happen. Take advantage of this reaction in region-specific manner, we can control the direction of Biomolecular Rocket.
    Introduction of a photo-switchable DNA system for the directional control, we conducted the following points.

1. Introduction of a photo-switchable DNA system for the directional control
2. Dissociation of photo-switchahble DNA duplex by UV light irradiation
3. Directional control of Biomolecular Rocket by detaching platinum catalytic
engines

3.1. Design of photoresponsive DNA

<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 of 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 was 22.6% lower. We thought this difference comes from formation of DNA duplex and interactions between base pairs, so the UV absorbance decreased 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.

a	b
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><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 observeded absorbance.

For investigating the relationship between the strength of UV light and the time for dissociation, to determine the valid time, we examined 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.

a	b
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 was achived.

a	b
c	d
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

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If you want to see all of our methods, click here

@@ Line 259: / Line 259: @@
 :&nbsp;&nbsp;&nbsp;&nbsp;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 Biomolecular Rocket.
-==2-2.Simulation for speeding-up of Biomolecular Rocket movement==
+==2.2. Comparison of the velocity among Biomolecular rocket and kinesin ==
-:&nbsp;&nbsp;&nbsp;&nbsp;<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>
+:&nbsp;&nbsp;&nbsp;&nbsp;<html><body><font size="5">Biomolecular Rocket moved at ten times faster than kinesin moves, twice faster than usual platinum motor without DNA in simulation, .</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>
 <br><br>

Biomod/2012/Titech/Nano-Jugglers/Results: Difference between revisions

Revision as of 08:54, 26 October 2012

Results

0. Construction of Biomolecular Rocket

0.1. Selective coating of the body

0.2. Design of linker DNA strands

0.3. DNA conjugation

0.3.1. Selective conjugation of DNA to polystyrene surface area

0.3.2. Selective conjugation of DNA to metal surface area

0.4. Catalyst attachment with DNA hybridization

1. Power supply for the rail-free movement of Biomolecular Rocket

1.1. DNA hybridization in solution of H₂O₂

1.2. Power supply for rail-free movement by using catalase catalytic engine

1.3. Power supply for rail-free movement by using platinum catalytic engine

2. Realization of high-speed movement of Biomolecular Rocket

2.1. Analyses of the speed of platinum in solution of H₂O₂ by High-speed camera

2.2. Comparison of the velocity among Biomolecular rocket and kinesin

3. Introduction of a photo-switchable DNA system for the directional control

3.1. Design of photoresponsive DNA

3.2. Directional control with photo-switchable DNA system

3-3.Simulation for directional control of Biomolecular Rocket

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Biomod/2012/Titech/Nano-Jugglers/Results: Difference between revisions

Revision as of 08:54, 26 October 2012

Results

0. Construction of Biomolecular Rocket

0.1. Selective coating of the body

0.2. Design of linker DNA strands

0.3. DNA conjugation

0.3.1. Selective conjugation of DNA to polystyrene surface area

0.3.2. Selective conjugation of DNA to metal surface area

0.4. Catalyst attachment with DNA hybridization

1. Power supply for the rail-free movement of Biomolecular Rocket

1.1. DNA hybridization in solution of H2O2

1.2. Power supply for rail-free movement by using catalase catalytic engine

1.3. Power supply for rail-free movement by using platinum catalytic engine

2. Realization of high-speed movement of Biomolecular Rocket

2.1. Analyses of the speed of platinum in solution of H2O2 by High-speed camera

2.2. Comparison of the velocity among Biomolecular rocket and kinesin

3. Introduction of a photo-switchable DNA system for the directional control

3.1. Design of photoresponsive DNA

3.2. Directional control with photo-switchable DNA system

3-3.Simulation for directional control of Biomolecular Rocket

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1.1. DNA hybridization in solution of H₂O₂

2.1. Analyses of the speed of platinum in solution of H₂O₂ by High-speed camera