Biomod/2014/fit Method.html

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<a name="header"></a> <img src="/images/4/46/Fitaologo.PNG" alt" alt="" width="422" height="98" hspace="0" align="left">




  • <a href="http://openwetware.org/wiki/Biomod/2014/Fukuoka#home">Home</a>
  • <a href="fit_Introduction.html#pro">Projects</a>
    • <a href="fit_Introduction.html#back">Background & Motivation</a>
    • <a href="fit_Introduction.html#goal">Project Goals</a>
  • <a href="fit_Approach and Goals.html#des">Design</a>
    • <a href="fit_Approach and Goals.html#ear">Early Design</a>
    • <a href="fit_Approach and Goals.html#fin">Final Design</a>
  • <a href="fit_Method.html#met">Method</a>
    • <a href="fit_Method.html#a">Preliminary Experiment</a>
    • <a href="fit_Method.html#b">Synthesis of the “Barrel” particles and the “Doll” particles</a>
    • <a href="fit_Method.html#c">Combining the Doll particles with the Barrels particles</a>
    •    
    • <a href="fit_Method.html#d">Pop-up of the doll particle</a>
    •    
    • <a href="fit_Method.html#e">Materials</a>
  • <a href="fit_Results and Discussion.html#">Result and Discassions</a>
    • <a href="fit_Results and Discussion.html#a">Preliminary Experiment</a>
    • <a href="fit_Results and Discussion.html#b">Synthesis of the “Barrel” particles and the “Doll” particles</a>
    • <a href="fit_Results and Discussion.html#c">Combining the Doll particles with the Barrels particles</a>
    • <a href="fit_Results and Discussion.html#d">Pop-up of the doll particle</a>
    •    
    • <a href="fit_Results and Discussion.html#e">Conclusion</a>
  • <a href="fit_Member.html#team">Team</a>
    • <a href="fit_Member.html#men">Member</a>
    • <a href="fit_Member.html#spo">Sponsor</a>


<a name="a"></a>

Preliminary Experiment

verification of FRET with the ssDNAs not modified to the silica

 
 We firstly confirmed that FRET is surely induced by hybridization of the sword-DNA and the barrel-DNA not yet modified on the silica. We prepared the mixed solutions of FITC/TAMRA-DNA and FITC-DNA/TAMRA-DNA. The concentration is set to 50pM. FITC-DNA/TAMRA-DNA solution was annealed: the solution was kept at 94ºC for 30 seconds and then the temperature was decreased at the rate of 6 ºC degrees per 15 minutes. Fluorescence spectra was measured on Hitachi F-2500 spectrophotometer.


<a name="b"></a>

Synthesis of the “Barrel” particles and the “Doll” particles

  <1> Synthesis of mesoporous silica modified with amino group by sol-gel method.
  We used mesoporous silica particles to use as “Barrel” and "Doll" bodies. We used sol-gel method to synthesize the mesoporous silica modified with amino group (MPS-NH2) as shown in Scheme 1. N-cetyltrimethylammonium bromide (CTAB, 1.0009 g) was first dissolved in 50 mL of pure hot water (100 ºC). After cooling to room temperature, aqueous ammonia (28wt%, 13 mL) and ethanol (75 mL) were added. The mixture was stirred for 15 min and tetraethoxysilane (TEOS ; 1.94 ml) was added rapidly while stirring was continued. After 30 min of stirring, TEOS (0.030 mL) and aminopropyltriethoxysilane (APTES; 0.030 mL) were added. The mixture was allowed to stir for 2 h to give rise to white precipitates, followed by washing with water by using Kiriyama Rohto.


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<2> Modification of the mesoporous silica particle with DNA
  Next, we modified a carboxyl group onto the mesoporous silica with amino group. The MPS-NH2 (50 mg) was reacted with succinic anhydride (1.00 g) in N,N-dimetylformamide solution (20 mL) under N2 atmosphere for 8 h with continuous stirring in a 50 mL screw pipe. After the reaction, we washed it carefully with pure water and the carboxylated mesoporous silica (MPS- COOH) was obtained.
Finally, we modified MPS-COOH with NH2-terminated ssDNAs. The MSN-COOH was dispersed in water and then activated by adding 0.1471 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) and 0.1532g of N-Hydroxysuccinimide (NHS) in a MES buffer (pH 6.0) for 15 min at room temperature with continuous stirring. 0.020 mL of PBS buffer (100mM, pH 7.4) was then added in the mixture. After adding 0.005 mL of an aqueous DNA solution, the mixture was continuously stirred for 6 h at room temperature with continuous stirring. At last, we washed the sample with PBS buffer (100mM,pH7.4) three times. EDC and NHS were used to catalyze the reaction and PBS was used just for pH adjustment.
<a name="c"></a>

Combining the Doll particles with the Barrels particles

<a name="d"></a> <p>   the Barrel particles (0.010mL) and the Doll particles (0.090mL) were mixed using a micropipette. After mixed solution was sonicated for twenty minutes

Pop-up of the doll particle by adding sword DNA

<p>   We added 0.010 mL of the sword-DNA aqueous solution to the aqueous dispersions of the barrel particles (0.010mL) followed by observation with the confocal laser scanning microscope.

<a name="e"></a>

Materials

<p>   The DNA samples shown in Table 2 were supplied from sigma-aldrich Japan. Other materials used in this project are summarized in Table 4.

<img alt="" src="/images/8/85/Fit0031.JPG" width="320" height="240" border="0" / >

<img alt="" src="/images/d/d0/Hy8.png" width="320" height="240" border="0" / ><p> Scheme 1. Synthetic scheme for the amino-funcionalized mesoporous silica (MPS-NH2), carboxylated mesoporous silica (MPS-COOH), and DNA-modified mesoporous silica.</p>
</td>
Table 3 .
Structural formula Name Manufacturer Grade
<img alt="" src="/images/2/2c/1.png.gif"/ width="160" height="100" border=""/> Sulfo-NHS DOUJINDO 200mg
<img alt="" src="/images/3/3b/FIT%EF%BC%92.png.gif" width="180" height="90" border=""/> Tetraethyl orthosilicate

(Tetraethoxysilane; TEOS)

Wako 500mg
<img alt="" src="/images/9/98/FIT%EF%BC%93.png.gif" width="180" height="90" border="" /> 3-aminopropyltriethoxysilane Wako 25g
<img alt="" src="/images/1/1f/FIT%EF%BC%94.png.png" width="230" height="120" border=""/> 1 - (3-dimethylaminopropyl) carbodiimide hydrochloride TGI 5g
<img alt="" src="/images/c/cf/FIT%E3%80%80%EF%BC%95.gif" width="150" height="100" border=""/> N-N-dimethylformamide TGI 200mg
<img alt="" src="/images/0/0a/FIT_6.gif" width="145" height="90" border=""/> Succinic anhydride Wako 25g
<img alt="" src="/images/7/73/FIT_7.gif" width="200" height="70" border=""/> Cetyltrimethylammonium bromide Wako 25g
<img alt="" src="/images/c/c2/FIT_8.gif" width="180" height="90" border=""/> MES DOUJINDO 25g




]



<center>Characterization

  The synthesized particles were observed with Keyence VE7800 scanning electron microscope (SEM). We observed the synthesized particles by Nikon A1R+ confocal laser scanning microscope operated with the Ar+ laser (488 nm) and with the Galvano mirror scanner at the scanning speed of 0.125 images per second. The appratus used in this project is summarized in Table 4.

                 
Table 4 .
Device name Manufacturer

Model number

Measurement condition Photograph Explanations of equipment
Spectrophotometer HITACHI F-2500 WL Range:

450nm~650nm Scanning Speed: 60nm/min

<img alt="" src="/images/6/6d/FIT_9.png" width="150" height="130" border=""/> This device can see the concentration of the solution, the material characteristics, the structure of the molecule.
SEM KEYENC VE-7800 WL Range:

450nm~650nm Scanning Speed: 60nm/min

<img alt="" src="/images/9/9f/FIT_10.png" width="150" height="130" border=""> It is possible to significantly change the magnification easily. In addition, photography is possible.
Confocal laser scanning microscope

(CLSM)

Nikon ECLIPSE-Ti Ar-Laser (488nm)

Garvano mirror scanner

<img alt="" src="/images/8/8e/FIT_11.png" width="150" height="130" border="">It is possible to obtain a high resolution image in focus in the field of view for the entire sample surface with irregularities.

Three-dimensional shape measurement non-destructive, non-contact is possible


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