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<2> Modification of the mesoporous silica particle with DNA<br>
<2> Modification of the mesoporous silica particle with DNA<br>
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.<br>
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.<br>
Finally, we modified MPS-COOH with NH2-terminated ssDNAs. The MSN-COOH was dispersed in ** mL of 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.<br>
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.<br>
<a name="c"></a>  
<a name="c"></a>  
<center><font size="6" color="#000022" face="Arial"><b> Combining the Doll particles with the Barrels particles</b></font></center>
<center><font size="6" color="#000022" face="Arial"><b> Combining the Doll particles with the Barrels particles</b></font></center>

Revision as of 18:06, 25 October 2014

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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">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">Barrel particle</a>
    • <a href="fit_Method.html#b">DNA modified</a>
    • <a href="fit_Method.html#c">樽と人形の結合</a>
      • 　　　
    • <a href="fit_Method.html#d">樽人形剣</a>
      • 　　　
    • <a href="fit_Method.html#e">Material</a>
  • <a href="fit_Results and Discussion.html#">Result and Discassions</a>
    • <a href="fit_Results and Discussion.html#a">Barrel particle and Doll particle</a>
    • <a href="fit_Results and Discussion.html#b">DNA modified</a>
    • <a href="fit_Results and Discussion.html#c">樽と人形の結合</a>
    • <a href="fit_Results and Discussion.html#d">樽人形剣</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">Menber</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.


<img alt="" src="/images/d/d0/Hy8.png" width="320" height="240" border="0" / >

Scheme 1. Synthetic scheme for the (a) amino-funcionalized mesoporous silica (MPS-NH2), (b) carboxylated mesoporous silica (MPS-COOH), and (c) DNA-modified mesoporous silica.




<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>

  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

  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

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


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