Difference between revisions of "Team:Pasteur Paris/Experiments"

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                 <div class="vignette_text">
 
                 <div class="vignette_text">
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                     <p style="margin:auto; text-align:center;font-weight:bold;" >PDMS Chips Fabrication</p>
+
                     <p style="margin:auto; text-align:center;font-weight:bold;" >PDMS Chip Fabrication</p>
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>
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                 <h3>Materials</h3>
 
                 <h3>Materials</h3>
 
                 <ul>
 
                 <ul>
−
                     <li> Sylgard 184 Elastomer Kit (Sigma-aldrich, 761036-5EA)  </li>
+
                     <li> Sylgard 184 Elastomer Kit   </li>
−
                     <li> Vacuum pump unit (Vacuubrand PC 3 RZ 2.5) </li>
+
                     <li> Vacuum pump unit </li>
−
                     <li> Stove (Memmert UM 400) </li>
+
                     <li> Stove </li>
 
                 </ul>
 
                 </ul>
 
                 <br>
 
                 <br>
 
                 <h3>Protocol</h3>
 
                 <h3>Protocol</h3>
 
                 <p>According to <a target="_blank" rel="noopener noreferrer" href="https://static.igem.org/mediawiki/igem.org/2/29/T--Technion_Israel-HardwarespecsPDMS.pdf">manufacturer's instruction</a>. <br>  
 
                 <p>According to <a target="_blank" rel="noopener noreferrer" href="https://static.igem.org/mediawiki/igem.org/2/29/T--Technion_Israel-HardwarespecsPDMS.pdf">manufacturer's instruction</a>. <br>  
−
                 <ul>
+
                 <ol>
−
                     <li> Mix monomer and curing agent (10:1 proportion) for 30 seconds  </li>
+
                     <li> Mix monomer and curing agent (10:1 proportion) for 30 seconds.   </li>
−
                     <li> Use a vacuum pump unit and a  vacuum bell jar to extract air bubbles until the mixture is clear </li>
+
                     <li> Use a vacuum pump unit and a  vacuum bell jar to extract air bubbles until the mixture is clear. </li>
−
                     <li> Pour mixture onto mold </li>
+
                     <li> Pour mixture onto mold. </li>
−
                     <li> Put mixture+mold in stove at 70 degrees Celsius for 3 hours </li>
+
                     <li> Put mixture+mold in stove at 70 degrees Celsius for 3 hours. </li>
−
                 </ul>  
+
                 </ol>  
 
                 <br>
 
                 <br>
 
                 <div class="protocol_box">
 
                 <div class="protocol_box">
−
                     <p> Get full protocol <a href="https://static.igem.org/mediawiki/2018/3/3d/T--Pasteur_Paris--Microfluidics-general-protocols.pdf" target="_blank">here</a> </p>
+
                     <p> <a href="https://static.igem.org/mediawiki/2018/3/3d/T--Pasteur_Paris--Microfluidics-general-protocols.pdf" target="_blank">Get full protocol here</a> </p>
 
                 </div>
 
                 </div>
 
                 <br>  
 
                 <br>  
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                 <h3>Materials</h3>
 
                 <h3>Materials</h3>
 
                 <ul>
 
                 <ul>
−
                     <li> Razor blade (OEMTOOLS 25181 Razor Blades, 100 Pack)  </li>
+
                     <li> Razor blade   </li>
−
                     <li> Biopsy puncher (Kai Biopsy Punch 4mm ) </li>
+
                     <li> Biopsy puncher 4mm  </li>
 
                 </ul>
 
                 </ul>
 
                 <br>
 
                 <br>
 
                 <h3>Protocol</h3>
 
                 <h3>Protocol</h3>
 
                 <br>
 
                 <br>
−
                 <ul>
+
                 <ol>
−
                     <li> Cut the borders of the chip with the razor blade  </li>
+
                     <li> Cut the borders of the chip with the razor blade.   </li>
−
                     <li> Extract the chip from its mold </li>
+
                     <li> Extract the chip from its mold. </li>
−
                     <li> Drill input and output holes with the biopsy puncher </li>
+
                     <li> Drill input and output holes with the biopsy puncher. </li>
−
                 </ul>
+
                 </ol>
 
                 <br>
 
                 <br>
 
                 <div class="protocol_box">
 
                 <div class="protocol_box">
−
                     <p> Get full protocol <a href="https://static.igem.org/mediawiki/2018/3/3d/T--Pasteur_Paris--Microfluidics-general-protocols.pdf" target="_blank">here</a> </p>
+
                     <p> <a href="https://static.igem.org/mediawiki/2018/3/3d/T--Pasteur_Paris--Microfluidics-general-protocols.pdf" target="_blank">Get full protocol here</a> </p>
 
                 </div>
 
                 </div>
 
                 <br>  
 
                 <br>  
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                 <h3>Materials</h3>
 
                 <h3>Materials</h3>
 
                 <ul>
 
                 <ul>
−
                     <li>  Plasma cleaner (Diener Pico PCCE)   </li>
+
                     <li>  Plasma cleaner  </li>
−
                     <li>  Distilled water (Fisherbrand, CAS number 7732-18-5)  </li>
+
                     <li>  Distilled water   </li>
−
                     <li>  Isopropanol (Fisherbrand, CAS number 67-63-0)  </li>
+
                     <li>  Isopropanol   </li>
 
                     <li>  Office duct tape </li>
 
                     <li>  Office duct tape </li>
−
                     <li>  Vertical laminar airflow cabinets (Euroclone aura vertical S.D.4) </li>
+
                     <li>  Fume hood </li>
 
                 </ul>
 
                 </ul>
 
                 <br>
 
                 <br>
 
                 <h3>Protocol</h3>
 
                 <h3>Protocol</h3>
 
                 <br>
 
                 <br>
−
                 <ul>
+
                 <ol>
−
                     <li> Take chip and the surface it needs to be bonded to into the airflow cabinet </li>
+
                     <li> Take chip and the surface it needs to be bonded to into the fume hood. </li>
−
                     <li> Clean chip with duct tape and isopropanol </li>
+
                     <li> Clean chip with duct tape and isopropanol. </li>
 
                     <li> Put the chip and the surface into the plasma cleaner. </li>
 
                     <li> Put the chip and the surface into the plasma cleaner. </li>
 
                     <li> Expose chip and surface 30 seconds to plasma. </li>
 
                     <li> Expose chip and surface 30 seconds to plasma. </li>
−
                     <li> Take the chip and the surface back in the airflow cabinet </li>
+
                     <li> Take the chip and the surface back in the fume hood. </li>
−
                     <li> Press the microfluidic chip against the surface </li>
+
                     <li> Press the microfluidic chip against the surface. </li>
−
                    <li> Insert distilled water into chip circuitry </li>
+
                </ol>
−
 
+
 
                 <br>
 
                 <br>
 
                 <div class="protocol_box">
 
                 <div class="protocol_box">
−
                     <p> Get full protocol <a href="https://static.igem.org/mediawiki/2018/3/3d/T--Pasteur_Paris--Microfluidics-general-protocols.pdf" target="_blank">here</a> </p>
+
                     <p> <a href="https://static.igem.org/mediawiki/2018/3/3d/T--Pasteur_Paris--Microfluidics-general-protocols.pdf" target="_blank">Get full protocol here</a> </p>
 
                 </div>
 
                 </div>
 
                     <br>  
 
                     <br>  
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                 <h3>Materials</h3>
 
                 <h3>Materials</h3>
 
                 <ul>
 
                 <ul>
−
                     <li>  Poly-D-Lysine solution 1.0 mg/mL (Sigma aldrich, A-003-E)  </li>
+
                     <li>  Poly-D-Lysine solution 1.0 mg/mL   </li>
−
                     <li>  Laminin (Sigma aldrich, Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, L2020-1MG) </li>
+
                     <li>  Laminin </li>
 
                 </ul>
 
                 </ul>
 
                 <br>
 
                 <br>
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                 <br>
 
                 <br>
  
−
                 <ul>
+
                 <ol>
−
                     <li> Pour poly-D-lysine with concentration 10 &mu g/mL into the chip  </li>
+
                     <li> Pour poly-D-lysine with concentration 10 &mu g/mL into the chip. </li>
−
                     <li> Incubate over night </li>
+
                     <li> Incubate over night. </li>
−
                     <li> Pour laminine with concentration 4 &mu g/mL </li>
+
                     <li> Pour laminine with concentration 4 &mu g/mL. </li>
−
                     <li> Incubate for a few hours</li>
+
                     <li> Incubate for a few hours.</li>
 +
                </ol>
 
                 <br>
 
                 <br>
 
                 <div class="protocol_box">
 
                 <div class="protocol_box">
−
                     <p> Get full protocol <a href="https://static.igem.org/mediawiki/2018/3/3d/T--Pasteur_Paris--Microfluidics-general-protocols.pdf" target="_blank">here</a> </p>
+
                     <p> <a href="https://static.igem.org/mediawiki/2018/3/3d/T--Pasteur_Paris--Microfluidics-general-protocols.pdf" target="_blank">Get full protocol here</a> </p>
 
                 </div>
 
                 </div>
 
                     <br>  
 
                     <br>  
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             <h2 style="order:1;width:100%">Microfluidics: well chip</h2>
 
             <h2 style="order:1;width:100%">Microfluidics: well chip</h2>
  
−
             <p style="text-indent:0px;order:2;margin:2em;width:100%"> The well chip was designed and assembled by our team. It was used to test the biocompatibility of our membranes, as well as the culture of bacteria in the presence of current. Here we show how the molds were made, how the chip itself was assembled, how well's conductivity was measured and how bio�lm culture was performed on it. </p>   
+
             <p style="text-indent:0px;order:2;margin:2em;width:100%"> The well chip was designed and assembled by our team. It was used to test the biocompatibility of our membranes, as well as the culture of bacteria in the presence of current. Here we show how the molds were made, how the chip itself was assembled, how well's conductivity was measured and how biofilm culture was performed on it. </p>   
  
 
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             <div class="vignette" id="vign_0100">
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                 </div>
 
                 </div>
 
             </div>
 
             </div>
 +
 +
            <div class="vignette" id="vign_0101">
 +
                <div class="vignette_for" id="for_0101">
 +
                </div>
 +
               
 +
                <div class="vignette_back" id="back_0101">
 +
                </div>
 +
               
 +
                <div class="vignette_text">
 +
                    <p style="margin:auto; text-align:center;font-weight:bold;">PDMS Well Chip Fabrication</p>
 +
                </div>
 +
            </div>
 +
  
  
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                 <br>
 
                 <br>
 
                 <br>
 
                 <br>
−
                 <p style="text-indent:0px;"> Molds were made of aluminium according to the following plans (Figure 1). Part 1 Mold's center cylinder part is detachable from the bottom to make the demolding ot PDMS easier.
+
                 <p style="text-indent:0px;"> Molds were made of aluminium according to the following plans (Figure 1). Part 1 Mold's center cylinder part is detachable from the bottom to make the demolding ot PDMS easier. </p>
−
</p>
+
 
                 <br>
 
                 <br>
 
                 <div style="position:relative;left:13em;">
 
                 <div style="position:relative;left:13em;">
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                 <br>
 
                 <br>
 
             </div>   
 
             </div>   
 +
 +
 +
          <div class="panel" id="pan_0101"  style="text-align:left;">
 +
                <div class="close_button">
 +
                </div>
 +
                <br>
 +
                <h3>Materials</h3>
 +
                <ul>
 +
                    <li>  Molds  </li>
 +
                    <li>  Syringe without needle </li>
 +
                    <li>  Platinum 24mm x 2 mm strip  </li>
 +
                    <li>  Circular 13mm diameter membrane </li>
 +
                </ul>
 +
                <br>
 +
                <h3>Protocol</h3>
 +
                <br>
 +
 +
                <ol>
 +
                    <li> Prepare 15g of PDMS monomer using our protocol (Link), section 1. Replace step 5 by : Fill the syringe with PDMS. Fill part 1 mold until it's full and part 2 mold until the PDMS layer is more or less 1 cm thick.  </li>
 +
                    <li> Demold the chip following our protocol (Link), section 2. Ignore step 2. </li>
 +
                    <li> Put membrane and platinum strip on PDMS part 1. Refer to figure 2 for their position. </li>
 +
                    <li> Refer to our protocol, section 3 to bond PDMS part 2 to the PDMS part prepared in the previous step. It should look like figure 3 and 4.</li>
 +
                    <li> Apply a small layer of PDMS with the syringe. Refer to figure 5 . This way, the well is watertight. </li>
 +
                    <li> Put the chip in the stove for 3 hours. </li>
 +
                </ol>
 +
                <br>
 +
                <div class="protocol_box">
 +
                    <p> Get full protocol here </p>
 +
                </div>
 +
                    <br>
 +
            </div>
 +
 +
 +
  
 
             <script>
 
             <script>

Revision as of 12:07, 23 August 2018

""

PROTOCOLS

Microfluidics: general protocols

PDMS (Polydimethylsiloxane) is a widely used polymer in microfluidics, for its biocompatibility and transparence, among other qualities. Here we show how to prepare PDMS for microfluidic chips, as well as how to demold them, bond them to other surfaces and treat them for neuron growth. Also, we explain how our molds and chips were fabricated.

PDMS Chip Fabrication

PDMS Chip Demolding

PDMS Chip Bonding

PDMS Chip Treatment for Nerve Growth


Materials

  • Sylgard 184 Elastomer Kit
  • Vacuum pump unit
  • Stove

Protocol

According to manufacturer's instruction.

  1. Mix monomer and curing agent (10:1 proportion) for 30 seconds.
  2. Use a vacuum pump unit and a vacuum bell jar to extract air bubbles until the mixture is clear.
  3. Pour mixture onto mold.
  4. Put mixture+mold in stove at 70 degrees Celsius for 3 hours.

Get full protocol here



Materials

  • Razor blade
  • Biopsy puncher 4mm

Protocol


  1. Cut the borders of the chip with the razor blade.
  2. Extract the chip from its mold.
  3. Drill input and output holes with the biopsy puncher.

Get full protocol here



Materials

  • Plasma cleaner
  • Distilled water
  • Isopropanol
  • Office duct tape
  • Fume hood

Protocol


  1. Take chip and the surface it needs to be bonded to into the fume hood.
  2. Clean chip with duct tape and isopropanol.
  3. Put the chip and the surface into the plasma cleaner.
  4. Expose chip and surface 30 seconds to plasma.
  5. Take the chip and the surface back in the fume hood.
  6. Press the microfluidic chip against the surface.

Get full protocol here



Materials

  • Poly-D-Lysine solution 1.0 mg/mL
  • Laminin

Protocol


  1. Pour poly-D-lysine with concentration 10 &mu g/mL into the chip.
  2. Incubate over night.
  3. Pour laminine with concentration 4 &mu g/mL.
  4. Incubate for a few hours.

Get full protocol here


Microfluidics: well chip

The well chip was designed and assembled by our team. It was used to test the biocompatibility of our membranes, as well as the culture of bacteria in the presence of current. Here we show how the molds were made, how the chip itself was assembled, how well's conductivity was measured and how biofilm culture was performed on it.

PDMS Well Chip Mold Fabrication

PDMS Well Chip Fabrication




Molds were made of aluminium according to the following plans (Figure 1). Part 1 Mold's center cylinder part is detachable from the bottom to make the demolding ot PDMS easier.


PDMS Well Chip Mold Plans



Materials

  • Molds
  • Syringe without needle
  • Platinum 24mm x 2 mm strip
  • Circular 13mm diameter membrane

Protocol


  1. Prepare 15g of PDMS monomer using our protocol (Link), section 1. Replace step 5 by : Fill the syringe with PDMS. Fill part 1 mold until it's full and part 2 mold until the PDMS layer is more or less 1 cm thick.
  2. Demold the chip following our protocol (Link), section 2. Ignore step 2.
  3. Put membrane and platinum strip on PDMS part 1. Refer to figure 2 for their position.
  4. Refer to our protocol, section 3 to bond PDMS part 2 to the PDMS part prepared in the previous step. It should look like figure 3 and 4.
  5. Apply a small layer of PDMS with the syringe. Refer to figure 5 . This way, the well is watertight.
  6. Put the chip in the stove for 3 hours.

Get full protocol here


Microfluidics: microchannel chip

We used the microchannel chip to test the effect of NGF on the neuron's growth.

PDMS Microchannel Chip Mold Fabrication




We were allowed to use the molds made by Institut Curie. We were not involved in the process of their fabrication. Here is a short video we made about how these molds were created.