Team:Pasteur Paris/Experiments/Microflu

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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: membrane filters

Soon enough we realized that we would need something to confine the bacteria, so that it doesn't attack the neurons during our experiments. The solution came as a nanoporous membrane, that would also be used as the conductive element in our system to transmit to the neuron's impulse.

Membrane PEDOT:PSS coating

Membrane PEDOT:Ts and PEDOT:Cl coating




An aqueous solution of PEDOT :PSS can be prepared [1]. We decided to dip the membranes in this solution during the polymerization.


[1] Jikui Wang, Guofeng Cai, Xudong Zhu, Xiaping Zhou, Oxidative Chemical Polymerization of 3,4-Ethylenedioxythiophene and its Applications in Antistatic coatings, Journal of Applied Polymer Science, 2012, Vol. 124, 109-115 .


Materials

  • 3,4-Ethylenedioxythiophene
  • Sodium 4-vinylbenzenesulfonate
  • Deionised water
  • Sodium persulfate
  • Iron(III) sulfate hydrate
  • Alumina Oxide Membrane Filters

Protocol


  1. Pour 0.8 g EDOT, 2g PSS and 208 mL water in the glass beaker.
  2. Put the membranes in the solution.
  3. Stir for 10 minutes.
  4. Add 2 g of sodium persulfate and 0.015 g of iron(III) sulfate hydrate.
  5. Stir for 24 hours.
  6. Wash membranes with water and let them dry at room temperature in a Petri dish.

Get full protocol here





PEDOT :Ts and PEDOT :Cl polymers can be obtained by vapor phase polymerization on alumina oxide membranes [1].


[1] Alexis E. Abelow, Kristin M. Persson, Edwin W.H. Jager, Magnus Berggren, Ilya Zharov, Electroresponsive Nanoporous Membranes by Coating Anodized Alumina with Poly(3,4ethylenedioxythiophene) and Polypyrrole. 2014, 299, 190-197.


Materials

  • 3,4-Ethylenedioxythiophene
  • Iron(III) p-toluenesulfonate hexahydrate for PEDOT :Ts or Iron(III) chloride for PEDOT :Cl
  • 1-butanol
  • Sodium persulfate
  • Iron(III) sulfate hydrate
  • Paper masks

Protocol


  1. Prepare homogenous oxidant solution (1.58 g Iron(III) p-toluenesulfonate hexahydrate and 10 mL butanol for PEDOT:Ts or 1.35 g Iron(III) chloride and 10 mL butanol for PEDOT:Cl)
  2. Dip membranes in oxydant solution.
  3. Let membranes dry at 40◦C.
  4. Place membranes in paper masks on Petri dish lids.
  5. Pour 200 µL EDOT in 50 mL beakers.
  6. Place Petri dish lids on top of the 50 mL beakers, membranes facing the inside of the beakers.
  7. Heat the beakers at 40◦C and stop when membranes darken (takes about 6 minutes).
  8. Wash membranes with butanol and water.
  9. Let membranes dry at room temperature.

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

PDMS Well Chip Conductivity Measurement




Molds were made of aluminium according to the following plans. 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, 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, section 2. Ignore step 2.
  3. Put membrane and platinum strip on PDMS part 1.
  4. Refer to our protocol, section 3 to bond PDMS part 2 to the PDMS part prepared in the previous step.
  5. Apply a small layer of PDMS with the syringe on the contact zone of the PDMS part 2 and the platinum strip.
  6. Put the chip in the stove for 3 hours.

Get full protocol here



Materials

  • Oscilloscope
  • Function generator
  • Solderless breadboard assembly
  • Electric wires with banana connectors
  • Coaxial cable
  • Male BCN to 2 female banana connectors converter
  • BNC Splitter
  • 1 kOhm resistor

Protocol


  1. Reproduce the following electric circuit.

    2. Electric circuit 1

  2. Set function generator on sine, no offset, 4.5 V amplitude.
  3. Measure Y2 peak-to-peak amplitude and Y2's phase relative to Y1.

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.