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.

PDMS Chip Fabrication

PDMS Chip Demolding

PDMS Chip Bonding

PDMS Chip Treatment for Nerve Growth


Materials


  • PDMS monomer and curing agent (Sigma-Aldrich, Sylgard 184, 761036-5EA)
  • Mold (epoxy resin or aluminium)
  • Isopropanol for cleaning purposes
  • Scale (Kern PCB 1000-2)
  • Plastic beaker
  • Vacuum pump unit (Vacuubrand PC 3 RZ 2.5)
  • Vacuum bell jar (Kartell desiccator)
  • Spatula
  • Stove (Memmert UM 400) at 70 degrees Celsius
  • Paper (Kimberly-Clark SCOTT Blue)
  • Gloves (Kimtech Science PFE)

Procedure

According to the Sylgard 184 manual.

  1. Pour PDMS monomer into a beaker.
  2. Pour curing agent into the same beaker (10:1 proportion: 1g for 10g of monomer).
  3. Mix with the spatula for 30 seconds. Spatula can be cleaned afterwards with some paper dipped in isopropanol.
  4. Put beaker into the vacuum bell jar connected to the vacuum pump unit in order to extract the air bubbles from the mixture (at least 10 minutes vacuum, look out for overflowings).
  5. Pour mixture onto mold.
  6. Put mold+mixture in stove at 70 degrees Celsius for 3 hours at least.



Materials


  • Razor blade (OEMTOOLS 25181 Razor Blades, 100 Pack)
  • Biopsy puncher (Kai Biopsy Punch 4mm )

Procedure


  1. Use a razor blade to cut the borders of the chip and extract the PDMS from its mold. Avoid touching the circuits on your chip to avoid unwanted fingerprints.
  2. Drill input and output holes with the biopsy puncher.

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In some cases, before using your chip, you'll need to seal the circuitry. In order to do that, it is common to use plasma bonding to glue the chip to another surface (PDMS or glass).


Materials


  • Plasma cleaner (Diener Pico PCCE)
  • Distilled water
  • Isopropanol for cleaning purposes
  • Office duct tape
  • Fume hood (Euroclone aura vertical S.D.4)

Procedure


  1. First, the chip needs to be cleaned in the fume hood. To do so, apply duct tape onto the surface of the chip you want to bond and remove it. Clean the chip with isopropanol.
  2. Put the chip and the surface you want to bond it to into the plasma cleaner. The surfaces you want to bond need to be facing up in the machine in order to be exposed to plasma.
  3. Expose chip and surface 30 seconds to plasma.
  4. Take the chip and the surface back in the fume hood.
  5. You have 20 minutes to execute this step. Press the microfluidic chip against the surface. The surfaces that need to be glued together need to face each other. If bonding failed, repeat from step 1.



Materials


  • Poly-D-Lysine (Sigma-Aldrich, Poly-D-Lysine solution, 1.0 mg/mL ,A-003-E)
  • Laminine (Sigma-Aldrich, Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, L2020-1MG)

Procedure


  1. Pour poly-D-lysine with concentration 10 $\mu$g/mL into the chip and incubate over night.
  2. Then pour laminine with concentration 4 $\mu$g/mL and incubate for a few hours.


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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 the neuron's impulse to an electrode. The goal here is to coat alumina oxide membranes with different types of conductive polymers.

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

  • EDOT (3,4-Ethylenedioxythiophene, Sigma-Aldrich, 483028-10G)
  • PSS (Sodium 4-vinylbenzenesulfonate, Sigma-Aldrich, 94904-100G )
  • Deionised water
  • Sodium persulfate (Sigma-Aldrich, 216232-500G)
  • Iron(III) sulfate hydrate (Sigma-Aldrich, F0638-250G)
  • Alumina Oxide Membrane Filters, 0.2 micron pores, 13 mm (Sterlitech) (figure 1)
    • Figure 1: White alumina oxide membranes before coating
    Figure 1: White alumina oxide membranes
    before coating
  • Stripette (Corning Costar, 5 mL) + pipette filler
  • Analytical balance (Mettler Toledo NewClassic MF ML204 /01)
  • Magnetic stirrer with heating plate (yellowline MSH basic)
  • Fume hood (Delagrave SA OPTIMUM 1500)
  • Gloves
  • Forceps (Bochem art. 1013)
  • Glass beaker (600 mL)
  • Petri dish

Procedure


  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 (figure 2).
    4. Figure 2: Solution after 20 minutes of stirring
    Figure 2: Solution after 20 minutes
    of stirring
  4. Add 2 g of sodium persulfate and 0.015 g of iron(III) sulfate hydrate.
  5. Stir for 24 hours (figure 3).
    6. Figure 3: Solution after 24 hours of stirring
    Figure 3: Solution after 24 hours
    of stirring
  6. Wash membranes with water and let them dry at room temperature in a Petri dish. (figure 4)
    7. Figure 4: PEDOT:PSS coated alumina oxide membranes
    Figure 4: PEDOT:PSS coated
    alumina oxide membranes







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

  • EDOT (3,4-Ethylenedioxythiophene, Sigma-Aldrich, 483028-10G)
  • Iron(III) p-toluenesulfonate hexahydrate for PEDOT :Ts (Sigma-Aldrich, 462861-25G) or Iron(III) chloride for PEDOT :Cl (Fischer Scientific, 217091000)
  • 1-butanol (Sigma-Aldrich, B7906-500ML)
  • Deionised water
  • Alumina Oxide Membrane Filters, 0.2 micron pores, 13 mm (Sterlitech) (figure 1)
    • Figure 1: White alumina oxide membranes before coating
    Figure 1: White alumina oxide membranes
    before coating
  • Paper masks (figure 2)
    • Figure 2: Paper mask
    Figure 2:
    Paper mask
  • Stripette (Corning Costar, 5 mL) + pipette filler
  • Analytical balance (Mettler Toledo NewClassic MF ML204 /01)
  • Magnetic stirrer with heating plate (yellowline MSH basic)
  • Fume hood (Delagrave SA OPTIMUM 1500)
  • Gloves
  • Forceps (Bochem art. 1013)
  • Glass beaker (600 mL)
  • Petri dish

Procedure


  1. Prepare homogenous oxidant solution (1.58 g Iron(III) p-toluenesulfonate hexahydrate and 10 mL butanol for PEDOT:Ts (figure 3) or 1.35 g Iron(III) chloride and 10 mL butanol for PEDOT:Cl (figure 4)
    2. Figure 3: Oxidant solution for PEDOT:Ts
    Figure 3: Oxidant solution
    for PEDOT:Ts
    Figure 4: Oxidant solution for PEDOT:Cl
    Figure 4: Oxidant solution
    for PEDOT:Cl
  2. Dip membranes in oxydant solution.
  3. Let membranes dry at 40◦C (figure 5).
    4. Figure 5: Membranes after being dipped in oxidant solution
    Figure 5: Membranes after being dipped
    in oxidant solution
  4. Place membranes in paper masks on Petri dish lids (figure 6) .
    5. Figure 6: Membrane in paper mask
    Figure 6:
    Membrane in paper mask
  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) (figure 7).
    8. Figure 7: Vapor phase polymerization of PEDOT :Ts
    Figure 7: Vapor phase
    polymerization of PEDOT :Ts
  8. Wash membranes with butanol and water.
  9. Let membranes dry at room temperature (figures 8 and 9).
    10. Figure 8: PEDOT:Ts coated membranes
    Figure 8: PEDOT:Ts
    coated membranes
    Figure 9: PEDOT:Cl coated membranes
    Figure 9: PEDOT:Cl
    coated membranes

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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.

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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.

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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.