Team:Pasteur Paris/Experiments/Microflu

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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 and bond them to surfaces.

PDMS Chip Fabrication

PDMS Chip Demolding

PDMS Chip Bonding


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 (1 g for 10 g 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.





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 the 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.


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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, or escape the device in a real prosthesis system. The solution came as a nanoporous membrane, that would also be used as the conductive element in our system to transmit the neuron's impulses 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 (Kimtech purple nitrile)
  • 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 10 minutes of stirring
    Figure 2: Solution and membranes after 10 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 (Kimtech purple nitrile)
  • 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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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

Biofilm culture




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.


Figure 1: PDMS Well Chip Mold Plans
Figure 1:
PDMS Well Chip Mold Plans
Figure 2: PDMS Well Chip Mold
Figure 2:
PDMS Well Chip Mold



Materials

  • Molds
  • Syringe (Terumo syringe without needle, 10 mL )
  • Platinum 24 mm x 2 mm strip (mechanically flattened 24mm long 0.7mm diameter platinum wire)
  • Polycarbonate gold-coated membrane filters, 0.4 micron, 13mm diameter (Sterlitech) or polymerized membrane see protocol
Refer to sections 1 and 2 of Microfluidics: general protocols for further needed materials.

Procedure


  1. Prepare 20 g of PDMS monomer using section 1 of Microfluidics: general protocols .
    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. Keep the PDMS that is left.
  2. Demold the chip following section 2 of Microfluidics: general protocols . Ignore step 2.
  3. Put membrane and platinum strip on PDMS part 1. Refer to figure 1 for their position.
    4. Figure 1: PDMS Parts 1 with platinum strip
    Figure 1: PDMS Parts 1
    with platinum strip
  4. Refer to section 3 of Microfluidics: general protocols to bond PDMS part 2 to the PDMS part prepared in the previous step. It should look like figure 2.
    5. Figure 2: PDMS Well Chip
    Figure 2:
    PDMS Well Chip
  5. Apply a small layer of PDMS with the syringe. Refer to figure 3. This way, the well is watertight.
    6. Figure 3: PDMS Deposit Zone
    Figure 2:
    PDMS deposit
    zone in red
  6. Put the chip in the stove for 3 hours.



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

Procedure


  1. Reproduce the following electric circuit.
    2. Figure 1: Electric circuit
    Figure 1: Electric circuit
  2. Set function generator on sine, no offset, 4.5 V amplitude.
    3. Figure 2: PDMS well chip on breadboard assembly
    Figure 2: PDMS well chip on breadboard assembly



Materials

  • BL21 liquid culture, see Molecular Biology: DNA Assembly and Microbiology
  • PDMS well chip
  • Crystal violet (Thermofisher, 0.1 % in water)
  • Distilled water
  • Acetone
  • Ethanol 96%
  • P1000, P200, P20 (Gilson) + tips
  • Gloves (Kimtech PFE)
  • Biochrom WPA CO8000 Cell density meter
  • Glass jar of bleach
  • Plastic jar
  • Falcon tube 15 mL

Procedure


According to Dr Jean-Marc Ghigo.


Biofilm formation

  1. Pour 600 µL of liquid culture in the well.
  2. Incubate well at 37 degrees Celsius for 24 hours.

Well wash

  1. Discard the supernatant in microbiological waste bin. Do not pipet.
  2. Immerse well in the plastic jar with distilled water (let the water softly enter the wells).
    3. Figure 1: Immersed well
    Figure 1: Immersed well
  3. Take the well out of the water and discard water sharply over the waste container.
  4. Repeat this operation twice.
  5. Bang on blotter paper to eliminate residual water.

Crystal violet staining

  1. Add 125 µL Crystal violet in the emptied well.
  2. Wait 15 minutes for staining.
    3. Figure 2: Wells with crystal violet
    Figure 2: Wells with crystal violet
  3. Wash 3 times with distilled water as described before.
  4. Bang on blotter paper to eliminate residual water.
  5. Suspend colored biofilm by adding 150 µL ethanol/acetone solution (80 :20).
  6. Transfer 50 µL of the solution in a falcon tube and add 1.5 mL of ethanol/acetone solution (80 :20).
    7. Figure 3: Solution ready for optical density measure
    Figure 3: Solution ready for optical density measure
  7. Read optical density of 1 mL of the falcon tube's solution at 600 nm.


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Microchannel Chip

We used the microchannel chip to test the effect of NGF on neuron's growth. Institut Curie allowed us to use their chip design and their molds for our experiments. The microchannel chip is composed of two chambers connected by microchannels of 5 micrometers width and 2 micrometers height, ideal to observe neuron growth. We then proceeded to enhance the chip with a few customizations. We integrated a nanoporous membrane in the chip to prevent our bacteria to come in contact with the neurons.

PDMS Microchannel Chip Mold Fabrication

Basic Microchannel Chip Fabrication

Membrane Microchannel Chip Fabrication

Microchannel Chip Bonding

Double Membrane Microchannel Chip

Chip Sterilization




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.





Materials


  • Mold

Refer to sections 1 and 2 of Microfluidics: general protocols for further materials.


Procedure


  1. Prepare 80 g of PDMS monomer using section 1 of Microfluidics: general protocols .
  2. Demold the chip following section 2 of Microfluidics: general protocols .




The goal here is to insert a membrane in one of the chambers of the microfluidic chip in order to isolate the neuron's chamber from the bacteria's one.


Materials


  • Basic microchannel chip
  • Polycarbonate gold-coated membrane filters, 0.4 micron, 13 mm diameter (Sterlitech)
  • Razor blade (OEMTOOLS 25181 Razor Blades, 100 Pack)
  • Scissors
  • Forceps

Procedure


  1. Make a cut in the basic microchannel chip with a razor blade (see figure below). Do not cut the chip in half!
    2. Figure 1: Make a cut in the microchannel chip along the red line
    Figure 1: Make a cut in the microchannel
    chip along the red line
    Figure 2: Cut microfluidic chip with membrane inserted
    Figure 2: Cut microfluidic chip
    with membrane inserted
  2. Stretch the cut and insert the membrane using the forceps. Cut with a razor blade the exceeding part of the membrane.



Materials


  • Basic or membrane microchannel chip
  • Distilled water
  • Imaging Dish (Ibidi &mu-dish 35 mm, high glass bottom)
  • Fridge

Refer to section 3 of Microfluidics: general protocols


Procedure


  1. Bond microfluidic chip to the bottom of an imaging dish using section 3 of Microfluidics: general protocols
  2. Fill the chip with distilled water. If water leaks out of the chip, unstick it from the imaging dish and retry step 1.
  3. Store in fridge.





Here we have to insert a membrane underneath the neuron's chamber of a membrane microchannel chip, in order to be able to expose the neurons to current.


Materials


  • Membrane microchannel chip
  • Distilled water
  • Imaging Dish (Ibidi $mu-dish 35 mm, high glass bottom)
  • Syringe (Terumo syringe without needle, 10 mL )
  • Conductive silver paste (MG Chemicals 8330S-21G)
  • Wooden toothpick
  • Petri dish
  • Stove
  • Fridge

Procedure


  1. Prepare 5g of PDMS following section 1 of Microfluidics: general protocols.
  2. Prepare 1 mL of conductive silver paste following the manufacturer's instructions (Preheat parts A and B in stove at 70 degrees Celsius, put 0.5 mL of part A and 0.5 mL part B in a Petri dish, mix with the toothpick).
  3. See figure below for more information about the position of each element. Deposit a small layer of conductive silver paste on the border of the bottom of the imaging dish. Stick a piece of gold membrane cut with the scissors in the silver paste. Put 10 minutes in the stove at 70 degrees Celsius. Put another small layer of silver paste on top of the previous one.
    4. Figure 1: Bonded double membrane microchannel chip
    Figure 1: Bonded double
    membrane microchannel chip
    Figure 2: Neuron and bacteria chambers
    Figure 2: Neuron
    and bacteria chambers
  4. Bond membrane microchannel chip to the bottom of the imaging dish following section 3 of Microfluidics: general protocols. The extremity of the gold membrane piece has to be in one of the holes of the neuron's chamber (see figure 3).
  5. Deposit a small layer of PDMS on the side where the membrane sticks out of the chip. Wait 2 minutes. Fill chip with distilled water. If water leaks out, unstick the chip, discard imaging dish and restart from step 3.
  6. Store in fridge.





Unwanted living organisms in microfluidic chips can be a big deal, especially when these chips have to stay for 3 days filled with cultur medium in an incubator. The chips need to be exposed to UV rays in order to eliminate these unwanted organisms. We took extra securitiy measures, because we also needed to transport our chips from Institut Curie's lab at IPGG to Institut Pasteur.


Materials


  • Bonded (to imaging dish) and water-filled microchannel chips
  • Big Petri sish (150 mm diameter)
  • Gloves (Kimtech PFE)
  • UV curing unit (DWS)
  • Wrapfilm for food use (Ecopla France film pro)
  • Parafilm (Bemis parafilm "M")
  • Fridge


Procedure


  1. Open imaging dishes containing bonded microchannel chips and put them in the UV curing unit with their corresponding lid.
    2. Figure 1: Imaging dishes in UV curing unit
    Figure 1: Imaging dishes
    in UV curing unit
  2. Expose to UV rays for 20 minutes.
  3. With gloves, put exposed dishes in a big Petri dish.
  4. Seal Petri dish with parafilm.
  5. Cover Petri dish with 3 layers of wrapfilm.
  6. Expose 15 minutes to UV rays.
  7. Cover Petri dish with 2 additional layers of wrapfilm
  8. Store in fridge.


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Vertical Chip

The vertical chip was one of the designs for the support of the final experiment, that would serve as a proof of concept. The idea is to isolate the modified bacteria from the neurons using a membrane. To simplify the membrane's integration in a PDMS chip, a vertical design instead of a microchannel horizontal one was proposed.

PDMS Vertical Chip Mold Fabrication

PDMS Vertical Chip Fabrication

PDMS Chip Sterilization



The mold was made of aluminium according to the following blueprint (figure 1).



Figure 1: Vertical chip mold blueprint
Figure 1: Vertical chip mold blueprint
Figure 2: Vertical chip mold
Figure 2: Vertical chip mold



Materials


  • Molds
  • Polycarbonate gold-coated membrane filters, 0.4 micron, 13mm diameter (Sterlitech) or polymerized membrane ( see protocol )
  • Biopsy puncher (Kai biopsy puncher 2 mm and 4 mm)
  • Conductive silver paste (MG Chemicals 8330S-21G)
  • Tissue culture dish (TPP 93060, 53 mm internal diameter) Refer to Microfluidics: general protocols for further materials.

Procedure


  1. Prepare 15 g of PDMS monomer using section 1 of Microfluidics: general protocols .
  2. Demold the PDMS layer following section 2 of Microfluidics: general protocols . Ignore step 2.
  3. Cut PDMS layer in two halves.
  4. Drill a hole in the center of each half with a 4 mm biopsy puncher. Drill holes in one of the two layer with a 2 mm biopsy puncher (see figure 1).
    5. Figure 1: Two halves of PDMS layer. Left : upper layer. Right : bottom layer
    Figure 1: Two halves of PDMS layer.
    Left : upper layer. Right : bottom layer
  5. Prepare 2 mL of conductive silver paste following the manufacturer's instructions (Preheat parts A and B in the stove at 70 degrees Celsius, put 0.5 mL of part A and 0.5 mL part B in a Petri dish, mix with the toothpick).
  6. Apply a path of silver paste on bottom layer starting from the center hole and going outwards of the layer (figure 2). Deposit one half of a membrane filter on center hole. Put in the stove at 70 degrees Celsius for 2 hours.
    7. Figure 2: Assembled vertical chip with gold-coated membrane filter
    Figure 2: Assembled vertical chip
    with gold-coated membrane filter
  7. Bond the two layers together (figure 3) according to section 3 of Microfluidics: general protocols .
  8. Take the bottom of a tissue culture dish and deposit the other half of the membrane filter in the center. Apply silver paste and put in the stove at 70 degrees Celsius for 2 hours (figure 3)
    9. Figure 3: Tissue culture dish with membrane and silver paste
    Figure 3: Bonded layers
  9. Bond prepared tissue culture dish with product of step 7 (figure 4), bottom layer (figure 1) facing the dish, refering to section 3 of Microfluidics: general protocols .
    10. Figure 4: Bonded vertical chip
    Figure 4: Bonded vertical chip



Materials


  • Bonded chip, product of section 2
  • Big Petri dish (150 mm diameter)
  • Gloves (Kimtech PFE)
  • UV curing unit (DWS)
  • Wrapfilm for food use (Ecopla France film pro)
  • Parafilm (Bemis parafilm "M")
  • Fridge

Procedure


  1. Open dishes containing bonded chips and put them in the UV curing unit with their corresponding lid.
  2. Expose to UV rays for 20 minutes.
  3. With gloves, put exposed dishes in a big Petri dish.
  4. Seal Petri dish with parafilm.
  5. Cover Petri dish with 3 layers of wrapfilm.
  6. Expose 15 minutes to UV rays.
  7. Cover Petri dish with 2 additional layers of wrapfilm.
  8. Store in fridge.


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