Difference between revisions of "Assignment 8, Part 2: fabricate a microfluidic device"

Revision as of 15:31, 30 October 2019

Overview

In this part of the assignment, you will make a microfluidic device out of double-sided tape sandwiched between a glass coverslip and a slab of PDMS. This procedure is incredibly simple, as far as microfluidics go, and was developed in Paul Blainey's lab to image the motion of transport molecules along DNA ^[1].

PDMS is a silicone elastomer made by mixing together a viscous liquid base with a crossliking agent. Once mixed and annealed at 60°C, the material will harden into a solid, rubber-like material that is optically clear, non-toxic, and chemically inert. Researchers typically use PDMS for microfluidics because they can use it to cast very small sharp features (down to ~1 micron), and they can covalently bond the PDMS to a glass coverslip, creating a sealed device that is readily compatible with most types of microscopy.

Since our experiment does not require extremely small features, we will simply cut the Y-shaped channel out of a piece of double-sided tape using a laser cutter, rather than casting PDMS over a mold. We're still going to take advantage of PDMS's properties, though. First, it will provide an optically clear structure for our device, making it compatible for microscopy, and second, because it is somewhat soft and flexible, we can easily punch holes in it (which is hard to do with something like glass) in order to connect our flow channel to inlet and outlet tubing.

Cast a slab of PDMS

Two notes before you begin:

• This protocol has two steps with 15-60 minute wait times. Part 2 of this assignment can be completed in parallel with Part 1, if you want to keep making progress during the downtime.
• PDMS is not harmful, but it is viscous and sticky. Work over a sheet of aluminum foil rather than directly on the work bench, wear gloves when pouring the elastomer base and crosslinking agent, and change them before touching other lab equipment. If you spill any PDMS, clean it up right away with a paper towel or kimwipe.

Onward!

1. Turn on the oven to 60°C (if it isn't on already).
2. Using the scale, measure 31.5 g of the Sylgard™ 184 Silicone Elastomer base into a paper cup.
3. Pour in 3.5 g of the curing agent to total 35 g. Note that the curing agent is much less viscous than the base, so pour extra carefully so you don't add too much.
4. Mix the elastomer base and curing agent REALLY WELL (i.e. for at least 2 minutes) using a plastic stirrer.
5. Pour the mixture into a square petri dish.
6. Degas the PDMS for 15 minutes using the vacuum desiccator. Make sure the red T-valve is closed before turning on the vacuum.
7. After 15 minutes, turn off the vacuum to the desiccator, and slowly vent the chamber using the red T-valve.
8. Remove any remaining bubbles with a plastic stirrer.
9. Place the petri dish in the 60°C oven for 1 hour.

The procedure can be stopped at this point and you may store the cured PDMS in the petri dish if you do not have time to complete the next steps in the procedure right away.

Assemble your device

Microfluidic device assembly.

• Work on a cutting mat, and not the bench, when punching holes and cutting tubing.

1. Pick up a rectangle of pre-cut double-sided tape and inspect it to make sure that the channels are clear of excess tape and debris. (Do not yet remove the clear backing of the tape!)
2. Cut out a slab of PDMS to be the same size as the tape. Leave it in the dish until you are ready to use it, to prevent dust accumulation.
   • You should be able to cut at least 6 devices out of a single petri dish. You will need to make more of the same devices in future assignments, so plan accordingly.
3. Use tweezers to peel off the clear backing from ONE side of the tape. Remove the cut PDMS slab from the petri dish, and stick the flat side (bottom) onto the green tape. Press everywhere to ensure a full seal.
4. Use a 1 mm biopsy punch to make holes for the two inlets, and a 0.5 mm biopsy punch to make the outlet hole.
   • Punch from the bottom side to avoid contaminating the fluidic channels with dust from the table.
   • After inserting the biopsy punch, remove the small core that it creates before extracting the punch from the PDMS.
5. Peel off the clear backing from the other side of the tape and seal the flow channel to a 22x40 mm glass coverslip. Apply pressure to the PDMS, rather than the coverslip, to prevent cracking of the coverslip.

Your device is now assembled!

Device Fabrication

Cast PDMS

1. If not already on, turn the oven on to heat to 60°C.
2. Pour 9 g PDMS base into a plastic cup.
3. Carefully pour 1 g of the crosslinker into the same cup, being careful to note that it is far less viscous than the base.
4. Use a plastic stirrer to mix the crosslinker and base together really well. Stir for at least 2 minutes.
5. Pour the mixed PDMS into a 3D printed master mold. The mold will make 3 devices.
6. Degas the poured PDMS in the vacuum desiccator for at least 15 minutes, or until all bubbles are removed.
7. Bake at 60°C for 1 hour.

Unmold the cured PDMS and punch inlet and outlet holes

1. Using a ceramic blade, carefully cut around the inner edge of the 3d printed mold to separate it from the PDMS. Gently pry the PDMS away from the walls with the blade and carefully peel it off of the 3D printed mold.
2. Place the cured PDMS with the molded channels facing up on a clean cutting mat.
3. Use the 1 mm biopsy punch to make the two inlet and one outlet holes at each end of the Y-shape. Each time you push the punch through the PDMS, make sure to remove the core.
4. Inspect the device to ensure that each hole is cleanly formed.
5. Store the PDMS in a clean petri dish with lid on to prevent too much dust from sticking to the surface.
6. Repeat the punching steps for your remaining two devices.

Seal flow channels with a glass cover slip

1. On a clean, clutter free section of the bench, lay out a 22 x 40 mm glass coverslip and the molded PDMS device (channel side up).
2. With supervision from an instructor, turn on the corona generator and pass it back and forth over the PDMS and coverslip for about 30 s.
3. Turn off the corona generator, then invert the PDMS onto the coverslip, trying to align the edges of the glass and PDMS as best you can.
4. Press down firmly onto the PDMS to form a seal with the coverslip.
5. Repeat with remaining PDMS devices
6. Place sealed devices in a 60°C oven for 5 minutes.
7. If possible, leave the devices overnight before use to ensure the best seal.

Connect tubing

Inlet and outlet tubing.

Using a scalpel, cut the following lengths of tubing as marked on their packages. A summary of the tubing needed is below. Cutting the tubing on an angle will make it easier to sleeve together.

Collect the following:

1. For each inlet, sleeve inlet tubing #1 into inlet tubing #2. Insert a luer-to-barbed fitting into the open end of inlet tubing #2.
2. Sleeve a third luer-to-barbed fitting into one end of the long outlet tubing.
3. Insert a bent pink needle into each of the PDMS inlet and outlet holes.
4. Twist on the barbed fitting of the inlet tubing to the inlet needles, and set the outlet tubing aside.

Your device is now ready! Keep all your parts stored in a clean petri dish with your microscope, until you are ready to take flow data in Part 3.

References

1. ↑ | K. Xiong and P. C. Blainey, “A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA,” J. Vis. Exp., no. 128, pp. 1–7, 2017

Navigation

• Overview
• Part 1: feedback systems
• Part 2: fabricate a microfluidic device
• Part 3: add flow control and test your device

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