The overall goal of this procedure is to fabricate a reconfigurable microfluidic device with sidewalls made of an array of pins. The main advantage of this microfluidic device is its ability to deal with difficult-to-flow object and situation that may be unknown at the channel design stage. Generally, individual new to this method will struggle to etch tiny pins into dog bone shapes.
Visual demonstration of this method is critical as the assembly of many microfluidic parts and mounting them on the base are difficult to learn from only the written protocol. This photo is of the completed microfluidic device with a reconfigurable channel. Here, the reconfigurable portion is along the top of the image.
An exploded view schematic reveals some of its principle components. In particular, note the microchannel and the pins used for reconfiguring it. As a first step, etch the pins.
Begin with degreased, 0.3-millimeter-thick, L-shaped, stainless steel pins of different lengths. Have ready four milliliters of 10%nitric acid, and immerse the pins in it. Then, place the container in a 65 degree Celsius oven for 30 minutes.
After retrieving the pins, transfer them with tweezers one by one to a container of deionized water. Next, sonicate them in the water for five minutes. When done, dry them with a paper towel.
Have ready 0.5 milliliters of mold release agent, and immerse the pins in it. After two hours, place the pins in deionized water and sonicate them for five minutes. Continuing requires an etching dish.
The etching dish is constructed of glass and silicone adhesive. Begin the etching process by dispensing silicone adhesive for the pin ends on the dish. Place the pins so that the tips on their straight ends are immersed in the adhesive.
This is the dish after all the pins are in place and ready for the next step. Transfer the etching dish to a humidified fermenter heated to 38 degrees Celsius, and wait overnight to cure the adhesive. When it is ready, place the etching dish with pins on a 65 degree Celsius hotplate.
Pour 0.8 milliliters of etching acid over the uncovered region of the pins. After 10 minutes, transfer the acid to a beaker using a micropipettor. Then, add more etching acid to the dish to repeat the etching step.
After etching, check that the width of the etched region is about 0.2 millimeters. If so, micropipette five milliliters of sodium bicarbonate solution onto the etching dish to neutralize the remaining acid. Next, use a pair of tweezers to remove the pins by pulling longitudinally.
Begin with a fabricated mold. As in this schematic, the mold is for a device with a fixed sidewall and space for pins. Create the mold on a glass slide using lithography.
Place the prepared slide at the bottom of a plastic dish. Now, pour prepolymer of PDMS onto the mold to a thickness of three millimeters. Place the dish into a vacuum desiccator for 10 minutes.
After that, move it to an oven at 65 degrees Celsius for one hour. The result is a partially cured slab. Use a scalpel to demold the slab for the next steps.
Place the slab into an oven at 120 degrees Celsius for an hour to fully cure it. Now, work with the fully-cured slab. It will have irregular edges along the guideline pattern.
Use a scalpel to precisely and cleanly trim the irregular edges. Pay special attention to the surface for the pin insertion slots to create the best device. Perforate holes for inlet/outlet of the main chamber and elsewhere using biopsy punches.
Now, fabricate a microchannel assembly. Heat a cleaning solution to 60 degrees Celsius, and obtain a number four coverglass. The coverglass measures 10 millimeters by 20 millimeters.
Immerse the coverglass in the solution for 10 minutes. Placed the rinsed and dried coverglass and the PDMS slab on the lower electrode of a sputter coater. Generate air vacuum plasma for 30 seconds.
Bond the retrieved item so the edges align and the channel features are visible through the coverglass. Use a sterile container to take the bonded layers to a laminar hood to sterilize them. When done, insert the pins into the slot.
Adjacent pins should be of different lengths. The pins form the other sidewall of the microchamber. Continue to work in the laminar hood.
For the next steps, have a base ready for the device. Prepare gap filler by homogenizing a mixture of white petrolatum and polytetrafluoroethylene powder. Pour the homogenized mixture into a dispenser syringe, and insert a plunger.
Push the plunger to fill the syringe tip. Then, attach a needle, and push the plunger until the needle is filled. Prepare a second syringe with silicone adhesive in a similar manner, and connect each to a pneumatic dispenser.
Now, dispense silicone adhesive onto the base. Put it along one of the pockets. Then, place a number four coverglass on the pocket, and press it firmly to bond it.
Next, dispense silicone adhesive along the two outer slots of the base by drawing segments about one millimeter deep. Along another slot at the middle, dispense gap filler in a one-millimeter-deep segment. Put more adhesive on the edge of the other pocket.
Place a microchannel assembly on the pocket, and press it firmly. Once again, put gap filler in the slot along the middle of the base. Follow this with putting adhesive on the adjacent slots.
Let the device stand for one day in the sterile hood. After curing, in the laminar hood, move each pin up to one millimeter along its adjacent pins to release them from the elastomeric barrier. Set up a fluorescence test for leakage by diluting green fluorescent dye in ionized water.
Then, open the microchannel to have a consistent width throughout. Use a micropipette to transfer dye to one end port of the microchannel, and fill it with the solution. In a large plastic dish, place two pieces of absorbent paper wet with deionized water and the device inside.
Incubate the dish in 5%carbon dioxide for at least 24 hours at 37 degrees Celsius. View the retrieved device with an inverted fluorescent microscope, and record images. Use these to confirm there is no leakage around the pins.
These images are of a leaking and non-leaking sample. Another test involves seeding cells. Open the microchannel to make a straight 400-micron-wide channel with the sidewall as flat as possible throughout.
Add cell suspension to one end port to fill the channel. Locate the pins that define the sidewall of the region to start the cell culture, and close them to enclose the cells in the region. Starting near the region, close all of the pins in order to expel the cells in the channel.
After aspirating suspension from the end ports, add medium. Place the device in a plastic container with wet absorbent paper. Incubate it for a minimum of 24 hours.
Look for the cells to be about 10 cells per in the enclosed area. Then, slowly retract a pin to widen the culture area. These images are of COS-7 cells grown in a reconfigurable microchannel.
Over the course of nine days, the microchannel culture area was expanded. The collected data allow a plot of the cell count and the density as functions of time. The blue points and curve fit are for the cell count.
The red symbols and fits are for the density. For the density, the vertical arrows indicate expansion of the cell culture area at two, five, and six days. Densities are calculated for the same culture areas.
After watching this video, you should have a good understanding of how to build a leakage-free sidewall of a microfluidic channel out of small pins. Once mastered, this technique can be done in one week if it is performed properly. While attempting this procedure, it's important to do each one of the protocol steps carefully and thoroughly.
Don't forget that working with strong acid can be extremely hazardous, and precautions such as preparation of adequate amount of neutralizing agent should always be taken. Following these procedures, other cells with fluid can be introduced in order to establish more complex cell cultures.
