This method can help address an important challenge in combining microscopy with microfluidics by enabling refractive index matching between the microchannel structure and aqueous media inside the channel. The main advantage of this technique is that it is compatible with fabrication methods commonly used to create microfluidic devices. Another key advantage of our approach is method used for sealing microchannels made of materials with low adhesion, which is a common problem among well refractive index polymers.
Visual demonstration of this method is crucial, as the steps to seal the channel can be difficult to get right. For example, it is important that reservoirs are unobstructed during clamping. Begin this procedure with fabrication of polydimethylsiloxane, or PDMS, negative, as described in the text protocol.
Then, fill the PDMS negative with 400 microliters of MY-133-V-2000. The negative must be slightly overfilled. Place the MY-133-V-2000 filled PDMS negative into the vacuum chamber for two hours to remove any bubbles.
After removing the MY-133-V-2000 from the vacuum chamber, press a glass slide against the top of the slightly overfilled negative to create a flat surface of MY-133-V-2000 and to prevent oxygen from inhibiting polymerization. Next, insert the MY-133-V-2000 into a 400 watt UV oven. Set the UV radiation to 50%of the maximum intensity for 300 seconds to cure the microchannel.
This is a power of approximately 4.5 joules per square centimeter, roughly double the minimum curing power recommended by the manufacturer. After cutting the acrylic as described in the text protocol, place two small drops on the edge of the acrylic and then use a disposable tool to evenly spread the glue. Place the glass substrate onto the acrylic and allow the glue to dry using the weight of the glass to hold it in place.
Coat the exposed glass with 100 microliters of PDMS using a positive displacement pipette. Then, insert the base layer into a vacuum spin coater to evenly coat the glass with a PDMS film approximately 10 micrometers thick using a speed of 1500 RPM for two minutes. Remove the base layer from the spin coater and carefully wipe away any excess PDMS that coated the acrylic with acetone and paper towel.
Be careful not to disturb the uncured PDMS. Now, bake the base layer with the PDMS in an oven at 65 degrees Celsius for two hours to cure the PDMS. Pipette one milliliter of PDMS onto a glass slide and then use another glass slide to evenly spread the PDMS until it starts to ooze out the sides.
Cure this on a hot plate at 150 degrees Celsius for 10 minutes. Cut the cured PDMS into a rectangle with the same dimensions as the MY-133-V-2000 device. Then, using the midlayer of the acrylic as a mold, punch holes for the reservoir and cut a square viewing window in the PDMS to make the PDMS gasket.
Now, remove the cured MY-133-V-2000 from the UV oven and remove the channel from the negative and place it onto a flat substrate with the channel side facing up. Remove the base layer containing the cured PDMS from the 65 degree oven and place it onto the substrate. Then, place the substrate with both the MY-133-V-2000 channel and the base layer into the oxygen plasma cleaner.
Set the vacuum pressure to 200 millitorr and the radio frequency level to high. Proceed to surface treat the channel and glass substrate for 30 seconds. The oxygen plasma should glow blue when the plasma cleaner is switched on.
After 30 seconds, remove both the microchannel and the base layer from the plasma cleaner. Using forceps, immediately place the MY-133-V-2000 channel side down in the rectangular cutout of the base layer acrylic, such that it contacts the PDMS. Now, place the PDMS gasket on top of the MY-133-V-2000 device, lining up the holes in the gasket with the reservoirs in the device.
The use of this PDMS gasket is crucial because PDMS is more elastic than MY-133-V-2000 and can therefore be compressed more. This allows the force to be transferred from the acrylic to the microchannel without breaking the glass. At this point, place the unfinished device on a raised platform above the bench to provide a clamping surface for device assembly.
Then, extract three milliliters of acrylic cement using a syringe. Distribute enough acrylic cement on top of the base layer to provide a thin coating. Make the coat as even as possible and do not let the material seep into the channel.
Now, place the mid layer acrylic piece on top of the base layer acrylic. Make sure that the holes line up with the reservoirs of the MY-133-V-2000 channel. Clamp the mid layer down onto the base layer as tightly as possible and hold it for two minutes while the cement hardens to bond the pieces of acrylic together.
Ensure that the reservoirs are not obstructed during this step to prevent air from being trapped underneath the MY-133-V-2000 device. Air trapped between the MY-133-V-2000 and the substrate will remain trapped if the reservoirs are obstructed. This will cause leaks in the finished device.
Therefore, it is critical that the holes are not obstructed during this step. After the cement hardens, remove the pressure from the device. Place the acrylic cement on the midlayer in the locations of anticipated contact with the top layer of the acrylic.
Now, place the top layer of the acrylic on the midlayer piece of the acrylic, ensuring that the holes are aligned with the reservoirs. Allow it to rest on the bench for two minutes while the acrylic cement dries. Test the MY-133-V-2000 device by adding 10 microliters of food dye or deionized water into one of the reservoir holes to test for the adhesion and flow.
Insert a tube into the opposite reservoir and connect the other end to a vacuum trap. Turn the vacuum on to pull the dye or water through the channel to ensure that a successful device was created. Check under a microscope to verify that there are no leaks in the channel or reservoir.
Using the vacuum, remove the dye from both reservoirs. Then, rinse the reservoirs with ethanol. Place the ethanol in one reservoir.
Pull the ethanol through using the vacuum. Allow the ethanol to sit at room temperature for 10 minutes. Spray the channel thoroughly with ethanol and put it into a sterile polystyrene dish.
Wrap it tightly with Parafilm and store until needed. The heat map shown here represents the mass of FOD adherent MCF-7 cells in a microchannel. Few artifacts are visible near the walls of the microchannel.
In this measurement, a single aderent MCF-7 cell can be seen on the edge of the microchannel next to the wall. The lack of artifacts near the wall allows precise quantitative measurements to be obtained, even when cells are in close proximity to the channel wall. This final figure shows a pair of MCF-7 cells seated near the microchannel wall.
The mass of these cells at each pixel can be precisely measured using quantitative phase microscopy. While following this protocol, it's important not to obstruct the reservoirs when clamping the device while the acrylic cement dries. Obstructing the holes traps air between the MY-133-V-2000 and the substrate.
This will cause leaks in the channel during flow. The use of MY-133-V-2000 as a fabrication material for microfluidic devices provides a distinct advantage over traditional materials by greatly reducing artifacts near microchannel walls. This allows quantitative measurements to be made in close proximity to microchannel structures.
Devices made with this protocol are compatible with any commonly used microscopy technique. This protocol is based on cell pathography so it's compatible with other advanced microfluidic techniques such as cell sorters or gradient generators for studying cell behavior. Overall, this technique arms researchers with a new tool for biomedical microfluidic assays based on microscopy.
The extra precision it grants to quantitative measurements in microchannels has important applications in many fields, including drug discovery and single cell analysis.
