This work was supported by the University of Utah office of the Vice President for Research, as well as by funds in conjunction with grant P30 CA042014 awarded to the Huntsman Cancer Institute and to the CRR Program at the Huntsman Cancer Institute.

1. Fabrication of the Polydimethylsiloxane Negative
<ol>
	<li>Preparation of polydimethylsiloxane
	<ol>
		<li>Measure 18 g of PDMS silicone elastomer and 1.8 g of the curing reagent. Pour the curing reagent into a measuring boat containing the elastomer.</li>
		<li>Mix the elastomer and the curing reagent vigorously for 1 min and put the mixture into a vacuum chamber for 30 min.</li>
		<li>Remove the PDMS from the vacuum, pour 15 g onto the negative using a cookie cutter (radius = 3.8 cm) to keep the P...

<ol>
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MY133-V2000 can be used as an alternative to traditional soft lithography fabrication materials such as PDMS. Previous work has shown that materials with a high index of refraction, such as PDMS, introduce significant artifacts near the channel walls due to the mismatching indices of refraction between the fabrication material and the aqueous solution inside the channel13. MY133-V2000 enables matching the refractive index of the microfluidic device to the aqueous solutions commonly used in biomedi...

The development of microfluidic technology has enabled a wide range of new biomedical techniques that leverage the unique physics of microscopic-scale flows1,2. This includes the diagnostic techniques built on microfluidic platforms that quantify clinically relevant biomarkers, including cell stiffness3, surface markers4, and growth5. By manipulating single cells, microfluidic devices can also be used to measure biomarker heterogeneity, for example as an indicator of malignancy6. The ability to combine microfluidic applications with microscopy has further increased the utility of these platforms by allowing for devices that measure multiple biomarkers simultaneously7.
QPM is a microscopy technique that measures the phase shift as light passes through and interacts with the matter inside transparent samples. The mass of individual cells can be calculated from QPM measurements, by using the known relationship between the refractive index and the biomass density8,9. Previous work has shown that QPM is capable of measuring clinically relevant parameters such as cell growth10,11 and cell mechanical properties via disorder strength12. When combined with microfluidics, QPM can potentially be used to measure cell behavior in a highly controlled environment in vitro. One of the primary challenges facing combining QPM with microfluidics is the high refractive index of most polymers used to construct microfluidic channels via soft lithography13.
An important challenge facing the combination of microfluidics with various microscopy techniques is the mismatch between the refractive index of the device material relative to the refractive index of water14,15. One method to address this is through the use of a low refractive index polymer such as CYTOP16 or MY133-V200013. The latter is a fluorinated ultraviolet (UV)-curable acrylate polymer that has a refractive index similar to water (n = 1.33) and that is compatible with soft lithography techniques, allowing for a smooth integration into many established microfluidic device fabrication workflows. This makes MY133-V2000 not only suitable for microfluidic device fabrication, but also allows it to be readily combined with QPM and other microscopy approaches, to measure cell behavior both at the colony and on a single-cell scale. MY133-V2000 eliminates artifacts due to phase unwrapping by producing little, if any, phase shift as light passes through the water-MY133 interface.
Although eliminating the mismatch in refractive index, one major challenge associated with the devices fabricated from fluorinated polymers, such as MY133-V2000, is the low adherence to other materials such as glass or PDMS. The present work demonstrates the fabrication of an MY133-V2000 microfluidic device using soft lithography. Using O2 plasma to treat the surface of both the channel and the PDMS substrate combined with a custom-fabricated acrylic holder ensures that the device adheres to the substrate, creating a sealed channel. This device is suitable for cell culture and QPM to measure the mass of cells in the channel, which has important applications for measuring the growth of live cells and the intracellular transport of cell biomass, both of which have clinical relevance in diagnostic medicine and drug discovery.

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fabrication of refractive-index-matched devices for biomedical microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of MY133-V2000, a fluorinated polymer with a low refractive index matching that of water. A key feature of this protocol is how to overcome the lack of adhesion that is characteristic of fluorinated polymers by using oxygen plasma and by fabricating the device within an acrylic holder to provide the extra mechanical force required to seal the channel against the PDMS substrate (Figure 1). The low refractive index of the...

Watch this Scientific Journal Video about Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics at JoVE.com

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...

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.

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7.3K vistas.  University of Utah. 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 refra...