Microfluidics deals with the behaviour, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale at which capillary penetration governs mass transport. This year, we designed a microfluidics-based biological application named “Christmas Tree” to be a platform of reaction to achieve concentration gradients automatically and save reacting time. Besides, we optimized the device to be detachable by by adding conical channels at the bottom of the reaction well, so that we can do parallel experiment by changing rows of well-plates with different OD values of bacteria. In addition,we can add pressure like voltage to control the velocity of the fluid and design various sizes of channel to achieve the concentration gradients we need through fluid simulation with application, ANSYS.
Fabrication of "Christmas Tree"
First Step: Photomask base plate
We connect with Nanjing Microcrystalline Electronic Technology limited liability company to manufacture the photomask base plate from chromium. The finished product is showed in Fig3.
Second Step: Lithography
The complete process is showed in Fig.4
A. Wafer pretreatment
The selected photoresist is EPG535. EPG535 is a positive photoresist, which has the advantage of mechanical properties. Good, chemical resistance, and excellent thermal stability. The substrate used in the experiment is 4 inches high purity. The single crystal silicon wafer is only polished on one side, and the thickness of the silicon wafer is 550 um. Before the lithography operation of the silicon wafer,Make sure that the polished surface of the wafer is clean, so the substrate is treated with acetone and absolute ethanol, and the ultrasonic is cleaned. The power of the washing machine is set to 100W and the cleaning time is 5min. After cleaning, the silicon wafer is blown dry with nitrogen and then placed Evaporate excess water on a hot plate to allow it to dry sufficiently. This process facilitates lithography in subsequent homogenization processes
The adhesion of the glue to the surface of the substrate prevents the occurrence of dirty spots due to the adhesion of the substrate to the photoresist.
B. Even photoresist
Using a centrifugal force to uniformly coat the photoresist dropped on the substrate covering the substrate, this method can obtain a better film. Before the homogenizing operation, the parameters of the homogenizer need to be adjusted well, because the viscosity of EPG535 is low, the speed should not be set too fast, so the parameters of the homogenizer are set to low speed (A speed) 500r / min, duration 7s; high speed (B speed) 800r / min, duration 40s. will be above. Place the dried silicon wafer on the rotating table of the homogenizer, open the vacuum pump, and gently push the silicon wafer from the side to confirm that the silicon wafer is completely adsorpted to ensure the safety of the experiment. The plastic dropper draws the entire tube of photoresist and drops from the center of the wafer to ensure photoresist coverage.
The entire wafer. Start the glue machine, after the spin coating is completed, a layer of uniform coating can be obtained on the surface of the substrate. A photoresist with good properties and appropriate thickness.
The silicon wafer completed by the above homogenization needs to be allowed to stand for a certain period of time, waiting for the photoresist to be leveled, and the silicon wafer coated with the photoresist transfer to a hot plate for pre-baking. The main function of pre-baking is to volatilize the solvent in EPG535. In addition, the adhesion of EPG535 to the substrate is effectively improved, and the tolerance in the developing solution during development is increased. The developed pattern is clean and complete. The temperature of the hot plate before the baking is set to 95°C, the duration is 5 min, the process should be in the yellow light. In the area, the film should avoid seeing light.
Exposing the pre-baked silicon wafer, placing the silicon wafer and the reticle on the lithography machine in sequence, and adjusting the connection between the two touch so that the two surfaces are in parallel contact. Turn on the UV light and set the exposure time to 8s. Ultraviolet light is irradiated through the reticle on the silicon wafer, the illuminated EPG535 chemically changes and the properties of the part change.
Remove the exposed portion of EPG535 with developer. The developer used in this article is NaOH solution. The degree is 5‰. The exposed silicon wafer was immersed in the developing solution and shaken slightly, and the development time was about 20 s. Take out the silicon wafer and put it back rinse repeatedly under flowing deionized water to remove residual developer on the surface. Complete the process to get the same pattern of the mask.
The developed substrate was baked again, which was called post-baking. The temperature of the hot plate was set to 110°C and the duration was 20 min. Post-baking, also known as hardening, removes residual developer and moisture, further enhancing the relationship between EPG535 and silicon wafers. Adhesion prevents it from falling off and prepares for subsequent reactive ion etching operations.
Third Step: Molding
Performing reactive ion etching on the silicon wafer after the above lithography operation, the etching depth is 50 um, and after etching, the result is a template that is complementary to the photodegrader flow path structure. Surface of perfluorosilane on silicon wafer with stencil pattern. Modification makes the subsequent demolding step easier to achieve. Micro-casting on the above template using PDMS to form microfluidic path.
Fourth Step: Package
Modification of the parts of the PDMS and glass substrate that require bonding before packaging.Place the glass substrate together with PDMS in a Petri dish, then place the Petri dish in etc. In the ion cleaner, be careful to have the microchannel facing up. At the end of this process, remove the Petri dish and place PDMS , remove, then attach the upward facing surface to the slide and gently press to remove the air bubbles. Then heat the plate in 100℃ for 1.5 h. The chip was removed and cooled to room temperature. At this point, the bonding is completed.
We optimize the device to be a detachable and more timesaving one by adding conical channels at the bottom of the reaction well, so that we can do parallel experiment by changing rows of well-plates with different OD values of bacteria（Fig.5). In addition,we can add pressure like voltage to control the velocity of the fluid and design various sizes of channel to achieve the concentration gradients we need through fluid simulation with applications such as ANSYS and COMSOL.
Toh, A.G.G., et al., Engineering microfluidic concentration gradient generators for biological applications. Microfluidics and Nanofluidics, 2014. 16(1-2): p. 1-18.