Microfluidics offers cell biologists, the technology to do high throughput experimentation where precise fluid handling is required. Hi, I'm Nin Chen Lee from Folk Lab at the Department of Bioengineering, university of Washington. Today I'm going to show you how to make microfluidic tips controlled by PDMS Micro valve array.
The device consists of three layers. First layer is the fluidic layer contains micro chambers in different sizes, and second layer is the control layer contains channels in between the two layers. There is a thin PMS membrane due to the hydrophobicity and compliance of PMS.
The membrane seals against its seed, so isolate the fluid chambers from each other. If we apply vacuums through the control channels, the PDMS membrane can then deflect and link the previously isolated fluid chambers. Today I'm going to show you a parallel mixer that allows to mix sub nanoliter volumes of aqui solutions at different mixing ratios.
And then crisp step from our lab is going to show you an integrated microfluidic system that allows to profuse multiple solutions into cell cultures. Let's get started. Here I show you a master with wrist features of A SUH on top of a silicon wafer.
The master was fabricated from standard SUH photography procedures from this masters. We can make PDMS replicas over and over again. To facilitate the release of PDMS from masters, we need to first ize the master.
We normally ize master using F foods because we are working with Florence Island, I first put the wafer into the ator and put the droplets of the forest and then close the desk skate chamber, turn on the vacuum, leave the vacuum for 1, 2, 3 minutes and then close the vacuum. Let the evaporate for half hour and then take the wave drop. Before replica MOD PDMS, we need to first pre-mix PDMS pre polymer and curing agents at 10 to one ratio.
So I weigh a pre polymer of 31 grams and then weigh the curing agents for 3.1 grams and mix them thoroughly for five minutes. The finished product looked at this. After that I put into a density heat to debub the PS for five to 10 minutes until it gets clear.
While waiting for PMS to debub, I can glue silicone tubings onto the region of the control layer. We choose silicone tubing because it's the same component of PMS, so later it can be embedded into the device and create a airtight and fluid type seal. Now I add a little bit of glue D cement onto the tip of the small piece of silicone tubing and press it onto the inlet area of the master.
So in order for the tubings not to far over the amps of those silicone tubing have to be very flat when you cut and you don't put too much glue on top, top of it, Explain just how you're pressing The glue. So let's see. Regions are created on both vent and take lid off.
Now the PS has been developed. I will pour PS on top of the master. Be careful to pull PGAs surrounding the tubings.
Now I'm pouring onto the other master which has the fluid layer features. After pouring PS to the masters, they have to be debub again in the dust kicker for five to 10 minutes. After bubbling, we cure PMS in oven at 65 to 70 degrees from one hour to 24 hours.
Okay, great. After two hours, the PMS has been cured. Now we cut the PMS device from the SU master and period of for the control layer.
With the tubings mode in, we remove the glue from the inlet areas. In our devices. Inlet regions are created on both three layers and control layers, but we only mode the silicone tubing onto one layer.
For example, here only on the control layer. To create access to the fluid layer, we can puncture the membrane underneath the silicone tubings to create access. So this we, we can access all those device from the top so it's easier to do mic microscopy onto stereoscope and the traditional inverted Microscope.
Now we are going into the C room. To prepare CTM S membranes, I'm going to show you to spin the PDFs membrane using the headway spinner. Before I put the wafer onto the top of the wafer check, I covered the ball in the plastic graph and place the paper towel underneath for easy cleaning of clean mess afterwards.
So if it has been finalized, just like what we did with the masters before, after cleaning on the vacuum, I dispense about two milliliters of the PMS taxi next on top of the silicon wafer because we want to have a 11 to 12 micron six membrane, the wafer will be spinned at 7, 000 RTM for 20 seconds it starts And now I would say something like, so here you can see the, the wafer spinning at. We will spin it for, for however long After spinning, we put the fer on the hot plate at 85 degrees For four minutes. Now the PDMS membrane has been cured.
We next oxidize the P DM S membrane and the control layer in a plasma oven. We affect the platinum power at 75%and use the ox oxygen pressure of 30 PSI and flu rate of five. Those parameters can always be adjusted according to different applications.
Turn on the tine, turn on the plasma for 30 seconds. Now place the control layer on top of the membrane. Bring them in contact in a couple minutes the control layer will be bonded to the membrane.
After a few minutes, remove the control layer with the membrane. Okay, now we are ready to align the Control layer to the, to the fully clear. Since we are not in the clean room anymore, we use the scotch tip to remove the dust on the fully clear.
We also need to remove membrane from the inlet area of the control layers. This is to make access to the underneath fluid layer. So put the control layer with the membrane on top of the fluid layer.
Look on all the chamber fluid chambers and the valves. Make sure they're all aligned from left to right. If it's not aligned, you can remove the control layer and redo it.
Here you can see the fluid layer and control Layer are aligned. Now I will insert some Thinner tubings into these inlets to make it connects to fluid sources and the pressure sources. Now connecting the valves to the pressure source and connect the fluid inlets to the fluid source.
Here we have two different dyes, one blue and one yellow to open and close the PDMS micro valves. We use solenoid valves connected to a vacuum source and air pressure force and valves are controlled by light view software. Now I'm opening the valve set number one.
We can see the PDMS membranes deflect and the valves are open. Now I'm closing the first first set of the valves close and open the second set open and Close. Now all the Micro valves are working.
I'm going to fill the micro fluidic chambers with two different dice. I will open valve set number one and also using a vacuum to pull the two dice into the chambers. After the chambers are filled with dice, I close valve set number one to isolate each chambers and then turn on the valve set.
Number two, to mix the pairs in the two arrays. Valve open and mixing mix generally take about from one to two minutes. Since we designed chamber sizes in 10 different sizes.
So we have a mixing ratio of 11 different ratios. So after mixing is finished, I close the valve and so you can see each individual chambers. There are different mixing ratios.
The color turned from blue to green to more to yellow. Once fabricated, those devices can potentially be used in biomedical essay such as drug screening or cell biology studies such as chemotaxis or cell response to different concentrations of chemicals and growth Factors or drugs. I'm Chris Sip and I'm gonna demonstrate a device that is an integrated microfluidic perfusion system and it's very similar to the device previously seen in fabrication.
The only, the main difference is that instead of having separate chambers separated by by a valve that mix via diffusion, we have multiple inlets that converge and are controlled by valves, a multiplex valving scheme. And what I'm gonna demonstrate is the selective actuation of valves, which will control different inlets on or off. In addition, I'll show the operation of an integrated mixing channel and steering of gradients.
On the top of the screen you see 16 inlets that are converging and flow is gonna come from the top down through this vertical channel and into this bifurcation network, which is the cell culture chamber. So now we're seeing the inlets of switch to a blue colored dye, which is flowing and you can see the laminar flow profile as it goes through the chamber. Now we're doing combination of blue and yellow and then we can switch this to create the opposite combination.
We can steer our gradient to the right side. We can create other types of gradients. Now this is showing the rapid switching of valves so we can alternate pretty quickly between different solutions.
Now we're feeding blue and yellow through this homogenizer and the solution comes out green in the chamber and we can also switch any number of different inlets. In this case we're we're feeding a red inlet through, which is taking the place of all the green solution. Today I have just showing you how to make micro footed valve based devices and demonstrated mixing of two color dyes in different ratios as sub liter volumes crisp.
Chris from our lab also demonstrated an integrated microfluidic systems for profusion of different solutions. Thank you for watching and good luck with your own experiment.
