Team:NCKU Tainan/Contribution
Overview
In the process of developing our project MenTAUR, we have also attempted new experiments, designs, and methods that can inspire and benefit other iGEM teams. In our oxidative stress sensing system experiments, we collected data that prove paraquat as a more suitable inducer that more closely resembles oxidative stress in the intestine. As the inventor of the microfluidic chip, we have also listed the step-by-step development methods, experiment process, and other tips to improve its function. We hope that our efforts and contributions can assist future teams in their projects to solve the urgent problems our world currently faces today.
Oxidative Stress Assay
Hydrogen peroxide (H2O2) was changed to paraquat (PQ) as the inducer for BBa_K2610031 in the oxidative stress sensing system for the following reasons:
Paraquat: a better inducer for the oxidative stress sensing system
1. PQ produces superoxide radical (O2¯·) catalyzed by NADPH-cytochrome P450 reductase[1]. Then, O2¯· is converted into hydrogen peroxide (H2O2) by the SOD enzyme system[2] or into hydroxyl radical (OH¯·) by the HWR enzyme system[2,3]. The pathway is shown in (Fig. 1).
2. Because chronic stress-induced depression (CSID) is related to a variety of abnormal changes in oxidative stress[4], a single kind of oxidative stress molecule cannot specifically mimic the changes in the human body. Therefore, PQ was chosen as the inducer in our project to more closely simulate oxidative stress in the intestine.
3. In our oxidative stress assay, we compared the strength of the oxidative stress sensing system when induced by PQ compared to when it is induced by hydrogen peroxide (H2O2). As shown in figure 2, sfGFP expression is greater when induced by PQ .(Fig. 2)
How to Microfluidics 101
Flow Chart
Fabrication of Microfluidic Chip
1. Place the PDMS mixture inside a vacuum chamber as long as possible.
Based on our own experience with a total PDMS value of 15 gr on a circular container (diameter: 9 cm), we put the solution in the vacuum chamber for at least 30 minutes (you do not have to pay very much attention to this progress, or even better if you leave it running for an extended duration). But your mileage might vary: a more robust vacuum pump, more extensive surface area of the container used, and less solution mixed will make this progress faster.
2. Make sure the silicon wafer is clean!
It is a good idea to use high-pressure nitrogen to clean any excess particles that may exist on the silicon wafer. However, if you do not have access to high-pressure nitrogen, high pressured air can be an alternative[5]. If there is any visible excess PDMS residue from the last iteration, it is a good idea to scrape it using any sharp object such as a cutter, then blow the wafer with high-pressure nitrogen or air. Close the lid as soon as possible to prevent any foreign particles from landing on the silicon wafer.
3. Hold the PDMS container on your dominant hand, maintain a very close distance to the silicon wafer, and pour it slowly!
Based on our own experience, it is better to hold PDMS container on your dominant hand and the silicon wafer on your less dominant hand (rather than placing the wafer on top of a table); this is to maintain a better flow of PDMS solution due to its high viscosity, which can be a little tricky to control. Then, pour the mixture slowly and maintain a very close distance without touching the silicon wafer, which will reduce the chance of bubble occurrence.
4. You have to pay attention during the second vacuum chamber process!
After pouring the PDMS mixture into the silicon wafer, there will be micro air bubble formation exist on the wafer (even that you already did your best to prevent bubble development, the perfect zero bubble condition is almost impossible to be reached), you have to perform the second vacuum chamber process, and you have to pay attention in this process (take a look every 15-20 seconds).
There will be several possibilities that will happen during this process, such as :
i. The air bubble propagates up normally, as shown in Video 4. (This condition is the best case scenario)
ii. The air bubbles propagate uncontrollably, as shown in Fig. 5 (This condition can be catastrophic or a very good implication).
a. If you only fabricate one chip, then it is disastrous because you need to wipe all PDMS that had spread all over the place.
b. If you cut the entire silicon wafer, the PDMS will spread very evenly after turning off the vacuum pump.
5. Bake the PDMS solution as long as possible!
Some research states[6] baking at 80 degrees celsius for 2.5 hours is enough to cure the PDMS, and some other[7] states 75 degrees celsius is already sufficient, but from our own experience with our precision oven is set to be at 65 degrees celsius it needs at least 3 hours to cure. But, our microfluidic end product can be considered very soft and easy to break; however, if we leave the microfluidic device to be baked overnight or more than 10 hours, the end product will be more solid and harder to break.
Microfluidic Chip Assembly
1. A single deep cut is favorable!
When slicing the PDMS with a knife (Especially when you have several microfluidic on a single silicon wafer), it is perfectly fine to cut with a little more force; this will make a cleaner cut and lower the chance of failure when extracting the chip.
2. Re-slice the edges before extracting the chamber from its mold.
Even after performing the deep cut, it is an excellent habit to re-slice(lightly) all edges you had cut to ensure that the chip is already perfectly detached from the mold.
3. Attach tape on the microfluidic side before performing any other job!
After extracting the microfluidic chamber from the mold, it is a good idea to immediately attach a tape on the working side of the microfluidic channel; this is to ensure that you do not misplace or even flipped the microfluidic chamber upside down, which will render your microfluidic chamber useless.
4. Immediately create holes after tape attachment!
Punching a hole into the microfluidic channel may be an effortless task, but sometimes we forget to perform this simple task that renders our chip unusable; this is why you should directly punch a hole immediately after tape attachment.
5. 8 minutes is the sweet spot duration for the oxygen plasma chamber.
Duration of oxygen plasma can vary a lot, such as due to the type of machine being used(our plasma machine reaches 27.6 watts in high configuration), or even the manufacturer of PDMS may have a slightly different effect on the proper duration. But, according to our senior experience, 8 minutes is enough to perform this step (Fun fact, even on the guidance attached to the machine, the duration stated is 15 minutes).
6. Bake the chip as long as possible after the bonding progress!
After bonding progress, the proper baking temperature and duration are at 150 degrees Celsius for 2 hours[8]. However, even baking at 65 degrees celsius is also viable to perform the proper bonding. Still, because of the lower temperature, it is necessary to bake the chip for a longer duration, and we always bake the chip for at least 8 hours.
7. Apply the epoxy resin to a more extensive area, as big as possible.
When attaching the needle into the microfluidic as the inlet and outlet, epoxy resin is usually used to seal any possible leakage from a small gap created. Therefore, applying the epoxy resin with the biggest surface area possible is good to prevent any leakage; this is mandatory, especially when experimenting with a very high flow rate or even very viscous liquid.
Microfluidic Experiment:
Based on our own experience, our the final volume of microfluidic chamber + outflow tube of 50 microliters; we got the best retention ratio result when BSA (Bovine Serum Albumin) is injected with 1 ml/h flow rate for 6 minutes or 0.1 ml, this is due to:
1. During microfluidic assembly (glass plate and the chip) using the oxygen plasma chamber[8], the oxygen plasma itself reduced the hydrophobicity of both the channel and glass plate. However, long baking progress makes the channel turn back into hydrophobic, making the chip useless if we directly use the chip without pre-application of BSA.
2. The BSA solution that we used has a concentration of 3.85 wt% (1 gr BSA + 25 ml DI water), and BSA acts as a primer that can let the inner channel walls become hydrophilic[9].
iGEM 3-D Printed Logo
Besides fabricating microfluidic channels, we did a mini side project which is 3-D printing iGEM logo using an Epoxy-based 3-D printer. We created iGEM logo using Autocad, which you can download, and our end product can be seen in Fig. 6.
References
- Gawarammana IB, Buckley NA. Medical management of paraquat ingestion. British Journal of Clinical Pharmacology. 2011;72(5):745-757. doi:10.1111/j.1365-2125.2011.04026.x
- Castello PR, Drechsel DA, Patel M. Mitochondria Are a Major Source of Paraquat-induced Reactive Oxygen Species Production in the Brain. Journal of Biological Chemistry. 2007;282(19):14186-14193. doi:10.1074/jbc.m700827200
- Loi VV, Rossius M, Antelmann H. Redox regulation by reversible protein S-thiolation in bacteria. Frontiers in Microbiology. 2015;6. doi:10.3389/fmicb.2015.00187
- Westfall S, Caracci F, Estill M, Frolinger T, Shen L, Pasinetti GM. Chronic Stress-Induced Depression and Anxiety Priming Modulated by Gut-Brain-Axis Immunity. Frontiers in Immunology. 2021;12. doi:10.3389/fimmu.2021.670500
- Generon. Using Nitrogen Gas in the Semiconductor Manufacturing Process. Nitrogen & Gas Solutions | GENERON. Published June 30, 2020.
- Burgoyne F. Rapid curing of PDMS for microfluidic applications – Chips and Tips. Rsc.org. Published 2011.
- Surface Treatment in PDMS-Microfluidic Devices. Encyclopedia.pub. Published 2021. Accessed September 12, 2021.
- Tan SH, Nguyen N-T, Chua YC, Kang TG. Oxygen plasma treatment for reducing hydrophobicity of a sealed polydimethylsiloxane microchannel. Biomicrofluidics. 2010;4(3):032204. doi:10.1063/1.3466882
- Tv Windvoel, M. Mbanjwa, N. Mokone, A. Mogale, K. Land. Surface Analysis of Polydimethylsiloxane Fouled with Bovine Serum Albumin. undefined. Published 2017.
