Team:Hainan China/Hardware

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1 What is a microfluidic chip?

The microfluidic chip is a device made up of many micro-channels and other functional structures. The channels in different types of microfluidic chips can be structured and shaped differently based on the needs for experiments and the desired features. Some features of the micro-fluidic chip can include: controlling a microenvironment, mixing reagents, and pumping certain fluids. The micro-channels vary in thickness, it can range from a few sub microns to millimeters. On top of everything, it also has the feature of allowing chemical and biological reactions and their products to be processed, visualized, and analyzed effectively^[1].

2 Microfluidics in our project

Microfluidics technology is introduced in our project for to create a coral-on-a-chip platform in which a dynamic fluid flows through the chip to provide sufficient nutrients and remove unnecessary metabolic wastes from the reservoirs along the stream lines where the coral polyps with symbiont Zooxanthellae sit. With microfluidic chip, we can also do long-term observation of zooxanthellae and coral polyps, which provides us more insights on realizing the goal of our project in real life. We designed two microfluidic chips, one is to do the long-term observation and the other is for finding the optimal nutrient concentration provided by our genetically engineered probiotics.

3 Fabrication procedure

materials: Silica, glass, elastomers, plastics, hydrogels, paper, hybrid and composite materials
1. Spin-coat a 4-inch silicon wafer with negative photoresist (SU-8 2005) and then bake the silicon wafer for a few minutes.
2. Expose the silicon wafer under a mask with an aligner. After that bake the silicon wafer on a hot plate for several minutes.
3. After letting the silicon wafer rest for few minutes at room temperature, rinse the silicon wafer with isopropyl alcohol.
4. Pattern SU-8 2050 on the second layer with the same 3 steps above for the deep channel.
5. Mix PDMS precursor and a curing agent. Then pour the mixture onto the master and it is cured at 95°C for 1 hour.
6. After a little of cooling, separate the solidified PDMS from the silicon wafer, carefully peel off the PDMS, and be careful not to damage the silicon wafer.
7. Carefully cut along the chip's outer frame with a cutting knife. Pay attention to cut it neatly.
8. Punch some holes with a punch. Pay attention to the position and size of the holes.
9. Bond the PDMS channel to a glass substrate.
10. Treat the PDMS channel with oxygen plasma for 40 seconds

4 Design of the two chips

4.1 The chip for long term cultivation

System: Our microfluidic system has three components: pump with injection poles, microfluidic chip, and a beaker to collect the metabolic wastes produced by coral polyps and zooxanthellae. The system is shown as in figure 1:

Figure 1. Microfluidic system: pump with injection poles, microfluidic chip, and a beaker (from the left to the right)

We used the pump and the injection poles to inject f/2 medium (a medium suitable for the growth of zooxanthellae) along with our probiotic bacteria into the microfluidic chip, where coral polyps tissue along with symbiotic zooxanthellae is placed.

Microfluidic chip: The microfluidic chip we designed is shown as in figure 2:

Figure 2. A photo of our microfluidic chip

From figure 2 we can see that after the the fluid was pumped into the microfluidic chip, they will flow through the channels and provide nutrients for the coral polyps and zooxanthellae.
This chip is small enough to be directly observed under the microscope.

Chambers: Single coral polyp is put into a chamber. Probiotic bacteria that we have engineered may be injected to provide VB₁₂ for the enhancement of coral symbiosis against environmental stress.

Figure 3. Working principle of the chambers

As the pumps pump in the medium along with our genetically engineered probiotics into the chip, the fluid will go flow through the channels and finally get to the chamber. The coral polyps tissue will be placed in the micro-chambers, which has a suitable size to fix the coral polyps tissue within the chamber. When the fluid arrives the chamber, the tentacles of the coral polyps can catch the nutrients.

4.2 Chip for finding the optimal nutrient concentration

System: Our micro-fluidic chip works under a system which can be illustrated as in figure 4:

Figure 4. An illustration of the micro-fluidic system

The system consists of two parts, the pump and the micro-fluidic chip. The f/2 medium along with different concentrations of nutrient (VB₁₂) is pumped into the chip, distributed into different concentrations through the channels and the zooxanthellae are cultivated in the chambers.

Micro-fluidic Chip: The chip has 9 channels(from A to I) and 20 chambers. The two-dimension illustration of the chip is shown as figure 5, it is illustrated using the software SolidWorks.

Figure 5. 2D design of the micro-fluidic chip: Alphabets A to I represent the 9 channels; C1 to C4 represent the 4 different concentrations of f/2 culture medium; W1 to W4 represent the 4 waste collectors.

Here we used S-shaped channels because fluid can be transported more fluently in S-shaped channels^[2]. We added f/2 medium of different nutrient concentrations into channel A and B. And the formation of different nutrient concentrations can be illustrated as figure 6:

Figure 6. An illustration of the principle of formation of different nutrient concentrations in the micro-fluidic chip

Standard f/2 medium means that all the nutrients are added at 1x concentration. 100x concentration means that one of the nutrients (VB₁₂ here) is added at 100x concentration, while the other nutrients are still added at 1x concentration.

The micro-fluidic chip can be divided into 2 main parts, the channels part and the chambers part. In the channel part, standard f/2 medium and f/2 medium of 100 times in VB₁₂ are added respectively from channel A and B. Then, as the fluid is distributed in along the channels, they will first form 1 time(standard), 50 times, and 100 times concentration, and they will be further distributed into 1 time, 25 times, 75 times, and 100 times concentration. Finally, these f/2 medium will flow into the chambers. Micro-chambers: We have 20 micro-chambers in total. The depth of the chambers is 1mm which makes sure that the coral polyps are fixed in the chambers while their tentacles can still get nutrients. The working principle of chambers can be illustrated as in figure 7:

Figure 7. An illustration of the chambers

After the fluid is distributed into the four different concentrations, they will arrive the chambers. In our micro-fluidic chip, we designed four parallel lines of chambers (from C1 to C4), with 5 micro-chambers on each line. Thus, 4 groups of zooxanthellae are cultivated in 4 different nutrient concentrations, and each group has 5 samples.

This design enables us to observe all the samples simultaneously. Moreover, the setting of 5 samples at each concentration make our results more universal.

Waste collectors: At the end of each line of chambers, we added waste collectors (from W1 to W4) to collect the metabolic wastes produced by the zooxanthellae. Thus, compared to traditional cultivation of zooxanthellae in conical flasks, the design of waste collectors can better mimic the actual situations in seawater as the wastes in seawater won’t stay at one spot but they will move along with the water currents.

6 Experiments and results

6.1 The chip for long term cultivation

The coral polyps and zooxanthellae were stained with fluorescent protein. After they were cultivated, we observed the chip under the microscope. We did heating on the corals and see whether the probiotics can help to retain the zooxanthellae after heating. The observation results are shown as in figure 8:

Figure 8. Coral polyps tissue and zooxanthellae under microscope

From figure 8 we can tell that when the coral polyps and zooxanthellae are cultivated at normal temperature (28°C), a large amount of zooxanthellae stay on the coral, indicating that the symbiosis is healthy. After we heated the coral polyps and zooxanthellae at 38°C for 2 hours, we found that the sample without our genetically engineered probiotics had almost no zooxanthellae on it, while the one with probiotic still had some zooxanthellae. This result clearly verifies that our probiotics can help to reinforce the symbiosis between coral polyps and zooxanthellae. Moreover, with the microfluidic system, the metabolic wastes won’t accumulate in the chip, thus we don’t need to change the medium like we did in the in vitro cultivation of zooxanthellae. Instead, we can do long-term cultivation with the microfluidic system.

Picture 1 refers to 28°C cultivation in microfluidic chip (control group); picture 2 refers to 38°C 2h heat shock in microfluidic chip; picture 3 refers to 38°C 2h heat shock and nutrition supply in microfluidic chip. Zooxanthellae was stained with red fluorescence, and the coral polyps tissue was stained with green fluorescence.

6.2 Chip for finding the optimal nutrient concentration

Unfortunately, due to limitation of time and lab usage restrictions during the pandemic, we were only able to finish the fabrication, which is shown as in figure 9:

Figure 9. A photo of the second microfluidic chip and its microfluidic system

7 Conclusion

The design of the two microfluidic chips and their systems are a good tool for long-term observation and finding optimal nutrient concentration respectively. The experiment finished with the first chip verified the success of our biotechnology. And it lays the foundation for the the realization of our project in real life in the future. In the future, we will first finish the experiments on the second microlfuidic chip to find the optimal nutrient concentration for coral polyps and zooxanthellae. Then we can analyze its results along with the results we got from the in vitro cultivation of zooxanthellae experiment.

8 Reference

[1] https://www.ufluidix.com/resources/definitions/
[2] https://www.comsol.com/microfluidics-module