Team:SUSTech Shenzhen/Hardware/Microfluidics

Team SUSTC-Shenzhen

Microfluidics

Create for wisdom of Life


1 The Selection Chip

Most researches about Caenorhabditis elegans show that inserted genes can express well in worms at L4 stages. Thus, we need to select appropriate stages of worms to get the best experimental results. The simple method is to distinguish them by sizes, because worms in L4 stage have medium sizes. There are two plans of selecting worms. The first one is using microfluidics. With the flow contained worms going through this chip, only the medium sized worms can remain in the medium chamber, and we could collect them by injecting the flow from the bottom and gather them in the top. (Fig. 1)

T--SUSTech Shenzhen--Microfluidics--fig1.png
Fig. 1 the selective chip after injecting worms. A) The whole worms are injected into microfluidics. B) Medium and small sized worms pass through the left chambers. Small sized worms go through the medium chambers, remaining medium sized worms. get them by injecting the flow from the bottom entry to the top exist.

The second plan is making the grows of worms synchronously, which is utilized to get a large number of worms at the same stage (链接到微流控protocol). After collecting embryos (Fig. 2) by bleaching adults, we culture them and get a large number of worms at the same stages after three days. our synchronous rate is calculated as the formula below.

\frac{(the\,number\,of\, the\,worms\,at\,L4)*100}{the\, number\, of\, all\, worms}

The successful rate can reach to about 80%.


T--SUSTech Shenzhen--2.png
Fig. 2 The Embryos after Bleaching Adults

Compared with those two methods, we think the synchronization method is better. You can get more detail results by clicking See Details.

2 The Gaussian Chip

In order to study locomotive behavior of C. elegans populations, we design the Gaussian Chip, a pillar-filled area, where the pillars are designed such that it allows crawling-like behaviors even though worms are immersed in liquid environment.(Fig. 3) [1].


T--SUSTech Shenzhen--3.gif
Fig. 3 Gaussian plate to study locomotion on-chip C. elegans crawl like a “sin” function, so the width and angle between pillars are so optimal that worms can more efficiently move.

After deciding to use the microfluidics to study the locomotive behavior, we are noticed that the shape of microfluidics is similar to the Galton board.[2]. (Fig. 4) Therefore, we assume that the probability for C. elegans choose to go left or right is equal when it passes a crossing. (Fig.5)

T--SUSTech Shenzhen--Microfuildics--gerdun.gif
Fig. 4 The simulation of Galton board
T--SUSTech Shenzhen--Microfluidics--fig5crossing.png
Fig. 5 The Crossing in the Gaussian Plate The probability is equal for worms to go left or right.</B>

Then, we can assume that C. elegans is just like balls in the Galton board. The force of slow buffer flow acting on worms is the same as the gravity acting on balls. Both of the distribution is Gaussian distribution. Given that we need to make sure that injecting the target genes in C. elegans will not affect its olfactory receptor neuron pair, we injected diacetyl (2-nonanone) that C. elegans prefers (repulse) into the right (left) channel to make a concentration gradient on Gaussian chip. Because of the gradient, worms tend to move to the side filled with diacetyl, causing Gaussian distribution changed. (Fig. 6)

T--SUSTech Shenzhen--Microfluidics--fig5.png
Fig. 6 The Gaussian distribution A) The ideal Gaussian distribution before adding chemicals. B) The changed Gaussian distribution after adding chemicals by using the first diffusion methods, which means diacetyl diffuse in the same plate as the Gaussian chip.

In order to make a concentration gradient, we come up with two methods to get it. (Fig. 7)

T--SUSTech Shenzhen--Microfluidics--fig7.png
Fig.7 Two methods to get a concentration gradient. A) Method 1: add chemicals on the side of layer 1. Chemicals can diffuse from one side of chip to another side of it. B) Method 2: add chemicals on the layer 2. Chemicals can diffuse downwards to make a concentration gradient on layer 1.

In order to simulate the process of diffusion, we make a diffusion model to guide us. More details.(https://2017.igem.org/Team:SUSTech_Shenzhen/Chemical_Diffusion_Model)


Both of those methods could be carried out theoretically. But in the process of experiment, we find the method 1 (Inject chemicals into the side of layer 1) is better.

3 The Immobilization Chip

After studying worms’ group behaviors and proving their olfactory neurons are not being affected by exogenous gene, we could study their individual neuron activity and behavioral response. Traditionally, anesthetics and glues are utilized to immobilize worms. However worms will be damaged in these and it will create difficulties in studying its behavioral response. Thus, we designed two kinds of microfluidic chips to allow high-resolution microscopic imaging on chip without damaging for worms. (Fig. 8)

T--SUSTech Shenzhen--Microfluidics--fig8.png
Fig. 8 The immobilization chip. The four channels at the top of figure are called worm clamps. A channel larger than the worm’s diameter is tapered to an opening of 30 um or less (for worms in the L4 stage). The four channels at the bottom of figure are parallel channels. A channel can restrict worms in z direction and worms can just move forwards or backwards.

The first one is used to research the imaging of neuronal response, called worm clamps or traps[3].Worms are trapped in the wedge-shaped channel. Trapped worms can then be released by reversing the flow. Traps allow worms’ movement in x-y plane but restrict it in z direction, and it is also utilized to study the contraction and elongation of their heads and research the neuronal activity by detecting calcium indicator GEM-GECO in imaging software.

The next channel is compressed and rectangular, called parallel channel. In order to immobilize worms in this channel, we design two gas valves to block the exit and entry by compressing PDMS (a flexible and Easily deformed material) under the pressure made by water or air. (Fig. 9) [4]

T--SUSTech Shenzhen--Microfluidics--fig9.png
Fig.9 the cross-section plane of gas valves A) A normal gas valves. B) When worms go into the parallel channel, we turn off the gas valves immediately by increasing the pressure in control layer. The width between control layer and flow layer is so narrow that it is easy to make flow layer bend and block the exit and entry.

The contents above are our all hardware parts in microfluidics. You can learn more details in results. (https://2017.igem.org/Team:SUSTech_Shenzhen/Results)

References

  1. ↑ Albrecht, D.R., and Bargmann, C.I. (2011). High-content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments. Nat. Methods 8, 599-605.
  2. ↑ Bean machine. (2017, October 5). In Wikipedia, The Free Encyclopedia. Retrieved 12:46, October 22, 2017, from https://en.wikipedia.org/w/index.php?title=Bean_machine&oldid=803992086
  3. ↑ Hulme, S. E., Shevkoplyas, S. S., Apfeld, J., Fontana, W., & Whitesides, G. M. (2007). A microfabricated array of clamps for immobilizing and imaging C. elegans. Lab on A Chip, 7(11), 1515.
  4. ↑ Unger, M.A., Chou, H.P., Thorsen, T., Scherer, A., and Quake, S.R. (2000). Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288, 113-116.

Made by from the elegans.Inc in SUSTech_Shenzhen.

Licensed under CC BY 4.0.

  • ©2017 SUSTech_Shenzhen