Microfluidics is both a science and a technology that is currently an active field of academic research and study. It consists of systems that work with small volumes of fluids in the nanoliter/microliter scale, through channels ranging from tens to hundreds of micrometers in diameter. It is well matched with the size of Caenorhabditis elegans. In this system, we can study a group of worms as well as an individual one clearly, efficiently and conveniently.

In order to test whether insertion affect C. elegans's previous function in population, we need to get their freely response. In addition, we want to test worms individually by activing the receptors on neurons. The semifixed is necessary in this test. In order to get an efficient experimental results, we need synchronous worms. Because of these demands above, we design three microfluidics: the selection chip, the Gaussian Plate, and the Immobilization chip. (Fig. 1)

Fig. 1 the flow chart of microfluidics We use the selection chip to select the appropriate sized worms, and then we use these worms to prove their previous olfactory function is not affected (The normal distribution is Gaussian distribution. When C. elegans feel odors they prefer (diacetyl) or repulse (2-nonanone), the distribution will change.) Finally, we immobilize worms to observe their neuron activity and behavioral response.

The Design of Microfluidics

1 The Selection Chip

Most researches about C. 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. 2)

Fig. 2 the selection 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. 3) 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%.

Fig. 3 The Embryos after Bleaching Adults

Compared with those two methods in experiment, we find that we can get almost 150 of 100 worms (One adult have no less than 3 embryos) in three days by the synchronization method, while we can just get 20 of 100 worms in one day by microfluidics method. Given that we need a large number of worms to do the following experiment, we think the synchronization method is better. You can get more detail results by clicking See Details.

2 The Gaussian Plate

In order to study locomotive behavior of C. elegans populations, we design the Gaussian Plate, 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. 4) ^[1].

Fig. 4 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. 5(A)) Therefore, we assume that the probability for C. elegans choose to go left or right is equal when it passes a crossing. (Fig. 5(B))

Fig. 5 A) The simulation of Galton board The distribution of balls is Gaussian distribution. B) The Gaussian Plate and the crossing in it The Gaussian Plate is simulate the Galton board and the probability is equal for worms to go left or right.

In addition, 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 Plate. Because of the gradient, worms tend to move to the side filled with diacetyl, causing Gaussian distribution changed. (Fig. 6)

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 Plate.

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

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. See Details.

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 affected by exogenous gene, we could study their individual neuron activity and behavioral response under a light stimulus of specific wavelength. Traditionally, anesthetics and glues are utilized to immobilize worms. However, worms will be damaged in this condition and it will make it difficult to study the behavioral response of worms. Thus, we designed two kinds of microfluidic chips to allow high-resolution microscopic imaging on chip without damaging for worms. (Fig. 8)

Fig. 8 The immobilization chip. The four channels on the top of figure are called worm clamps. A channel with diameter that 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’ movement in z direction and worms only cound move forwards or backwards after turning off gas valves. When worms go into these channels, the gas valves on the entry and exit will be turned off to restrict worms in the channels.

The first kind of channel is trapping worms in the wedge-shaped channel, called worm clamps. It is 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 second kind of channel is compressed and rectangular called parallel channel. Worms can be restricted by turning off gas valves in this compressing channel.

Both of these channels can restrict worms. But in case that they go away from channels, we designed gas valves to block their entry and exit by compressing PDMS (a flexibility and Easily deformed material) under the pressure made by water or air. The principle of gas valves is as below. (Fig. 9) ^[3]

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 Fabricating And Using of Microfluidics

After designing these microfluidics, we need to fabricate them and utilize them. The microfluidics chips used in our project were all produced using this protocol by SUSTech_Shenzhen last year. (Fig. 10) Detailed Protocol

Fig. 10 the overview of bonding process SU-8 Photoresist can cross-link to a network of polymer when exposed to short wavelength light. If the cross-linked network has formed, it cannot be dissolved by the SU-8 developer. So if we place a photo mask to shade the light shined on the photoresist, we can make a copy of the pattern on the photo mask. Both PDMS and glass contain silicon element. When they were treated by oxygen plasma, unstable hydroxyl group will form on the treated surface. When they get close enough, two hydroxyl group will dehydrate and bond together covalently.

When injecting the flow and worms into microfluidics, We use pump to inject them and use microscope to observe the condition in chips. (Fig. 11)

Fig. 10 A) the whole devices used in microfluidic experiment. B) the pump and microscope used in our experiment. C) the microfluidics and hose used in our experiment.

The contents above are our all hardware parts in microfluidics. You can learn more details in results. Detailed 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. ↑ 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.