Micro-fluidic chip for capturing and fixing nematodes in unified body position and preparation method of micro-fluidic chip

Abstract

The invention provides a micro-fluidic chip for capturing and fixing nematodes in a unified body position and a preparation method, and relates to the technical field of model animal nematode research, the micro-fluidic chip comprises a square structure, an adsorption channel, a sample inlet hole and a sample outlet hole, the square structure is internally provided with a main channel, the main channel is communicated with the sample outlet hole and the sample inlet hole, and the adsorption channel is communicated with the sample inlet hole and the sample outlet hole. The main channel is communicated with the adsorption channel, one side, deviating from the adsorption channel, of the main channel of the square structure is provided with two nematode U-turn single channels, and the number of the nematode U-turn single channels is two; the two nematode U-turn single channels are located between the sample inlet hole and the adsorption channel and between the sample outlet hole and the adsorption channel respectively, and the nematode U-turn single channels are used for being communicated with a 40 KPa negative pressure source. In the use process of the micro-fluidic chip, nematodes can be firmly adsorbed and fixed, and the time can be as long as 2 hours; during adsorption, nematode activity is less affected. And long-term imaging analysis, precise microinjection and the like can be implemented.

Description

Technical Field

[0001] The invention relates to the technical field of model animal nematode research, and in particular to a microfluidic chip for capturing and fixing nematodes in a uniform position and a preparation method thereof. Background Art

[0002] The model animal Caenorhabditis elegans has the advantages of rapid growth and reproduction, easy cultivation, small size, simple structure, and convenient molecular biology operations. The nematode body is tiny, with an adult body length of 1mm and a width of about 80μm. It is difficult to capture and fix it for observation. The booming microfluidic chip technology has been widely used in the manipulation of nematodes with its excellent manipulation ability at the micro-nano scale. The use of microfluidic chips to fix nematodes can better maintain their activity and is currently recognized as the least harmful fixation technology to nematodes. The small size and good transparency of nematodes and microfluidic chips are both suitable for microscopic imaging. Optical imaging is a common method for studying nematodes. The micron-level size of nematodes is just within the working range of microfluidic chips and optical imaging. The three are well matched. For example, microfluidic chips can fix nematodes and perform optical imaging analysis on nematodes. While microfluidic chip technology has greatly promoted the development of nematode research, it has also expanded its own application space. Many microfluidic chips used in nematode research can be developed into nematode research tools.

[0003] Common methods for fixing nematodes on microfluidic chips include single-channel method, side adsorption channel adsorption fixation method, and film pressing method, among which the latter two are more commonly used. The adsorption method is easy to operate, has a good fixation effect, and only affects the local surface of the nematode. The film pressing method affects the entire nematode. The adsorption fixation method has unique advantages.

[0004] In a liquid environment, nematodes will instinctively perform a "C"-shaped swinging motion with a fixed swinging frequency, which is reflected as an "S"-shaped (sinusoidal function curve) crawling motion on a solid culture medium. The swinging behavior of nematodes is related to the longitudinal muscles parallel to the spine and ventral lines. Here, unlike common reptiles, when nematodes crawl, their spine and ventral lines are on both sides of the body, rather than distributed up and down. Their lateral lines are distributed up and down. In the natural state, nematodes crawl sideways. This is the natural position of nematodes and is a habitual characteristic of nematodes. Controlled by neurons, the back and abdominal muscles distributed on both sides contract and relax alternately, forming the swinging motion of nematodes. With this feature, the microfluidic air chip is adsorbed and fixed when it swings naturally, which will keep the lateral line in the upper and lower dorsal and ventral lines on the left and right sides. In this way, different nematode individuals can be fixed in the same position (natural position), and the experimental data between different nematode individuals are more comparable. Summary of the invention

[0005] The purpose of the present invention is to solve the technical problem that nematodes cannot be fixed in the same body position in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A microfluidic chip comprises a square structure, an adsorption channel, an injection hole and an outlet hole, wherein the injection hole and the outlet hole are located at both ends of the length direction of the square structure, a main channel is arranged in the square structure, the main channel is connected with the outlet hole and the injection hole, the main channel is connected with the adsorption channel, the adsorption channel is used to connect to a negative pressure source with adjustable pressure of 40 to 70 KPa, a nematode turning single channel is arranged on the side of the main channel of the square structure away from the adsorption channel, two nematode turning single channels are arranged, the two nematode turning single channels are respectively located between the injection hole and the adsorption channel and between the outlet hole and the adsorption channel, the nematode turning single channel is used to connect to a negative pressure source of 40 KPa.

[0008] Preferably, the width of the nematode turning single channel at the opening close to one end of the square structure is 20 μm, the width of the remaining area is greater than 20 μm, and the height is 40 μm.

[0009] Preferably, the adsorption channel includes a main adsorption channel and an auxiliary adsorption channel. Two auxiliary adsorption channels are provided. The two auxiliary adsorption channels are respectively located on both sides of the width direction of the main adsorption channel. The two auxiliary adsorption channels form an angle of 45° with the main adsorption channel, and the total width of the main adsorption channel and the auxiliary adsorption channel is 1 mm.

[0010] Preferably, the main adsorption channel is perpendicular to the edge of the square structure, the width of the main adsorption channel is 740μm, the height of the main adsorption channel within 400μm close to the edge of the square structure is 40μm, and the height of the rest of the main adsorption channel is 80μm. The width of the auxiliary adsorption channel is consistent with that of the main adsorption channel, and the opening width close to the edge of the square structure is 20μm, and the width of the rest of the area is greater than 20μm. The height of the auxiliary adsorption channel is 40μm.

[0011] Preferably, it further comprises a PDMS film, which is cut out in the square structure, the bottom surface of the square area is a glass substrate, and the opening of the adsorption channel is 70 to 80 μm above the glass substrate.

[0012] Preferably, the main channel has a channel width of 120 μm within a range of 900 μm on both sides of the left and right sides of the nematode turning single channel opening, and the remaining parts are 80 μm in height and 80 μm in width.

[0013] Preferably, the injection hole is circular, and the diameter of the injection hole is 4 mm. Starting from the injection hole, the channel width gradually narrows from 150 μm to 80 μm, and then connects to the main channel, and then is connected to the outlet hole.

[0014] Preferably, a buffer channel is connected between the sample inlet and the sample outlet, the buffer channel is parallel to the main channel, and the buffer channel is located below the square structure.

[0015] Preferably, it also includes a flushing channel, which is located on the side of the square structure away from the adsorption channel. Two flushing channels are provided, and the two flushing channels are located between the nematode turning single channels on both sides.

[0016] The present application also provides a method for preparing a microfluidic chip, which is used to prepare the microfluidic chip described above. PDMS and a 24*40mm cover glass are selected, and the chip is made according to the process. First, the PDMS below the square structure area is cut off under a stereo microscope using a No. 11 surgical blade. When cutting, care should be taken to keep the main adsorption channel and the left and right auxiliary adsorption channel openings cut flat.

[0017] A microfluidic chip provided in the present application realizes efficient capture of nematodes on the microfluidic chip, and fixation of different nematode individuals in the same position (natural position), eliminating the differences in experimental results caused by different positions of nematodes, and the experimental data is more accurate. The chip device is "tailored" around the individual physiological characteristics of nematodes to complete specific experimental operations on nematodes: the physiological characteristics of the nematodes in their natural position when they swing freely are converted into experimental operation results of fixing nematodes in a unified natural position on the microfluidic chip. It is a refinement and further division of labor of microfluidic technology for the operation of nematodes. It has complete and independent functions and can be developed into a nematode research tool. This technical method is conducive to the automation, intelligence and high throughput of nematode operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the structure of a microfluidic chip in one embodiment of the present invention;

[0019] Figure 2 This is a real picture of the nematode adsorption and fixation module and square structure of the microfluidic chip in one embodiment of the present invention. Dark field, the channel contains fluorescent solution;

[0020] Figure 3The microfluidic chip adsorption and fixation module in one embodiment of the present invention adsorbs and fixes nematodes (the square structure where the nematodes are located is dug down 70-80μm). After being captured by the auxiliary adsorption channel, the nematodes will swing and move. The larger square structure creates space for their swinging. After their body position is adjusted to a natural crawling position, they are adsorbed and fixed by the adjacent main adsorption channel. The scale is 200μm on the left and 80μm on the right;

[0021] Figure 4 This is a graph showing the results of calcium ion imaging analysis using the microfluidic chip to adsorb and immobilize nematodes in one embodiment of the present invention.

[0022] Legend:

[0023] 1. Main adsorption channel; 2. Sample injection hole; 3. Nematode turning channel; 4. Flushing channel; 5. Auxiliary adsorption channel; 6. Auxiliary adsorption channel; 7. Flushing channel; 8. Second nematode turning channel; 9. Sample outlet hole. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific embodiments.

[0025] See also Figure 1 and Figure 2 A microfluidic chip device for capturing and fixing nematodes in a unified position comprises a square structure, an adsorption channel, a sampling hole 2, a sampling hole 9, nematode turning single channels 3, 8 and flushing channels 4, 7, wherein the square structure is a place for nematodes to be adsorbed and fixed, and the adsorption channel, flushing channels 4, 7, the sampling hole 2, the sampling hole 9 and the nematode turning single channels 3, 8 are all connected to the square structure; specifically, in one embodiment, the adsorption channel and the flushing channels 4, 7 are located on two opposite sides of the square structure, the sampling hole 2 and the sampling hole 9 are respectively located on the other two opposite sides, and two nematode turning single channels 3, 8 are provided, and the two nematode turning single channels 3, 8 are respectively located between the sampling hole 2 and the adsorption channel and between the sampling hole 9 and the adsorption channel.

[0026] See also Figure 1 and Figure 2 The square structure is a square structure with a length of 1 mm, a width of 0.8 mm, and a height of about 160 mm. A main channel is arranged in the square structure along its length direction. Both ends of the main channel are connected to the sample outlet 9 and the sample inlet 2 respectively. The nematode turning single channels 3 and 8 are located above the two main channels. Figure 3After the nematode enters the square structure, the tail end of the nematode is caught by the auxiliary adsorption channels 5 and 6 on one side. It is the instinct of the nematode to break free. After the tail end is caught, the nematode will swing and swim in the opposite direction without stopping. The square structure provides it with a swinging space, so that it can adjust its natural body position. The tail position is also adjusted by appropriately reducing the force on the tail.

[0027] The adsorption channel includes a main adsorption channel 1 and two auxiliary adsorption channels 5 and 6, and the two auxiliary adsorption channels 5 and 6 are respectively located on both sides of the width direction of the main adsorption channel 1. In one embodiment, the main adsorption channel 1 is perpendicular to the edge of the square structure, the width of the main adsorption channel 1 is 740μm, and the height of the main adsorption channel 1 at 400μm near the edge of the square structure is 40μm. In order to provide a sufficiently high suction force and prevent the main adsorption channel 1 from being blocked, the remaining part of the main adsorption channel 1 is 80μm high. The main adsorption channel 1 is set to open at one end of the square structure, and the opening is divided into 10 parallel thin channels, each of which is 20μm wide, and the interval between adjacent main adsorption channels 1 is 60μm.

[0028] There are two auxiliary adsorption channels 5 and 6, which are located on both sides of the main adsorption channel 1. The two auxiliary adsorption channels 5 and 6 form an angle of 45° with the main adsorption channel 1, and the total width of the main adsorption channel 1 and the auxiliary adsorption channels 5 and 6 is 1 mm. The width of the auxiliary adsorption channels 5 and 6 is consistent with that of the main adsorption channel 1. The opening width near the edge of the square structure is 20 μm, and the width of the remaining area is greater than 20 μm. The height of the auxiliary adsorption channels 5 and 6 is 40 μm. When the nematode enters the main channel mouth in the square structure, the tail end of the nematode can be easily captured by the auxiliary adsorption channels 5 and 6 near the side of the injection hole 2. The auxiliary adsorption channels 5 and 6 on this side are next to the main adsorption channel 1, and the two form an angle of 45 degrees. The captured nematode is at an angle of about 45 degrees with the edge of the square frame, which is conducive to the subsequent adsorption and fixation of the nematode by the main adsorption channel 1. The adjacent functional structure makes the nematode injection, tail capture, and adsorption and fixation operations smooth, stable and efficient.

[0029] During application, the end of the main adsorption channel 1 away from the square structure is connected to a negative pressure source with an adjustable pressure of 40 to 70 KPa, and the ends of the auxiliary adsorption channels 5 and 6 away from the square structure are connected to a negative pressure source with a pressure of 40 to 70 KPa.

[0030] The injection hole 2 is circular, and the diameter of the injection hole 2 is 4 mm. Starting from the injection hole 2, the channel width gradually narrows from 150 μm to 80 μm, and then connects to the main channel, and then connects to the sample outlet 9. The main channel has a channel width of 120 μm in the range of 900 μm on both sides of the opening of the nematode turning single channel 3 and 8, and the rest of the channel is 80 μm in height and 80 μm in width.

[0031] In one embodiment, since the form of force in the microscopic environment is very different from the macroscopic world that people are accustomed to, when the liquid flow is slightly disturbed and unstable, the nematodes positioned in the main channel will disappear instantly. In order to achieve controllable movement speed of the nematodes in the main channel, a buffer channel is also connected between the injection hole 2 and the outlet hole 9. The buffer channel is parallel to the main channel and is located below the square structure. The buffer channel is used to divert the main channel and reduce the liquid flow speed of the main channel. At the same time, it can balance the pressure in the main channel at both ends of the adsorption and fixing module, stabilize the liquid in the main channel, and reduce the occurrence of turbulence. At the same time, the buffer channel can also serve as a second injection channel. The nematodes can enter the square structure through the buffer channel. The stability of the system operation is improved by the design of the buffer channel.

[0032] There are two single nematode turning channels 3, 8, which are located on the side of the square structure away from the adsorption channel. The openings of the single nematode turning channels 3, 8 are connected to the main channel. The width of the single nematode turning channels 3, 8 at the opening close to one end of the square structure is 20 μm, and the width of the remaining areas is greater than 20 μm, and the height is 40 μm.

[0033] In one embodiment, in order to reduce the contact area between the chip and the nematode body when the nematodes are adsorbed and fixed, a 70-80 μm PDMS (Polydimethylsiloxane) film is laid under each channel structure, and the film is cut in a square structure. The bottom surface of the square area is a glass substrate, and the opening of the adsorption channel is 70 to 80 μm above the glass substrate. When the nematodes are adsorbed and fixed, they have less contact with the bottom glass, thereby reducing the affected area of ​​their surface.

[0034] The flushing channels 4 and 7 are located on the side of the square structure away from the adsorption channel. In one embodiment, two flushing channels 4 and 7 are provided, and the two flushing channels 4 and 7 are used to flush the buffer solution to flush the chip, thereby avoiding the retention of nematodes in the square structure.

[0035] The microfluidic chip uses PDMS and a 24*40mm cover glass. The chip is manufactured according to the process. First, the PDMS below the square structure area is cut off under a stereo microscope using a No. 11 surgical blade. When cutting, care should be taken to keep the main adsorption channel 1 and the left and right auxiliary adsorption channels 5 and 6 openings cut smoothly. Other areas are not required, and even if the cutting crosses the line, the impact is not significant.

[0036] The method of using the microfluidic chip is as follows:

[0037] The solution containing adult nematodes is added into the injection hole 2. If fixation is required, the head of the nematode points to the direction of liquid flow, and the negative pressure (-40KPa) of the nematode turning single channel 3, 8 closer to the nematode is started. Conversely, the negative pressure of the nematode turning single channel 3, 8 on the other side is started.

[0038] Through the nematode turning single channel 3, 8, one end of the nematode is captured. If the adsorbed end is the nematode's tail, the nematode is released first, and then the closer auxiliary adsorption channels 5, 6 (-40KPa) are activated to capture the nematode's tail. If the nematode turning single channel 3, 8 adsorbs the nematode's head, the closer auxiliary adsorption channels 5, 6 are activated first, and the nematode is waited for to complete a 180° turn in the main channel, and then released to complete the capture of its tail. This is the process of the nematode turning and being captured in the channel. In this paper, the auxiliary adsorption channels 5, 6 tend to capture the nematode's tail, and its head has the function of initiating a swinging movement, so it is not suitable to be adsorbed and fixed first.

[0039] After the auxiliary adsorption channels 5 and 6 capture the nematode, the nematode enters the square structure and begins to swing freely. Its body position is a natural body position with the ventral line on both sides and the lateral line above and below. The negative pressure of the auxiliary adsorption channels 5 and 6 is reduced (-5 to -20 KPa), and the suction force on the tail is reduced to maintain the nematode being captured. The nematode continues to swing. Since the nematode's tail is soft and has a wedge-shaped structure that gradually becomes thinner, when the suction force is reduced, the swing of its body will gradually correct the tail position, which is consistent with the overall body position of the nematode.

[0040] The auxiliary adsorption channels 5 and 6 are adjacent to the main adsorption channel 1 and are at an angle of 45°. When the nematode swings, its long body is easily captured and adsorbed by the main adsorption channel 1. Start the main adsorption channel 1 (-50KPa) to capture and adsorb. The length of the adult nematode is about 1 mm. The 740μm opening of the main adsorption channel 1 plus the total width of the auxiliary adsorption channels 5 and 6 is about 1mm. After starting the main adsorption channel 1, all 10 openings will capture the nematode body. If the nematode curls up, some openings will remain open, and the main adsorption channel 1 can be re-operated.

[0041] At this time, the auxiliary channel that captured the tail end of the nematode can release the tail end of the nematode. The head and tail of the nematode can also be adsorbed at the same time to achieve a more secure fixation effect.

[0042] After the imaging analysis is completed, the nematodes are released and drawn out from the sample outlet 9. During this period, buffer can be introduced from the flushing channels 4 and 7 to clean the square structure and other channel structures. After the operation is completed, the chip is rinsed and stored for next use.

[0043] like Figure 4 As shown, the microfluidic chip provided in this application can be used for calcium ion imaging in intestinal cells of cultured nematodes: by labeling intestinal cells with calcium ion fluorescent probes, the change process of intestinal cell calcium ion levels can be observed, which is conducive to the study of calcium ion signals, calcium oscillations and calcium transmission. It can also be used for fluorescent imaging of nematode neurons.

[0044] In summary, the microfluidic chip provided by the present application can realize that all nematode individuals are adsorbed and fixed in a unified position (natural position), and then perform imaging analysis, which can reduce the error caused by position differences and improve the accuracy of experimental data. This performance feature is conducive to the development of nematode manipulation technology towards automation, intelligence, and high throughput. This technical equipment fully utilizes the advantages of microfluidic chips that can efficiently capture nematodes, and can flexibly manipulate nematodes in microscale space, improving the manipulability of nematodes. The chip structure is simple in function, low in production cost, and the design of the buffer channel improves the stability of the chip performance. It is easy and smooth to operate and can be used as a common tool for nematode research.

Claims

1. A microfluidic chip, comprising a square structure, an adsorption channel, an injection hole and an outlet hole, wherein the injection hole and the outlet hole are located at both ends of the length direction of the square structure, a main channel is provided in the square structure, the main channel is connected with the outlet hole and the injection hole, the main channel is connected with the adsorption channel, the adsorption channel is used to connect a negative pressure source with an adjustable pressure of 40 to 70 KPa, characterized in that: A nematode turning single channel is arranged on the side of the main channel of the square structure away from the adsorption channel. Two nematode turning single channels are arranged. The two nematode turning single channels are respectively located between the sampling hole and the adsorption channel and between the sampling hole and the adsorption channel. The nematode turning single channel is used to connect to a 40KPa negative pressure source.

2. A microfluidic chip according to claim 1, characterized in that: The width of the nematode turning single channel at the opening close to one end of the square structure is 20 μm, and the width of the remaining area is greater than 20 μm, and the height is 40 μm.

3. A microfluidic chip according to claim 1, characterized in that: The adsorption channel includes a main adsorption channel and an auxiliary adsorption channel. Two auxiliary adsorption channels are provided. The two auxiliary adsorption channels are respectively located on both sides of the width direction of the main adsorption channel. The two auxiliary adsorption channels form an angle of 45° with the main adsorption channel, and the total width of the main adsorption channel and the auxiliary adsorption channel is 1 mm.

4. A microfluidic chip according to claim 3, characterized in that: The main adsorption channel is perpendicular to the edge of the square structure, has a width of 740 μm, a height of 400 μm within 400 μm close to the edge of the square structure, and a height of the remaining part of the main adsorption channel is 80 μm. The width of the auxiliary adsorption channel is consistent with that of the main adsorption channel, and its opening width close to the edge of the square structure is 20 μm, and the width of the remaining area is greater than 20 μm. The height of the auxiliary adsorption channel is 40 μm.

5. The microfluidic chip according to claim 1, characterized in that: It also includes a PDMS film, which is cut in the square structure. The bottom surface of the square area is a glass substrate, and the opening of the adsorption channel is 70 to 80 μm above the glass substrate.

6. A microfluidic chip according to claim 1, characterized in that: The main channel has a channel width of 120 μm in the range of 900 μm on both sides of the opening of the nematode turning single channel, and the rest of the channel has a height of 80 μm and a width of 80 μm.

7. A microfluidic chip according to claim 1, characterized in that: The injection hole is circular and has a diameter of 4 mm. Starting from the injection hole, the channel width gradually narrows from 150 μm to 80 μm, then connects to the main channel, and then connects to the outlet hole.

8. The microfluidic chip according to claim 1, characterized in that: A buffer channel is connected between the sample inlet and the sample outlet. The buffer channel is parallel to the main channel and is located below the square structure.

9. The microfluidic chip according to claim 1, characterized in that: It also includes a flushing channel, which is located on the side of the square structure away from the adsorption channel. Two flushing channels are provided, and the two flushing channels are located between the nematode turning single channels on both sides.

10. A method for preparing a microfluidic chip, for preparing the microfluidic chip according to any one of claims 1 to 9, characterized in that: Select PDMS and a 24*40mm cover glass, and make the chip according to the process. First, use a No. 11 surgical blade to cut off the PDMS below the square structure area under a stereo microscope. When cutting, pay attention to keeping the main adsorption channel and the left and right auxiliary adsorption channel openings cut smoothly.

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