Microfluidic chip for capturing and fixing nematodes in a uniform position and preparation method thereof

Abstract

This invention provides a microfluidic chip and its preparation method for capturing and immobilizing nematodes in a uniform position, relating to the field of nematode model animal research technology. The chip includes a square structure, an adsorption channel, an inlet port, and an outlet port. The square structure contains a main channel, which is connected to both the inlet and outlet ports. The main channel is also connected to the adsorption channel. Two nematode-turning channels are provided on the side of the main channel opposite to the adsorption channel. These two channels are located between the inlet port and the adsorption channel, and between the outlet port and the adsorption channel, respectively. Each nematode-turning channel is connected to a 40 kPa negative pressure source. During use, the microfluidic chip allows for relatively firm adsorption and immobilization of nematodes for up to 2 hours, with minimal impact on nematode viability. Long-term imaging analysis and precise microinjection can be implemented.

Description

Technical Field

[0001] This invention relates to the field of model animal nematode research technology, and in particular to a microfluidic chip for capturing and immobilizing nematodes in a uniform position and its preparation method. Background Technology

[0002] The model organism *Caenorhabditis elegans* offers advantages such as rapid growth and reproduction, ease of culture, tiny size, simple structure, and convenient molecular biological manipulation. However, the nematodes are extremely small, with adults measuring only 1 mm in length and approximately 80 μm in width, making capture, fixation, and observation challenging. The rapidly developing microfluidic chip technology, with its excellent manipulation capabilities at the micro- and nano-scale, has been widely applied to nematode manipulation. Using microfluidic chips to fix nematodes effectively maintains their viability and is currently recognized as the least damaging fixation technique. The small size and good transparency of nematodes, along with microfluidic chips, make them suitable for microscopic imaging. Optical imaging is a common method for nematode research, and the micrometer-scale size of nematodes falls perfectly within the working range of microfluidic chips and optical imaging, resulting in a harmonious combination. For example, microfluidic chips can fix nematodes for optical imaging analysis. Microfluidic chip technology has significantly promoted the development of nematode research while also expanding its own application scope; many microfluidic chips used in nematode research can be developed into nematode research tools.

[0003] Common methods for immobilizing nematodes on microfluidic chips include the single-channel method, the lateral adsorption channel adsorption method, and the thin-film pressing method, with the latter two being more frequently used. The adsorption method is simple to operate, has a good immobilization effect, and only affects the local surface layer of the nematode, while the thin-film pressing method affects the entire nematode. The adsorption method has unique advantages.

[0004] In a liquid environment, nematodes instinctively exhibit a "C"-shaped swaying motion with a fixed frequency, while on a solid culture medium, this manifests as an "S"-shaped (sine function curve) crawling motion. This swaying behavior is related to the longitudinal muscles parallel to the dorsal and ventral lines. Unlike common reptiles, nematodes' dorsal and ventral lines are located on either side of their bodies during crawling, rather than vertically; their lateral lines are vertically distributed. In their natural state, nematodes crawl sideways, a characteristic of their natural body position. Controlled by neurons, the dorsal and ventral muscles on both sides alternately contract and relax, creating the swaying motion. This characteristic allows for the fixation of nematodes on a microfluidic chip during their natural swaying motion, maintaining a position with lateral lines vertically distributed and dorsal and ventral lines horizontally distributed. This enables different nematode individuals to be fixed in the same position (natural position), improving the comparability of experimental data between different nematode individuals. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A microfluidic chip includes a square structure, an adsorption channel, an inlet port, and an outlet port. The inlet port and the outlet port are located at opposite ends of the length of the square structure. A main channel is provided within the square structure, communicating with the outlet port and the inlet port. The main channel is also communicating with the adsorption channel. The adsorption channel is used to connect to an adjustable negative pressure source of 40 to 70 kPa. Two nematode-turning single channels are provided on the side of the main channel of the square structure opposite to the adsorption channel. The two nematode-turning single channels are located between the inlet port and the adsorption channel, and between the outlet port and the adsorption channel, respectively. The nematode-turning single channel is used to connect to the 40 kPa negative pressure source.

[0008] Preferably, the nematode turning channel has a width of 20 μm at the opening near one end of the square structure, a width greater than 20 μm in the remaining area, and a height of 40 μm.

[0009] Preferably, the adsorption channel includes a main adsorption channel and an auxiliary adsorption channel. Two auxiliary adsorption channels are provided, which are located on both sides of the width direction of the main adsorption channel. The two auxiliary adsorption channels form a 45° angle with the main adsorption channel, and the total width of the main adsorption channel and the auxiliary adsorption channels 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 740 μm, the height of the main adsorption channel within 400 μm of the edge of the square structure is 40 μm, the height of the remaining part of the main adsorption channel is 80 μm, the width of the auxiliary adsorption channel is the same as that of the main adsorption channel, the opening width near the edge of the square structure is 20 μm, the width of the remaining area is greater than 20 μm, and the height of the auxiliary adsorption channel is 40 μm.

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

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

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

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

[0015] Preferably, it also includes a rinsing channel, which is located on the side of the square structure opposite to the adsorption channel. There are two rinsing channels, which are located between the two nematode turning channels on both sides.

[0016] This application also provides a method for fabricating a microfluidic chip, which is used to fabricate the microfluidic chip described above. PDMS and a 24*40mm cover glass are selected, and the chip is fabricated according to the procedure. First, the PDMS below the square structure area is removed under a stereomicroscope using a No. 11 scalpel blade. When cutting, care should be taken to keep the openings of the main adsorption channel and the left and right auxiliary adsorption channels flat.

[0017] The microfluidic chip provided in this application enables efficient capture of nematodes on a microfluidic chip, fixing different nematodes in the same position (natural position), eliminating the difference in experimental results caused by different nematode positions, and making the experimental data more accurate. The chip device is "tailor-made" based on the individual physiological characteristics of nematodes to complete specific effects experiments on them: the physiological characteristic that the nematode's natural position when freely swinging is transformed into the experimental result of fixing the nematode in a uniform natural position on the microfluidic chip. This represents a refinement and further specialization of microfluidic technology for manipulating nematodes, possessing 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 manipulation. Attached Figure Description

[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 physical image of a microfluidic chip nematode adsorption and fixation module and a square structure, according to one embodiment of the present invention. Dark field, with fluorescent solution inside the channel;

[0020] Figure 3In one embodiment of the present invention, a microfluidic chip adsorption and fixation module is used to adsorb and fix nematodes (the square structure containing the nematodes is excavated 70-80 μm below the surface). After being captured by the auxiliary adsorption channel, the nematodes will wriggle. The larger square structure provides space for their wriggling. After adjusting their body position to a natural crawling position, they are adsorbed and fixed by the adjacent main adsorption channel. The scale bar is 200 μm on the left and 80 μm on the right.

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

[0022] Legend:

[0023] 1. Main adsorption channel; 2. Sample inlet; 3. Nematode turning single channel; 4. Rinsing channel; 5. Auxiliary adsorption channel; 6. Auxiliary adsorption channel; 7. Rinsing channel; 8. Second nematode turning single channel; 9. Sample outlet. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments.

[0025] Please see Figure 1 and Figure 2 A microfluidic chip device for capturing and immobilizing nematodes in a uniform position includes a square structure, an adsorption channel, an inlet 2, an outlet 9, nematode turning channels 3 and 8, and rinsing channels 4 and 7. The square structure is the site for nematode adsorption and immobilization. The adsorption channel, rinsing channel 4 and 7, inlet 2, outlet 9, and nematode turning channels 3 and 8 are all connected to the square structure. Specifically, in one embodiment, the adsorption channel and rinsing channel 4 and 7 are located on opposite sides of the square structure, the inlet 2 and outlet 9 are located on the other opposite sides, and there are two nematode turning channels 3 and 8, located between the inlet 2 and the adsorption channel and between the outlet 9 and the adsorption channel, respectively.

[0026] Please see Figure 1 and Figure 2 The square structure is 1mm long, 0.8mm wide, and approximately 160mm high. A main channel is provided along the length of the square structure. Both ends of the main channel are connected to the sample outlet 9 and the sample inlet 2, respectively. The nematode turning channels 3 and 8 are located above the two main channels. Please refer to [link / reference]. Figure 3After entering the square structure, the nematode is gripped at its tail end by auxiliary suction channels 5 and 6 on one side. Breaking free is an instinct for the nematode; after being captured at its tail end, it will continuously swing and swim in the opposite direction. The square structure provides space for this swinging motion, allowing it to adjust its natural body position. Appropriately reducing the force applied to the tail end will also adjust its tail position.

[0027] The adsorption channel includes a main adsorption channel 1 and two auxiliary adsorption channels 5 and 6, which are located on opposite sides of the main adsorption channel 1 in the width direction. In one embodiment, the main adsorption channel 1 is perpendicular to the edge of the square structure. The main adsorption channel 1 has a width of 740 μm and a height of 40 μm at a point 400 μm from the edge of the square structure. To provide sufficiently high adsorption force and prevent clogging of the main adsorption channel 1, the remaining portion of the main adsorption channel 1 has a height of 80 μm. The main adsorption channel 1 is open at one end of the square structure, and the opening is divided into 10 parallel fine channels, each with a width of 20 μm. The interval between adjacent main adsorption channels 1 is 60 μm.

[0028] Two auxiliary adsorption channels 5 and 6 are provided, located on either side of the main adsorption channel 1, forming a 45° angle with the main adsorption channel 1. The total width of the main adsorption channel 1 and the auxiliary adsorption channels 5 and 6 is 1 mm, and the width of the auxiliary adsorption channels 5 and 6 is the same as 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 a nematode enters the main channel opening in the square structure, its tail end can be easily captured by the auxiliary adsorption channels 5 and 6 on the side near the sample inlet 2. This side of the auxiliary adsorption channels 5 and 6 is adjacent to the main adsorption channel 1, forming a 45-degree angle. The captured nematode forms an angle of approximately 45 degrees with the edge of the square, which is beneficial for the subsequent adsorption and fixation of the nematode by the main adsorption channel 1. The adjacent functional structure makes the nematode sample introduction, tail capture, and adsorption fixation operations smooth, stable, and highly efficient.

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

[0030] The inlet port 2 is circular with a diameter of 4 mm. Starting from the inlet port 2, the channel width gradually narrows from 150 μm to 80 μm, then connects to the main channel and subsequently to the outlet port 9. The main channel has a width of 120 μm within a 900 μm range on either side of the openings of the nematode turning channels 3 and 8, with the remaining portion having a height and width of 80 μm.

[0031] In one embodiment, since the force in the microscopic environment is very different from that in the macroscopic world, when the liquid flow is slightly disturbed and unstable, the nematodes located in the main channel will disappear instantly. In order to make the movement speed of the nematodes in the main channel controllable, a buffer channel is connected between the sample inlet 2 and the sample outlet 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, reduce the liquid flow velocity in the main channel, and 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 sample inlet channel. The nematodes can enter the square structure through the buffer channel. The design of the buffer channel improves the stability of the system operation.

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

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

[0034] The rinsing channels 4 and 7 are located on the side of the square structure away from the adsorption channel. In one embodiment, there are two rinsing channels 4 and 7. The two rinsing channels 4 and 7 are used to flush the chip with buffer solution, 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 fabricated according to the procedure. First, the PDMS under the square structure area is removed under a stereomicroscope using a No. 11 scalpel blade. When cutting, care should be taken to keep the openings of the main adsorption channel 1 and the left and right auxiliary adsorption channels 5 and 6 flat. The requirements for other areas are not high, and even if the cutting goes beyond the line, it will not have a significant impact.

[0036] The microfluidic chip is used as follows:

[0037] Add the solution containing adult nematodes into the injection port 2. If fixation is required, point the nematode head in the direction of liquid flow and activate the negative pressure (-40KPa) of the nematode turning channel 3 and 8 on the closer side. Conversely, activate the nematode turning channel 3 and 8 on the other side.

[0038] The nematode is captured at one end through single-channel 3 and 8 when it turns around. If the adsorbed end is the tail, the nematode is released first, and then the nearby auxiliary adsorption channels 5 and 6 (-40 kPa) are activated to capture the tail. If the nematode turns around and its head is adsorbed through single-channel 3 and 8, the nearby auxiliary adsorption channels 5 and 6 are activated first. The nematode is allowed to complete a 180° turn within the main channel before being released, thus completing the tail capture. This describes the process of the nematode turning around and being captured within the channels. In this study, auxiliary adsorption channels 5 and 6 tend to capture the tail of the nematode, as its head has a swinging motion and is not suitable for being adsorbed and fixed first.

[0039] After the nematodes are captured by auxiliary adsorption channels 5 and 6, they enter the square structure and begin to swing freely, adopting a natural body position with the ventral and dorsal lines on both sides and the lateral lines above and below. Reducing the negative pressure in auxiliary adsorption channels 5 and 6 (from -5 to -20 kPa) decreases the suction force on the tail, maintaining the nematode's capture. The nematode continues to swing; because its tail is relatively soft and has a wedge-shaped, tapering structure, the body's swinging motion gradually corrects the tail's position, aligning it 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 form a 45° angle with it. When the nematode undulates, its elongated body is easily captured and fixed by the main adsorption channel 1. The main adsorption channel 1 is activated (-50 kPa) to capture and fix the nematode. The adult nematode is approximately 1 mm long. The total width of the 740 μm opening of the main adsorption channel 1, plus the auxiliary adsorption channels 5 and 6, is about 1 mm. After activating the main adsorption channel 1, all 10 openings will capture and suck the nematode's body. If the nematode is curled up, some openings will remain open; simply restart the main adsorption channel 1.

[0041] At this point, the auxiliary channel that previously captured the nematode's tail can release the tail. Alternatively, both the head and tail of the nematode can be adsorbed simultaneously for a more secure fixation.

[0042] After imaging analysis is completed, the nematodes are released and removed through sample outlet 9. During this process, buffer solution can be introduced through rinsing channels 4 and 7 to clean the square structure and other channel structures. After the operation, the chip is rinsed and stored for future use.

[0043] like Figure 4 As shown, the microfluidic chip provided in this application can perform calcium ion imaging in cultured nematode intestinal cells: by labeling intestinal cells with calcium ion fluorescent probes, the changes in calcium ion levels in intestinal cells can be observed, which is beneficial for studies on calcium ion signals, calcium oscillations, and calcium transfer. It can also be used for fluorescence imaging of nematode neurons.

[0044] In summary, the microfluidic chip provided in this application enables all individual nematodes to be adsorbed and immobilized in a uniform (natural) position for imaging analysis, reducing errors caused by positional differences and improving the accuracy of experimental data. This performance characteristic facilitates the development of nematode manipulation techniques towards automation, intelligence, and high throughput. This technology fully utilizes the advantages of microfluidic chips in efficiently capturing nematodes and flexibly manipulating them within a microscale space, improving manipulability. The chip has a simple structure and low manufacturing cost; the buffer channel design improves the stability of chip performance; and operation is simple and smooth, making it a commonly used tool for nematode research.

Claims

1. A microfluidic chip, comprising a square structure, an adsorption channel, an inlet port, and an outlet port, wherein the inlet port and the outlet port are located at opposite ends along the length of the square structure, a main channel is provided within the square structure, the main channel is connected to the outlet port and the inlet port, and the main channel is connected to the adsorption channel, wherein the adsorption channel is used to connect to an adjustable negative pressure source of 40 to 70 kPa, characterized in that: On the main channel of the square structure, a nematode turning channel is provided on the side opposite to the adsorption channel. There are two nematode turning channels, which are located between the sample inlet and the adsorption channel and between the sample outlet and the adsorption channel, respectively. The nematode turning channel is used to connect to a 40 kPa negative pressure source.

2. A microfluidic chip according to claim 1, characterized in that: The nematode turning channel has a width of 20 μm at the opening near one end of the square structure, a width greater than 20 μm in the remaining area, and a height of 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. There are two auxiliary adsorption channels, which are located on both sides of the width direction of the main adsorption channel. The two auxiliary adsorption channels form a 45° angle with the main adsorption channel, and the total width of the main adsorption channel and the auxiliary adsorption channels 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. The width of the main adsorption channel is 740 μm. The height of the main adsorption channel within 400 μm of the edge of the square structure is 40 μm, and the height of the remaining part of the main adsorption channel is 80 μm. The width of the auxiliary adsorption channel is the same as that of the main adsorption channel. 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 channel is 40 μm.

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

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

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

8. A microfluidic chip according to claim 1, characterized in that: A buffer channel is also 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. A 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. There are two flushing channels, which are located between the two nematode turning channels.

10. A method for fabricating a microfluidic chip, used to fabricate the microfluidic chip according to any one of claims 1-9, characterized in that: PDMS and 24*40 mm coverslips were selected, and the chip was fabricated according to the procedure. First, the PDMS below the square structure area was removed under a stereomicroscope using a No. 11 scalpel blade. When cutting, care should be taken to keep the openings of the main adsorption channel and the left and right auxiliary adsorption channels flat.

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