Microfluidic chip device for culture, storage, capture and imaging analysis of nematodes

Abstract

The invention provides a micro-fluidic chip device for nematode culture, storage, capture and imaging analysis, and relates to the technical field of model animal nematode research methods, the micro-fluidic chip device comprises a culture pool, the top of the culture pool is open, the length and width of the culture pool are 1-2 cm, and the depth of the culture pool is 0.3-0.8 cm. The nematode culture method is the same as the conventional nematode culture method, and the conventional chip internal closed nematode culture method is changed, so that the culture environment is the same as that of the conventional culture, and the influence of a chip technology on nematode culture is eliminated. Meanwhile, open culture is more flexible in operation, accepts external operation and is high in plasticity. According to the device, efficient capturing, fixing, optical imaging analysis and the like of the nematodes cultured in real time as well as recycling and re-culturing can be completed. The imaging analysis can be used for neuron structure imaging, fluorescence labeling imaging, probe molecule imaging, ion level dynamic monitoring, drug stimulation reaction analysis and the like. The design can also be integrated with other nematode operation analysis modules to be developed into a multifunctional nematode analysis micro-fluidic chip.

Description

Technical Field

[0001] The present invention relates to the technical field of model animal nematode research methods, and in particular to a microfluidic chip device for nematode culture, storage, capture and imaging analysis. 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. Nematodes are taken from the soil environment, have strong vitality, and are easier to cultivate than cells. Nematode cultivation methods can be divided into solid culture medium and liquid culture medium. The solid culture medium is mainly agar, and Escherichia coli is cultured on the surface as food for nematodes, and the nematodes grow on the surface of the culture medium. The liquid culture medium contains salts, amino acids, nucleotides, vitamins, energy substances and other nutrients necessary for the growth and development of nematodes. For example, the C.elegans Maintenance Medium (CeMM) liquid culture medium proposed by Lu and Goetsch can also be artificially added with nematode food Escherichia coli at regular intervals for nematodes to feed and grow. Liquid culture nematode technology has promoted the development of microfluidic chip nematode cultivation.

[0003] Nematodes are tiny, with an adult body length of 1 mm and a width of about 80 microns. It is difficult to capture, fix, and observe them. The booming microfluidic chip technology has been widely used in the manipulation of nematodes due to its excellent manipulation capabilities at the micro-nano scale. Using microfluidic chips to fix nematodes can better maintain their activity. After the experiment, the nematodes can be released and cultured again. This is a low-damage nematode fixation technology. The physiological functions of the nematodes in culture are in a natural state, and they can be fixed immediately for optical imaging analysis, which can minimize the impact of the fixation operation on their natural physiological state. Both nematodes and microfluidic chips are suitable for microscopic imaging. Using conventional fixation techniques, such as glue adhesion method, the nematodes cannot be recovered after being fixed.

[0004] Microfluidic chip technology has greatly promoted the development of nematode research and expanded its own application space. Many microfluidic chips used in nematode research can be developed into nematode research tools. Therefore, expanding the application field of microfluidic chips will not only help deepen the research on nematode biology, but also open up new development directions and potential market space for microfluidic technology itself. Summary of the invention

[0005] The purpose of the present invention is to realize liquid nematode culture in an open environment and natural state on a microfluidic chip, which is a non-chip channel internal culture technology, which is no different from conventional liquid culture, and can realize fine operations such as nematode capture and fixation, and realize high-resolution imaging analysis of nematodes.

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

[0007] A microfluidic chip device for nematode culture, storage, capture and imaging analysis, the microfluidic chip device is made of PDMS and is characterized by comprising a culture pool with an opening at the top, a length and width of 1-2 cm, and a depth of 0.3-0.8 cm.

[0008] Preferably, it also includes a nematode adsorption and fixation module, which includes a connecting channel and an adsorption channel, wherein the connecting channel is connected to the culture pool, both ends of the connecting channel are opened, one end of the connecting channel is opened and connected to the culture pool, and the adsorption channel is located on one side of the connecting channel in the width direction and is connected to the connecting channel.

[0009] Preferably, the connecting channel is 80 microns high and 80 microns wide. The adsorption channel includes a main adsorption channel, which is arranged perpendicular to the connecting channel. The side of the main adsorption channel facing away from the connecting channel is used to be connected to a negative pressure source of 50Kpa, and a plurality of fine channels arranged side by side are arranged on the side pointing to the connecting channel. The fine channels are perpendicular to the connecting channel, and the main adsorption channel is connected to the connecting channel through the fine channels.

[0010] Preferably, there are 10 fine channels, the width of a single fine channel is 20 microns, the interval between adjacent fine channels is 60 microns, and the width of the main adsorption channel is 740 microns.

[0011] Preferably, the adsorption channel also includes an auxiliary adsorption channel, and two of the auxiliary adsorption channels are provided. The two auxiliary adsorption channels are located on both sides of the width direction of the main adsorption channel. The side of the auxiliary adsorption channel away from the connecting channel is used to connect to the negative pressure. The auxiliary adsorption channel is connected to the connecting channel, and the height of the auxiliary adsorption channel is 40 microns.

[0012] Preferably, the main adsorption channel, the auxiliary adsorption channel and the bottom of the connecting channel are arranged on the same plane.

[0013] Preferably, the microfluidic chip operation process is as follows:

[0014] When in use, the culture pool is filled with liquid culture medium for culturing nematodes, and experimental operations can be carried out after the nematodes adapt to the new environment;

[0015] Observe the opening of the microfluidic chip channel at the bottom of the "culture pool" under a microscope. When a nematode approaches, close the connecting channel and start the auxiliary adsorption channel of the imaging analysis module, and the end of the nematode is captured.

[0016] Then the main adsorption channel is started. If there are other nematodes in the main channel, the excess nematodes are pushed out by applying pressure from the connecting channel.

[0017] The nematodes are allowed to move freely for a while before being adsorbed and fixed. The connecting channel is opened to communicate with the atmosphere to reduce the disturbance of the liquid flow in the channel and the position of the nematodes changes little. Then the negative pressure of the main adsorption channel and the auxiliary adsorption channel is released. The nematodes move freely for a while, and the negative pressure of the main adsorption channel starts the nematodes to be adsorbed and fixed.

[0018] Imaging analysis was then performed.

[0019] Preferably, if the nematodes migrate before the main adsorption channel is activated, their position can be adjusted left and right through the auxiliary adsorption channel.

[0020] The present application provides a microfluidic chip device for nematode culture, storage, capture and imaging analysis. Its nematode culture method is no different from conventional nematode culture. It changes the customary closed nematode culture method in the chip, makes the culture environment the same as conventional culture, and eliminates the influence of chip technology on nematode culture. At the same time, open culture is more flexible in operation, accepts external operations, and has strong plasticity. This device can complete the efficient capture, fixation and optical imaging analysis of real-time cultured nematodes, as well as recycling and re-cultivation. When the nematodes are adsorbed and fixed, they are still surrounded by nematode culture medium and have food supply, reducing the amount of environmental changes. Imaging analysis can perform neuronal structure imaging, fluorescent labeling imaging, probe molecule imaging, dynamic monitoring of ion levels, drug stimulation response analysis, etc. This design can also be integrated with other nematode operation and analysis modules to develop into a multifunctional nematode analysis microfluidic chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic structural diagram of a microfluidic chip device for nematode culture, storage, capture and imaging analysis in one embodiment of the present invention;

[0022] Figure 2 This is a physical picture of a microfluidic chip device for nematode culture, storage, capture and imaging analysis in one embodiment of the present invention;

[0023] Figure 3 An adsorption and fixation module of a microfluidic chip device for nematode culture, storage, capture and imaging analysis in one embodiment of the present invention;

[0024] Figure 4 This is a diagram of an experiment of fluorescent imaging analysis of nematode motor neurons using an adsorption and fixation module of a microfluidic chip device for nematode culture, storage, capture and imaging analysis in one embodiment of the present invention.

[0025] Legend:

[0026] 1. Main adsorption channel; 2 / 3. Auxiliary adsorption channel; 4. Connecting channel; 5. Culture tank;. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments.

[0028] See also Figure 1 A microfluidic chip device for nematode culture, storage, capture and imaging analysis, wherein the chip material is PDMS (Polydimethylsiloxane), and the chip device comprises a culture pool 5 and a nematode adsorption and fixation module, wherein the top of the culture pool 5 is open and connected to the nematode adsorption and fixation module.

[0029] See also Figure 1 and Figure 2 In one embodiment, the culture pool 5 is roughly square, with a length and width of 1-2 cm and a depth of 0.3-0.8 cm. The culture pool 5 is located on the chip and has an opening at the top. A microfluidic chip channel opening is provided at the bottom edge of the culture pool 5, and the microfluidic chip channel opening is connected to the nematode adsorption and fixation module. Through the setting of the microfluidic chip channel opening, the nematodes can enter the nematode adsorption and fixation module through the microfluidic chip channel opening. The culture pool 5 has the functions of culturing, storing and recovering nematodes.

[0030] See also Figure 1 and Figure 2 In one embodiment, the nematode adsorption and fixation module includes a connecting channel 4 and a plurality of adsorption channels. In one embodiment, the connecting channel 4 is used to connect with the microfluidic chip channel opening at the bottom of the culture pool 5. The connecting channel 4 is 80 microns high and 80 microns wide. Both ends of the connecting channel 4 are opened, one end of which is used to connect with the microfluidic chip channel opening, and the other end is used for the nematode discharge.

[0031] The adsorption channel includes a main adsorption channel 1 and two auxiliary adsorption channels 2 and 3, wherein the main adsorption channel 1 is arranged perpendicular to the connecting channel 4, and the two auxiliary adsorption channels 2 and 3 are located on both sides of the width direction of the main adsorption channel 1. In one embodiment, the bottom of the main adsorption channel 1 and the auxiliary adsorption channels 2 and 3 are arranged coplanar with the bottom of the connecting channel 4.

[0032] The height of the main adsorption channel 1 and the auxiliary adsorption channels 2 and 3 is 40 microns, wherein the width of the main adsorption channel 1 is 740 microns, and it is used to adsorb and fix the main structure of the nematodes. The side of the main adsorption channel 1 away from the connecting channel 4 is used to connect to the negative pressure source of 50Kpa, and the side pointing to the connecting channel 4 is provided with a plurality of fine channels arranged side by side, and the fine channels are perpendicular to the connecting channel 4. In one embodiment, there are 10 fine channels, and the width of a single fine channel is 20 microns, and the interval between adjacent fine channels is 60 microns. The main adsorption channel 1 is connected to the connecting channel 4 through the fine channel. When the main adsorption channel 1 is working, it is difficult for the nematodes to pass through the 20-micron fine channel, thereby preventing the nematodes from being adsorbed.

[0033] The two auxiliary adsorption channels 2 and 3 are located on both sides of the main adsorption channel 1 in the width direction. The auxiliary adsorption channels 2 and 3 are used for capturing and micro-nanomanipulating nematodes. Specifically, the auxiliary adsorption channels 2 and 3 can be used to attract and capture nematodes, adjust the position of nematodes, and implement chemical drug stimulation on nematodes. Chemical drugs are introduced from the auxiliary adsorption channels 2 and 3 near the head of the nematodes, and the entered chemical drugs can flow out through the connecting channel 4 away from the end of the culture tank 5.

[0034] In other embodiments, the remaining parts of the microfluidic chip device can be designed into other functional modules to achieve functional integration.

[0035] Usage and operation process:

[0036] The culture pool 5 is made by hand cutting, not micro-nano processing.

[0037] When in use, the culture pool 5 is filled with liquid culture medium for culturing nematodes. The nematodes should be early adults, and the nematodes are more active during this period. After they adapt to the new environment, they can be captured and optically imaged for analysis. The opening of the microfluidic chip channel at the bottom of the "culture pool 5" is observed under a microscope. When a nematode approaches, the connecting channel 4 is closed, and the auxiliary adsorption channel 3 of the imaging analysis module is activated, and the end of the nematode is captured. The capture efficiency is extremely high, up to 100%.

[0038] Then the main adsorption channel 1 is started. If there are other nematodes in the main channel, the excess nematodes are pushed out by applying pressure from the connecting channel 4. This is also a method for recovering nematodes after the experiment.

[0039] Since the nematode has an S-shaped swinging movement characteristic, when it swings, its abdomen and back are on the side, and the lateral line is on the upper and lower sides. This is its movement characteristic. In this way, when the nematode moves freely, its body position is relatively fixed. If the nematode is adsorbed during its free movement, its body position will remain consistent, which is conducive to the comparison and analysis between nematode individuals. Therefore, it is necessary to let the nematode move freely for a moment before adsorbing and fixing it. The connecting channel 4 is opened to communicate with the atmosphere to reduce the disturbance of the liquid flow in the channel, and the position of the nematode changes little. Then the negative pressure of the main adsorption channel 1 and the auxiliary adsorption channels 2 and 3 is released. The nematode moves freely for a moment, and the negative pressure of the main adsorption channel 1 starts the nematode to be adsorbed to achieve its fixation operation. If the nematode migrates before starting the main adsorption channel 1, its position can be adjusted left and right through the auxiliary adsorption channels 2 and 3.

[0040] Then, the imaging analysis is performed. If the chip substrate uses a cover glass, it supports the use of an optical microscope with a 100x oil objective lens for imaging, which is highly magnified. The optical microscope is an inverted microscope.

[0041] Finally, the nematodes are released and sent back to the "cultivation pool 5" if further cultivation is required. If subsequent operations are required or they are abandoned, they continue to move forward along the connecting channel 4.

[0042] After the operation, rinse and save the chip for next use.

[0043] The nematodes can be firmly adsorbed and fixed for up to 2 hours; within 2 hours of adsorption and fixation, the vitality of the nematodes is less affected; if a more firm fixation effect is required, the negative pressure of the auxiliary adsorption channels 2 and 3 on both sides can be activated to suck the head and tail of the nematodes.

[0044] See also Figure 4 The microfluidic chip device provided in this application can perform fluorescence imaging of nematode neurons in culture. The nematodes in culture are captured and adsorbed and fixed, and their motor neurons are labeled with green fluorescent protein, which can be used to monitor the growth, development, damage and regeneration of neurons. Femtosecond laser ablation technology can be used to ablate nematode motor neurons at specific points to study nerve damage and regeneration. Applications also include imaging of calcium ions, cAMP levels, protein expression localization, and the effects of drug stimulation on neuronal function.

[0045] The present application provides a microfluidic chip device for nematode culture, storage, capture and imaging analysis. Its nematode culture method is no different from conventional nematode culture. It changes the customary closed nematode culture method in the chip, makes the culture environment the same as conventional culture, and eliminates the influence of chip technology on nematode culture. At the same time, open culture is more flexible in operation, accepts external operations, and has strong plasticity. This device can complete the efficient capture, fixation and optical imaging analysis of real-time cultured nematodes, as well as recycling and re-cultivation. When the nematodes are adsorbed and fixed, they are still surrounded by nematode culture medium and have food supply, reducing the amount of environmental changes. Imaging analysis can perform neuronal structure imaging, fluorescent labeling imaging, probe molecule imaging, dynamic monitoring of ion levels, drug stimulation response analysis, etc. This design can also be integrated with other nematode operation and analysis modules to develop into a multifunctional nematode analysis microfluidic chip.

Claims

1. A microfluidic chip device for nematode culture, storage, capture and imaging analysis, wherein the microfluidic chip device is made of PDMS, and is characterized by: The invention comprises a culture pool, the top of which is open, the length and width of the culture pool are 1-2 cm, and the depth is 0.3-0.8 cm.

2. A microfluidic chip device for nematode culture, storage, capture and imaging analysis according to claim 1, characterized in that: It also includes a nematode adsorption and fixation module, which includes a connecting channel and an adsorption channel, wherein the connecting channel is connected to the culture pool, both ends of the connecting channel are opened, one end of the connecting channel is opened and connected to the culture pool, and the adsorption channel is located on one side of the connecting channel in the width direction and is connected to the connecting channel.

3. A microfluidic chip device for nematode culture, storage, capture and imaging analysis according to claim 2, characterized in that: The connecting channel is 80 microns high and 80 microns wide. The adsorption channel includes a main adsorption channel, which is arranged perpendicular to the connecting channel. The side of the main adsorption channel away from the connecting channel is used to be connected to a negative pressure source of 50Kpa. A plurality of fine channels arranged side by side are arranged on the side pointing to the connecting channel. The fine channels are perpendicular to the connecting channel, and the main adsorption channel is connected to the connecting channel through the fine channels.

4. A microfluidic chip device for nematode culture, storage, capture and imaging analysis according to claim 3, characterized in that: There are 10 fine channels, the width of a single fine channel is 20 microns, the interval between adjacent fine channels is 60 microns, and the width of the main adsorption channel is 740 microns.

5. A microfluidic chip device for nematode culture, storage, capture and imaging analysis according to claim 4, characterized in that: The adsorption channel also includes an auxiliary adsorption channel, two of which are located on both sides of the width direction of the main adsorption channel. The side of the auxiliary adsorption channel away from the connecting channel is used to connect to negative pressure. The auxiliary adsorption channel is connected to the connecting channel, and the height of the auxiliary adsorption channel is 40 microns.

6. A microfluidic chip device for nematode culture, storage, capture and imaging analysis according to claim 5, characterized in that: The main adsorption channel, the auxiliary adsorption channel and the bottom of the communication channel are arranged on the same plane.

7. A microfluidic chip device for nematode culture, storage, capture and imaging analysis according to claim 6, characterized in that: The microfluidic chip operation process is as follows: When in use, the culture pool is filled with liquid culture medium for culturing nematodes, and experimental operations can be carried out after the nematodes adapt to the new environment; Observe the opening of the microfluidic chip channel at the bottom of the "culture pool" under a microscope. When a nematode approaches, close the connecting channel and start the auxiliary adsorption channel of the imaging analysis module, and the end of the nematode is captured. Then the main adsorption channel is started. If there are other nematodes in the main channel, the excess nematodes are pushed out by applying pressure from the connecting channel. The nematodes are allowed to move freely for a while before being adsorbed and fixed. The connecting channel is opened to communicate with the atmosphere to reduce the disturbance of the liquid flow in the channel and the position of the nematodes changes little. Then the negative pressure of the main adsorption channel and the auxiliary adsorption channel is released. The nematodes move freely for a while, and the negative pressure of the main adsorption channel starts the nematodes to be adsorbed and fixed. Imaging analysis was then performed.

8. A microfluidic chip device for nematode culture, storage, capture and imaging analysis according to claim 7, characterized in that: If the nematodes migrate before the main adsorption channel is activated, their position can be adjusted left and right through the auxiliary adsorption channel.

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