Interference-free zebra fish micro-fluidic chip

Abstract

The utility model belongs to the technical field of micro-fluidic chips, and particularly relates to an interference-free zebra fish micro-fluidic chip which comprises a channel assembly, a limiting opening and a containing channel. The channel assembly is connected with one side of the limiting opening; the accommodating channel is connected with the other side of the limiting opening; wherein the minimum length and width size of the channel assembly is larger than the maximum size of the limiting opening, and a fillet is formed at the transition position; the channel assembly is configured to be capable of conducting self-adaptive adjustment on the height and the width according to the size of the zebra fish. According to the interference-free micro-fluidic chip for the zebra fish, stress reaction to the zebra fish in the process of fixing the zebra fish through manual operation is avoided, the use of anesthetic is avoided, the physiological state of the zebra fish is not influenced, the accuracy and the objectivity of experimental data are ensured, the micro-fluidic chip can be used for adapting to juvenile zebra fish at different development time, and the working efficiency of the zebra fish is improved. Or the micro-fluidic chip is used for observation and shooting recording of other fingerlings, and the universality of the micro-fluidic chip is improved.

Description

Technical Field

[0001] The utility model belongs to the technical field of microfluidic chips, and particularly relates to an interference-free zebrafish microfluidic chip. Background Art

[0002] Zebrafish, due to their high transparency, strong permeability, small size, and ease of manipulation, have been widely used in a wide range of research applications, including research on vertebrate life systems, complex diseases, and drug screening. Manipulation and imaging of zebrafish are key enabling technologies for these studies.

[0003] The existing traditional methods for zebrafish manipulation mainly involve fixation with agarose and methylcellulose sodium and anesthesia with the anesthetic tricaine. However, the use of agarose and methylcellulose fixation is not only cumbersome, but also prone to introducing human interference errors and causing stress reactions in zebrafish, affecting the objectivity of the data and not meeting the requirements for long-term observation. The use of tricaine can also cause stress reactions in zebrafish, affecting the objectivity of the data, especially in the field of brain and nerve research. At the same time, the use of tricaine is also not sufficient for long-term observation. Therefore, how to achieve long-term zebrafish fixation and culture without affecting the physiological state of zebrafish is one of the key challenges in zebrafish-related research.

[0004] Therefore, providing a non-interference zebrafish microfluidic chip and its application is an urgent problem to be solved by those skilled in the art. Utility Model Content

[0005] In order to overcome the deficiencies of the prior art, the present invention provides an interference-free zebrafish microfluidic chip to solve the problems of the prior art.

[0006] One solution of the present invention provides a non-interference zebrafish microfluidic chip, including a channel assembly, a restriction port, and a receiving channel;

[0007] The channel assembly is connected to one side of the restriction port;

[0008] The accommodating channel is connected to the other side of the restriction opening;

[0009] The minimum length and width of the channel component are greater than the maximum size of the restriction port, and a rounded corner is formed at the transition between the channel component and the restriction port; the channel component is configured to be able to adaptively adjust the height and width according to the size of the zebrafish.

[0010] In one embodiment of the present invention, a liquid inlet is further included, wherein the liquid inlet is connected to the channel assembly;

[0011] and a liquid outlet, wherein the accommodating channel is connected to the liquid outlet.

[0012] In this solution, the water in the water flow channel flows into the channel assembly from the liquid inlet and flows out of the accommodating channel after passing through the restriction port. Under the continuous action of the water flow, the zebrafish fry transported through the water flow channel are pushed toward the channel assembly by the water flow, thereby achieving directional fixation of the zebrafish fry. This solution avoids the stress response caused to the zebrafish during the manual operation of fixing the zebrafish, as well as the use of anesthetics, does not affect the physiological state of the zebrafish, and ensures the accuracy and objectivity of the experimental data.

[0013] In one embodiment of the present invention, the channel component includes an identification component and a channel wall, the identification component is configured to identify the zebrafish entering the channel component, and the channel wall is configured to adjust the height and width of the channel component according to the identification result of the identification component.

[0014] In one solution of the present invention, the channel wall includes a fixed portion and a movable portion, and the movable portion adjusts its position relative to the fixed portion according to the recognition result of the recognition component.

[0015] In one solution of the present invention, the identification component is configured as a pressure sensor, which is disposed on the channel wall and is used to detect pressure changes generated by the fish body on the channel wall;

[0016] The movable part is connected to a micro-driving element and is used to adjust the position of the movable part after the pressure of the pressure sensor exceeds a preset value.

[0017] In one embodiment of the present invention, the identification component is configured as a miniature camera for capturing real-time images of the zebrafish in the channel component to identify the size information of the fish, and the identification component is further configured to collect the size information of the current channel component;

[0018] The movable part is connected to the micro-driving element and is used to adjust the position of the movable part when the size information of the fish body identified by the identification component does not match the size information of the current channel component.

[0019] In one solution of the present invention, the identification component is configured as a sensing electrode for detecting bioelectrical signals of the zebrafish in the channel component;

[0020] The movable part is used to adjust the position of the movable part when the strength and duration of the electrical signal detected by the identification component exceed standard values.

[0021] In one solution of the present invention, the identification component is configured as a flow rate sensor and a pressure sensor, and the identification component is used to monitor the flow rate and pressure changes of the fluid in the channel component;

[0022] The movable part is used to adjust the position of the movable part when the recognition component detects that the flow rate suddenly drops and the pressure rises beyond a preset value.

[0023] In one solution of the present invention, the cross-sectional shape of the restriction opening is set to any one of a bar, an ellipse or a rounded rectangle;

[0024] The accommodating channel is configured as an activity chamber for allowing the zebrafish tail to move, or the accommodating channel is configured as a restriction chamber for restricting the zebrafish tail from swinging.

[0025] In one embodiment of the present invention, the channel assembly, the restriction port, and the accommodating channel constitute a microstructure unit, and the non-interference zebrafish microfluidic chip includes a plurality of the microstructure units;

[0026] A portion of the accommodating channels of a plurality of the microstructure units is configured as a restriction cavity, and another portion of the accommodating channels is configured as an active cavity, so as to be combined to form a first type of zebrafish microfluidic chip;

[0027] Alternatively, the accommodating channels in a plurality of the microstructure units are configured as limiting cavities, so as to be combined to form a second type of zebrafish microfluidic chip;

[0028] Alternatively, the accommodating channels in several of the microstructure units are configured as active cavities, so as to form a third type of zebrafish microfluidic chip in combination.

[0029] In one solution of the present invention, the limiting opening corresponding to the limiting cavity is arranged in a horizontal direction or a vertical direction, and the cross-sectional shape of the limiting cavity is the same as the cross-sectional shape of the limiting opening.

[0030] In one solution of the present invention, the restriction opening corresponding to the movable cavity is set in a horizontal direction or a vertical direction.

[0031] The non-interference zebrafish microfluidic chip provided by this solution has the following beneficial effects:

[0032] 1. The hatched zebrafish larvae are transported to the microfluidic chip through a delivery pipe. Under the propulsion of the water flow, the zebrafish larvae are naturally pushed into the microstructure unit. The channel assembly is used to fix the zebrafish's head, and the restriction port is used to restrict the zebrafish's head from entering the accommodating channel. The accommodating channel is used to accommodate the zebrafish's tail. In addition, the channel assembly is configured to be able to adaptively adjust the height and width according to the size of the zebrafish. It can be used to accommodate zebrafish larvae at different developmental times, or for the observation and recording of other fish species, thereby improving the versatility of the microfluidic chip.

[0033] 2. It can achieve long-term zebrafish fixation and culture. While fixing the zebrafish larvae, water can still flow through the restricted mouth, keeping the zebrafish in a swimming physiological state. At the same time, it avoids the stress response caused by manual manipulation of the zebrafish and avoids the use of anesthetics, without affecting the physiological state of the zebrafish, ensuring the accuracy and objectivity of the experimental data. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0035] Figure 1 A schematic diagram showing the structure of a zebrafish microfluidic chip according to Example 1 of the present invention;

[0036] Figure 2 A schematic diagram showing the structure of another zebrafish microfluidic chip according to Example 1 of the present invention;

[0037] Figure 3 Figures showing the skeleton imaging of zebrafish in Example 3 of the present utility model on a common glass slide and a zebrafish microfluidic chip;

[0038] Figure 4 Schematic diagrams showing cardiac imaging of a zebrafish in Example 4 of the present invention on a common glass slide and a zebrafish microfluidic chip;

[0039] Figure 5 Graphs showing heart rate changes of zebrafish in Example 4 of the present invention on a common glass slide and a zebrafish microfluidic chip;

[0040] Figure 6 Showing images of the intestine of the zebrafish in Example 5 of the present invention on a common glass slide and a zebrafish microfluidic chip;

[0041] Figure 7 1. In vivo images of neutrophils in zebrafish of Example 6 of the present invention on a common glass slide and a zebrafish microfluidic chip;

[0042] Figure 8 Schematic diagram showing the in vivo vascular images of zebrafish in Example 7 of the present invention on a common glass slide and a zebrafish microfluidic chip;

[0043] Figure 9In vivo ROS (green fluorescence) images of zebrafish in Example 8 of the present invention on a common glass slide and a zebrafish microfluidic chip, respectively;

[0044] Figure 10 In vivo ROS (red fluorescence) images of zebrafish in Example 8 of the present invention on a common glass slide and a zebrafish microfluidic chip, respectively;

[0045] Figure 11 Showing the brain images of the zebrafish of Example 9 of the present invention on a common glass slide and a zebrafish microfluidic chip respectively.

[0046] Figure Numbers

[0047] 1-channel assembly, 2-restriction port, 3-accommodating channel, 4-liquid inlet, 5-liquid outlet. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), such directional indications are only used to explain the relative position relationship, movement status, etc. between the various components in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0050] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of such features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel schemes. Taking "A and / or B" as an example, it includes scheme A, or scheme B, or a scheme in which A and B are satisfied at the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0051] the term

[0052] As used herein, the terms “chip” and “microfluidic chip” are used interchangeably;

[0053] As used in this article, 7 dpf (days post fertilization), where dpf is the abbreviation for days after fertilization, for example 7 dpf means seven days after fertilization.

[0054] Example 1

[0055] Please refer to Figure 1 and Figure 2 , this embodiment provides a non-interference zebrafish microfluidic chip, including a channel component 1, a restriction port 2, and a receiving channel 3;

[0056] The channel assembly 1 is connected to one side of the restriction port 2;

[0057] The accommodating channel 3 is connected to the other side of the limiting opening 2;

[0058] Among them, the minimum length and width of the channel component are greater than the maximum size of the restriction port, and a rounded corner is formed at the transition between the channel component and the restriction port to prevent the zebrafish fry from being injured; the channel component 1 is configured to be able to adaptively adjust the height and width according to the size of the zebrafish.

[0059] In this embodiment, the interference-free zebrafish microfluidic chip includes a channel assembly 1, a restriction port 2, and a receiving channel 3. The channel assembly 1 is used to fix the head of the zebrafish fry, the restriction port 2 is used to fix the yolk sac of the zebrafish fry, and the receiving channel 3 is used to fix the tail of the zebrafish fry, so that the zebrafish fry is oriented and confined in the chip in a relatively fixed posture.

[0060] The channel assembly 1 is configured to be able to adaptively adjust its height and width according to the size of the zebrafish. It can be used to adapt to zebrafish juveniles at different developmental times, or for observing and photographing other fish species, thereby improving the versatility of the microfluidic chip.

[0061] At the same time, a milder fluid environment is provided for the zebrafish, which reduces the stimulation to the zebrafish larvae, allowing them to be more stably confined inside the microstructure unit, thereby achieving long-term zebrafish fixation and culture. While the zebrafish larvae are fixed, water can still flow through the restriction port 2, keeping the zebrafish in a swimming physiological state. At the same time, after the drug application is stopped, the water flow can promptly discharge the drugs or metabolic wastes in the microfluidic chip without affecting the physiological state of the zebrafish.

[0062] Furthermore, the fillet radius at the transition is 50-100 μm (the fillet is not shown in the figure in this embodiment).

[0063] Please refer to Figure 1and Figure 2 In one embodiment of the present utility model, it further includes a liquid inlet 4, and the liquid inlet 4 is connected to the channel assembly 1;

[0064] and a liquid outlet 5 , the accommodating channel 3 is connected to the liquid outlet 5 .

[0065] In this embodiment, the water in the water flow channel flows into the channel assembly 1 from the liquid inlet 4 and flows out of the accommodating channel 3 after flowing through the restriction port 2. Under the continuous action of the water flow, the zebrafish fry transported through the water flow channel are pushed toward the channel assembly 1 by the water flow, thereby achieving directional fixation of the zebrafish fry; the liquid outlet 5 is used to be connected to the water outlet channel in the water flow channel. The main function of the liquid outlet 5 is to make the internal channels of the microfluidic chip in a flowing state, so that the zebrafish is in a physiological state of swimming, and at the same time discharge the liquid inside the chip, including possible waste, unused drug solution or excrement produced by the zebrafish fry after the experiment.

[0066] In one embodiment of the present invention, the channel component 1 includes an identification component and a channel wall, the identification component is configured to identify the zebrafish entering the channel component 1, and the channel wall is configured to adjust the height and width dimensions of the channel component 1 according to the identification result of the identification component.

[0067] In one embodiment of the present invention, the channel wall includes a fixed portion and a movable portion, and the movable portion adjusts its position relative to the fixed portion according to the recognition result of the recognition component.

[0068] In one implementation scenario of this embodiment, the channel wall includes an upper channel wall, a lower channel wall, a left channel wall, and a right channel wall; wherein, when the upper channel wall and the lower channel wall are configured as fixed parts, the left channel wall and the right channel wall are configured as movable parts, and the width of the channel assembly is adjusted by the position of the movable parts; or, when the left channel wall and the right channel wall are configured as fixed parts, the upper channel wall and the lower channel wall are configured as movable parts, and the height of the channel assembly is adjusted by the position of the movable parts. This embodiment allows for flexible adjustment of the height and width of the channel assembly.

[0069] In one embodiment of the present invention, the identification component is configured as a pressure sensor, and the identification component is disposed on the channel wall to detect pressure changes generated by the fish body on the channel wall;

[0070] The movable part is connected to a micro-driving element and is used to adjust the position of the movable part after the pressure of the pressure sensor exceeds a preset value.

[0071] In this embodiment, a pressure sensor is installed within the channel. When a zebrafish enters the channel, the sensor detects the pressure changes exerted by the fish on the channel walls. For example, if the pressure exceeds a preset threshold, the control system activates an adjustment mechanism, such as using a micro-actuator to move the movable portion of the channel wall outward, increasing the channel's height and width to accommodate the fish's size. Assuming the preset threshold is a first numerical range, when the detected pressure reaches 1.5 times or more of the first numerical range, the motor activates, increasing the channel's width by 0.2 mm and its height by 0.1 mm.

[0072] In one embodiment of the present invention, the identification component is configured as a miniature camera for capturing real-time images of the zebrafish in the channel component 1 to identify the size information of the fish, and the identification component is further configured to collect the size information of the current channel component 1;

[0073] The movable part is connected to the micro-driving element and is used to adjust the position of the movable part when the size information of the fish body identified by the identification component does not match the size information of the current channel component 1.

[0074] In this embodiment, a high-resolution miniature camera is used to capture real-time images of zebrafish within the channel. The fish's dimensions are then analyzed using an image recognition algorithm. For example, parameters such as the fish's length, width, and height are identified. Based on these parameters, the intelligent control system calculates the required channel adjustment dimensions and controls the corresponding mechanical structure to make the adjustments. For example, if a fish's width is detected to be 1.1 mm, while the current channel width is only 1 mm, the system automatically expands the channel width to 1.2 mm to ensure that the fish can successfully enter the channel assembly 1 and pass through the restriction port 2 for positioning.

[0075] In one embodiment of the present invention, the identification component is configured as a sensing electrode for detecting the bioelectrical signal of the zebrafish in the channel component 1;

[0076] The movable part is used to adjust the position of the movable part when the strength and duration of the electrical signal detected by the identification component exceed standard values.

[0077] Zebrafish generate weak bioelectrical signals while swimming. In this embodiment, special electrodes are placed on the channel walls to detect these electrical signals, and the size of the fish is determined based on the signal's strength and characteristics. For example, if the detected electrical signal is strong and lasts for a long time, it indicates that the fish is large, and the system will increase the channel size accordingly. If the detected electrical signal strength exceeds a certain standard value, it will trigger an adjustment of the channel wall, increasing the channel height by 20% and the width by 15%.

[0078] In one embodiment of the present invention, the identification component is configured as a flow rate sensor and a pressure sensor, and the identification component is used to monitor the flow rate and pressure changes of the fluid in the channel component 1;

[0079] The movable part is used to adjust the position of the movable part when the recognition component detects that the flow rate suddenly drops and the pressure rises beyond a preset value.

[0080] In this embodiment, according to the principles of fluid mechanics, objects of different sizes produce different resistance and pressure changes in a fluid with the same flow rate. By monitoring the flow rate and pressure changes in the fluid within the channel, the size of the zebrafish can be indirectly inferred. For example, if the flow rate suddenly drops and the pressure rises, it may mean that the fish is too large and blocking the channel. In this case, the system will automatically adjust the channel size to restore normal flow rate and pressure. For example, the channel width can be instantly increased by 0.2 mm and the height by 0.1 mm.

[0081] In one embodiment of the present invention, the channel assembly 1, the restriction port 2, and the accommodating channel 3 constitute a microstructure unit. The microstructure unit is used to individually and directional fix the zebrafish. By arranging a plurality of microstructure units in a horizontal array or in a radial pattern, an interference-free zebrafish microfluidic chip is formed. The multiple microstructure units are arranged in parallel via one or more water flow channels to make each microstructure unit relatively independent, so that the zebrafish fry can be independently confined within the microstructure unit, facilitating the observation of the real-time status of the zebrafish fry.

[0082] A portion of the accommodating channels 3 of several of the microstructure units is configured as a restriction chamber, and another portion of the accommodating channels 3 is configured as an active chamber, so as to form a first type of zebrafish microfluidic chip in combination.

[0083] In this embodiment, by integrating the confinement chamber and the activity chamber on the same chip, multifunctionality is achieved, allowing for simultaneous immobilization and behavioral analysis of zebrafish larvae. Furthermore, integrating channels with different functions reduces the need to switch between different chips, allowing researchers to assess multiple physiological responses of zebrafish within the same experimental setup, simplifying experimental design and operational workflow, and improving experimental efficiency and continuity. Furthermore, the responses of zebrafish larvae to drug stimulation under different confinement conditions can be more intuitively demonstrated.

[0084] In another implementation scenario of this embodiment, the accommodating channels 3 in several of the microstructure units are configured as confining cavities, thereby forming a second type of zebrafish microfluidic chip. Alternatively, the accommodating channels 3 in several of the microstructure units are configured as active cavities, thereby forming a third type of zebrafish microfluidic chip.

[0085] In this implementation scenario, the second and third types of zebrafish microfluidic chips can be used for specialized research. For example, when the containing channels 3 of all microstructure units are configured as confined chambers, zebrafish image acquisition is facilitated. Another example is when the containing channels 3 of all microstructure units are configured as activity chambers. This design allows zebrafish to move freely in a less restrictive environment, facilitating the observation and analysis of their natural behavior, and focusing on studying the zebrafish's response to specific conditions (such as drug stimulation).

[0086] Therefore, this embodiment can achieve high-throughput screening, and the uniformly configured microstructure units can improve the throughput of the experiment, allowing multiple samples to be tested under the same experimental conditions at the same time.

[0087] In one embodiment of the present invention, the cross-sectional shape of the restriction opening 2 is set to be any one of a bar, an ellipse or a rounded rectangle;

[0088] The accommodating channel 3 is configured as an activity chamber for allowing the zebrafish's tail to move, or the accommodating channel 3 is configured as a restriction chamber for restricting the zebrafish's tail from swinging.

[0089] In one implementation scenario of this embodiment, the limiting opening 2 corresponding to the limiting cavity is set in a horizontal direction or a vertical direction, and the cross-sectional shape of the limiting cavity is the same as the cross-sectional shape of the limiting opening 2;

[0090] Alternatively, the restriction opening 2 corresponding to the movable cavity is set in a horizontal direction or a vertical direction.

[0091] In this embodiment, the cross-sectional shape of the restraining port 2 is diverse to accommodate different experimental requirements and the biological characteristics of zebrafish. A strip-shaped restraining port 2 provides ample lateral space for the zebrafish while limiting its forward and backward movement, while an elliptical or rounded rectangular cross-section provides a more uniform spatial distribution. A rounded rectangular cross-section reduces potential stress from sharp corners on the zebrafish, as the rounded corners reduce fluid shear forces. An elliptical restraining port 2 provides better visual quality, allowing for a clearer field of view when observing the zebrafish under a microscope.

[0092] In combination with the cross-sectional shape of the restriction opening 2 being set to any one of a bar, an ellipse, or a rounded rectangle, by setting the restriction opening 2 corresponding to the restriction chamber to a horizontal direction, the side of the zebrafish larvae fixed in the microstructure unit can be used as the main observation surface, making it easier for researchers to observe the internal tissue status of the zebrafish larvae or take side images of the zebrafish larvae;

[0093] For another example, by setting the restriction port 2 corresponding to the restriction chamber to a vertical direction, the top view of the zebrafish fry fixed in the microstructure unit can be used as the main observation surface, making it easier for researchers to observe the brain state of the zebrafish fry or take a top view image of the zebrafish fry.

[0094] Furthermore, the restriction port 2 corresponding to the activity chamber is set in a horizontal direction, which facilitates researchers to observe the physiological state of the zebrafish larvae's internal tissues under drug stimulation or in a natural state. Alternatively, the restriction port 2 corresponding to the activity chamber is set in a vertical direction, which facilitates researchers to observe and analyze the tail swinging state or brain activity of the zebrafish larvae under drug stimulation or in a natural state.

[0095] In one embodiment of the present invention, a non-interference zebrafish microfluidic chip is also provided, comprising a liquid inlet 4, a channel assembly 1, a restriction port 2, a receiving channel 3 and a liquid outlet 5;

[0096] The channel assembly 1 is connected to one side of the restriction port 2, and the liquid inlet 4 is connected to the channel assembly 1;

[0097] The accommodating channel 3 is connected to the other side of the restriction port 2, and the accommodating channel 3 is connected to the liquid outlet 5;

[0098] The channel assembly 1 and the accommodating channel 3 are configured to be able to adaptively adjust the height and width according to the size of the zebrafish.

[0099] In this embodiment, the technical solution for adaptively adjusting the height and width of the accommodating channel 3 is similar to the adjustment solution of the channel assembly 1 in the above-mentioned embodiments. It can adaptively adjust the height and width according to the size of the zebrafish, and can be used to adapt to zebrafish fry with different developmental times, or for observation and filming of other fish species, thereby improving the versatility of the microfluidic chip.

[0100] In one embodiment of the present invention, hatched zebrafish fry are transported to a microfluidic chip via a delivery pipe. Under the impetus of the water flow, the zebrafish fry are naturally pushed into the channel assembly 1 for fixing the zebrafish head. The restriction port 2 is used to restrict the zebrafish head from entering the accommodating channel 3. The accommodating channel 3 is used to accommodate the zebrafish tail, thereby achieving long-term zebrafish fixation and cultivation. While fixing the zebrafish fry, water can still flow through the restriction port 2, keeping the zebrafish in a swimming physiological state. At the same time, after stopping the application of the drug, the water flow can promptly discharge the drug or metabolic waste in the microstructure unit without affecting the physiological state of the zebrafish. This embodiment, based on the non-interference zebrafish microfluidic chip, avoids the stress response caused to the zebrafish during the manual operation of fixing the zebrafish, and avoids the use of anesthetics, without affecting the physiological state of the zebrafish, ensuring the accuracy and objectivity of the experimental data.

[0101] Example 2

[0102] Based on the interference-free zebrafish microfluidic chip described in any one of the above embodiments, the channel component 1 is configured in a conical shape, and the tip of the channel component 1 is connected to the restriction port 2 .

[0103] The conical channel assembly 1 can effectively guide and fix the zebrafish's head, ensuring its stable position during the experiment and reducing motion artifacts. At the same time, the conical channel helps smooth fluid flow, reduce turbulence and eddies, provide a milder fluid environment for the zebrafish, reduce the stimulation to the zebrafish larvae, and enable them to be more stably confined inside the microstructure unit. At the same time, the tip of the channel assembly 1 is connected to the restriction port 2, allowing the zebrafish larvae's tail to flow naturally into the accommodating channel 3 as pushed by the water flow, while the zebrafish larvae's head and yolk sac are retained by the restriction port 2 and remain in the channel assembly 1.

[0104] The restriction chamber is configured to be conical and contract outwards, for restricting the tail swing of the zebrafish; or the activity chamber is configured to be conical and expand outwards, with the tip of the activity chamber connected to the restriction port 2, for allowing the tail of the zebrafish to move.

[0105] The restriction chamber can be used to control the freedom of movement of the zebrafish larvae's tail. The restriction chamber's outward contraction design can limit unnecessary movement of the zebrafish's tail and improve the accuracy of experimental observation and measurement.

[0106] In addition, the activity chamber can be used to simulate the physiological behavior of zebrafish larvae. The outward-expanding conical design of the activity chamber can sufficiently expand the swing range of the zebrafish's tail in the natural environment, allowing them to exhibit more natural behavior in experiments. This is very useful for accurately studying the response of fish to specific stimuli, especially when observing the response of fish to drugs or environmental changes and taking multiple exposure images.

[0107] In one implementation scenario of this embodiment, the channel component 1 and the accommodating channel 3 are configured to be pyramidal or conical, so as to guide the zebrafish fry to enter and fix them in a directional manner; and the edges of the channel component 1 and the accommodating channel 3 are both provided with rounded corners to prevent the drug from remaining in the dead corners of the channel.

[0108] In this embodiment, the fixed channel height gradually decreases from the liquid inlet (900 μm) to the restriction port 2 (400 μm); the fixed channel length is 5 mm and the width is 1000 μm. Furthermore, the restriction channel height gradually decreases from the restriction port 2 (400 μm) to the liquid outlet 5 (100 μm); the restriction channel length is 5 mm and the width is 1000 μm.

[0109] Example 3

[0110] Based on the microfluidic chip of the utility model, an embodiment further provides an application scenario for collecting bone images, including:

[0111] (1) Select AB zebrafish that have developed to 7 dpf (days post fertilization) and place them in a 2 mL centrifuge tube. Add 1 mL of 0.2% calcein aqueous solution (Beijing Solebow Technology Co., Ltd.) and incubate at 28°C for 20 min.

[0112] (2) Wash three times with E3 water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4), 5 min each time, until the eluate is colorless;

[0113] (3) Place them on a regular glass slide and load them into a zebrafish microfluidic chip, and observe and image them under a microscope. The one placed on the regular glass slide is the effect control group.

[0114] The experimental results are as follows Figure 3 The images show the skeleton of a zebrafish on a regular glass slide and on a zebrafish microfluidic chip. The zebrafish skeleton cannot be imaged on the regular glass slide, but the skeleton of the zebrafish mounted on the zebrafish microfluidic chip is imaged (dashed box). This example demonstrates that the zebrafish microfluidic chip can be used for research on the zebrafish skeleton.

[0115] Example 4

[0116] Based on the microfluidic chip of the utility model, an embodiment further provides an application scenario for collecting cardiac images, including:

[0117] (1) AB zebrafish of 7 dpf (days post fertilization) were selected and placed on a glass slide. Tricaine was added to anesthetize the fish and the fish were placed under a microscope. The images were taken continuously for 1 min at a rate of 21 frames per second.

[0118] (2) AB zebrafish that developed to 7 dpf (days post fertilization) were selected, fixed using a zebrafish microfluidic chip, and placed under a microscope at 21 frames per second for 1 minute.

[0119] (3) Data statistics: GraphPad Prism 6.0 software was used to statistically process the data. The experimental data were expressed as mean ± SEM and analyzed by unpaired t-test. Compared with the 5th s, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0120] The results are Figure 4 The zebrafish heart images on a regular glass slide and a zebrafish microfluidic chip are shown. The zebrafish heart on the regular glass slide cannot be fully imaged, while the zebrafish heart fixed in the zebrafish microfluidic chip can be fully imaged (dashed box).

[0121] The results are Figure 5 The graphs showing changes in zebrafish heart rate on a conventional glass slide and on a zebrafish microfluidic chip show that, in the tricaine group, the zebrafish heart rate decreased significantly from the 25th second compared to the 5th second (p < 0.05). In the microfluidic wire group, the zebrafish heart rate remained unchanged compared to the 5th second (p > 0.05). This example demonstrates that immobilizing the zebrafish on the zebrafish microfluidic chip does not significantly affect the fish's heart rate, indicating that the zebrafish microfluidic chip can be used for research on the zebrafish heart.

[0122] Example 5

[0123] Based on the microfluidic chip of the utility model, an embodiment further provides an application scenario for collecting intestinal images, including:

[0124] (1) AB zebrafish that developed to 7 dpf (days post fertilization) were selected, placed on a glass slide, anesthetized with tricaine, and placed under a microscope to collect images.

[0125] (2) AB zebrafish that have developed to 7 dpf (days post fertilization) were selected, fixed using a zebrafish microfluidic chip, and placed under a microscope to capture images.

[0126] The results are Figure 6Images of the zebrafish intestine on a regular glass slide and on a zebrafish microfluidic chip show that the zebrafish intestine cannot be fully imaged on the regular glass slide. However, the zebrafish intestine immobilized on the zebrafish microfluidic chip can be fully imaged (dashed box). This example demonstrates that the zebrafish microfluidic chip can be used for research on the zebrafish intestine.

[0127] Example 6

[0128] Based on the microfluidic chip of the utility model, an embodiment further provides an application scenario for collecting neutrophil images, including:

[0129] (1) Select Tg(mpx:EGFP) transgenic zebrafish that have developed to 7 dpf (days post fertilization), place them on a glass slide, anesthetize them with tricaine, and place them under a microscope to collect images.

[0130] (2) Tg(mpx:EGFP) transgenic zebrafish that developed to 7 dpf (days post fertilization) were selected, fixed using a zebrafish microfluidic chip, and placed under a microscope to capture images.

[0131] The results are Figure 7 Images of zebrafish neutrophils on a regular glass slide and on a zebrafish microfluidic chip show that neutrophils (green fluorescence) in the zebrafish's head and tail (dashed boxes) are not fully imaged on the regular glass slide. However, neutrophils (green fluorescence) in the zebrafish immobilized on the zebrafish microfluidic chip can be fully imaged. This example demonstrates that the zebrafish microfluidic chip can be used to study specific cells in the zebrafish body.

[0132] Example 7

[0133] Based on the microfluidic chip of the utility model, an embodiment further provides an application scenario for collecting blood vessel images, including:

[0134] (1) Select Tg(fli1:EGFP) transgenic zebrafish that have developed to 7 dpf (days post fertilization), place them on a glass slide, anesthetize them with tricaine, and place them under a microscope to collect images.

[0135] (2) Tg(fli1:EGFP) transgenic zebrafish that developed to 7 dpf (days post fertilization) were selected, fixed using a zebrafish microfluidic chip, and placed under a microscope to capture images.

[0136] The results are Figure 8Images of zebrafish vasculature on a regular glass slide and on a zebrafish microfluidic chip show that the zebrafish vasculature (green fluorescence) (dashed box) is not fully imaged on the regular glass slide. However, the zebrafish vasculature (green fluorescence) (dashed box) can be fully imaged when mounted on the zebrafish microfluidic chip. This example demonstrates the applicability of the zebrafish microfluidic chip for zebrafish vascular research.

[0137] Example 8

[0138] Based on the microfluidic chip of the utility model, an embodiment further provides an application scenario for collecting reactive oxygen species images, including:

[0139] (1) AB zebrafish that developed to 7 dpf (days post fertilization) were selected and placed in a 24-well cell culture plate. The zebrafish were stained with 2 μM DCFH-DA reactive oxygen species (ROS) fluorescent probe (green fluorescence) (Beijing Solebold Technology Co., Ltd.) and 5 μM reactive oxygen species (ROS) detection reagent (red fluorescence) (Beijing Solebold Technology Co., Ltd.), respectively. After incubation at 28.5±1°C in the dark for 30 min,

[0140] (2) Wash the zebrafish three times with E3 water to remove the dye reagent, and then place the zebrafish under a fluorescence microscope for observation and photography.

[0141] The results are Figure 9 Images of ROS (green fluorescence) in vivo in zebrafish on a regular glass slide and in a zebrafish microfluidic chip show that ROS (green fluorescence) in the zebrafish intestine (dashed box) on the regular glass slide cannot be fully imaged, while ROS (green fluorescence) in the zebrafish intestine (dashed box) fixed in the zebrafish microfluidic chip can be fully imaged.

[0142] The results are Figure 10 Images of ROS (red fluorescence) in zebrafish on a regular glass slide and in a zebrafish microfluidic chip show that ROS (red fluorescence) in the zebrafish on the regular glass slide (dashed box) is not fully imaged. However, ROS (red fluorescence) in the zebrafish immobilized in the zebrafish microfluidic chip (dashed box) can be fully imaged. This example demonstrates that the zebrafish microfluidic chip can be used for fluorescent dye imaging studies in zebrafish.

[0143] Example 9

[0144] Based on the microfluidic chip of the utility model, an embodiment further provides an application scenario for collecting brain images, including:

[0145] (1) Tg(elavl3:H2B-GCaMP6f) transgenic zebrafish that developed to 7 dpf (days post fertilization) were selected, placed on a glass slide, anesthetized with tricaine, and placed under a microscope to collect images.

[0146] (2) Tg(elavl3:H2B-GCaMP6f) transgenic zebrafish that developed to 7 dpf (days post fertilization) were selected, fixed using a zebrafish microfluidic chip, and placed under a microscope to collect images.

[0147] The results are Figure 11 Images of a zebrafish brain mounted on a regular glass slide and on a zebrafish microfluidic chip show that the zebrafish brain cannot be fully imaged on the regular glass slide. However, the zebrafish brain mounted on the zebrafish microfluidic chip can be fully imaged. This example demonstrates the applicability of the zebrafish microfluidic chip for zebrafish brain neuroscience research.

[0148] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made by using the contents of the present invention specification and drawings under the utility model concept, or direct / indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A non-interference zebrafish microfluidic chip, characterized in that It includes a channel assembly, a restriction port, and an accommodating channel; The channel assembly is connected to one side of the restriction port, the channel assembly is tapered, and the tip of the channel assembly is connected to the restriction port. The tapered channel assembly is used to guide and fix the head of the zebrafish, and the restriction port is used to position the head and yolk sac of the zebrafish; The accommodating channel is connected to the other side of the restriction port, and the accommodating channel is used to accommodate the tail of the zebrafish; The chip further comprises a liquid inlet and a liquid outlet, wherein the liquid inlet is connected to the channel assembly; the accommodating channel is connected to the liquid outlet, and water flows from the liquid inlet into the channel assembly and flows out of the accommodating channel after passing through the restriction port, so as to keep the zebrafish in a physiological state of swimming and discharge the liquid inside the chip at the same time; Among them, the minimum length and width of the channel component are greater than the maximum size of the restriction port, and a rounded corner is formed at the transition between the channel component and the restriction port; the height of the channel component gradually decreases from the liquid inlet to the restriction port, and the width of the channel component is fixed.

2. The interference-free zebrafish microfluidic chip according to claim 1, wherein The cross-sectional shape of the restriction opening is set to any one of a bar, an ellipse or a rounded rectangle; The accommodating channel is configured as an activity chamber for allowing the zebrafish tail to move, or the accommodating channel is configured as a restriction chamber for restricting the zebrafish tail from swinging.

3. The interference-free zebrafish microfluidic chip according to claim 2, wherein The channel assembly, the restriction port and the accommodating channel constitute a microstructure unit, and the interference-free zebrafish microfluidic chip includes a plurality of the microstructure units.

4. The interference-free zebrafish microfluidic chip according to claim 3, wherein A portion of the accommodating channels of a plurality of the microstructure units is configured as a restriction cavity, and another portion of the accommodating channels is configured as an active cavity, so as to be combined to form a first type of zebrafish microfluidic chip; Alternatively, the accommodating channels in a plurality of the microstructure units are configured as limiting cavities, so as to be combined to form a second type of zebrafish microfluidic chip; Alternatively, the accommodating channels in several of the microstructure units are configured as active cavities, so as to form a third type of zebrafish microfluidic chip in combination.

5. The interference-free zebrafish microfluidic chip according to claim 2, wherein The limiting opening corresponding to the limiting cavity is arranged in a horizontal direction or a vertical direction, and a cross-sectional shape of the limiting cavity is the same as a cross-sectional shape of the limiting opening.

6. The interference-free zebrafish microfluidic chip according to claim 2, wherein: The restriction opening corresponding to the active cavity is set in a horizontal direction or a vertical direction.

Images