Non-interference zebrafish microfluidic chip and image acquisition method

Abstract

The present invention belongs to the technical field of microfluidic chips, and specifically relates to a non-interfering zebrafish microfluidic chip and an application method thereof, wherein the microfluidic chip comprises a channel component, a restriction port, and a receiving channel; the channel component is connected to one side of the restriction port; the receiving channel is connected to the other side of the restriction port; wherein the channel component is configured to be able to perform adaptive adjustment of height and width according to the size of the zebrafish. The non-invasive monitoring method realized by the non-interfering zebrafish microfluidic chip of the present invention avoids stress response to zebrafish during manual operation and fixation of zebrafish, and avoids the use of anesthetics, does not affect the physiological state of zebrafish, ensures the accuracy and objectivity of experimental data, can be used to adapt to zebrafish fry of different developmental times, or for observation and shooting and recording of other fish species, and improves the versatility of microfluidic chips.

Description

Technical Field

[0001] The invention belongs to the technical field of microfluidic chips, and in particular relates to a non-interference zebrafish microfluidic chip and an application method thereof. Background Art

[0002] Zebrafish have been widely used in many studies on vertebrate life systems, complex diseases, and drug screening due to their high transparency, strong permeability, small size, and easy operation. Among them, manipulation and imaging of zebrafish is one of the important enabling technologies for various studies.

[0003] The existing traditional methods for zebrafish manipulation are mainly agarose, methylcellulose sodium fixation and tricaine anesthesia. However, the use of agarose and methylcellulose fixation is not only cumbersome to operate, but also easy to introduce human interference errors, and cause zebrafish to have stress reactions, affecting the objectivity of the data, and cannot meet long-term observation. The use of tricaine will also cause zebrafish to have stress reactions, affecting the objectivity of the data, especially in the field of cranial nerve research. At the same time, the use of tricaine cannot meet 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 problems 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. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a non-interference zebrafish microfluidic chip and an application method thereof to solve the problems existing in 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 limiting opening;

[0009] Wherein, 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, it further comprises a liquid inlet, 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, water in the water flow channel flows into the channel assembly from the liquid inlet and flows out of the containing 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 stress reactions to zebrafish caused by manual manipulation of zebrafish fixation and the use of anesthetics, does not affect the physiological state of zebrafish, and ensures the accuracy and objectivity of experimental data.

[0013] In one of the embodiments 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 dimensions of the channel component according to the identification result of the identification component.

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

[0015] In one of the solutions 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 the pressure change caused 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 real-time image acquisition of zebrafish in the channel component to identify size information of the fish body, and the identification component is further configured to acquire 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 of the schemes 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;

[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 of the solutions 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 of the solutions of the present invention, the cross-sectional shape of the restriction opening is set to any one of a strip, an ellipse or a rounded rectangle;

[0024] The accommodating channel is configured as an activity chamber to allow the zebrafish tail to move, or the accommodating channel is configured as a restriction chamber to restrict 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 all configured as limiting chambers, 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 all configured as active chambers, so as to form a third type of zebrafish microfluidic chip in combination.

[0029] In one of the solutions 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 of the solutions of the present invention, the limiting opening corresponding to the active cavity is set in a horizontal direction or a vertical direction.

[0031] In one of the schemes of the present invention, an image acquisition method is also pointed out, comprising:

[0032] Fixing the zebrafish by a microfluidic chip, wherein the microfluidic chip is a non-interfering zebrafish microfluidic chip as described in any one of the above-mentioned schemes;

[0033] Based on the fixed posture of the zebrafish, performing zebrafish image acquisition or zebrafish real-time information comparison;

[0034] The zebrafish image acquisition includes at least one or more of: acquiring bone images, acquiring heart images, acquiring intestinal images, acquiring neutrophil images, acquiring blood vessel images, acquiring active oxygen images, and acquiring brain images.

[0035] The non-interference zebrafish microfluidic chip and image acquisition method provided in this scheme have the following beneficial effects:

[0036] 1. The hatched zebrafish fry are transported to the microfluidic chip through a conveying pipeline. The zebrafish fry are naturally pushed into the microstructure unit under the impetus of the water flow. The channel component is used to fix the head of the zebrafish, and the restriction port is used to restrict the head of the zebrafish from entering the accommodating channel. The accommodating channel is used to accommodate the tail of the zebrafish. In addition, the channel component is configured to be able to perform adaptive adjustment of height and width according to the size of the zebrafish. It can be used to adapt to zebrafish fry at different development times, or for the observation and shooting of other fish species, thereby improving the versatility of the microfluidic chip.

[0037] 2. It can achieve long-term fixation and culture of zebrafish. While fixing the zebrafish fry, the water flow can still flow through the restricted mouth, so that the zebrafish is in a swimming physiological state. At the same time, it avoids the stress response of zebrafish caused by manual operation of fixing zebrafish, and avoids the use of anesthetics, does not affect the physiological state of zebrafish, and ensures the accuracy and objectivity of experimental data. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0039] Figure 1 A schematic diagram showing the structure of a zebrafish microfluidic chip according to Example 1 of the present invention;

[0040] Figure 2 A schematic diagram showing the structure of another zebrafish microfluidic chip according to Example 1 of the present invention;

[0041] Figure 3 Skeletal imaging diagrams of zebrafish in Example 3 of the present invention in a common glass slide and a zebrafish microfluidic chip respectively;

[0042] Figure 4 The zebrafish heart imaging diagrams of Example 4 of the present invention in a common glass slide and a zebrafish microfluidic chip are shown;

[0043] Figure 5 Graphs showing changes in the heart rate of the zebrafish of Example 4 of the present invention in a common glass slide and in a zebrafish microfluidic chip;

[0044] Figure 6Showing the intestinal images of zebrafish in Example 5 of the present invention in a common glass slide and a zebrafish microfluidic chip respectively;

[0045] Figure 7 The images of in vivo neutrophils of zebrafish in Example 6 of the present invention in a common glass slide and a zebrafish microfluidic chip are shown;

[0046] Figure 8 In vivo vascular images of zebrafish in Example 7 of the present invention in a common glass slide and a zebrafish microfluidic chip are shown;

[0047] Fig. 9 The in vivo ROS (green fluorescence) images of zebrafish in Example 8 of the present invention in a common glass slide and a zebrafish microfluidic chip are shown;

[0048] Fig.10 The in vivo ROS (red fluorescence) images of zebrafish in Example 8 of the present invention in a common glass slide and a zebrafish microfluidic chip are shown;

[0049] Fig.11 The brain images of the zebrafish of Example 9 of the present invention are shown in a common glass slide and a zebrafish microfluidic chip respectively.

[0050] Reference Numbers

[0051] 1-channel assembly, 2-restriction port, 3-accommodating channel, 4-liquid inlet, 5-liquid outlet. DETAILED DESCRIPTION

[0052] 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 described embodiments 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 creative work are within the scope of protection of the present invention.

[0053] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the components in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0054] 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 used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or solutions that satisfy both A and B. 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 contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0055] the term

[0056] As used herein, the terms “chip” and “microfluidic chip” are used interchangeably;

[0057] As used herein, 7 dpf (days post fertilization), where dpf is the abbreviation for days after fertilization, for example 7 dpf means seven days after fertilization.

[0058] Example 1

[0059] 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;

[0060] The channel assembly 1 is connected to one side of the restriction opening 2;

[0061] The accommodating channel 3 is connected to the other side of the limiting opening 2;

[0062] The channel component 1 is configured to be able to adaptively adjust the height and width according to the size of the zebrafish.

[0063] In this embodiment, the interference-free zebrafish microfluidic chip includes a channel component 1, a restriction port 2 and a receiving channel 3. The channel component 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.

[0064] The channel component 1 is configured to be able to perform adaptive adjustment of height and width according to the size of the zebrafish, and can be used to adapt to zebrafish fry of different developmental times, or to be used for observation and photographing and recording of other fish species, thereby improving the versatility of the microfluidic chip;

[0065] At the same time, a milder fluid environment is provided for the zebrafish, which reduces the stimulation to the zebrafish fry, so that the zebrafish fry can be more stably confined inside the microstructure unit, and long-term zebrafish fixation and culture can be achieved. While the zebrafish fry are fixed, the water flow can still flow through the restriction port 2, so that the zebrafish is in a swimming physiological state. At the same time, after the application of the drug is stopped, the water flow can timely discharge the drug or metabolic waste in the microfluidic chip without affecting the physiological state of the zebrafish.

[0066] Please refer to Figure 1 and Figure 2 In one embodiment of the present invention, it further comprises a liquid inlet 4, wherein the liquid inlet 4 is connected to the channel assembly 1;

[0067] and a liquid outlet 5 , wherein the accommodating channel 3 is connected to the liquid outlet 5 .

[0068] In this embodiment, the water flow in the water flow channel flows into the channel assembly 1 from the liquid inlet 4, and flows out from 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 realizing the 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 water state, so that the zebrafish is in a physiological state of swimming, and at the same time, the liquid inside the chip is discharged, including possible waste, unused drug solutions or excrement produced by the zebrafish fry after the experiment.

[0069] 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.

[0070] In one of the embodiments 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.

[0071] 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 set as fixed parts, the left channel wall and the right channel wall are set as movable parts, and the width dimension of the channel component is adjusted by the position of the movable part; or, when the left channel wall and the right channel wall are set as fixed parts, the upper channel wall and the lower channel wall are set as movable parts, and the height dimension of the channel component is adjusted by the position of the movable part. This embodiment can flexibly adjust the height dimension and width dimension of the channel component.

[0072] 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 the pressure change caused by the fish body on the channel wall;

[0073] 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.

[0074] In this embodiment, by setting a pressure sensor in the channel, when the zebrafish enters the channel, the sensor will detect the pressure change caused by the fish body on the channel wall. For example, if the pressure value exceeds the preset threshold, the control system will start the adjustment mechanism, such as driving the movable part of the channel wall outward through the micro-drive element to increase the height and width of the channel to adapt to the size of the fish body. Assuming that the preset threshold is a first numerical interval, when the detected pressure reaches more than 1.5 times the first numerical interval, the motor starts to work, increasing the channel width by 0.2 mm and the height by 0.1 mm.

[0075] In one embodiment of the present invention, the identification component is configured as a miniature camera for real-time image acquisition of the zebrafish in the channel component 1 to identify the size information of the fish body, and the identification component is also configured to acquire the size information of the current channel component 1;

[0076] 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.

[0077] In this embodiment, a high-resolution micro camera is used to collect real-time images of zebrafish in the channel, and then the size of the fish body is analyzed by an image recognition algorithm. For example, the length, width, and height of the fish are identified. Based on these parameters, the intelligent control system calculates the required channel adjustment size and controls the corresponding mechanical structure to adjust it. For example, when the fish body width is detected to be 1.1 mm, and the current channel width is only 1 mm, the system will automatically expand the channel width to 1.2 mm to ensure that the fish can smoothly enter the channel component 1 and be limited by the restriction port 2.

[0078] 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;

[0079] 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.

[0080] Zebrafish generate weak bioelectric signals while swimming. In this embodiment, these electrical signals are detected by arranging special electrodes on the channel wall, and the size of the fish body is judged based on the strength and characteristics of the signal. For example, if the detected electrical signal is strong and lasts for a long time, it means that the fish body is large, and the system will increase the channel size accordingly. Assuming that when the detected electrical signal strength exceeds a specific standard value, it will trigger the adjustment of the channel wall, increasing the channel height by 20% and the width by 15%.

[0081] 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;

[0082] 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.

[0083] In this embodiment, according to the principles of fluid mechanics, objects of different sizes will produce different resistance and pressure changes in the fluid with the same flow rate. By monitoring the flow rate and pressure changes of the fluid in 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 large and blocks the channel. At this time, the system will automatically adjust the channel size to restore normal flow rate and pressure. For example, the channel width is instantly increased by 0.2 mm and the height is increased by 0.1 mm.

[0084] In one embodiment of the present invention, the channel assembly 1, the restriction port 2 and the accommodating channel 3 constitute a microstructure unit, and the microstructure unit is used to individually and directional fix the zebrafish. A plurality of microstructure units are arranged in a horizontal array or in a radial shape to form an interference-free zebrafish microfluidic chip, wherein a plurality of microstructure units are arranged in parallel through one or more water flow channels to make each microstructure unit relatively independent, so that the zebrafish fry can be independently confined in the microstructure unit, which is convenient for observing the real-time status of the zebrafish fry;

[0085] A portion of the accommodating channels 3 of the plurality of 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.

[0086] In the implementation scenario of this embodiment, by integrating the confinement chamber and the activity chamber on the same chip, multifunctionality can be achieved, and the fixation and behavior analysis of zebrafish larvae can be performed simultaneously. In addition, the integration of channels with different functions can reduce the need to switch between different chips. Researchers can evaluate multiple physiological responses of zebrafish in the same experimental setting, simplifying the experimental design and operation process, and improving the efficiency and continuity of the experiment; and more intuitively showing the response of zebrafish larvae to drug stimulation under different confinement conditions.

[0087] In another implementation scenario of this embodiment, the accommodating channels 3 in several of the microstructure units are all configured as restricted cavities, which are used to form a second type of zebrafish microfluidic chip in combination. Or the accommodating channels 3 in several of the microstructure units are all configured as active cavities, which are used to form a third type of zebrafish microfluidic chip in combination.

[0088] In this implementation scenario, the second and third types of zebrafish microfluidic chips can be used for specific research. For example, when the accommodating channels 3 of all microstructure units are set as restricted chambers, it is convenient to collect zebrafish images. For another example, when the accommodating channels 3 of all microstructure units are set as activity chambers, this design allows zebrafish to move freely in a less restricted environment, which is convenient for observing and analyzing their natural behaviors, and focusing on studying the response of zebrafish to specific conditions (such as drug stimulation).

[0089] 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.

[0090] In one embodiment of the present invention, the cross-sectional shape of the limiting opening 2 is set to be any one of a strip, an ellipse or a rounded rectangle;

[0091] The accommodating channel 3 is configured as an activity chamber to allow the zebrafish's tail to move, or the accommodating channel 3 is configured as a restriction chamber to restrict the zebrafish's tail from swinging.

[0092] 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;

[0093] Alternatively, the limiting opening 2 corresponding to the active cavity is arranged in a horizontal direction or a vertical direction.

[0094] In this embodiment, the cross-sectional shapes of the restriction port 2 are diversified to meet different experimental requirements and the biological characteristics of zebrafish; among them, the restriction port 2 with a strip cross-section can provide sufficient lateral space for the zebrafish while limiting its forward and backward movement, while the elliptical or rounded rectangular cross-section can provide a more uniform spatial distribution. The rounded rectangular cross-section can reduce the potential stress caused by sharp corners to the zebrafish, because the rounded corners can reduce the shear force of the fluid. The elliptical restriction port 2 can provide a better visual effect, so that the zebrafish has a clearer field of view when observed under a microscope.

[0095] In combination with the cross-sectional shape of the restriction opening 2 being set to any one of a strip, 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 fry fixed in the microstructure unit can be used as the main observation surface, which is convenient for researchers to observe the in vivo tissue state of the zebrafish fry or take a side image of the zebrafish fry;

[0096] 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, which is convenient for researchers to observe the brain state of the zebrafish fry or take a top view image of the zebrafish fry.

[0097] Also, the restriction port 2 corresponding to the activity chamber is set in a horizontal direction, which is convenient for researchers to observe the physiological state of the zebrafish fry's in vivo 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 is convenient for researchers to observe and analyze the tail swinging state or brain activity state of the zebrafish fry under drug stimulation or in a natural state.

[0098] In one embodiment of the present invention, a non-interference zebrafish microfluidic chip is also disclosed, comprising a liquid inlet 4, a channel component 1, a restriction port 2, a receiving channel 3 and a liquid outlet 5;

[0099] The channel component 1 is connected to one side of the restriction port 2, and the liquid inlet 4 is connected to the channel component 1;

[0100] The accommodating channel 3 is connected to the other side of the limiting opening 2, and the accommodating channel 3 is connected to the liquid outlet 5;

[0101] 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.

[0102] In this embodiment, the technical solution for realizing adaptive adjustment of 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 at different developmental times, or for observation and photographing and recording of other fish species, thereby improving the versatility of the microfluidic chip.

[0103] In one embodiment of the present invention, an image acquisition method is also provided, comprising:

[0104] S10, fixing the zebrafish by a microfluidic chip, wherein the microfluidic chip is the non-interfering zebrafish microfluidic chip described in any one of the above embodiments;

[0105] S20, based on the fixed posture of the zebrafish, performing zebrafish image acquisition or zebrafish real-time information comparison;

[0106] The zebrafish image acquisition includes at least one or more of: acquiring bone images, acquiring heart images, acquiring intestinal images, acquiring neutrophil images, acquiring blood vessel images, acquiring active oxygen images, and acquiring brain images.

[0107] In this embodiment, the hatched zebrafish fry is transported to the microfluidic chip through a delivery pipeline, and the zebrafish fry is naturally pushed to the channel component 1 under the impetus of the water flow, which is used to fix the zebrafish head, and the restriction port 2 is used to limit the zebrafish head from entering the accommodating channel 3, and the accommodating channel 3 is used to accommodate the tail of the zebrafish, so as to achieve long-term zebrafish fixation and cultivation, and while fixing the zebrafish fry, the water flow can still flow through the restriction port 2, so that the zebrafish is in a swimming physiological state, and after stopping the application of the drug, the water flow can timely discharge the drug or metabolic waste in the microstructure unit, without affecting the physiological state of the zebrafish. Based on the non-interference zebrafish microfluidic chip, this embodiment avoids the stress response caused to the zebrafish during the manual operation of fixing the zebrafish, and avoids the use of anesthetics, does not affect the physiological state of the zebrafish, and ensures the accuracy and objectivity of the experimental data.

[0108] Example 2

[0109] Based on the non-interference zebrafish microfluidic chip described in any one of the above-mentioned embodiments, the channel component 1 is arranged in a conical shape, and the tip of the channel component 1 is connected to the restriction port 2.

[0110] The conical channel component 1 can effectively guide and fix the head of the zebrafish, ensure its stable position during the experiment, and reduce motion artifacts; at the same time, the conical channel helps to smooth the flow of fluid, reduce turbulence and eddy currents, provide a milder fluid environment for the zebrafish, reduce the stimulation to the zebrafish fry, and enable it to be more stably confined inside the microstructure unit; at the same time, the tip of the channel component 1 is connected to the restriction port 2, so that the tail of the zebrafish fry can naturally flow into the accommodating channel 3 as pushed by the water flow, while the head and yolk sac of the zebrafish fry are blocked by the restriction port 2 and stay in the channel component 1.

[0111] The restriction chamber is configured in an outwardly contracting cone shape to restrict the tail swing of the zebrafish; or, the activity chamber is configured in an outwardly expanding cone shape, and the tip of the activity chamber is connected to the restriction port 2 to allow the tail of the zebrafish to move.

[0112] The restriction chamber can be used to control the freedom of movement of the tail of a zebrafish larva. The restriction chamber can limit unnecessary movement of the zebrafish tail through its outward contraction design, thereby improving the accuracy of experimental observation and measurement.

[0113] In addition, the activity chamber can be used to simulate the physiological behavior of zebrafish larvae. The outward expansion cone design of the activity chamber can be sufficient for the swing range of the zebrafish's tail movement in the natural environment, allowing them to exhibit more natural behaviors 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.

[0114] In one implementation scenario of the present embodiment, the channel component 1 and the receiving 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 receiving channel 3 are both provided with rounded corners to prevent the drug from remaining in the dead corners of the channel.

[0115] In this embodiment, the height of the fixed channel gradually decreases from the liquid inlet (900 μm) to the restriction port 2 (400 μm); the length of the fixed channel is 5 mm and the width is 1000 μm. Also, the height of the restriction channel gradually decreases from the restriction port 2 (400 μm) to the liquid outlet 5 (100 μm); the length of the restriction channel is 5 mm and the width is 1000 μm.

[0116] Example 3

[0117] In an embodiment, a method for acquiring a bone image is also provided, comprising:

[0118] (1) AB zebrafish at 7 dpf (days post fertilization) were selected and placed in a 2 mL centrifuge tube. 1 mL of 0.2% calcein aqueous solution (Beijing Solebow Technology Co., Ltd.) was added and incubated at 28°C for 20 min.

[0119] (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 eluent is colorless;

[0120] (3) The cells were placed on a regular glass slide and loaded into a zebrafish microfluidic chip, respectively, and observed and imaged using a microscope. The cells placed on the regular glass slide served as the effect control group.

[0121] The experimental results are as follows Figure 3 The zebrafish skeleton images shown in the figure are respectively on a common glass slide and a zebrafish microfluidic chip. The skeleton of the zebrafish cannot be imaged on the common glass slide. However, the skeleton of the zebrafish fixed in the zebrafish microfluidic chip is imaged (dashed box). In this embodiment, it is shown that the zebrafish microfluidic chip can be used in the research of the zebrafish skeleton field.

[0122] Example 4

[0123] In an embodiment, a method for acquiring a cardiac image is also provided, comprising:

[0124] (1) AB zebrafish that had developed to 7 dpf (days post fertilization) were selected, placed on a glass slide, anesthetized with tricaine, and placed under a microscope at 21 frames per second for 1 min.

[0125] (2) AB zebrafish that had 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 min.

[0126] (3) Data statistics: GraphPad Prism 6.0 software was used to 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.

[0127] Results from Figure 4 The zebrafish heart images on a regular glass slide and a zebrafish microfluidic chip are shown in the figure. 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).

[0128] Results from Figure 5 As shown in the graphs of the heart rate changes of zebrafish in ordinary glass slides and zebrafish microfluidic chips, in the tricaine group, compared with the 5th second, the heart rate of zebrafish was significantly reduced from the 25th second (p<0.05). In the microfluidic wire group, compared with the 5th second, the heart rate of zebrafish did not change (p>0.05). In this embodiment, it is shown that the zebrafish microfluidic chip does not significantly affect the heart rate of zebrafish after fixing the zebrafish, that is, the zebrafish microfluidic chip can be used in the research of zebrafish heart field.

[0129] Example 5

[0130] In an embodiment, a method for collecting intestinal images is also provided, including:

[0131] (1) AB zebrafish that had 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.

[0132] (2) AB zebrafish that had developed to 7 dpf (days post fertilization) were selected, fixed using a zebrafish microfluidic chip, and placed under a microscope to collect images.

[0133] Results from Figure 6 As shown in the images of zebrafish intestine on a common glass slide and in a zebrafish microfluidic chip, the intestine of zebrafish cannot be completely imaged on a common glass slide. However, the intestine of zebrafish fixed in the zebrafish microfluidic chip can be completely imaged (dashed box). In this embodiment, it is shown that the zebrafish microfluidic chip can be used in the research of zebrafish intestine.

[0134] Example 6

[0135] In an embodiment, a method for acquiring a neutrophil image is also provided, comprising:

[0136] (1) Tg(mpx:EGFP) transgenic zebrafish that had 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.

[0137] (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 collect images.

[0138] Results from Figure 7As shown in the images of zebrafish neutrophils in vivo on a common glass slide and in a zebrafish microfluidic chip, the neutrophils (green fluorescence) in the head and tail (dashed box) of the zebrafish in the common glass slide cannot be fully imaged. However, the neutrophils (green fluorescence) in the zebrafish fixed in the zebrafish microfluidic chip can be completely imaged. In this embodiment, it is shown that the zebrafish microfluidic chip can be used to study specific cells in zebrafish.

[0139] Example 7

[0140] In an embodiment, a method for acquiring a blood vessel image is also provided, comprising:

[0141] (1) Tg(fli1:EGFP) transgenic zebrafish that had 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.

[0142] (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 collect images.

[0143] Results from Figure 8 As shown in the images of zebrafish blood vessels in the ordinary glass slide and the zebrafish microfluidic chip, the blood vessels (green fluorescence) (dashed line frame) in the zebrafish body on the ordinary glass slide cannot be fully imaged. However, the blood vessels (green fluorescence) (dashed line frame) in the zebrafish body fixed in the zebrafish microfluidic chip can be completely imaged. In this embodiment, it is shown that the zebrafish microfluidic chip can be used in the research of zebrafish blood vessels.

[0144] Example 8

[0145] In an embodiment, a method for collecting an active oxygen image is also provided, comprising:

[0146] (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℃ in the dark for 30 min,

[0147] (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.

[0148] Results from Fig. 9 The images of ROS (green fluorescence) in vivo in zebrafish on a regular glass slide and in a zebrafish microfluidic chip show that the ROS (green fluorescence) in the zebrafish intestine on the regular glass slide (dashed box) cannot be fully imaged. However, the ROS (green fluorescence) in the zebrafish intestine fixed in the zebrafish microfluidic chip (dashed box) can be fully imaged.

[0149] Results from Fig.10 As shown in the images of ROS (red fluorescence) in zebrafish on a common glass slide and in a zebrafish microfluidic chip, the ROS (red fluorescence) (dashed line frame) in the zebrafish on the common glass slide cannot be fully imaged. However, the ROS (red fluorescence) (dashed line frame) in the zebrafish fixed in the zebrafish microfluidic chip can be completely imaged. In this embodiment, it is shown that the zebrafish microfluidic chip can be used in the study of fluorescent dye imaging of zebrafish.

[0150] Example 9

[0151] In an embodiment, a method for acquiring a brain image is also provided, comprising:

[0152] (1) Tg(elavl3:H2B-GCaMP6f) transgenic zebrafish that had 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.

[0153] (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.

[0154] Results from Fig.11 As shown in the images of the zebrafish brain on a common glass slide and in a zebrafish microfluidic chip, the zebrafish brain cannot be completely imaged on the common glass slide. However, the zebrafish brain fixed in the zebrafish microfluidic chip can be completely imaged. In this embodiment, it is shown that the zebrafish microfluidic chip can be used in the research of zebrafish brain nerves.

[0155] 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 changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly / indirectly applied 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 a receiving channel; The channel component is connected to one side of the restriction port, and 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; The accommodating channel is connected to the other side of the limiting opening; Also includes a liquid inlet, the liquid inlet is connected to the channel assembly; and a liquid outlet, the accommodating channel being connected to the liquid outlet; Wherein, the channel component is configured to be able to adaptively adjust the height and width according to the size of the zebrafish.

2. The interference-free zebrafish microfluidic chip according to claim 1, characterized in that: 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.

3. The interference-free zebrafish microfluidic chip as claimed in claim 2, characterized in that: The identification component is configured as a pressure sensor, which is disposed on the channel wall and is used to detect the pressure change caused by the fish body on the channel wall. 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. Alternatively, the identification component is configured as a micro camera for real-time image acquisition of the zebrafish in the channel component to identify the size information of the fish body, and the identification component is further configured to acquire the size information of the current channel component, and the movable part is connected to a micro driving element for adjusting 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; Alternatively, the identification component is configured as a sensing electrode for detecting the bioelectrical signal of the zebrafish in the channel component, and for adjusting the position of the movable part when the intensity and duration of the electrical signal detected by the identification component exceed a standard value; Alternatively, 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, and to adjust the position of the movable part when the identification component detects a sudden drop in flow rate and a pressure increase exceeding a preset value.

4. The interference-free zebrafish microfluidic chip according to claim 1, characterized in that: The cross-sectional shape of the limiting opening is set to any one of a strip, an ellipse or a rounded rectangle; The accommodating channel is configured as an activity chamber to allow the zebrafish tail to move, or the accommodating channel is configured as a restriction chamber to restrict the zebrafish tail from swinging.

5. The interference-free zebrafish microfluidic chip according to claim 4, characterized in that: 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; 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 all configured as limiting chambers, 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 all configured as active chambers, so as to form a third type of zebrafish microfluidic chip in combination.

6. The non-interference zebrafish microfluidic chip according to claim 4, characterized in that: 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.

7. The interference-free zebrafish microfluidic chip according to claim 4, characterized in that: The limiting opening corresponding to the active cavity is arranged in a horizontal direction or a vertical direction.

8. An image acquisition method, characterized in that: include: Fixing the zebrafish by a microfluidic chip, wherein the microfluidic chip is the non-interfering zebrafish microfluidic chip according to any one of claims 1 to 7; Based on the fixed posture of the zebrafish, performing zebrafish image acquisition or zebrafish real-time information comparison; The zebrafish image acquisition includes at least one or more of: acquiring bone images, acquiring heart images, acquiring intestinal images, acquiring neutrophil images, acquiring blood vessel images, acquiring active oxygen images, and acquiring brain images.

Images