An experimental zebrafish drug screening auxiliary device

Abstract

This invention discloses an experimental zebrafish drug screening auxiliary device in the field of biological experimental auxiliary devices. It includes a carrier with several culture wells circumferentially opened at the top. A first mixing chamber and a second mixing chamber are formed within the carrier. The first mixing chamber is connected to an inlet mechanism for delivering drug solution into the first mixing chamber. A transfer mixing mechanism is provided within the first mixing chamber to homogenize the drug solution based on the boundary layer effect and deliver the drug solution to the second mixing chamber. A transfer mechanism is connected to the end of the second mixing chamber away from the first mixing chamber to connect the first and second mixing chambers. An isolation mechanism connects the culture wells and the second mixing chamber to control the connection between the two chambers. This invention can quickly and efficiently achieve uniform dispersion of the drug solution throughout the entire well area, thereby improving the reliability of experimental data.

Description

Technical Field

[0001] This invention belongs to the field of biological experimental auxiliary devices, specifically an experimental zebrafish drug screening auxiliary device. Background Technology

[0002] Zebrafish, due to their high genetic homology with humans (approximately 87%), easily observable transparent bodies, high reproductive capacity, and low experimental costs, have become an indispensable model organism in life science research and early drug screening. In the drug discovery stage, high-throughput screening using juvenile zebrafish allows for rapid and intuitive evaluation of the efficacy and potential toxicity of compounds on specific physiological and pathological processes (such as angiogenesis, tumor metastasis, neurodevelopment, and inflammatory responses), significantly improving the efficiency of new drug development.

[0003] Currently, drug screening based on zebrafish larvae typically follows this basic procedure: zebrafish larvae at a specific post-fertilization period (e.g., 2-5 days) are randomly assigned to the wells of a multi-well plate (e.g., a 96-well or 384-well plate) using manual pipetting. Then, different concentrations or types of the test compound are added to each well. After a period of exposure, the multi-well plate is observed, photographed, and the data analyzed under a stereomicroscope or high-content imaging system.

[0004] However, the dissolution process of the drug stock solution after being added to the aqueous culture medium is entirely passive and random. The dispersion of drug molecules is highly dependent on their own free diffusion and weak convection, which is an extremely slow and uncontrollable physical process. Hydrophobic drugs have poor diffusion ability in the aqueous phase, making it difficult to quickly and uniformly distribute them throughout the entire pore space. The system takes a long time to reach concentration equilibrium after drug addition, meaning that in the critical initial exposure phase, zebrafish are constantly in an environment of unknown and continuously changing concentrations, making it impossible to accurately define the onset time and kinetic characteristics of drug action, severely affecting the precise evaluation of efficacy and toxicity timelines. Furthermore, after manual addition, the high-concentration stock solution droplet point will form a temporary, locally extremely high concentration zone. Without immediate, gentle, and effective active mixing, zebrafish swimming through this area will suffer acute "drug shock." The acute toxicity, stress response, or tissue damage caused by this shock is not a specific pharmacological / toxicological effect of the drug itself at a uniform concentration, but rather an experimental bias introduced by human operation, thus misleading the judgment of the drug's true safety and efficacy.

[0005] Given the shortcomings of existing technologies, there is an urgent need for a new type of auxiliary device that can achieve rapid, gentle, controllable and thorough mixing of drug solution and culture medium, ensuring that a highly uniform drug concentration field can be established in the zebrafish's living environment from the beginning of exposure, thereby fundamentally improving the quality and scientific validity of zebrafish drug screening models. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide an experimental zebrafish drug screening auxiliary device that can quickly and efficiently achieve uniform dispersion of the drug solution throughout the entire well range, thereby improving the reliability of experimental data.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] An experimental zebrafish drug screening auxiliary device includes a carrier with several culture wells circumferentially opened at the top of the carrier. A first mixing chamber and a second mixing chamber are opened inside the carrier. The first mixing chamber is connected to a liquid inlet mechanism for delivering drug solution into the first mixing chamber. A transfer mixing mechanism is provided in the first mixing chamber for mixing the drug solution based on the boundary layer effect and delivering the drug solution to the second mixing chamber. A transfer mechanism is connected to the end of the second mixing chamber away from the first mixing chamber for connecting the first mixing chamber and the second mixing chamber.

[0009] An isolation mechanism is connected between the culture well and the second mixing chamber, and the isolation mechanism is used to control the opening and closing of the culture well and the second mixing chamber.

[0010] Working Principle: The drug stock solution and deionized water are introduced into the first mixing chamber in a specific ratio through the liquid inlet mechanism. A transfer mixing mechanism within the first mixing chamber actively mixes the drug solution based on the boundary layer effect (a hydrodynamic phenomenon where fluid velocity decreases near a solid surface, forming a gradient and enhancing mixing efficiency). This mechanism breaks up the aggregation of drug molecules, accelerating their dispersion in the aqueous phase, which is faster and more uniform than passive diffusion. The mixed drug solution is pumped into the second mixing chamber by the transfer mixing mechanism, and then returned to the first mixing chamber via a transfer mechanism, forming a closed loop. This loop design allows the drug solution to pass through the mixing area multiple times, ensuring a highly uniform drug concentration throughout the chamber and avoiding localized concentration fluctuations.

[0011] After mixing, the isolation mechanism opens, connecting the second mixing chamber to the culture wells. Because the mixing mechanism draws in a small amount of gas during operation, a slight positive pressure is created in both the first and second mixing chambers, allowing the medication to be smoothly injected into each culture well under pressure. This injection method avoids turbulence or impact, ensuring that the zebrafish juveniles are directly exposed to a uniform concentration environment.

[0012] The above approach has the following beneficial effects:

[0013] 1. This protocol, through an active mixing and circulation system, ensures thorough mixing of the drug solution before injection into the culture wells, establishing a highly uniform drug concentration field from the outset of exposure. This eliminates the problems of unknown and fluctuating concentrations, making the assessment of efficacy and toxicity timelines more accurate and improving the reliability and repeatability of experimental data.

[0014] 2. In traditional methods, manually added drops can create localized high-concentration areas that may induce acute stress in zebrafish, leading to experimental bias. This invention, through a stable injection method, prevents these localized high-concentration shocks, ensuring that the observed effects are due to the drug's specific action rather than human error, thereby improving the accuracy of assessing drug safety and efficacy.

[0015] 3. This approach enables rapid, gentle, and controllable mixing, shortening the time required for drug diffusion and making high-throughput screening more efficient. Simultaneously, the uniform concentration field reduces experimental variables, enhancing the scientific rigor of the zebrafish model and aiding in early drug discovery decisions.

[0016] Automated mixing and dispensing processes reduce human error and make experimental results from different batches or different laboratories more comparable.

[0017] The mixing process avoids severe mechanical impact (such as excessive shearing by eddies), and the medication is injected into the culture wells in a controlled manner to prevent physical damage to the zebrafish fry. The isolation mechanism is designed to ensure a smooth transition of the medication and maintain the normal physiological state of the zebrafish.

[0018] Furthermore, the liquid inlet mechanism includes a drug inlet tube, one end of which is connected to the first mixing chamber, and the other end of which is provided with a sealing plug.

[0019] Beneficial effects: Removing or opening the sealing plug creates an open channel for the drug inlet. Precisely measured doses of the drug stock solution and deionized water can then be injected through this inlet using tools such as pipettes. Immediately replace the sealing plug afterward to re-establish a closed system (first mixing chamber, second mixing chamber, and inlet mechanism).

[0020] The sealing plug design completely isolates the system from external air and environmental contaminants such as bacteria and fungi after chemical dosing. This sealing plug ensures the system's airtightness, thereby guaranteeing a stable and controllable internal pressure environment.

[0021] Furthermore, the transmission and mixing mechanism includes a drive component, a transmission shaft, and several discs; the drive component is located at the bottom of the first mixing chamber and is used to drive the transmission shaft to rotate; one end of the transmission shaft is rotatably connected to the bottom of the first mixing chamber, and the other end of the transmission shaft extends through the first mixing chamber and into the second mixing chamber, and the transmission shaft is rotatably connected to the carrier; all discs are rotatably fitted into the first mixing chamber, and all discs are coaxially fixedly connected to the transmission shaft, and a preset distance is provided between adjacent discs and between the discs and the sidewall of the first mixing chamber, the preset distance being less than twice the thickness of the fluid boundary layer; a transmission channel is axially opened inside the transmission shaft, and several openings and several discharge channels are respectively connected to both ends of the transmission channel, the openings being located between adjacent discs, and the discharge channels being used to connect the transmission channel to the second mixing chamber.

[0022] Beneficial effect: When a fluid (in this case, a liquid medicine) flows over a solid surface (such as a disc), the fluid layer close to the surface has a velocity of almost zero, while the velocity increases as you move further away from the surface. This velocity gradient region is the boundary layer.

[0023] When the drive shaft rotates the disks at high speed, the surface of each disk causes the surrounding fluid to move. Due to the extremely small gaps, the boundary layers generated by multiple disks overlap and shear each other, forming an extremely high velocity gradient within a very small space. This gradient generates an extremely strong and uniform shear force, which can efficiently separate and spread the drug mother liquor into layers like "threads," thereby achieving rapid and uniform mixing at the molecular level. This is far more efficient and thorough than passive mixing that relies on the free diffusion of molecules.

[0024] The mixing process primarily occurs in the gap region between the disks. The rotation of the disks creates a centrifugal force that "throws" the fluid in the gaps outward, thus forming a low-pressure zone in the axial region near the disks (i.e., at the opening of the transmission channel). This low-pressure zone, like a vacuum, draws the surrounding pre-mixed liquid into the transmission channel.

[0025] The fluid drawn into the transmission channel, under the influence of the angular momentum brought by the rotating shaft, will flow along the channel to the discharge channel located in the second mixing chamber, and be thrown out under the action of centrifugal force, thus completing the transfer from the first mixing chamber to the second mixing chamber.

[0026] By utilizing the boundary layer effect and ultra-high shear force, this design can thoroughly and uniformly disperse high-concentration drug stock solution in water in a very short time.

[0027] Furthermore, the transfer mechanism includes several transfer channels opened on the carrier, one end of each transfer channel is connected to the side of the first mixing chamber, and the other end of each transfer channel is connected to the top of the second mixing chamber.

[0028] Beneficial effects: The liquid medicine is initially mixed by the transmission mixing mechanism in the first mixing chamber and then pumped into the transmission channel inside the drive shaft.

[0029] The liquid medicine is discharged into the second mixing chamber through the discharge channel at the end of the transmission channel.

[0030] The medicine continues to diffuse and mix in the second mixing chamber.

[0031] Due to the continuous pumping of the mixing mechanism, flow force is generated within the system. Under the action of pressure difference, the liquid medicine naturally flows back to the side of the first mixing chamber through the transfer channel located at a high position.

[0032] The refluxed drug solution re-enters the mixing zone to undergo another round of shear mixing.

[0033] This process repeats itself until the concentration of the drug solution throughout the system reaches a high degree of uniformity.

[0034] Furthermore, the isolation mechanism includes several sliding frames slidably connected to each culture well, and several third channels are provided on the side walls of the sliding frames; several first channels are connected between the second mixing chamber and the adjacent culture wells, and each sliding frame is also provided with a shifting mechanism, which is used to adjust the connection and disconnection between the third channels and the first channels based on the displacement of the sliding frame.

[0035] Beneficial effects: When the sliding frame slides to a specific position, the solid portion of its sidewall completely blocks the outlet of the first channel. At this time, the third channel is misaligned with the first channel, the passage is interrupted, and the second mixing chamber is isolated from the culture wells. This is the state during drug circulation mixing, ensuring that the drug does not leak into the culture wells prematurely. When the shifting mechanism pushes the sliding frame to another predetermined position, the third channel on the sliding frame aligns and connects with the first channel on the carrier. This opens a channel connecting the second mixing chamber and the culture wells. Since a positive pressure has been established in the second mixing chamber, the uniformly mixed drug will instantly and smoothly flow into all the culture wells through this open channel.

[0036] This design ensures that the zebrafish in the culture wells remain completely immersed in the original, clean culture medium, undisturbed, until the drug solution is thoroughly mixed. Only after mixing is confirmed is the valve of all wells opened simultaneously via a single command, exposing all zebrafish to the target concentration of drug solution at the same time.

[0037] Furthermore, a second channel is connected between the adjacent transfer channel and the culture well, and the shifting mechanism is also used to adjust the connection between the second channel and the first channel based on the displacement of the sliding frame.

[0038] Beneficial effects: The transfer mixing mechanism circulates and mixes the drug solution in the first and second mixing chambers. At this time, the isolation mechanism is in the closed state (the sliding frame blocks the first and second channels), and the culture wells contain pure culture medium and zebrafish, completely isolated from the mixing system.

[0039] Once the mixture is homogeneous, the shifting mechanism activates, pushing all the sliding frames to move synchronously to a specific position. At this position, the channels on the sliding frames simultaneously connect the first channel (connecting to the second mixing chamber) and the second channel (connecting to the transfer channel / first mixing chamber system) to the culture wells.

[0040] The moment the channel opens, the entire system (first mixing chamber, second mixing chamber, all intermediate channels) and all culture wells form a connected container. According to the principles of hydrostatics and communicating vessels, the liquid will flow instantaneously from the higher liquid level to the lower liquid level under the influence of gravity until the liquid levels in all connected parts are equal.

[0041] This solution eliminates the need for complex pumps and valves to control the distribution process to each orifice, achieving optimal results solely through a simple synchronous switching action and basic physical principles. The simpler the mechanical structure, the lower the failure rate, and the better the stability and repeatability of the device.

[0042] Furthermore, the shifting mechanism includes a groove on the side wall of the culture well, a spring and a slider are installed in the groove, the slider is fixedly connected to the side wall of the sliding frame, and the slider slides in cooperation with the groove. The two ends of the spring are fixedly connected to the bottom of the groove and the bottom of the slider, respectively. A turntable is also rotatably connected inside the carrier. The side wall of the turntable has a closed zigzag groove, and the slider slides in cooperation with the zigzag groove. The slider contacts the top of the zigzag groove under the action of the spring. When the slider contacts the lowest point of the top of the zigzag groove, the third channel is simultaneously connected to the first channel and the second channel. When the slider contacts the highest point of the top of the zigzag groove, the third channel is simultaneously misaligned with the first channel and the second channel.

[0043] Beneficial effect: Under the elastic force of the spring, the slider is lifted up and pressed tightly against the top of the zigzag groove.

[0044] At this point, the slider is at the highest point of the zigzag groove. The position of this highest point, through the connection between the slider and the sliding frame, determines the depth of the sliding frame within the culture well.

[0045] At this depth, the solid portion of the sliding frame sidewall completely blocks (misaligns) the first and second channels, achieving isolation. This is the safe initial state.

[0046] The operator presses the sliding frame, which causes the slider to overcome the spring force and slide downwards along the zigzag groove.

[0047] This downward movement forces the turntable to start rotating.

[0048] When the hand is released, the spring pushes the slider back, attempting to return to the top of the zigzag groove. However, since the turntable has already rotated, the slider will now slide into a new position at the top of the zigzag groove—the lowest point.

[0049] This lowest point corresponds to a shallower depth of the sliding frame within the culture well. At this depth, the third channel on the sliding frame is perfectly aligned (connected) with both the first and second channels.

[0050] After the experiment, press the sliding frame again, and the slider will move down again, causing the turntable to continue rotating.

[0051] After you release your hand, the spring will push the slider back again, and this time the slider will slide back to the highest point at the top along the closed zigzag groove path.

[0052] The sliding frame then returns to its initial depth, re-cutting off all channels and completing the reset.

[0053] This solution enables precise, synchronized control with a single button. Operation is extremely simple and reliable; the operator requires no fine-tuning, simply pressing a button to simultaneously and precisely open all the valves in the culture wells to their predetermined positions. Pressing it again will simultaneously close them all.

[0054] Furthermore, a tangential groove is connected between the transfer channel and the first mixing chamber. The tangential groove is used to connect the transfer channel with the first mixing chamber along the tangential direction of the first mixing chamber.

[0055] Beneficial effect: When the mixed liquid flows at high speed from the transfer channel into the first mixing chamber through this tangential channel, the fluid acquires a tangential velocity component. According to the principles of fluid mechanics, this will forcibly form a rotating vortex, or "vortex" or "tangential flow," within the cylindrical first mixing chamber.

[0056] The resulting vortex generates a pressure gradient from the outside in (from the cavity wall to the axis), which helps to guide the liquid medicine more smoothly into the disk gap and the entrance of the transmission channel located in the axis region, thereby optimizing the path of the liquid medicine into the next cycle and improving the transmission efficiency of the entire circulation system.

[0057] Furthermore, an arc-shaped cover plate is provided at the top of the second mixing chamber, and several conduits are circumferentially connected to the arc-shaped cover plate, which are respectively connected to the top of the transfer channel.

[0058] Beneficial effects: The arc-shaped cover at the top of the second mixing chamber is a streamlined enclosure. When the liquid enters the second mixing chamber from below and impacts upwards, the arc-shaped structure can very smoothly convert the upward kinetic energy into radial flow in all directions.

[0059] Several circumferentially distributed conduits have their inlets facing the aforementioned radial flow path. These conduits serve as predetermined channels, uniformly capturing the diverted drug solution and guiding it into its corresponding transfer channels.

[0060] By distributing the drug solution evenly to each transfer channel through conduits, the flow rate and pressure of each circulation loop can be kept essentially consistent. This avoids the "short circuit" or "dead zone" phenomenon caused by uneven flow, where some paths have good mixing effects while others have poor effects. From a system perspective, this ensures that the concentration of the drug solution injected into all culture wells is highly uniform.

[0061] Furthermore, the carrier is made of a transparent material.

[0062] Beneficial effects: By making the carrier that supports the entire fluid pathway (culture wells, first and second mixing chambers, and various channels) transparent, real-time, non-destructive visual monitoring of the internal state of the device can be achieved. Operators can directly observe the mixing state of the drug solutions in the first and second mixing chambers.

[0063] For colored drugs or drugs containing tracer dyes, the process of the drug stock solution changing from an initial uneven stripe pattern to a completely uniform and transparent state can be clearly observed, thus accurately determining when the mixing is complete and avoiding exposure experiments being conducted too early or too late.

[0064] It can also visually confirm whether the transmission and mixing mechanism (disc) is operating normally, and whether the eddy current effect introduced by the tangential through groove is good. Attached Figure Description

[0065] Figure 1 This is a three-dimensional structural schematic diagram of the zebrafish drug screening auxiliary device used in the experiment of this invention.

[0066] Figure 2 for Figure 1 A top view of the experimental zebrafish drug screening auxiliary device.

[0067] Figure 3 for Figure 2 Sectional view along the AA direction.

[0068] Figure 4 for Figure 2 Sectional view along the BB direction.

[0069] Figure 5 for Figure 4 A magnified view of a portion of point M in the middle.

[0070] Figure 6 for Figure 3 A three-dimensional structural diagram of the middle disk.

[0071] The reference numerals in the accompanying drawings include: 1. Carrier; 2. Inlet tube; 3. Guide tube; 4. Cover plate; 5. Culture well; 101. First mixing chamber; 102. Servo motor; 103. Tangential groove; 104. Disc; 105. Drive shaft; 106. Second mixing chamber; 107. Transmission channel; 108. Excretion channel; 109. First channel; 110. Ultrasonic vibration unit; 111. Second channel; 112. Third channel; 301. Transfer channel; 501. Sliding frame; 502. Slider; 503. Turntable; 504. Zigzag groove; 505. Slide groove; 506. Spring. Detailed Implementation

[0072] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0073] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "vertical", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0074] In the description of this invention, unless otherwise specified and limited, it should be noted that the terms "installation", "connection" and "linking" should be interpreted broadly. For example, they can refer to mechanical or electrical connections, or internal connections between two components. They can be direct connections or indirect connections through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms according to the specific circumstances.

[0075] The following detailed description illustrates the specific implementation method:

[0076] The basic implementation examples are as follows: Figures 1-6 The image shows an experimental zebrafish drug screening auxiliary device, comprising a carrier 1, preferably made of a transparent material. The top of the carrier 1 has several circumferentially oriented culture holes 5. A first mixing chamber 101 and a second mixing chamber 106 are formed inside the carrier 1. Preferably, the first mixing chamber 101 is located at the bottom of the second mixing chamber 106. The first mixing chamber 101 has a cylindrical structure. A liquid inlet mechanism for delivering drug solution into the first mixing chamber 101 is connected to the side of the first mixing chamber 101. Specifically, the liquid inlet mechanism includes a drug inlet tube 2, one end of which is connected to the first mixing chamber 101, and the other end of which is provided with a sealing plug (not shown in the figure). The sealing plug is hung on the side of the carrier 1 by a traction rope. When delivering drug solution into the first mixing chamber 101, the sealing plug is simply pulled out from the inlet of the drug inlet tube 2, and after drug delivery, the sealing plug is reinserted into the inlet of the drug inlet tube 2.

[0077] The first mixing chamber 101 is provided with a transmission mixing mechanism, which is used to mix the liquid medicine based on the boundary layer effect and transport the liquid medicine to the second mixing chamber 106.

[0078] Specifically, the transmission and mixing mechanism includes a drive component, a drive shaft 105, and several disks 104. The drive component is installed at the bottom of the first mixing chamber 101 and is a servo motor 102. The drive component drives the drive shaft 105 to rotate, and the output shaft of the servo motor 102 is coaxially and fixedly connected to the drive shaft 105 via a coupling. The bottom of the drive shaft 105 is rotatably connected to the bottom of the first mixing chamber 101, the top of the drive shaft 105 extends through the first mixing chamber 101 and into the second mixing chamber 106, and the side of the drive shaft 105 is rotatably connected to the carrier 1 (at the junction of the first mixing chamber 101 and the second mixing chamber 106). All discs 104 are rotatably fitted within the first mixing chamber 101. Each disc 104 is coaxially and keyed to the drive shaft 105. A preset distance is provided between adjacent discs 104 and between each disc 104 and the sidewalls (top and bottom) of the first mixing chamber 101. This preset distance is less than twice the boundary layer thickness of the fluid (in this embodiment, the fluid is a mixture of drug mother liquor and deionized water, i.e., drug solution). A transmission channel 107 is axially formed within the drive shaft 105, with several openings connected to both ends of the transmission channel 107. The system includes several discharge channels 108 with openings located between adjacent disks 104. The discharge channels 108 are used to connect the transmission channel 107 with the second mixing chamber 106. The discharge channels 108 discharge liquid towards the second mixing chamber 106 in a downward tilted manner. When the liquid enters the second mixing chamber 106 from the discharge channels 108, the liquid will exhibit a spiral downward trend and accumulate in the second mixing chamber 106 until the second mixing chamber 106 is filled, and then flow back into the first mixing chamber 101.

[0079] The second mixing chamber 106 is connected to a transfer mechanism at one end away from the first mixing chamber 101. The transfer mechanism is used to connect the first mixing chamber 101 and the second mixing chamber 106. Specifically, the transfer mechanism includes a plurality of transfer channels 301 circumferentially opened on the carrier 1. One end of each transfer channel 301 is connected to the side of the first mixing chamber 101, and the other end of each transfer channel 301 is connected to the top of the second mixing chamber 106.

[0080] Preferably, a tangential groove 103 is also provided between the transfer channel 301 and the first mixing chamber 101. The tangential groove is used to connect the transfer channel 301 to the first mixing chamber 101 along the tangential direction of the first mixing chamber 101. (See attached diagram) Figure 3 As shown, in this embodiment, the bottom of the drug inlet tube 2 is also connected to the first mixing chamber 101 through the tangential through groove 103.

[0081] Preferably, the top of the second mixing chamber 106 is provided with an arc-shaped cover plate 4, which is integrated with the carrier 1. The arc-shaped cover plate 4 is circumferentially connected with a plurality of conduits 3, and the two ends of the conduits 3 are respectively connected to the top of the transfer channel 301 and the top of the second mixing chamber 106.

[0082] An isolation mechanism is connected between the culture well 5 and the second mixing chamber 106. The isolation mechanism is used to control the opening and closing of the culture well 5 and the second mixing chamber 106.

[0083] Specifically, the isolation mechanism includes several sliding frames 501 slidably connected to each culture well 5. In this embodiment, the sliding frame 501 is a cylindrical structure that fits the shape and structure of the culture well 5. Several third channels 112 are provided on the side wall of the sliding frame 501. Several first channels 109 are connected between the second mixing chamber 106 and the adjacent culture well 5. Preferably, a second channel 111 is also connected between the adjacent transfer channel 301 and the culture well 5. In this embodiment, the liquid content injected into the first mixing chamber 101 and the second mixing chamber 106 is at least greater than the total volume of the first mixing chamber 101 and the second mixing chamber 106.

[0084] Each sliding frame 501 is also equipped with a shifting mechanism, which is used to adjust the connection and disconnection between the third channel 112 and the first channel 109 based on the displacement of the sliding frame 501.

[0085] Specifically, in conjunction with the appendix Figure 4 and attached Figure 5 As shown, the shifting mechanism includes a groove 505 formed on the side wall of the culture well 5. A spring 506 and a slider 502 are provided in the groove 505. The slider 502 is integrally formed with the side wall of the sliding frame 501. The slider 502 slides in cooperation with the groove 505. The two ends of the spring 506 are respectively bonded and fixed to the bottom of the groove 505 and the bottom of the slider 502. A turntable 503 is also rotatably connected in the carrier 1. A closed zigzag groove 504 is formed on the circumferential side wall of the turntable 503. The slider 502 slides in cooperation with the zigzag groove 504. The slider 502 contacts the top of the zigzag groove 504 under the action of the spring 506. When the slider 502 contacts the lowest point of the top of the zigzag groove 504, the third channel 112 is simultaneously connected to the first channel 109 and the second channel 111. When the slider 502 contacts the highest point of the top of the zigzag groove 504, the third channel 112 is simultaneously misaligned with the first channel 109 and the second channel 111.

[0086] Preferably, an ultrasonic vibration unit 110 is further provided inside the side wall of the first mixing chamber 101. The ultrasonic vibration unit 110 includes at least one piezoelectric ceramic transducer encapsulated inside the side wall material of the first mixing chamber 101. The transducer is connected to an external ultrasonic generator (or driving power supply) via an insulated wire.

[0087] For highly hydrophobic and easily aggregated drug molecules, fluid shear force alone may not be sufficient to completely depolymerize and disperse them. Ultrasonic energy is converted into high-frequency mechanical vibration through a transducer, generating a cavitation effect in the liquid. This effectively breaks up drug clumps or large particles, dispersing them into nano-sized droplets or molecules, greatly increasing the specific surface area and thus significantly improving their apparent solubility and dispersion stability in the aqueous phase.

[0088] The shear flow and eddy current generated by the ultrasonic vibration and transmission hybrid mechanism complement and synergize with each other.

[0089] The specific implementation process is as follows:

[0090] Initial state of the device

[0091] All sliding frames 501 are in their highest position under the action of their respective springs 506 (i.e., slider 502 is at the highest point of the zigzag groove 504). At this time, the third channel 112 on the sliding frame 501 is completely misaligned with the first channel 109 and the second channel 111 on the carrier 1, and the culture well 5 is isolated from the second mixing chamber 106. The inlet of the drug inlet tube 2 is tightly plugged by a sealing plug.

[0092] Step 1: Loading Zebrafish

[0093] Add an appropriate amount of culture water to each culture well 5 and place the zebrafish embryos or juveniles used in the experiment into the well.

[0094] At this time, since the isolation mechanism is closed, culture well 5 is an independent static space, and the zebrafish will not enter the mixed circulation system.

[0095] Step 2: Preparation and mixing of the medicine solution

[0096] The operator removes the sealing plug and adds the drug stock solution and deionized water (or diluent) sequentially through the drug inlet pipe 2 according to the predetermined concentration ratio. The total amount added is calculated to ensure that the liquid level can be balanced to the predetermined height after the internal circulation system is completely filled. Since the drug inlet pipe 2 is connected to the first mixing chamber 101 through the tangential channel 103, the drug solution generates a preliminary tangential vortex when it flows in, which is beneficial for premixing.

[0097] The servo motor 102 is turned on (if necessary, the ultrasonic vibration unit 110 can be activated; according to the preset program, the ultrasonic vibration unit 110 operates with specific parameters to assist the transmission mixing mechanism in efficiently mixing the liquid medicine). The servo motor 102 drives the transmission shaft 105 to rotate at high speed, and the multiple disks 104 fixed on the transmission shaft 105 rotate synchronously.

[0098] Because the preset distance between adjacent disks 104 and between disk 104 and the top / bottom wall of the first mixing chamber 101 is less than twice the fluid boundary layer thickness, the liquid medicine in this space is subjected to extremely strong shearing action. The velocity gradient (boundary layer effect) generated on the surface of the rotating disk 104 is maximized, achieving intense, rapid, and uniform mixing of the liquid medicine in a very small space, avoiding dead zones that may occur with traditional stirring.

[0099] Under the centrifugal force and pressure difference generated by the rotation of the disc 104, the uniformly mixed liquid in the first mixing chamber 101 is forcibly drawn into the opening between the discs 104 on the side wall of the drive shaft 105 and enters the transmission channel 107.

[0100] The liquid medicine flows upward along the transmission channel 107 and enters the second mixing chamber 106 through the discharge channel 108.

[0101] The downward-sloping design of the discharge channel 108 allows the liquid to be sprayed out at a certain speed and direction, forming a spiral downward liquid flow in the second mixing chamber 106, which further ensures the uniformity of the liquid after entering the second mixing chamber 106 and prevents excessively high local concentrations.

[0102] The medicinal solution accumulates in the second mixing chamber 106. When the liquid level exceeds the inlet of the transfer channel 301 (conduit 3) at the top (cover plate 4) of the second mixing chamber 106, the medicinal solution flows back to the first mixing chamber 101 through the conduit 3 and the transfer channel 301 under the action of gravity and pressure difference (the disc 104 continuously sends liquid from the first mixing chamber 101 to the second mixing chamber 106). Similarly, the transfer channel 301 is connected to the first mixing chamber 101 through the tangential groove 103, and the medicinal solution flows back tangentially, merging with the medicinal solution being mixed in the first mixing chamber 101, forming a continuous and efficient closed-loop mixing path of "first mixing chamber 101 → transfer channel 107 → second mixing chamber 106 → transfer channel 301 → first mixing chamber 101". This process lasts for several minutes to ensure that the medicinal solution is completely mixed.

[0103] Through the synergistic effect of microscopic shearing and macroscopic eddy currents, the drug solution continuously circulates in a closed loop. Real-time observation can be achieved through the wall of the transparent carrier 1; once the drug solution becomes uniformly transparent (within a preset time), the mixing process has reached its endpoint.

[0104] Step 3: Infuse the culture well 5 with the drug solution (exposure begins)

[0105] After mixing, the operator simultaneously presses down on the top of all sliding frames 501 with their fingertips (a ring can be welded in series on the top of the sliding frames 501, and the sliding frames 501 move synchronously by pressing the ring). The sliding frames 501 drive the sliders 502 to move down synchronously, compressing the springs 506 and sliding along the zigzag grooves 504 on the turntable 503, thus driving the turntable 503 to rotate.

[0106] After the pressure is released, the spring 506 rebounds, pushing all the sliders 502 to simultaneously engage at the lowest point of the top of the zigzag groove 504. This action causes all the sliding frames 501 to move up to the predetermined position simultaneously, with the third channel 112 on them precisely aligned with the first channel 109 (connecting to the second mixing chamber 106) and the second channel 111 (connecting to the transfer channel 301 system).

[0107] Instantly, the internal mixing system and all culture wells 5 form a large communicating vessel. Under the established positive pressure and gravity within the system, the uniformly mixed drug solution flows smoothly and synchronously into all culture wells 5 until the liquid levels in all connected sections reach the same height. This process ensures that all zebrafish larvae are exposed to a completely consistent drug concentration environment at the same millisecond level.

[0108] Throughout the exposure period (e.g., 24 hours), the behavior, physiological responses (e.g., heartbeat, movement) and phenotypic changes of zebrafish larvae in each culture well 5 can be observed directly, in situ, and in real time through the transparent carrier 1, and images can be acquired.

[0109] After the exposure is complete, press down evenly on the top of the sliding frame 501 again and then release it. The slider 502 moves along the zigzag groove 504 and bounces back to the highest position, causing the sliding frame 501 to reset. The third channel 112 is repositioned with the first channel 109 and the second channel 111, cutting off the connection between the culture well 5 and the internal system.

[0110] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific structures and / or characteristics in the solutions are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An experimental zebrafish drug screening auxiliary device, comprising a carrier (1), wherein the top of the carrier (1) has a plurality of culture wells (5) circumferentially open, characterized in that: The carrier (1) has a first mixing chamber (101) and a second mixing chamber (106) inside. The first mixing chamber (101) is connected to a liquid inlet mechanism for conveying the liquid medicine into the first mixing chamber (101). The first mixing chamber (101) is provided with a transfer mixing mechanism for mixing the liquid medicine based on the boundary layer effect and conveying the liquid medicine to the second mixing chamber (106). The end of the second mixing chamber (106) away from the first mixing chamber (101) is connected to a transfer mechanism for connecting the first mixing chamber (101) and the second mixing chamber (106). An isolation mechanism is connected between the culture well (5) and the second mixing chamber (106), and the isolation mechanism is used to control the opening and closing of the culture well (5) and the second mixing chamber (106); The transmission and mixing mechanism includes a drive component, a drive shaft (105), and several discs (104). The drive component is located at the bottom of the first mixing chamber (101), and its output shaft is coaxially and fixedly connected to the drive shaft (105). The drive component is used to drive the drive shaft (105) to rotate. One end of the drive shaft (105) is rotatably connected to the bottom of the first mixing chamber (101), and the other end of the drive shaft (105) extends through the first mixing chamber (101) and into the second mixing chamber (106). The drive shaft (105) is rotatably connected to the carrier (1). The discs (104) are all rotatably equipped with... Within the first mixing chamber (101), the discs (104) are all coaxially and fixedly connected to the drive shaft (105). A preset distance is provided between adjacent discs (104) and between the discs (104) and the side wall of the first mixing chamber (101). A transmission channel (107) is axially opened inside the drive shaft (105). Several openings and several discharge channels (108) are respectively connected to both ends of the transmission channel (107). The openings are located between adjacent discs (104), and the discharge channels (108) are used to connect the transmission channel (107) with the second mixing chamber (106). The transfer mechanism includes several transfer channels (301) opened on the carrier (1). One end of the transfer channel (301) is connected to the side of the first mixing chamber (101), and the other end of the transfer channel (301) is connected to the top of the second mixing chamber (106). The isolation mechanism includes several sliding frames (501) that are slidably connected to each culture well (5). Several third channels (112) are provided on the side walls of the sliding frames (501). Several first channels (109) are connected between the second mixing chamber (106) and the adjacent culture well (5). A shifting mechanism is also provided on each sliding frame (501). The shifting mechanism is used to adjust the connection between the third channel (112) and the first channel (109) based on the displacement of the sliding frame (501). A second channel (111) is also connected between the adjacent transfer channel (301) and the culture well (5). The shifting mechanism is also used to adjust the connection between the second channel (111) and the first channel (109) based on the displacement of the sliding frame (501). The shifting mechanism includes a groove (505) opened on the side wall of the culture well (5), a spring (506) and a slider (502) are provided in the groove (505), the slider (502) is fixedly connected to the side wall of the sliding frame (501), the slider (502) slides in cooperation with the groove (505), and the two ends of the spring (506) are fixedly connected to the bottom of the groove (505) and the bottom of the slider (502) respectively; a turntable (503) is also rotatably connected in the carrier (1), and a closed zigzag groove (504) is opened on the side wall of the turntable (503), and the slider (502) slides in cooperation with the zigzag groove (504).

2. The experimental zebrafish drug screening auxiliary device according to claim 1, characterized in that: The liquid inlet mechanism includes a drug inlet tube (2), one end of which is connected to the first mixing chamber (101), and the other end of which is provided with a sealing plug.

3. The experimental zebrafish drug screening auxiliary device according to claim 2, characterized in that: The slider (502) contacts the top of the zigzag groove (504) under the action of the spring (506). When the slider (502) contacts the lowest point of the top of the zigzag groove (504), the third channel (112) is simultaneously connected to the first channel (109) and the second channel (111). When the slider (502) contacts the highest point of the top of the zigzag groove (504), the third channel (112) is simultaneously misaligned with the first channel (109) and the second channel (111).

4. The experimental zebrafish drug screening auxiliary device according to claim 3, characterized in that: A tangential through groove (103) is also connected between the transfer channel (301) and the first mixing chamber (101). The tangential through groove (103) is used to connect the transfer channel (301) with the first mixing chamber (101) along the tangential direction of the first mixing chamber (101).

5. The experimental zebrafish drug screening auxiliary device according to claim 4, characterized in that: The second mixing chamber (106) is provided with an arc-shaped cover plate (4) at the top. Several conduits (3) are connected around the arc-shaped cover plate (4) in the circumferential direction. The conduits (3) are respectively connected to the top of the transfer channel (301).

6. The experimental zebrafish drug screening auxiliary device according to claim 5, characterized in that: The carrier (1) is made of transparent material.

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