A fully automated zebrafish chip system for microscale sample screening

Abstract

The application discloses a kind of full-automatic zebra fish chip systems for micro sample screening, the system includes the microfluidic chip of fixed zebra fish seedlings, the chip includes one or more array arrangement microfluidic chip unit of fixed zebra fish seedlings, and the microfluidic chip unit of fixed zebra fish seedlings includes reaction channel;Reaction channel includes fluid communication first seedling fixed chamber, second seedling fixed chamber and main seedling channel, zebra fish seedling sample inlet for zebra fish seedling into reaction channel is arranged on main seedling channel;Wherein, first seedling fixed chamber and second seedling fixed chamber are symmetrically arranged and same structure, and first seedling fixed chamber and second seedling fixed chamber include fixed chamber, restriction channel and movable chamber, and the width of restriction channel is less than the width of fixed chamber and movable chamber.The system realizes high-throughput multi-information acquisition and zebra fish automatic loading fixation, and can greatly reduce sample amount, and greatly reduce drug development cost.

Description

Technical Field

[0001] This application relates to the field of microfluidic chip technology, and in particular to a fully automated zebrafish chip system for screening trace samples. Background Technology

[0002] Traditional large-scale drug screening primarily uses in vitro assays based on cellular or molecular targets for evaluation. However, this is limited by the finite number of targets, the difficulty in simulating specific systemic microenvironments (such as the gut), and the inability to continuously observe pathological changes. Furthermore, traditional methods using small animals (such as mice and rats) for drug screening are typically limited by low throughput and slow speed, resulting in extremely low drug screening efficiency.

[0003] Zebrafish, with their 87% genetic homology to humans and their advantages such as high embryonic and larval transparency, small size, and ease of manipulation, are widely used in numerous research fields, including the construction of complex disease pathological models and high-throughput drug screening. Among these, the manipulation, fixation, processing, and real-time observation of zebrafish are crucial enabling technologies for these studies. Traditional zebrafish fixation often employs artificial agarose embedding. This method is complex, time-consuming, and prone to errors, contradicting the current demands for high-throughput, high-precision recording.

[0004] Current advanced zebrafish immobilization technologies are based on microfluidic chip manipulation and fixation. These technologies primarily utilize the microfluidic chip as the main component, supplemented by various external devices. For example, a recently published zebrafish manipulation system employs a complex external control system, including light-blocking sensors and controllers, to determine the zebrafish's orientation and position. Only through cumbersome manipulation using pumps and valves can the zebrafish be positioned tail-forward and secured within the immobilization chamber. However, due to the high transparency of zebrafish larvae, the light-blocking sensors often fail to accurately determine their orientation, leading to immobilization errors. Furthermore, the complex external devices, large size, and high cost of this automated system make it unsuitable for commercial application. Additionally, the immobilization of live zebrafish within the microfluidic chip requires continuous infusion via an injection pump, resulting in significant drug waste during drug screening and greatly increasing drug development costs.

[0005] Therefore, there is an urgent need in this field to develop a fully automated zebrafish microarray system for screening micro-samples that is suitable for high-throughput micro-sample administration, simple to operate, and capable of acquiring multiple physiological information. This system is of great significance for drug performance evaluation and disease research and treatment, and can greatly reduce sample volume, significantly reduce drug development costs, and facilitate commercialization. Summary of the Invention

[0006] The purpose of this invention is to provide a fully automated zebrafish microarray system for screening trace samples. This system is of great significance for drug performance evaluation and disease research and treatment, and can greatly reduce sample volume, significantly reduce drug development costs, and facilitate commercialization.

[0007] This invention provides a microfluidic chip unit for immobilizing zebrafish larvae.

[0008] It includes a first liquid channel, a second liquid channel, and a reaction channel, wherein the reaction channel is disposed between the first liquid channel and the second liquid channel;

[0009] The reaction channel includes a first seedling fixation chamber, a second seedling fixation chamber, and a main seedling channel that are in fluid communication. The main seedling channel is located between the first seedling fixation chamber and the second seedling fixation chamber. A zebrafish seedling inlet is provided on the main seedling channel for zebrafish seedlings to enter the reaction channel.

[0010] The first seedling fixing chamber and the second seedling fixing chamber have the same structure and are symmetrically arranged. Both the first seedling fixing chamber and the second seedling fixing chamber include a fixing chamber, a restricting channel and a movable chamber along the axial direction of the main seedling channel. The fixing chamber is fluidly connected to the main seedling channel. The width of the restricting channel is smaller than the width of the fixing chamber and the movable chamber.

[0011] In another preferred embodiment, the first seedling fixing chamber is in fluid communication with the first liquid channel, and the second seedling fixing chamber is in fluid communication with the second liquid channel.

[0012] In another preferred embodiment, the limiting channel is located between the fixed chamber and the movable chamber.

[0013] In another preferred embodiment, the movable chamber is fluidly connected to the first liquid channel and / or the second liquid channel.

[0014] In another preferred embodiment, the first liquid channel and the second liquid channel are arranged in parallel.

[0015] In another preferred embodiment, the reaction channel is perpendicular to the first liquid channel and the second liquid channel.

[0016] In another preferred embodiment, the main seedling channel has a length of 8-12 mm and a width of 900-1000 μm.

[0017] In another preferred embodiment, the length of the main seedling channel is 9 mm.

[0018] In another preferred embodiment, the movable chamber is used for observing the tail movement of the zebrafish, the movable chamber has a fan-shaped structure, and the corners of the movable chamber are connected to the limiting channel.

[0019] In another preferred embodiment, the radius of the movable chamber is 2.5 mm.

[0020] In another preferred embodiment, the fixing chamber is used to fix the head of the zebrafish larva, and the fixing chamber has a conical structure, the tip of which is fluidly connected to the restricting channel.

[0021] In another preferred embodiment, the conical structure is designed according to the physiological characteristics of the zebrafish head in order to collect data on various organs of the head.

[0022] In another preferred embodiment, the opposite end of the fixed chamber is fluidly connected to the main seedling channel.

[0023] In another preferred embodiment, the restrictive channel only allows the tail of the zebrafish fry to pass through, and the restrictive channel has a length of 300μm-450μm and a width of 200μm-300μm.

[0024] In another preferred embodiment, it includes: one or more microfluidic chip units arranged in an array according to any one of claims 1-4.

[0025] In another preferred embodiment, the height of the microfluidic chip is 600μm-800μm.

[0026] In another preferred embodiment, the diameter of the zebrafish larvae inlet is matched to the width of the microfluidic channel.

[0027] In another preferred embodiment, the microfluidic chip further includes a plug that mates with the zebrafish larvae inlet.

[0028] Another aspect of the present invention provides an automatic loading and securing system for zebrafish.

[0029] Including the aforementioned microfluidic chips,

[0030] A zebrafish larvae feeding chamber, wherein the zebrafish larvae feeding chamber contains zebrafish larvae and their culture medium.

[0031] A micropump is fluidly connected to the zebrafish larvae feeding chamber. The micropump is used to inject larvae from the zebrafish larvae feeding chamber into the microfluidic chip through the zebrafish larvae inlet of the microfluidic chip.

[0032] A pressure-applying device is used to apply water pressure toward the head of the zebrafish larvae, causing the tail of the larvae to sequentially enter the fixed chamber, the confinement channel, and the movable chamber of the first or second larvae fixation chamber, thereby completing the fixation of the zebrafish larvae.

[0033] In another preferred embodiment, the zebrafish automatic loading system further includes a fish delivery pipeline, the inlet of which is fluidly connected to the micro-pump, and the outlet of which is aligned with the zebrafish larvae inlet.

[0034] In another preferred embodiment, the system further includes a three-dimensional moving device for moving the microfluidic chip so that the outlet of the fish delivery tube is aligned with the next zebrafish larvae inlet of the microfluidic chip. Therefore, this system eliminates the need for a complex external control system; it simply and automatically delivers zebrafish to each inlet. Once the zebrafish enters the microfluidic chip channel, regardless of its orientation, a gentle water pressure is applied towards its head, and the zebrafish is easily secured using fluid dynamics.

[0035] Another aspect of the present invention provides a fully automated zebrafish microarray system for screening trace samples, the fully automated zebrafish microarray system including the above-described automatic zebrafish loading and fixing system.

[0036] In another preferred embodiment, the fully automated zebrafish chip system further includes:

[0037] A micro-sample injector, wherein the micro-sample injector is filled with a micro-sample to be screened, and the operation of the micro-sample injector is controlled by the micro-pump;

[0038] A micro-sample input line is provided for inputting a micro-sample from the micro-sample syringe into the microfluidic chip.

[0039] The acquisition device is used to capture images of zebrafish larvae within a microfluidic chip, and

[0040] An analysis device is used to receive image information acquired by the acquisition device and analyze the image information.

[0041] In another preferred embodiment, the acquisition device includes an optical microscope equipped with a high-definition camera for capturing images of zebrafish larvae in the microfluidic chip;

[0042] The analysis device includes a computer, which is communicatively connected to the high-definition camera. The computer is used to receive and store image information transmitted by the high-definition camera.

[0043] In another preferred embodiment, one end of the microsample input line is fluidly connected to the microsample syringe, and the other end of the microsample input line is fluidly connected to the first liquid channel and the second liquid channel.

[0044] In another preferred embodiment, the fully automated zebrafish chip system further includes a fluorescence excitation device for exciting fluorescence in zebrafish larvae labeled with fluorescent proteins.

[0045] Another aspect of the present invention provides a method for automatically loading and securing zebrafish, the method comprising the step of loading and securing zebrafish using the above-described automatic zebrafish loading and securing system.

[0046] In another preferred embodiment, the method for automatically loading and securing zebrafish includes the following steps:

[0047] (a) Provide the above-mentioned microfluidic chip,

[0048] (b) Start the micropump to inject zebrafish larvae from the zebrafish larvae feeding chamber into the microfluidic chip through a zebrafish larvae inlet, and then close the zebrafish larvae inlet.

[0049] (c) Activate the pressure application device and, according to the zebrafish's orientation, apply pressure directly to the head of the zebrafish larvae through the first or second liquid channel, causing the tail of the zebrafish larvae to sequentially enter the fixed chamber, the confinement channel, and the movable chamber of the first or second larvae fixation chamber, thereby completing the fixation of the zebrafish larvae.

[0050] (d) Move the microfluidic chip.

[0051] (e) Repeat steps (b), (c), and (d) to inject and fix the zebrafish larvae into the next zebrafish larvae inlet, thereby completing the loading and fixation of all zebrafish. Therefore, compared to existing technologies that require continuous drug administration via an injection pump to fix the zebrafish, the zebrafish fixation of this invention does not require prior drug treatment, and the required drug sample volume can be reduced to 20-30 μL each time, significantly reducing drug sample consumption and lowering costs.

[0052] Another aspect of the present invention provides a method for screening trace samples, including the step of screening trace samples using the micro-fully automated zebrafish chip system described above.

[0053] In another preferred embodiment, the trace sample screening method includes the following steps:

[0054] (a) Provide the above-mentioned microfluidic chip,

[0055] (b) Start the micropump to inject zebrafish larvae from the zebrafish larvae feeding chamber into the microfluidic chip through a zebrafish larvae inlet, and then close the zebrafish larvae inlet.

[0056] (c) Move the microfluidic chip.

[0057] (d) Repeat steps (b) and (c) to inject and fix zebrafish larvae into the next zebrafish larvae inlet, thus completing the loading of all zebrafish.

[0058] (e) Activate the pressure application device and apply pressure to the head of the zebrafish larvae through the first or second liquid channel, depending on the orientation of the zebrafish, to fix the zebrafish larvae. At the same time, collect tail wagging data of the control group zebrafish larvae using an optical microscope and a high-speed camera.

[0059] (f) Using a microsample injector, a trace sample to be screened is injected into the reaction channel of the microfluidic chip via the first liquid channel or the second liquid channel.

[0060] (g) Repeat step (e) to collect tail wagging data from the zebrafish fry in the experimental group.

[0061] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0062] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. It should be understood that the accompanying drawings described below are merely some implementation examples of the present invention, and those skilled in the art can obtain other implementation examples based on these drawings without creative effort.

[0063] Figure 1 This is a top view of the microfluidic chip according to this application;

[0064] Figure 2 This is a top view of the microfluidic chip unit according to this application;

[0065] Figure 3a This is a structural schematic diagram of the zebrafish automatic loading and fixing system according to this application;

[0066] Figure 3b The image shown is a physical diagram of the microfluidic chip according to this application.

[0067] Figure 4This is a schematic diagram of the process by which zebrafish enters the microfluidic chip according to this application;

[0068] Figure 5 This is a physical image of a zebrafish entering a microfluidic chip according to this application;

[0069] Figure 6 This is a schematic diagram of the fluid dynamics simulation of the microfluidic chip according to this application;

[0070] Figure 7 yes Figure 6 A magnified schematic diagram of the fluid dynamics simulation of a microfluidic chip unit;

[0071] Figure 8 This is a schematic diagram of a zebrafish fixed in a microfluidic chip according to this application;

[0072] Figure 9 and Figure 10 These are behavioral readings of zebrafish induced by MK-801 according to Example 3 of this application.

[0073] The labels in each of the attached figures are as follows:

[0074] 1-First Liquid Channel

[0075] 2-Second Liquid Channel

[0076] 3-Reaction Channel

[0077] 31-First Seedling Fixation Chamber

[0078] 311-Fixed Chamber

[0079] 312 - Restricted Channel

[0080] 313-Active Chamber

[0081] 32-Second seedling fixation chamber

[0082] 33-Main Seedling Channel

[0083] 331-Zebrafish larvae inlet Detailed Implementation

[0084] Through extensive and in-depth research, the inventors have for the first time disclosed a fully automated zebrafish microarray system for screening micro-samples. This system features relatively simple external devices, eliminating the need for complex positioning systems. Based on the unique structural design of the microfluidic chip channels, and utilizing a droplet-based zebrafish injection method, the system can immobilize zebrafish without the need for a continuous water flow, eliminating the need for an injection pump. This allows for automated zebrafish immobilization using simple external devices. Furthermore, this system enables fully automated zebrafish immobilization, high-throughput micro-sample delivery, and multi-information screening, which is of great significance for drug performance evaluation and disease research and treatment. Moreover, the sample volume required for each drug processing step can be reduced to 20-30 μL, significantly reducing sample volume and substantially lowering drug development costs.

[0085] the term

[0086] As used in this article, the terms “juvenile,” “young fish,” and “larvae” are used interchangeably;

[0087] As used herein, the terms “chip,” “microfluidic chip,” and “microfluidic chip” are used interchangeably.

[0088] As used in this article, the terms "zebrafish larvae inlet" and "zebrafish larvae sprue inlet" are used interchangeably;

[0089] As used herein, the terms “micropump” and “microfluidic pump” are used interchangeably;

[0090] It should be noted that in this patent application, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0091] In this invention, all directional indicators (such as up, down, left, right, front, back, etc.) are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indicator will also change accordingly.

[0092] In the following description, many technical details are presented to help the reader better understand this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0093] Microfluidic chip for fixing zebrafish larvae

[0094] like Figure 1-2 As shown, the microfluidic chip includes multiple arrayed microfluidic chip units for fixing zebrafish larvae, comprising: one or more arrayed microfluidic chip units for fixing zebrafish larvae.

[0095] The microfluidic chip unit for fixing zebrafish larvae includes a first liquid channel 1, a second liquid channel 2, and a reaction channel 3, with the reaction channel 3 disposed between the first liquid channel 1 and the second liquid channel 2; preferably, the first liquid channel 1 and the second liquid channel 2 are arranged in parallel. Preferably, the reaction channel 3 is perpendicular to the first liquid channel 1 and the second liquid channel 2.

[0096] The reaction channel 3 includes a first seedling fixation chamber 31, a second seedling fixation chamber 32, and a main seedling channel 33, which are fluidly connected. The first seedling fixation chamber 31 and the second seedling fixation chamber 32 have the same structure and are symmetrically arranged. The main seedling channel 33 is located between the first seedling fixation chamber 31 and the second seedling fixation chamber 32. The first seedling fixation chamber 31 is fluidly connected to the first liquid channel 1, and the second seedling fixation chamber 32 is fluidly connected to the second liquid channel 2. The main seedling channel 33 is provided with a zebrafish seedling inlet 331 for zebrafish seedlings to enter the reaction channel 3. The first seedling fixation chamber 31 and the second seedling fixation chamber 32 each include a fixed chamber 311, a restrictive channel 312, and a movable chamber 313 in sequence along the axial direction of the main seedling channel 33. The fixed chamber 311 is fluidly connected to the main seedling channel 33, and the width of the restrictive channel 312 is smaller than the width of the fixed chamber 311 and the movable chamber 313. The first seedling fixing chamber 31 is in fluid communication with the first liquid channel 1, and the second seedling fixing chamber 32 is in fluid communication with the second liquid channel 2.

[0097] Preferably, the movable chamber 313 is used to observe the tail movement of the zebrafish. The movable chamber 313 has a fan-shaped structure, and its corners are connected to the restrictive channel 312. Preferably, the radius of the movable chamber 313 is 2.5 mm. Preferably, the fixed chamber 311 is used to fix the head of the zebrafish larva. The fixed chamber 311 has a conical structure, and its tip is fluidly connected to the restrictive channel 312. The conical structure is designed according to the physiological characteristics of the zebrafish head to collect data on various organs of the head. Preferably, the restrictive channel 312 only allows the tail of the zebrafish larva to pass through. The length of the restrictive channel 312 is 300 μm-450 μm, and its width is 200 μm-300 μm.

[0098] See Figure 1 As shown in Figure 3, the microfluidic chip serves as the core of this fully automated zebrafish chip system for screening trace samples. A physical image of the chip is shown below. Figure 3b As shown. The entire chip is 600–800 μm high, and its top view is as follows. Figure 1 As shown, Figure 2 This is a top view of a microfluidic chip unit;

[0099] The microfluidic chip consists of multiple symmetrical and specially designed single or array-type microstructures. Each microstructure unit includes two symmetrical and identical zebrafish larvae fixation chambers (i.e., the first larvae fixation chamber 31 and the second larvae fixation chamber 32 mentioned above), used to fix zebrafish larvae (5-8 dpf). Figure 1 As shown, the zebrafish larvae inlet (zebrafish larvae sample inlet 331) is located in the middle of the main larvae channel 33, which connects two symmetrical zebrafish fixation chambers (i.e., the first larvae fixation chamber 31 and the second larvae fixation chamber 32 mentioned above). The main larvae channel is 900-1000 μm wide and 9 mm long to ensure that the larvae can smoothly enter the microfluidic channel through the automatic loading method designed in this invention.

[0100] The symmetrical juvenile fixation chambers include a conical structure (e.g., fixation chamber 311) for head fixation, a narrow, elongated restrictive structure (e.g., restrictive channel 312), and a fan-shaped structure (e.g., active chamber 311) for observing tail movement. The conical structure is designed based on the physiological characteristics of the zebrafish head to collect data on various head organs, such as the brain and heart, as well as behavioral information such as eye movement frequency, fin sliding frequency, and amplitude. Behind the conical structure is a narrow, elongated restrictive structure, crucial for zebrafish fixation, measuring 300–450 μm in length and 200–300 μm in width, allowing only the tail of the juvenile to pass through. Behind the restrictive structure is a spacious fan-shaped chamber with a radius of 2.5 mm, used to collect data on tail movement. The fan-shaped chamber allows the zebrafish juvenile's tail to exhibit various movement states, thus recording behavioral information such as tail movement frequency and amplitude.

[0101] The symmetrical design of the zebrafish larvae fixation chambers makes the fixation technique simpler and easier to operate, eliminating the need for a complex external zebrafish control system. It simply requires automatically transferring the zebrafish to each inlet (e.g., Figure 3a As shown, once the zebrafish enters the microfluidic chip channel, regardless of its orientation, a gentle water pressure is applied directly in front of its head, and the zebrafish can be easily fixed using fluid dynamics.

[0102] Zebrafish Automatic Loading and Fixing System

[0103] The system includes the aforementioned microfluidic chip.

[0104] The zebrafish larvae feeding chamber contains zebrafish larvae and their culture medium.

[0105] The micropump is fluidly connected to the zebrafish larvae feeding chamber. The micropump is used to inject the larvae in the zebrafish larvae feeding chamber into the microfluidic chip through the zebrafish larvae inlet of the microfluidic chip.

[0106] A pressure-applying device is used to apply water pressure towards the head of the zebrafish larvae, causing the tail of the larvae to sequentially enter the fixed chamber 311, the confinement channel 312, and the movable chamber 313 of the first larvae fixing chamber 31 or the second larvae fixing chamber 32, thereby completing the fixing of the zebrafish larvae. Preferably, the zebrafish automatic loading system also includes a fish delivery pipeline, the inlet of which is fluidly connected to a micro-pump, and the outlet of which is aligned with the zebrafish larvae inlet. Preferably, the system also includes a three-dimensional moving device, which is used to align the outlet of the fish delivery pipeline with the next zebrafish larvae inlet of the microfluidic chip.

[0107] When the zebrafish automatic loading system provided by the present invention is as follows Figure 3a As shown, after zebrafish are sequentially introduced into each zebrafish larvae's inlet, a microdroplet containing the zebrafish forms above the inlet. As the volume of the microdroplet gradually decreases due to gravity, the zebrafish will enter the microfluidic chip through the larvae's inlet. Figure 4 and 5 As shown, plug the entrance of the zebrafish fry with a stopper. At this point, regardless of the zebrafish's orientation, gently apply water pressure directly towards its head. Based on fluid dynamics, this easily achieves the zebrafish's orientation and fixation. Figure 6 and 7 As shown, subsequent experimental observations were then conducted. In particular, when prolonged drug administration is required before formal observation and recording, the technology provided by this invention no longer relies on syringes or glass capillaries for prolonged drug administration to immobilize live zebrafish and prevent them from escaping from the zebrafish immobilization chamber. Only an extremely small amount of drug is needed to fill the internal channels of the microfluidic chip to complete prolonged drug administration, greatly reducing drug consumption and further saving costs.

[0108] Automatic loading method for fixed zebrafish

[0109] The method includes the following steps:

[0110] (a) Provide the above-mentioned microfluidic chip,

[0111] (b) Start the micropump to inject zebrafish larvae from the sample chamber into the microfluidic chip through a zebrafish larvae inlet 331, and then close the zebrafish larvae inlet 331.

[0112] (c) Activate the pressure application device and, according to the zebrafish's orientation, apply pressure directly to the head of the zebrafish larvae through the first liquid channel 1 or the second liquid channel 2, causing the tail of the zebrafish larvae to sequentially enter the fixed chamber 311, the restriction channel 312, and the movable chamber 313 of the first larvae fixing chamber 31 or the second larvae chamber 32, thereby completing the fixing of the zebrafish larvae.

[0113] (d) Mobile microfluidic chip,

[0114] (e) Repeat steps (b), (c) and (d) to inject and fix zebrafish larvae into the next zebrafish larvae inlet 331, thereby completing the loading and fixation of all zebrafish.

[0115] Fully Automated Zebrafish Chip System for Micro-sample Screening

[0116] Including the aforementioned zebrafish automatic loading and securing system,

[0117] The micro-sample injector is filled with a micro-sample to be screened, and its operation is controlled by a micro-pump.

[0118] The microsample input line is used to input a microsample from the microsample syringe into the microfluidic chip.

[0119] The acquisition device is used to capture images of zebrafish larvae within a microfluidic chip, and

[0120] An analysis device is used to receive image information acquired by the acquisition device and analyze the image information.

[0121] Preferably, the acquisition device includes an optical microscope equipped with a high-definition camera for capturing images of zebrafish larvae in the microfluidic chip;

[0122] Preferably, the analysis device includes a computer that is communicatively connected to a high-definition camera, and the computer is used to receive and store image information transmitted by the high-definition camera.

[0123] Preferably, one end of the micro-sample input tubing is fluidly connected to the micro-sample syringe, and the other end of the micro-sample input tubing is fluidly connected to the first liquid channel 1 and the second liquid channel 2.

[0124] Preferably, the fully automated zebrafish chip system also includes a fluorescence excitation device for exciting the fluorescence of zebrafish larvae labeled with fluorescent proteins in vivo.

[0125] Setting up external system devices:

[0126] The external devices of this fully automated zebrafish chip system are the foundation for the automated loading and securing of zebrafish. For example... Figure 3a As shown, the external equipment of the system includes a zebrafish larvae feeding chamber, a microfluidic pump, a three-dimensional moving device, and silicone tubing for connecting the various components to the microfluidic chip. The zebrafish larvae feeding chamber contains zebrafish larvae (5-8 dpf) and their culture medium. A microfluidic pump connects the feeding chamber to the zebrafish larvae inlet in the central channel of the microfluidic chip via the silicone tubing. During operation, the micropump draws zebrafish larvae from the feeding chamber and delivers them through the silicone tubing to the zebrafish larvae inlet in the microfluidic chip. A microdroplet containing the zebrafish is formed above the inlet. The volume of the microdroplet gradually decreases due to gravity, and the zebrafish enters the microfluidic chip along the zebrafish larvae inlet. Figure 4 and 5As shown, as zebrafish larvae enter the microfluidic channel, a three-dimensional moving device aligns the next zebrafish larvae inlet with the fish delivery tubing outlet, and a micro-pump operates to continue injecting zebrafish larvae. This process is repeated to complete the automatic sampling of zebrafish larvae. The microfluidic chip has four liquid inlets and outlets at its four corners. Two of these openings are selectively connected to silicone tubing as liquid inlets, with the other end of the tubing connected to a syringe. The syringe's operation is controlled by the micro-pump. After the zebrafish larvae enter the microfluidic chip channel, the zebrafish larvae inlet is plugged. Figure 6 and Figure 7 As shown, regardless of the zebrafish's orientation, applying a gentle water pressure directly in front of its head allows for easy orientation and fixation of the zebrafish based on fluid dynamics, enabling subsequent experimental observations.

[0127] In addition, depending on information recording requirements, the external equipment configured in this zebrafish microarray system may also include an optical microscope, a fluorescence excitation device, a high-definition camera, an external photoacoustic-electrostimulation device, and a computer. The optical microscope is equipped with a high-definition camera for capturing images of zebrafish larvae within the microfluidic chip. The camera is connected to the computer, allowing for precise control of image capture parameters via corresponding software, and real-time transmission and storage of image information to facilitate the recording and subsequent processing of zebrafish larvae behavioral information. The fluorescence excitation device is used to excite fluorescence in zebrafish larvae labeled with fluorescent proteins, facilitating observation and recording of features such as the brain. The external photoacoustic-electrostimulation device, through program control, selectively stimulates zebrafish larvae within the microfluidic chip, facilitating the evaluation of the effects of specific light, sound, and electrical stimuli on various organs and systems of the zebrafish larvae.

[0128] Fabrication of microfluidic chips:

[0129] This invention employs a manufacturing method based on high-precision computer numerical control (CNC) machining or 3D printing to fabricate a microfluidic chip mold, and uses polydimethylsiloxane (PDMS) to replicate the microfluidic hollow channels from the mold to prepare a microfluidic chip with fluid channels.

[0130] 2.1.3 Fabrication of microfluidic chip templates:

[0131] The process involves preparing a template, designing the microfluidic chip structure using 3D engineering software such as SolidWorks, and then machining it on a metal plate (such as brass) of a certain thickness using a CNC machine tool. Alternatively, the mold can be directly 3D printed. If using a CNC machine tool to carve a brass plate to create the microfluidic chip mold, the machined copper plate should first be soaked in anhydrous ethanol overnight. Then, remove the copper plate and gently wipe its surface with lint-free alcohol wipes or lint-free paper soaked in alcohol to remove dirt from corners and crevices. Next, ultrasonically clean for 20 minutes and dry with an air gun. Wrap the bottom and four sides of the copper plate with aluminum foil to form a closed enclosure around the mold surface, completing the preparation of the microfluidic chip template.

[0132] 2.1.4 Fabrication of microfluidic chips:

[0133] PDMS(A) and PDMS(B) were mixed in a 10:1 ratio in the same container and thoroughly stirred. Air bubbles were then removed under vacuum at room temperature. The mixed PDMS was then poured onto a cleaned copper template, and vacuum was maintained until all air bubbles were removed from the PDMS. The template was then placed in a vacuum drying oven at 70°C for 4-6 hours. After the PDMS was completely cured, the sample was removed and cooled to room temperature. The cured PDMS was then removed from the mold, and holes were drilled according to the inlet and outlet locations. The interface between the PDMS and the glass substrate was treated with a plasma cleaner to make it hydrophilic for subsequent bonding. After cleaning, the interface was pressed together and a weight was applied to ensure tight bonding, thus completing the fabrication of the microfluidic chip.

[0134] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be understood that these are merely examples provided to the reader of possible implementations of the present invention and are not intended to limit the scope of the invention.

[0135] Example 1:

[0136] Internal structure of microfluidic chips

[0137] See Figure 1 Figure 3 shows the microfluidic chip, which serves as the core of this fully automated zebrafish chip system for screening trace samples. A physical image of the chip is shown below. Figure 3b As shown. The entire chip is 600–800 μm high, and its top view is as follows. Figure 1 As shown, Figure 2 This is a top view of a microfluidic chip unit; the microfluidic chip consists of multiple symmetrical and specially designed single or array-type microstructures. Each microstructure unit includes two symmetrical and identical zebrafish larvae fixation chambers (first larvae fixation chamber 31 and second larvae fixation chamber 32) used to fix zebrafish larvae (5-8 dpf). Figure 1 As shown, the zebrafish larvae inlet (zebrafish larvae sample inlet 331) is located in the middle of the main larvae channel 33, which connects two symmetrical zebrafish fixation chambers (first larvae fixation chamber 31 and second larvae fixation chamber 32). The main larvae channel 33 is 900-1000 μm wide and 9 mm long to ensure that the larvae can smoothly enter the microfluidic channel through the automatic loading method designed in this invention.

[0138] The symmetrically designed fixed chambers include a conical structure (fixed chamber 311) for securing the head, a narrow, elongated restrictive structure (restrictive channel 312), and a fan-shaped structure (movable chamber 311) for observing tail movements. The conical structure is designed based on the physiological characteristics of the zebrafish head to collect data on various head organs, such as the brain and heart, as well as behavioral information such as eye movement frequency, fin sliding frequency, and amplitude. Behind the conical structure is a narrow, elongated restrictive structure, crucial for securing the zebrafish; it is 300–450 μm long and 200–300 μm wide, allowing only the tail of the juvenile fish to pass through. Behind the restrictive structure is a spacious fan-shaped chamber with a radius of 2.5 mm, used to collect data on the zebrafish's tail movements. The fan-shaped chamber allows the juvenile zebrafish's tail to exhibit various movement states, thus recording behavioral information such as the frequency and amplitude of tail movements.

[0139] The symmetrical design of the zebrafish fixation chambers makes the zebrafish fixation technology simpler and easier to operate, eliminating the need for a complex external zebrafish control system. It simply requires automatically transporting the zebrafish to each inlet... Figure 3a Once the zebrafish enters the microfluidic chip channel, regardless of its orientation, a gentle water pressure is applied directly to its head, and the zebrafish can be easily secured using fluid dynamics.

[0140] Example 2:

[0141] Automated loading and fixation of zebrafish larvae in a microfluidic chip

[0142] The fully automated zebrafish loading system provided by this invention is as follows: Figure 3aAs shown, the system includes a zebrafish larvae feeding chamber, a microfluidic pump, a three-dimensional moving device, and silicone tubing for connecting the various components to the microfluidic chip. The zebrafish larvae feeding chamber contains zebrafish larvae (5-8 dpf) and their culture medium. The microfluidic pump, connected to the feeding chamber at one end via the silicone tubing, and the other end, in conjunction with the three-dimensional moving device, sequentially delivers samples to the zebrafish larvae inlet of the main seedling channel of the microfluidic chip. During operation, the micropump draws zebrafish larvae from the feeding chamber and, with the help of the three-dimensional moving device, transports them through the silicone tubing to the inlet of the microfluidic chip (i.e., the zebrafish seedling inlet). Above this inlet, a microdroplet containing the zebrafish is formed. The volume of the microdroplet gradually decreases due to gravity, and the zebrafish enters the microfluidic chip through the inlet. Figure 5 and 6 As shown, as zebrafish larvae enter the microfluidic channel, the three-dimensional moving device aligns the next zebrafish larvae inlet with the fish delivery tubing outlet, and the micro-pump operates to continue injecting zebrafish larvae. This process is repeated to complete the automatic sampling of zebrafish larvae. The microfluidic chip has four liquid inlets and outlets at its four corners. Two of these openings are selectively connected to a silicone tubing as liquid inlets, with the other end of the tubing connected to a syringe. The syringe's operation is controlled by an injection pump. After the zebrafish larvae enter the microfluidic chip channel, the zebrafish larvae inlet is plugged. For zebrafish fixation, the technology provided by this invention abandons a complex peripheral directional fixation system. When the zebrafish enters the main larval channel, regardless of its orientation, a gentle water pressure is applied directly towards its head, such as... Figure 6 and Figure 7 By following the arrows indicating the direction of the fluid dynamics, one can easily select one of the zebrafish fixation chambers to complete the directional fixation of the zebrafish, and then carry out subsequent experimental observations.

[0143] In particular, when long-term drug administration is required before formal observation and recording, the technology provided by this invention no longer relies on syringes or glass capillaries to administer drugs for a long time to fix live zebrafish and prevent them from escaping from the zebrafish fixation chamber. Only an extremely small amount of drug is needed to fill the internal channels of the microfluidic chip to complete long-term drug administration.

[0144] Example 3: Behavioral phenotypes of zebrafish exposed to MK-801

[0145] See Figures 8-10 To test the capabilities of the fully automated microfluidic chip system for microsample screening provided by this invention in drug screening and behavioral phenotypic assessment, zebrafish were treated with the MK-801 for up to ten minutes before formal behavioral recording. Subsequently, the tail movements of the zebrafish exposed to the MK-801 were recorded using an optical microscope and a high-speed camera. Figure 8As shown, the zebrafish is fixed inside the chip.

[0146] First, the zebrafish were loaded using the automatic zebrafish loading technology mentioned in Example 2. Then, based on the zebrafish's orientation, a gentle water pressure (control group: E3 water) was applied directly towards the head, causing the zebrafish's tail to first enter the fixation chamber and then pass through the restrictive structure to reach the open fan-shaped structure behind it. This completed the fixation of the zebrafish, and tail movement data of the control group was collected using an optical microscope and a high-speed camera. After the control group information was collected, a small amount of MK-801 (20 μM) was injected into the injection pump to fill the entire microfluidic channel. In this case, only a very small amount of drug was needed to complete the processing before formal data collection. When observation was required, a gentle water pressure (experimental group: 20 μM MK-801) was applied again directly towards the head to fix the zebrafish and collect relevant data.

[0147] MK-801, also known as dizocilpine, is a central nervous system medication with anti-epileptic and neuroprotective effects. Studies have shown that acute treatment with MK-801 under light conditions can significantly reduce spontaneous movement in juvenile fish. After screening using the fully automated zebrafish screening system provided in this invention, tail behavior readings are as follows: Figure 9 and 10 As shown, zebrafish treated with 20 μM MK-801 exhibited significantly reduced tail wagging amplitude and frequency compared to the control group. This result demonstrates the high-throughput drug screening and behavioral phenotypic evaluation capabilities of the technology provided by this invention. The fully automated zebrafish loading and fixation system offers strong technical support for commercialization, and the reduction of drug sample consumption to minimal levels greatly saves on drug screening costs.

[0148] Main advantages of the invention

[0149] (a) The single-layer PDMS chip structure of the microfluidic chip of the present invention is simpler and more economical to manufacture than the complex multilayer structure, and is easier to promote and apply.

[0150] (b) The fully automated zebrafish loading and fixing system of the present invention provides strong technical support for commercial promotion, and the consumption of drug samples is reduced to a tiny amount, which greatly saves the cost of drug screening.

[0151] (c) The fully automated zebrafish chip for screening trace samples of the present invention can reduce the sample volume required for each drug processing to 20-30uL, which greatly reduces the consumption of drug samples and lowers the cost.

[0152] (d) The fully automated zebrafish loading and fixation system of the present invention (automatic zebrafish loading and fixation system) has a symmetrical design of zebrafish fixation chamber, which makes the zebrafish fixation technology simpler and easier to operate. It abandons the complex external zebrafish control system (i.e. the external fixation device is simple). It only requires the zebrafish to be automatically delivered to the inlet of each microfluidic chip unit. After the zebrafish enters the microfluidic chip channel, regardless of the zebrafish’s orientation, a water pressure is gently applied in the direction facing the head. According to fluid dynamics, the zebrafish can be easily fixed without the need for continuous drug administration to fix the zebrafish.

[0153] (e) The fully automated zebrafish loading and fixing system of the present invention can still perfectly fix zebrafish in the microfluidic chip array when the continuous hydraulic pressure is removed, and hydraulic pressure is only applied during observation. This not only allows the microfluidic chips to be removed from the external continuous hydraulic system at any time, but also greatly reduces the operating cost and the amount of drugs to be screened.

[0154] (f) The fully automated zebrafish chip system for micro-sample screening of the present invention has simple equipment. The system can perform fully automated zebrafish fixation, micro-sample delivery, high-throughput screening of multi-information zebrafish, which is of great significance for drug performance evaluation and disease research and treatment.

[0155] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A microfluidic chip unit for immobilizing zebrafish larvae, characterized in that, It includes a first liquid channel (1), a second liquid channel (2), and a reaction channel (3), wherein the reaction channel (3) is disposed between the first liquid channel (1) and the second liquid channel (2); The reaction channel (3) includes a first seedling fixation chamber (31), a second seedling fixation chamber (32), and a main seedling channel (33) that are in fluid communication. The main seedling channel (33) is located between the first seedling fixation chamber (31) and the second seedling fixation chamber (32). A zebrafish seedling inlet (331) for zebrafish seedlings to enter the reaction channel (3) is provided on the main seedling channel (33). The first seedling fixing chamber (31) and the second seedling fixing chamber (32) have the same structure and are symmetrically arranged. The first seedling fixing chamber (31) and the second seedling fixing chamber (32) each include a fixing chamber (311), a restricting channel (312) and a movable chamber (313) along the axial direction of the main seedling channel (33). The fixing chamber (311) is fluidly connected to the main seedling channel (33). The width of the restricting channel (312) is smaller than the width of the fixing chamber (311) and the movable chamber (313).

2. The microfluidic chip unit as described in claim 1, characterized in that, The length of the main seedling channel (33) is 8-12 mm and the width is 900-1000 μm.

3. The microfluidic chip unit as described in claim 1, characterized in that, The movable chamber (313) is used to observe the tail swing of the zebrafish. The movable chamber (313) has a fan-shaped structure, and the corner of the movable chamber (313) is connected to the limiting channel (312).

4. The microfluidic chip unit as described in claim 1, characterized in that, The fixing chamber (311) is used to fix the head of the zebrafish fry. The fixing chamber (311) has a conical structure, and the tip of the conical structure is fluidly connected to the restricting channel (312).

5. A microfluidic chip for immobilizing zebrafish larvae, characterized in that, include: One or more microfluidic chip units arranged in an array according to any one of claims 1-4.

6. The microfluidic chip as described in claim 5, characterized in that, The height of the microfluidic chip is 600μm-800μm.

7. An automatic loading and securing system for zebrafish, characterized in that, Including the microfluidic chip as described in claim 5 or 6, A zebrafish larvae feeding chamber, wherein the zebrafish larvae feeding chamber contains zebrafish larvae and their culture medium. A micropump is fluidly connected to the zebrafish larvae feeding chamber and is used to inject larvae from the zebrafish larvae feeding chamber into the microfluidic chip through the zebrafish larvae inlet of the microfluidic chip. The pressure device is used to apply water pressure toward the head of the zebrafish fry, so that the tail of the zebrafish fry enters the fixed chamber (311), the restriction channel (312) and the movable chamber (313) of the first fry fixing chamber (31) or the second fry chamber (32) in sequence, thereby completing the fixing of the zebrafish fry.

8. The zebrafish automatic loading and securing system as described in claim 7, characterized in that, The zebrafish automatic loading system also includes a fish delivery pipeline, the inlet of which is fluidly connected to the micro-pump, and the outlet of which is aligned with the zebrafish larvae inlet.

9. The zebrafish automatic loading and securing system as described in claim 8, characterized in that, The system also includes a three-dimensional moving device for moving the microfluidic chip so that the outlet of the fish delivery pipeline is aligned with the next zebrafish larvae inlet of the microfluidic chip.

10. A fully automated zebrafish microarray system for screening trace samples, characterized in that, The fully automated zebrafish chip system includes the zebrafish automatic loading and fixing system as described in any one of claims 7-9.

Images