Micro-fluidic chip system for fixing juvenile zebrafish as well as preparation method and application of micro-fluidic chip system

Abstract

The invention provides a micro-fluidic chip system for fixing juvenile zebrafish with a matched injection pump facing upwards on the side / back surface as well as a preparation method and application of the micro-fluidic chip system. The micro-fluidic chip system for fixing the juvenile zebra fish with the matched injection pump facing upwards on the side / back surface comprises a conveying device, the juvenile zebra fish micro-fluidic chip and an analysis device, the conveying device comprises an injection pump and a conveying catheter. According to the side / back upward type micro-fluidic chip system for fixing the juvenile zebrafish by matching with the injection pump, and the preparation method and the application of the micro-fluidic chip system, provided by the invention, the juvenile zebrafish can be automatically fixed through automatic water flow injection under the condition of no anesthetic treatment by combining a fluid dynamic method with the shape of the juvenile zebrafish; meanwhile, the system has the advantages that high throughput can be realized, and potential technical support can be provided for further realizing drug screening, poison screening and toxicological research and related research of zebra fish.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, and in particular to a microfluidic chip system for fixing zebrafish larvae with a matching syringe pump and its preparation method and application. Background Technology

[0002] Zebrafish, due to their high genetic homology with humans, strong reproductive capacity, ease of manipulation, transparent embryos and larvae with short developmental cycles, and high permeability, have been widely used in numerous research fields, including elucidating embryonic development mechanisms, constructing complex disease models, establishing drug screening platforms, and environmental toxicology studies. Among these, fixed imaging of zebrafish larvae is one of the key techniques for achieving these research goals.

[0003] Current mainstream methods for manipulating zebrafish juveniles involve fixation with 1%-3% methylcellulose. However, methylcellulose fixation is not only cumbersome, has low throughput, and is time-consuming, often requiring the use of anesthetics, but is also prone to introducing human error. Therefore, achieving rapid, long-lasting, and high-throughput fixation of zebrafish juveniles is one of the key challenges in current experimental research on zebrafish juveniles.

[0004] Currently, there are many automated fixation technologies for zebrafish. For example, the VAST bioImager system can automatically load, position, and rotate juvenile zebrafish, and can also perform high-throughput image acquisition when combined with microscope imaging. However, it can only be performed after anesthesia, which can greatly interfere with the observation of neural activity. In addition, the high cost of commercial products also limits its application in toxicology research.

[0005] Therefore, it is necessary to provide a new microfluidic chip system for fixing zebrafish larvae with a matching injection pump and its preparation method and application to solve the above-mentioned technical problems. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a microfluidic chip system for fixing zebrafish larvae with a side / back-facing injection pump, which combines fluid dynamics methods with the body shape of zebrafish larvae, and achieves automated fixation of zebrafish larvae through automated water injection without the need for anesthetic treatment. At the same time, the system can achieve high throughput and provides highly promising technical support for further drug screening, toxicology screening and toxicological research, as well as zebrafish-related research. The system also includes its preparation method and application.

[0007] To solve the above-mentioned technical problems, the present invention provides a matching injection pump side / back side facing upward fixed zebrafish larvae microfluidic chip system, which includes: a delivery device, a zebrafish larvae microfluidic chip, and an analysis device;

[0008] The delivery device includes an injection pump and a delivery conduit, and is used to maintain a continuous water flow in the microfluidic chip and to assist in fixing zebrafish fry.

[0009] The zebrafish juvenile microfluidic chip includes several microstructural units; each microstructural unit is sequentially provided with an auxiliary channel, an inlet channel, a fixed chamber, and an outlet channel.

[0010] Preferably, the auxiliary channel parameters have a height of 800 μm and a width of 423-1000 μm.

[0011] Preferably, the height and width of the inlet channel and the outlet channel are both 800 μm.

[0012] Preferably, the microstructure unit is further provided with a fixing cavity between the liquid inlet channel and the liquid outlet channel, the fixing cavity having a height of 800 μm and a width of 180-1700 μm.

[0013] This invention also provides a method for fabricating a microfluidic chip system for fixing zebrafish larvae with the side / back facing upwards of the syringe pump. The method comprises the following steps:

[0014] S1: Use the delivery device to inject 75% ethanol into the inlet channel of the zebrafish larvae microfluidic chip to remove air bubbles;

[0015] S2: Use the delivery device to inject PBS buffer into the inlet channel of the zebrafish larvae microfluidic chip, repeat 2-3 times, and remove ethanol;

[0016] S3: Load the zebrafish fry into the fixed chamber of the zebrafish microfluidic chip, and use the injection pump to stabilize the fluid flow rate to assist in fixation;

[0017] S4: Connect to the analysis device to obtain a matching injection pump side / back side facing up fixed zebrafish larvae microfluidic chip system.

[0018] This invention also provides an application of a microfluidic chip system for fixing zebrafish juveniles with a matching syringe pump (side / back facing up) in drug screening, toxicology screening, and toxicological research.

[0019] Compared with related technologies, the microfluidic chip system for fixing zebrafish larvae with a matching syringe pump facing upwards, its preparation method, and its application provided by this invention have the following beneficial effects:

[0020] This invention provides a microfluidic chip system for immobilizing zebrafish larvae with a side / back-facing syringe pump, along with its preparation method and application. The invention employs a simple microfluidic channel structure design combined with a syringe pump to immobilize zebrafish larvae. For high-throughput zebrafish experiments, this invention is simpler, more economical, and easier to promote and apply. This invention enables automated immobilization of large numbers of zebrafish larvae without methylcellulose embedding or anesthesia, significantly reducing immobilization time compared to traditional manual methods. This invention provides a high-throughput, automated microfluidic chip for zebrafish larvae and its associated system, offering highly promising technical support for further drug screening, toxicology research, and zebrafish-related studies. Attached Figure Description

[0021] Figure 1 These are schematic diagrams of the different microfluidic chips used in Examples 1(A) and 2(B);

[0022] Figure 2 This is a schematic diagram of the automatic loading and fixation of zebrafish larvae using the microfluidic chip in Example 1;

[0023] Figure 3 The images show the effect of fixing zebrafish juveniles in a microfluidic chip. In Example 1, A shows the fixing effect of the microfluidic chip with the zebrafish side up; in Example 2, B shows the fixing effect of the microfluidic chip with the zebrafish back up.

[0024] Figure 4 The present invention provides the results of collecting physiological information such as heart, blood vessels, and brain data from zebrafish larvae using a microfluidic chip system for immobilizing zebrafish larvae.

[0025] Figure 5 The present invention provides a microfluidic chip system for zebrafish larvae to perform vascular imaging on Tg(fli1a:eGFP) transgenic zebrafish (acquired under a fluorescence microscope with an excitation wavelength of 488 nm).

[0026] Figure 6 This is a comparison diagram showing the long-term fixation effect of the zebrafish juvenile microfluidic chip system of the present invention on zebrafish juveniles and the long-term fixation effect of using methylcellulose embedding on zebrafish juveniles. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Please refer to the following: Figures 1-6 The matching injection pump side / back-facing fixed zebrafish larvae microfluidic chip system includes: a delivery device, the aforementioned zebrafish larvae microfluidic chip, and an analysis device;

[0029] The aforementioned delivery device includes an injection pump and a delivery conduit for transporting fluid and zebrafish larvae. By connecting with the aforementioned zebrafish larvae microfluidic chip, the zebrafish larvae can be automatically delivered into the fixed chamber of the aforementioned zebrafish larvae microfluidic chip. Combined with the stabilizing effect of the injection pump on the fluid flow rate, the zebrafish larvae can be fixed.

[0030] The analysis device includes a microscope and a computer connected to it, used to acquire image information of zebrafish juveniles;

[0031] The microfluidic chip for zebrafish larvae has several microstructural units;

[0032] The micro-structural unit is sequentially provided with an auxiliary channel, a liquid inlet channel, a fixed chamber, and a liquid outlet channel;

[0033] The auxiliary channel is located above the inlet channel and is used to provide additional fluid dynamics when the juvenile fish enter, to help the juvenile fish enter the fixed chamber and fix the body position of the zebrafish juveniles;

[0034] The fixed chamber, through height and / or width differences, enables zebrafish larvae to maintain their position within a fluid dynamic microfluidic chip;

[0035] The fixed chamber and auxiliary channels, combined with continuous water flow, enable zebrafish larvae to maintain their body position within the fluid dynamics-compliant microfluidic chip.

[0036] The microfluidic chip for zebrafish larvae can be used to image the zebrafish larvae in a side-up position to observe the physiological condition of organs such as the heart and blood vessels; or it can be used to fix the zebrafish in a back-up position to collect brain information (brain imaging and observation and recording of neural signals) and behavioral movement.

[0037] The auxiliary channel, inlet channel, fixed chamber, and outlet channel are all 800 μm high and 180-1700 μm wide. These dimensions ensure unobstructed passage for zebrafish juveniles (7-10 dpf). Furthermore, the connection between the inlet channel and the fixed chamber is designed as an arc-shaped channel to ensure the juveniles can smoothly enter each fixed chamber and avoid sharp turns that could cause injury.

[0038] In an embodiment of the present invention, the fixing chamber is designed as three parts: head, constriction section and tail. The head is designed as an oval or rectangular shape to better fix the head of the zebrafish fry. The constriction section is designed as a tube with a narrowing width to fix the tail of the zebrafish fry. The constriction section and the tail are used together for the placement of the tail of the zebrafish fry.

[0039] In an embodiment of the present invention, from a top view, the width of the fixed chamber gradually decreases along the direction of water flow, then gradually increases, and then gradually decreases again, in order to connect the liquid outlet channel.

[0040] In the embodiments of the present invention, the following are specifically illustrated: Figure 1 The two types of zebrafish larvae microfluidic chips shown are not included in this invention; however, the zebrafish larvae microfluidic chip claimed in this invention includes, but is not limited to, the following: Figure 1 The two types shown.

[0041] In one embodiment of the present invention, the zebrafish larvae microfluidic chip described above has several microstructural units, each of which is sequentially provided with an auxiliary channel, a liquid inlet channel, a fixed chamber, and a liquid outlet channel. The auxiliary channel, the liquid inlet channel, the fixed chamber, and the liquid outlet channel are all 800 μm high and 180-1700 μm wide. The auxiliary channel has a width of 423-1000 μm. The connection between the auxiliary channel and the liquid inlet channel is a 500 μm wide pipe to provide additional power for fluid flow, assisting the larvae in entering the fixed chamber. The liquid inlet channel is 800 μm wide, and its connection with the fixed chamber is designed as an arc-shaped channel. The aforementioned fixation chamber is divided into three parts: a head, a constricting section, and a tail. The head is rectangular with a width of 800 μm. The constricting section, where the tail connects to the head, narrows from 800 μm to 360 μm and then widens back to 800 μm in the direction of fluid flow to secure the zebrafish juvenile's tail. The tail, where the inlet channel connects to the fixation chamber, is an arc-shaped channel with a width that narrows from 1700 μm to 800 μm in the direction of fluid flow. The constricting section and tail of the fixation chamber work together to position the zebrafish juvenile's tail, conforming to the structural characteristics of the zebrafish's tail and providing better fixation. The head of the fixation chamber is connected to the outlet channel via a slender pipe with a width of 180 μm, and the outlet channel has a width of 800 μm. The chamber is designed for side-facing fixation of the zebrafish juvenile.

[0042] In another embodiment of the present invention, the zebrafish larvae microfluidic chip described above has several microstructural units, each of which is sequentially provided with an auxiliary channel, a liquid inlet channel, a fixed chamber, and a liquid outlet channel. The auxiliary channel, the liquid inlet channel, the fixed chamber, and the liquid outlet channel are all 800 μm high and 180-1700 μm wide. The auxiliary channel has a width of 423-1000 μm. The connection between the auxiliary channel and the liquid inlet channel is a 500 μm wide pipe to provide additional power for fluid flow, assisting the larvae in entering the fixed chamber. The liquid inlet channel is 800 μm wide, and its connection with the fixed chamber is designed as an arc-shaped channel. The aforementioned fixation chamber is divided into three parts: a head, a constricting section, and a tail. The head is elliptical, with its width narrowing from 800 μm to 180 μm in the direction of fluid flow. The constricting section, where the tail connects to the head, narrows from 800 μm to 360 μm and then widens back to 800 μm in the direction of fluid flow to secure the zebrafish juvenile's tail. The tail, where the inlet channel connects to the fixation chamber, is an arc-shaped channel with its width narrowing from 1700 μm to 800 μm in the direction of fluid flow. The constricting section and tail of the fixation chamber are used together to position the zebrafish juvenile's tail, conforming to the structural characteristics of the zebrafish's tail and providing better fixation. The head of the fixation chamber is connected to the outlet channel via a slender pipe with a width of 180 μm, and the outlet channel has a width of 800 μm. The chamber is designed for fixation of zebrafish juveniles with their backs facing upwards.

[0043] Example 1:

[0044] This embodiment provides a microfluidic chip for zebrafish juveniles and its preparation method:

[0045] Among them, the microfluidic chip for zebrafish larvae, such as Figure 1 As shown in Figure A, the zebrafish juvenile microfluidic chip is composed of multiple microstructural units, each of which is sequentially provided with an auxiliary channel, a liquid inlet channel, a fixed chamber, and a liquid outlet channel.

[0046] The height of the auxiliary channel, the inlet channel, the fixed chamber, and the outlet channel is 800 μm.

[0047] The auxiliary channel is designed as a combination of trapezoid and rectangle, with a length of 4000μm, a width of 423μm at its narrowest point, and a width of 1000μm at its widest point.

[0048] The connection between the auxiliary channel and the inlet channel is designed as a 500μm wide pipe to provide additional power for the fluid to flow through and for the juvenile fish to enter the fixed chamber.

[0049] The fixation chamber is designed with three parts: a head, a constricting section, and a tail. The head is rectangular, 800 μm long and 800 μm wide. The constricting section is a long, narrow tube, 1840 μm long, narrowing in width from 800 μm to 360 μm and then widening back to 800 μm in the direction of fluid flow. The tail is an arc-shaped tube, narrowing in width from 1700 μm to 800 μm in the direction of fluid flow. The head of the fixation chamber is used to fix the head of the zebrafish juveniles; the constricting section and tail are used together to position the tail of the zebrafish juveniles. This chamber design is for the side-facing fixation of the zebrafish juveniles.

[0050] The liquid inlet channel is located at one end of the microfluidic chip, and the liquid outlet channel is located at the other end of the microfluidic chip; the connection between the liquid inlet channel and the fixed chamber is rounded; the liquid outlet channel and the fixed chamber are connected by a slender pipe with a length of 800 μm and a width of 180 μm.

[0051] In this embodiment, the zebrafish larvae microfluidic chip was prepared by replicating the microfluidic hollow channel in the zebrafish larvae microfluidic chip using polydimethylsiloxane (PDMS) through 3D printing technology.

[0052] The zebrafish larvae microfluidic chip prepared in this embodiment is a side-up microfluidic chip for fixing zebrafish larvae, which can be used to observe the physiological conditions of organs such as the heart and blood vessels. Figure 2 The diagram shows the automatic loading and fixation of the zebrafish juvenile microfluidic chip.

[0053] Example 2:

[0054] This embodiment provides another type of microfluidic chip for zebrafish juveniles and its preparation method:

[0055] Among them, the microfluidic chip for zebrafish larvae, such as Figure 1 As shown in B, the microfluidic chip for zebrafish larvae consists of multiple microstructural units, each of which is sequentially equipped with an auxiliary channel, an inlet channel, a fixed chamber, and an outlet channel.

[0056] The height of the auxiliary channel, the inlet channel, the fixed chamber, and the outlet channel is 800 μm.

[0057] The auxiliary channel is designed as a combination of trapezoidal and rectangular superpositions, with a length of 4000μm, a width of 423μm at its narrowest point, and a width of 1000μm at its widest point.

[0058] The connection between the auxiliary channel and the inlet channel is designed as a 500μm wide pipe to provide additional power for the fluid to flow through and for the juvenile fish to enter the fixed chamber.

[0059] The fixation chamber is designed with three parts: a head, a constricting section, and a tail. The head is elliptical, 700 μm long, and its width narrows from 800 μm to 180 μm in the direction of fluid flow. The constricting section is a long, narrow channel, 1840 μm long, with its width narrowing from 800 μm to 360 μm and then widening back to 800 μm in the direction of fluid flow. The tail is an arc-shaped channel, with its width narrowing from 1700 μm to 800 μm in the direction of fluid flow. The head of the fixation chamber is used to fix the head of the zebrafish juveniles; the constricting section and the tail of the fixation chamber are used together to position the tail of the zebrafish juveniles.

[0060] The liquid inlet channel is located at one end of the microfluidic chip, and the liquid outlet channel is located at the other end of the microfluidic chip; the connection between the liquid inlet channel and the fixed chamber is rounded; the liquid outlet channel and the fixed chamber are connected by a slender pipe with a length of 800μm and a width of 180μm.

[0061] The fabrication method of the microfluidic chip in this embodiment is as shown in Example 1.

[0062] The zebrafish larvae microfluidic chip prepared in this embodiment is a back-facing microfluidic chip for fixing zebrafish larvae and can be used to collect brain information (brain imaging and observation and recording of neural signals) and behavioral movement.

[0063] Example 3:

[0064] This embodiment provides a microfluidic chip system for fixing zebrafish larvae with a matching syringe pump, where the side / back side faces upwards.

[0065] The microfluidic chip system for fixing zebrafish larvae with a side / back-facing injection pump in this embodiment mainly includes the microfluidic chip, delivery device, and analysis device (including an optical microscope and a connected computer) as described in Embodiment 1 or Embodiment 2. The microfluidic chip and delivery device are the core technologies of this system and are crucial for the automated loading and fixation of zebrafish larvae. Loading of the zebrafish larvae and addition of toxic drugs are both achieved by controlling the flow rate through the delivery device. The microscope is equipped with a high-definition camera to capture images and videos of the zebrafish larvae during the research process.

[0066] The specific assembly method of the matching injection pump-side / backside-facing fixed zebrafish larvae microfluidic chip system in this embodiment is as follows:

[0067] Connect the microfluidic chip inlet to the delivery device, and the outlet to a circulation system or waste tank. Before loading zebrafish larvae, first inject 75% ethanol into the chip inlet to fill the entire channel and remove air bubbles. Then inject PBS buffer into the chip inlet to fill the entire channel, repeating 2-3 times to remove the ethanol. Next, replace the fluid in the delivery device with fluid containing zebrafish larvae. Utilizing the principles of fluid dynamics, the chip will automatically and orderly load the zebrafish larvae into each fixed chamber. Simultaneously, maintain a stable fluid flow rate using a syringe pump to assist in fixation.

[0068] With the help of this matching microfluidic chip system for fixing zebrafish larvae with the side / back facing upwards of the syringe pump, the live zebrafish larvae are stably fixed in the correct position in an array to facilitate subsequent research and applications. The effect is shown in the image below. Figure 3 As shown.

[0069] Example 4:

[0070] This embodiment provides an application of a microfluidic chip system for fixing zebrafish larvae with a side / back-facing injection pump in the acquisition of physiological information such as brain, heart, and blood vessels in zebrafish larvae:

[0071] This embodiment combines the side / back-facing microfluidic chip system for fixing zebrafish larvae with the microfluidic chips for zebrafish larvae in Embodiments 1 and 2. It includes a side-facing microfluidic chip for observing organs such as the heart and blood vessels (Embodiment 1) and a back-facing microfluidic chip for observing organs such as the brain, eyes, and mouth (Embodiment 2), thereby fixing zebrafish larvae in multiple directions to collect information parameters of different physiological structures.

[0072] The specific assembly method of the microfluidic chip system for fixing zebrafish juveniles with the side / back facing upwards and the matching injection pump is shown in Example 3.

[0073] Information parameters collected from different physiological structures, such as Figure 4 As shown, when the matching injection pump side / back-facing fixed zebrafish larvae microfluidic chip system is used in conjunction with the zebrafish larvae microfluidic chips in Examples 1 and 2, the hearts of zebrafish larvae can be successfully collected. Figure 4 A, Figure 4 D) Blood vessels Figure 4 B. Figure 4 E) and brain ( Figure 4 C Figure 4 Physiological information such as F).

[0074] Example 5:

[0075] This embodiment provides an application of a microfluidic chip system for fixing zebrafish larvae with a matching syringe pump (side / back facing up) in fluorescence imaging:

[0076] In this embodiment, the zebrafish larvae microfluidic chip in Example 1 and the matching injection pump in Example 3 of the present invention were used to fix the zebrafish larvae microfluidic chip system and perform whole-body vascular fluorescence imaging of Tg(fli1a:eGFP) transgenic zebrafish.

[0077] First, the Tg(fli1a:eGFP) transgenic zebrafish larvae were automatically loaded using the zebrafish larvae microfluidic chip described in Example 1. Then, the microfluidic chip was connected to an injection pump for fixation, and whole-body vascular fluorescence imaging images of the zebrafish larvae were acquired under a 10x lens using a laser scanning confocal microscope. The results are as follows: Figure 5 As shown.

[0078] In this embodiment, whole-body vascular imaging was performed on untreated zebrafish larvae. The imaging was maintained in a lateral view to ensure the most comprehensive observation of blood vessels throughout the body, while maintaining clear and reliable imaging results. This result demonstrates the feasibility and stability of the microfluidic chip system for immobilizing zebrafish larvae with the accompanying injection pump provided by this invention. Furthermore, this microfluidic chip system for immobilizing zebrafish larvae with the accompanying injection pump is highly scalable (extended to screening for more types of drugs and toxins) and economical (requiring only micro-drug injection). For example, it can be used in conjunction with different strains of transgenic zebrafish to observe changes in heart rate, blood flow velocity, and behavior in zebrafish larvae after acute exposure to toxic drugs; to observe the development of organs such as the heart, blood vessels, and liver; or to acquire brain data using the microfluidic chip in Example 2. It can provide strong technical support for large-scale, high-throughput drug screening and toxin screening.

[0079] Comparative Example 1:

[0080] Comparison of zebrafish juvenile fixation capabilities between existing zebrafish juvenile fixation technologies and the zebrafish juvenile microfluidic chip system of this invention:

[0081] Existing zebrafish juvenile fixation techniques used in the comparison include:

[0082] 1. Directly anesthetize zebrafish fry (MS-222 anesthesia).

[0083] 2. Use methylcellulose or other gelling materials to fix the zebrafish fry.

[0084] 3. Use a methylcellulose mixed anesthetic (MS-222) to fix the zebrafish fry.

[0085] 4. VAST technology

[0086] The comparison items included whether anesthesia was required, the degree of automation, the throughput, the ease with which the zebrafish juveniles' body position could be changed, and the ease with which the zebrafish juveniles were subjected to vibration.

[0087] The results are shown in the table below.

[0088] Table 1. Comparison of zebrafish juvenile fixation capabilities between existing technologies and the zebrafish juvenile microfluidic chip system of this invention.

[0089]

[0090] As shown in Table 1, compared with other existing technologies, the zebrafish larvae microfluidic chip system of the present invention does not require manual fixation of zebrafish larvae and can achieve high throughput. Furthermore, this technology does not require anesthesia of the zebrafish larvae, avoiding interference from anesthetics during neurological experiments. The accompanying injection pump fixation method facilitates microfluidic drug perfusion of the zebrafish larvae, and reagent consumption can be greatly reduced by controlling the fluid flow rate. Most importantly, the zebrafish larvae microfluidic chip system of the present invention eliminates the problem of difficult directional fixation in manual embedding techniques, achieving both automated loading and directional fixation of zebrafish larvae, demonstrating strong scalability.

[0091] Comparative Example 2:

[0092] Comparative experiment of the microfluidic chip system for zebrafish larvae of this invention with existing methylcellulose immobilization technology:

[0093] Immobilization experiments were conducted on zebrafish juveniles using the microfluidic chip system of the present invention and existing methylcellulose immobilization technology, respectively.

[0094] The results are as follows Figure 6 As shown. Figure 6 B and Figure 6 As shown in Figure D, during prolonged methylcellulose embedding and fixation, the viscous nature of methylcellulose can exert continuous mechanical pressure on the surface of zebrafish larvae, causing compression and damage to relatively vulnerable areas such as spinal twisting and eye rupture. Furthermore, existing methylcellulose fixation techniques for zebrafish larvae require manual embedding and constant adjustments to achieve directional fixation. In contrast, the zebrafish larvae microfluidic chip system of this invention can directionally fix zebrafish larvae through the chip's special structure without causing the aforementioned damage. Figure 6 A, Figure 6 C). In comparison, the zebrafish larvae microfluidic chip system of this invention is irreplaceable by existing methylcellulose embedding and fixation technology, and has certain application advantages.

Claims

1. A microfluidic chip system for fixing zebrafish larvae with a matching syringe pump, characterized in that, include: Delivery device, zebrafish larvae microfluidic chip, and analysis device; The delivery device includes an injection pump and a delivery conduit, and is used to maintain a continuous water flow in the microfluidic chip and to assist in fixing zebrafish fry. The zebrafish juvenile microfluidic chip includes several microstructural units; each microstructural unit is sequentially provided with an auxiliary channel, an inlet channel, a fixed chamber, and an outlet channel.

2. The microfluidic chip system for fixing zebrafish larvae with the side / back facing upwards, as described in claim 1, is characterized in that... The auxiliary channel parameters are 800μm in height and 423-1000μm in width.

3. The microfluidic chip system for fixing zebrafish larvae with the side / back facing upwards and the matching injection pump as described in claim 1, characterized in that, The height and width of the inlet and outlet channels are both 800 μm.

4. The microfluidic chip system for fixing zebrafish larvae with the side / back facing upwards, as described in claim 1, is characterized in that... The microstructure unit also has a fixing cavity between the liquid inlet channel and the liquid outlet channel. The height of the fixing cavity is 800 μm and the width of the fixing cavity is 180-1700 μm.

5. A method for fabricating a microfluidic chip system for fixing zebrafish larvae with a matching syringe pump and side / back facing upwards, characterized in that, To prepare the microfluidic chip system for fixing zebrafish larvae with a matching syringe pump as described in any one of claims 1-4, the following steps are included: S1: Use the delivery device to inject 75% ethanol into the inlet channel of the zebrafish juvenile microfluidic chip to remove air bubbles; S2: Use the delivery device to inject PBS buffer into the inlet channel of the zebrafish larvae microfluidic chip, repeat 2-3 times, and remove ethanol; S3: Load the zebrafish fry into the fixed chamber of the zebrafish microfluidic chip, and use the injection pump to stabilize the fluid flow rate to assist in fixation; S4: Connect to the analysis device to obtain a matching injection pump side / back side facing up fixed zebrafish larvae microfluidic chip system.

6. The application of the matching injection pump side / back-facing fixed zebrafish larvae microfluidic chip system according to any one of claims 1-5 in drug screening, toxicology screening and toxicology research.

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