阅读说明：本技术 一种多样品斑马鱼幼苗高通量微流控芯片、筛选系统及其应用 (Multi-sample zebra fish fry high-flux microfluidic chip, screening system and application of multi-sample zebra fish fry high-flux microfluidic chip ) 是由 林旭东 唐明卉 于 2020-11-02 设计创作，主要内容包括：本发明公开了一种多样品斑马鱼幼苗高通量微流控芯片、筛选系统及其应用。所述斑马鱼微流控芯片具有若干个微型结构单元；所述微型结构单元依次设置有进液通道、固定腔、限制通道以及出液通道。该芯片结构简单、制作方便、成本低、易于操作,能够在极短的时间内实现斑马鱼幼苗的自动定向固定和装载,并能够实现在单一采集窗口下多样品的斑马鱼幼苗操控,极大地减少了时间损耗,进一步提升了通量和效率。(The invention discloses a multi-sample zebra fish fry high-flux microfluidic chip, a screening system and application thereof. The zebra fish microfluidic chip is provided with a plurality of micro-structure units; the micro-structure unit is sequentially provided with a liquid inlet channel, a fixed cavity, a limiting channel and a liquid outlet channel. The chip has the advantages of simple structure, convenience in manufacture, low cost and easiness in operation, can realize automatic directional fixation and loading of zebra fish fries in a very short time, can realize control of the zebra fish fries of multiple samples under a single acquisition window, greatly reduces time loss, and further promotes flux and efficiency.)

1. The zebra fish microfluidic chip is characterized by comprising a plurality of micro structural units;

the micro structure unit is sequentially provided with a liquid inlet channel, a fixed cavity, a limiting channel and a liquid outlet channel;

the height of the limiting channel is smaller than the height of the fixing cavity and/or the width of the limiting channel is smaller than the width of the fixing cavity;

the inlet channel connects 2 or more fixed chambeies simultaneously, fixed chamber with the inlet channel is the centre of a circle to the different angles encircle the expansion.

2. The zebrafish microfluidic chip according to claim 1, wherein a movable cavity is further arranged between the limiting channel and the liquid outlet channel, and a supporting backflow baffle perpendicular to the fluid flowing direction is arranged in the movable cavity.

3. The zebrafish microfluidic chip of claim 2, wherein the movable cavity is a sector-shaped cavity with a radius of 2.5-3.0 mm.

4. The zebrafish microfluidic chip of claim 2 or 3, wherein the movable chambers are communicated with each other.

5. The zebrafish microfluidic chip of claim 2, wherein the thickness of the support return baffle is 300-500 μm.

6. The zebrafish microfluidic chip according to claim 4 or 5, wherein the liquid inlet channel, the fixing cavity and the liquid outlet channel have a height of 500-1000 μm and a width of 800-1500 μm.

7. A zebra fish microfluidic system is characterized by comprising a loading device, a zebra fish microfluidic chip as claimed in any one of claims 1 to 6, an observation device and an analysis device;

the zebra fish microfluidic chip can be used for loading a plurality of zebra fish samples in a single visual field observed or imaged by the observation device.

8. The zebrafish microfluidic system of claim 7, wherein the zebrafish samples comprise zebrafish fries and zebrafish roes.

9. Use of the zebra fish microfluidic chip according to any one of claims 1 to 6 or the zebra fish microfluidic system according to claim 6 or 7 for real-time information acquisition or comparison of multiple zebra fish samples.

10. Use of a zebrafish microfluidic chip according to any one of claims 1 to 6 or a zebrafish microfluidic system according to claim 6 or 7 in drug screening.

Technical Field

The invention relates to the technical field of microfluidic chips, in particular to a multi-sample zebra fish fry high-flux microfluidic chip, a screening system and application thereof.

Background

Zebrafish, as an excellent model organism, is widely used in various life system researches, and especially plays an increasingly important role in various complex human disease researches, such as the development and screening of brain disease drugs, the rapid detection of CODVI-19, the vaccine development and the like, because the zebrafish is highly similar to human in terms of the organ structure and biochemical level.

In recent years, microfluidic technology has become one of the most popular techniques due to its advantages of high integration and miniaturization, precisely controllable liquid flow, and very small sample reagent consumption. The zebra fish fries are small in size, large in number and the like, and the perfect combination of the microfluidic technology promotes the progress of multiple scientific fields such as genetics, neuroscience and pathology to a great extent.

However, most of the existing researches based on the microfluidic technology and using zebra fish as the object require that the zebra fish is directionally fixed and then various physiological parameters are acquired. The traditional method has certain defects in animal control and data acquisition: for example, in the aspect of animal manipulation, tedious manual operations are usually required to directionally fix the zebra fish, such as hydrogel embedding and anesthesia treatment, which not only causes huge time loss, but also greatly limits the experimental throughput, and more importantly, the manual operations are often accompanied by the introduction of individual differences, which directly hinders the evaluation of various physiological information of the zebra fish and further influences the research on human diseases and the development of related drugs; in the aspect of data acquisition, it is difficult for the current research to simultaneously capture physiological information of multiple zebra fishes in a single microscope visual field on the premise of realizing anesthesia-free and automatic directional fixation of the zebra fishes, while the data monitoring and observation of a single sample can be up to tens of minutes or even tens of minutes under the common condition, if the simultaneous recording of the information of the multiple samples cannot be realized, huge time and expense are brought, and meanwhile, the research with high flux and high efficiency is limited. Therefore, it is crucial to develop a multi-sample control high-throughput zebra fish fry screening system based on microfluidics and capable of realizing multi-sample observation under a single visual field.

Disclosure of Invention

The invention aims to provide a zebra fish microfluidic chip;

another objective of the present invention is to provide a zebrafish microfluidic system;

the invention also aims to provide the application of the zebra fish micro-fluidic chip or the zebra fish micro-fluidic system in the real-time information acquisition or comparison of multiple zebra fish samples;

the invention also aims to provide the application of the zebra fish microfluidic chip or the zebra fish microfluidic system in drug screening.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a zebra fish microfluidic chip is provided with a plurality of micro-structure units;

the micro-structure unit is sequentially provided with a liquid inlet channel, a fixed cavity, a limiting channel and a liquid outlet channel;

the height of the limiting channel is smaller than the height of the fixing cavity and/or the width of the limiting channel is smaller than the width of the fixing cavity;

the liquid inlet channel is simultaneously connected with 2 or more fixed cavities, and the fixed cavities are circularly unfolded at different angles by taking the liquid inlet channel as a circle center.

The zebra fish microfluidic chip in the embodiment of the invention is composed of a plurality of specially designed micro-structure units, each micro-structure unit is formed by communicating an inlet channel and an outlet channel with a plurality of zebra fish fixing chambers, the head and part of the body of the zebra fish fry are automatically fixed under the continuous action of hydrodynamics, and organ parts such as the tail, the fins and the mouth are in a free moving state. Moreover, each fixed cavity is unfolded towards the same circle center (liquid inlet channel) at different angles, so that the number of zebra fish in a single visual field is expanded to 5, 7 or more, and the information of a plurality of different zebra fish can be acquired simultaneously under the visual field of a single microscope or other observation devices.

The zebra fish microfluidic chip can be used for directionally fixing zebra fish in an upward back manner and can be used for collecting head information (observing brain nerve signals) and behavioural movement; or the zebra fish microfluidic chip is imaged laterally, so that the zebra fish microfluidic chip can be used for observing the physiological conditions of organs of the heart and blood vessels.

In an embodiment of the invention, the fixation cavity is designed to be tapered for better fixation of the head.

Furthermore, the micro-structure unit is also provided with a movable cavity between the limiting channel and the liquid outlet channel, and a support backflow baffle perpendicular to the flowing direction of the fluid is arranged in the movable cavity.

The movable cavity can enable organ parts such as tails of zebra fish fries, fins and the like to be in a free moving state, so that test information of related tests taking the organ parts such as the tails of the zebra fish fries, the fins and the like as observation objects can be obtained, and corresponding analysis data can be provided.

Furthermore, the movable cavity is a fan-shaped cavity with the radius of 2.5-3.0 mm.

Furthermore, the movable cavities are communicated with each other.

Of course, the skilled person can also set the active cavities in a non-communicating manner according to the actual use requirement.

Furthermore, the thickness of the supporting reflow baffle plate is 300 to 500 μm.

Support the backward flow baffle and not only can play the supporting role in order to prevent that large tracts of land hollow structure from collapsing, more importantly, support the backward flow baffle and can make microfluid form a backward flow before the baffle, make liquid can be full of whole cavity more fully, if do not have this support backward flow baffle, very big bubble can appear in the activity chamber region usually, not only influence the motion of zebra fish afterbody and the collection of each item signal, still can cause the injury to the zebra fish.

Furthermore, the height of the liquid inlet channel, the height of the fixing cavity and the height of the liquid outlet channel are 150-1000 μm, and the width of the liquid inlet channel is 800-900 μm.

In an embodiment of the invention, the zebra fish microfluidic chip comprises a plurality of micro-structural units, and each micro-structural unit is sequentially provided with a liquid inlet channel, a fixed cavity, a limiting channel, a movable cavity (including a supporting backflow baffle) and a liquid outlet channel. The height of the liquid inlet channel, the height of the micro valve generating cavity and the height of the liquid outlet channel are all 500-600 mu m, and the width of the liquid inlet channel is 900-1000 mu m. The liquid inlet channel is simultaneously connected with 2 or more fixed cavities, and the fixed cavities are circularly unfolded at different angles by taking the liquid inlet channel as a circle center. The joint of the liquid inlet channel and the fixed cavity is set to be an arc with the radius of 100-150 mu m. The fixed cavity is tapered, the width of the fixed cavity is narrowed from 700-1000 μm to 200-300 μm along the flowing direction of the fluid, and the height of the fixed cavity is 500-600 μm. The tail end of the fixed cavity is connected with the limiting channel, the width of the limiting channel is 200-300 mu m, so that the tail part of the zebra fish is consistent with the structural characteristics of the tail part of the zebra fish, and a better fixing effect is achieved. The height of the limiting channel is 500 to 600 μm. The tail end of the limiting channel is connected with a movable cavity, the movable cavity is a fan-shaped cavity with the radius of 2.5-3.0 mm, so that zebra fish can show various different motion states (such as J-shaped bending and C-shaped bending) at the movable cavity, the height of the movable cavity is 500-600 mu m, and the movable cavities are communicated with one another.

In another embodiment of the present invention, the zebra fish microfluidic chip has a plurality of micro-structural units, and each micro-structural unit is sequentially provided with a liquid inlet channel, a fixed cavity, a limiting channel and a liquid outlet channel. The height of the liquid inlet channel and the height of the fixed cavity are both 500-600 microns, and the width of the liquid inlet channel and the fixed cavity is 900-1000 microns. The liquid inlet channel is simultaneously connected with 2 or more fixed cavities, and the fixed cavities are circularly unfolded at different angles by taking the liquid inlet channel as a circle center. The joint of the liquid inlet channel and the fixed cavity is set to be an arc with the radius of 100-150 mu m. The fixed cavity is tapered, the width of the fixed cavity is narrowed from 800 to 1000 μm to 250 to 300 μm along the flow direction of the fluid, and the height of the fixed cavity is 500 to 600 μm. The tail end of the fixed cavity is connected with the limiting channel, and the width of the limiting channel is 150-300 mu m.

In an embodiment of the present invention, the zebrafish microfluidic chip is prepared by copying a microfluidic hollow channel from a mold using Polydimethylsiloxane (PDMS).

In a second aspect of the present invention, there is provided:

a zebra fish microfluidic system comprises a loading device, the zebra fish microfluidic chip, an observation device and an analysis device;

the zebra fish microfluidic chip can be used for loading a plurality of zebra fish samples in a single visual field observed or imaged by the observation device.

At present, agarose manual embedding, continuous water flow injection or long-term anesthesia treatment is basically implemented for micro-control and fixation of zebra fish, the control process is too complicated, time and labor are wasted, samples are difficult to move, excessive errors and uncertainty are introduced to experimental and application results due to manual control, agarose curing temperature, anesthesia treatment and the like, and the zebra fish micro-fluidic chip system provided by the invention can realize automatic fixation of the zebra fish through a fluid dynamics method and a micro-structure design under the condition that the long-term anesthesia treatment is not performed.

Further, the zebra fish sample comprises zebra fish fries and zebra fish roes.

In a third aspect of the present invention, there is provided:

the zebra fish microfluidic chip or the zebra fish microfluidic system is applied to the real-time information acquisition or comparison of multiple zebra fish samples.

In a fourth aspect of the present invention, there is provided:

the zebra fish microfluidic chip or the zebra fish microfluidic system is applied to drug screening.

Of course, according to actual needs, the physiological information collecting and screening system of the present invention can be applied to biomedical basic research in the field.

The invention has the beneficial effects that:

1. the micro-fluidic chip has the advantages of simple structure, convenient manufacture, low cost and easy operation, and can realize the automatic directional fixation and loading of zebra fish fries in a very short time;

2. the micro-fluidic system can realize the control of the zebra fish fries with multiple samples under a single acquisition window, greatly reduces time loss, and further improves flux and efficiency;

3. in order to better realize multi-sample information acquisition, the micro-fluidic chip is additionally provided with the supporting backflow baffle in the movable cavity, and the supporting backflow baffle can enable micro-fluid to form backflow in front of the baffle, so that liquid can fully fill the whole cavity, bubbles are reduced, the adverse effects on the movement of the tail of the zebra fish and the acquisition of various signals are reduced, and the zebra fish is prevented from being damaged;

4. the microfluidic system can acquire physiological information of various different parts and organs, and is favorable for promoting researchers to further research and understand complex systems in animal bodies.

Drawings

Fig. 1 is a schematic design diagram of a microfluidic chip in example 1;

FIG. 2 is a schematic diagram of the microfluidic chip of example 1 for automatically loading and directionally fixing zebra fish fries;

fig. 3 is a schematic design diagram of the microfluidic chip in examples 2 and 3, wherein a is example 2 and b is example 3;

FIG. 4 is a schematic design diagram of the microfluidic chip of example 4;

FIG. 5 is a schematic representation of the multi-sample signal acquisition under a single acquisition window, wherein A is a open-field image and B is an image of transgenic zebrafish (elavl3: GCaMP5G) with genetically encoded calcium indicators under a fluorescence microscope (excitation light: 488 nm);

fig. 6 is a comparison of a fluid dynamics simulation of a supporting return baffle, wherein a is a microfluidic chip with a supporting return baffle structure and b is a microfluidic chip without a supporting return baffle structure;

fig. 7 is a schematic diagram of the actual effect of the supporting backflow baffle, wherein a is a microfluidic chip with a supporting backflow baffle structure, and b is a microfluidic chip without a supporting backflow baffle structure;

FIG. 8 is a schematic view of multi-sample multi-information acquisition under a microscope;

FIG. 9 is a graph of the effect of different concentrations of ethanol solutions on the behavioural characteristics of zebrafish, wherein a is the effect of different concentrations of ethanol solutions on tail oscillations; b is the effect of ethanol solutions of different concentrations on the movement of the mouth; c is the effect of eye rotation of different concentrations on tail swing;

FIG. 10 is a graph of the effect of ethanol solutions of different concentrations on the heart beat of zebra fish, wherein a is a line graph of the dynamic effect within 15min from the start of the test, and b is a histogram of the statistical analysis of the whole test process.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Polydimethylsiloxane (PDMS) used in the examples of the present invention was purchased from the PDMS used in the examples of the present invention

The silicone (PDMS) was purchased from DOW CORNING SYLGARD 184, and includes a prepolymer A and a crosslinker B.

Preparation of zebra fish microfluidic chip

The zebra fish microfluidic chip provided by the invention has the following specific structure:

the zebra fish microfluidic chip consists of a plurality of micro structural units, wherein each micro structural unit is sequentially provided with a liquid inlet channel, a fixed cavity, a limiting channel and a liquid outlet channel;

the height of the limiting channel is smaller than that of the fixing cavity and/or the width of the limiting channel is smaller than that of the fixing cavity, so that automatic partial fixing of the zebra fish fries is achieved based on continuous action of hydrodynamics (the fixing cavity is mainly used for fixing the heads of the zebra fish fries, and the limiting channel is mainly used for limiting the movement of the tails of the zebra fish).

The inlet channel connects 2 or more fixed chambeies simultaneously, fixed chamber uses the inlet channel is the centre of a circle to the expansion is encircleed to different angles to the real-time physiological state of a plurality of zebra fish samples of simultaneous presentation in single field of vision.

Example 1

The zebra fish microfluidic chip in example 1 is shown in fig. 1, and is composed of a plurality of micro structural units, and each micro structural unit is sequentially provided with a liquid inlet channel, a fixed cavity, a limiting channel, a movable cavity (including a supporting backflow baffle), and a liquid outlet channel.

The height of the liquid inlet channel and the liquid outlet channel is 500 micrometers, and the width of the liquid inlet channel and the liquid outlet channel is 900 micrometers.

The liquid inlet channel is simultaneously connected with 3 fixed cavities, and the fixed cavities are circularly unfolded by taking the liquid inlet channel as a circle center at different angles. The joint of the liquid inlet channel and the fixed cavity is set to be an arc with the radius of 150 mu m.

The fixed cavity is tapered, the width of the fixed cavity is narrowed from 900 μm to 260 μm along the flowing direction of the fluid, and the height of the fixed cavity is 500 μm. The tail end of the fixing cavity is connected with the limiting channel, the width of the limiting channel is 260 mu m, so that the tail part of the zebra fish can be met, and a better fixing effect can be achieved. The height of the above-mentioned restricted channel was 500. mu.m. The tail end of the limiting channel is connected with a movable cavity, the movable cavity is a sector cavity with the radius of 2.5mm, so that zebra fish can show various different motion states (such as J-shaped bending, C-shaped bending and the like) at the movable cavity, the height of the movable cavity is 500 mu m, and the movable cavities are communicated with one another.

And a supporting backflow baffle plate perpendicular to the fluid flowing direction is arranged in the movable cavity, and the thickness of the supporting backflow baffle plate is 400 micrometers.

The zebra fish microfluidic chip described in example 1 was prepared using Polydimethylsiloxane (PDMS) by replicating the microfluidic hollow channels in the zebra fish microfluidic chip from the mold.

The method comprises the following specific steps:

preparing a mould: machining the copper plate by using a CNC (computer numerical control) machine tool; and (3) cleaning the template, soaking the processed copper plate in absolute ethyl alcohol for one night, then clamping an alcohol cotton sheet by using a pair of tweezers to slightly wipe the copper plate, removing stains at corners and gaps, then ultrasonically cleaning for 20min, and blow-drying by using an air gun to finish the preparation of the microfluidic chip template.

PDMS (prepolymer A) and PDMS (crosslinking agent B) are put into the same container according to the proportion of 10:1, fully and uniformly stirred, and then the bubbles are removed under vacuum at room temperature. The mixed PDMS was then poured onto a cleaned copper template and again evacuated to completely remove air bubbles. Baking in oven at 80 deg.C for 4-6 hr. And after the PDMS is completely solidified, taking out and cooling to room temperature, taking the PDMS out of the mold, and punching the PDMS by using a puncher according to the positions of an inlet and an outlet.

Preparing a glass microfluidic negative plate by adopting a conventional method in the field according to the size of the designed microfluidic chip; and (3) treating the joint surface of the PDMS microfluidic chip substrate and the glass cover plate by using plasma, and then combining the two together to finish the preparation of the microfluidic chip.

The zebra fish micro-fluidic chip based on the gel micro-valve prepared by the embodiment is a back-up type zebra fish micro-fluidic chip fixed directionally, and can be used for collecting head information (observing brain nerve signals) and behavioural movements. As shown in fig. 2, the automatic loading and orientation fixing diagram based on the zebra fish microfluidic chip is provided.

Example 2

The zebra fish microfluidic chip in example 2 is shown in fig. 3a, and the zebra fish microfluidic chip in example 2 is different from the zebra fish microfluidic chip in example 1 in that a liquid inlet channel is simultaneously connected with 5 fixed cavities.

The preparation method of the zebra fish microfluidic chip in example 2 is shown in example 1.

Example 3

The zebra fish microfluidic chip in example 3 is shown in fig. 3b, and the zebra fish microfluidic chip in example 3 is different from the zebra fish microfluidic chip in example 1 in that a liquid inlet channel is simultaneously connected with 7 fixed cavities.

The preparation method of the zebra fish microfluidic chip in example 3 is shown in example 1.

Example 4

The zebra fish microfluidic chip in example 4 is shown in fig. 4, and is composed of a plurality of micro structural units, and each micro structural unit is provided with a liquid inlet channel, a fixed cavity, a limiting channel, and a liquid outlet channel in sequence. The liquid inlet channel is arranged at one end of the microfluidic substrate, and the liquid outlet channel is arranged at the other end of the microfluidic substrate; the liquid inlet channel and the liquid outlet channel are connected with the reaction channel through fillets, and the radius of each fillet is 150 micrometers. The widths of the liquid inlet channel, the micro valve generating cavity and the liquid outlet channel are all 900 micrometers. The height of the front ends of the liquid inlet channel and the fixed cavities is 800 micrometers, the liquid inlet channel can be simultaneously connected with 3, 5 and 7 fixed cavities, the fixed cavities are arranged around the liquid inlet channel as a circle center at different angles, and the fixed cavities are not communicated with each other. The height of the fixed chamber gradually decreases (from 800 μm to 250 μm) in the direction of fluid flow, and thus it assumes a tapered structure in side view, with a fixed chamber width of 900 μm. The tail end of the fixed cavity is connected with a limiting channel, the height of the limiting channel is reduced to 150 mu m from 250 mu m along the flowing direction of the fluid, the limiting channel is 4mm long and 900 mu m wide, and therefore the tail of the zebra fish fry can only be allowed to pass through. The above-described chamber design is critical to the lateral immobilization of zebrafish. The tail end of the limiting channel is connected with the liquid outlet channel, and the height of the liquid outlet channel is 150 mu m.

The method for preparing the microfluidic chip in example 4 is as shown in example 1.

The zebra fish microfluidic chip based on the gel microvalve prepared by the embodiment is a zebra fish microfluidic chip with a side surface upward type fixed in an oriented manner, and can be used for observing the physiological conditions of organs of heart and blood vessel. The side-up chip is equivalent to a configuration in which the head-up microfluidic chip structure is rotated by 90 degrees along an XZ plane in a three-dimensional space so that zebra fish fries are side-up.

Embodiment 5 zebra fish microfluidic system

The zebra fish microfluidic system in the embodiment mainly comprises any one of the microfluidic chips in embodiments 1-4, an automatic zebra fish loading device (comprising various conveying devices), a drug injection module, and an optical microscope (observation device) connected with a computer (analysis device). The micro-fluidic chip is a core technology of the system and is also a vital part for zebra fish automatic control and single-view multi-sample information real-time acquisition. The medicine is added and is realized through the control of flow rate of binary channels syringe pump, and the microscope is furnished with high definition digtal camera and is used for catching zebra fish juvenile fish's image and video in the research process.

Before loading zebra fish, water is firstly injected into the inlet of the liquid inlet channel and fills the whole channel. By utilizing the principle of fluid dynamics, zebra fish can enter each micro array in order, and for each micro array, according to the distribution of a flow velocity field (as shown in figure 2), the zebra fish can enter different fixing chambers in sequence, so that the automatic loading and fixing of the zebra fish are completed. In order to keep the living zebra fish firmly fixed at the correct position to complete subsequent brain information and behavior data acquisition, after the zebra fish is loaded, a fine positive pressure needs to be continuously applied to the chamber at an inlet by 10-20 mL/h of water flow, and after the zebra fish is loaded, experiments and researches can be carried out in an expanded mode.

FIG. 5 records high throughput zebrafish fry manipulation of multiple samples at a single collection window (where A is the open field image and B is the image under fluorescent microscope (excitation light: 488nm) of transgenic zebrafish (elavl3: GCaMP5G) with genetically encoded calcium indicator), respectively. Under a common optical microscope, the zebra fish microfluidic system can realize the collection of multi-sample neural activity calcium ion fluorescence signals, and by combining optical imaging with transgenic zebra fish (elavl3: GCaMP5G) with a genetic coding calcium indicator, real-time whole brain nerve imaging is allowed, brain nerve signals are collected, and the change of neuron activity is analyzed.

Example 6 testing of the Effect of the supporting reflux baffle of the Zebra fish microfluidic chip

The zebra fish microfluidic chip is prepared according to the method in the embodiment 1, wherein the zebra fish microfluidic chip without the supporting backflow baffle structure is used as a control, and the using effect of the zebra fish microfluidic chip in the embodiment 1 and the zebra fish microfluidic chip in the control group is detected.

As shown in fig. 6, by comparing the fluid dynamics simulation of the zebra fish microfluidic chip in example 1 with that of the control group, it can be found that the flow direction of the fluid in the zebra fish microfluidic chip in example 1 is affected by the supporting backflow baffle, so that the microfluidic forms a backflow in front of the baffle, so that the liquid can fill the whole chamber more fully, and if the supporting backflow baffle is not provided (as shown in fig. 6b), a plurality of areas with slow flow are generated in the microfluidic chip, so that air bubbles may not be taken away by the water flow and be deposited. And according to repeated tests, the probability that large bubbles appear in the moving cavity area of the zebra fish microfluidic chip without the supporting backflow baffle structure is high (as shown in fig. 7), and the situation not only influences the movement of the tail of the zebra fish and the acquisition of various signals, but also can injure the zebra fish.

Moreover, verification shows that the supporting backflow baffle can also play a supporting role to prevent the collapse of a large-area hollow structure, and the supporting backflow baffle plays an important role in the structural stability and the test accuracy of the zebra fish microfluidic chip.

Example 7 application of zebra fish microfluidic chip and zebra fish microfluidic system in zebra fish fry multi-sample real-time information acquisition

The loading and immobilization of zebrafish was performed as described in example 5, and the loaded fish chips were placed under a microscope and connected to a waste reservoir, one of which was filled with E3(5mM NaCl; 0.17mM KCl; 0.33mM CaCl; inlet of inlet channel and two-channel syringe pump₂；0.33mM MgSO₄(ii) a 0.00001% (w/v) methyl Blue) connected (using water as a control group (CTRL)), continuously injecting E3 water into the chip at a flow rate of 10-20 mL/h, simultaneously recording a light field image as a control group data by using photomicrographs, then switching to ethanol solutions with different volume ratios (1%, 2%, 3%, v/v respectively), and then placing the chip under a microscope and recording the movement of the head and tail of the zebra fish fry.

The zebra fish microfluidic chip disclosed by the invention uses a 4-time microscope lens, and can be used for simultaneously acquiring data of at least two samples, so that the efficiency is increased to two times of the original efficiency.

The region of interest is then selected by the Image processing software Image J for subsequent processing and analysis of all the pictures taken, the results of which are shown in fig. 8.

Acute ethanol treatment adversely affects the development of zebrafish nerves, and exposure of zebrafish to ethanol solution can affect the histaminergic and dopaminergic systems, thereby stimulating the motility of zebrafish larvae. After the zebra fish is treated by ethanol with different concentrations, the behavioral movements of the tail, eyes and mouth can be analyzed. The rotation frequency of the zebra fish eyes relative to the control group is found to increase along with the increase of the ethanol concentration; for movements of the mouth, the movement frequency is maximum in an ethanol solution with the volume ratio of 2 percent; for tail waggles, the ethanol in each of the different volume ratios showed no significant difference relative to the control.

Example 8 simultaneous and long-time information collection capability test of a zebra fish microfluidic chip and a zebra fish microfluidic system zebra fish load zebra fish in the manner of example 7, and the same experimental group and control group are set.

The chip was placed under a microscope and the movements of the head and tail of the zebrafish fry were recorded (5 samples were recorded in each case); the Image processing software Image J then selects a cardiac region for subsequent processing and analysis, and the results are shown in fig. 9.

Due to its high solubility in aqueous environments, ethanol is able to diffuse extremely easily through biological membranes and rapidly affects the tissues and organs of zebrafish fry, in particular the cardiovascular and central nervous systems. After ethanol with a certain concentration is added, the heart beat of the zebra fish in the experimental group is increased compared with that in the control group, but the heart beat frequency of the zebra fish is gradually reduced along with the increase of the ethanol concentration (volume ratio).

According to the dynamic effect of ethanol solutions with different concentrations on the heart beat of the zebra fish (figure 10), the E3 water is continuously infused into the chip for the first 5min, and ethanol solutions with different concentrations are added from the 6 th min, and the heart beat frequency is also rapidly increased from the 6 th min. The beating frequency showed instability within 10min of continuous perfusion for 1% and 2% ethanol solutions, but no significant decrease, while for 3% ethanol solutions the beating frequency showed a significant decrease. After the data collection was completed, the zebra fish was pushed out of the liquid channel and placed into E3 water.

And continuously recording the state of the zebra fish fries in E3 water in 72 hours after data acquisition, and finding that the zebra fish fries still keep a normal physiological state and do not die or are abnormal. The zebra fish micro-fluidic chip and the zebra fish micro-fluidic system can ensure that the survival rate of experimental zebra fish is 100 percent.

Comparative example 1 comparison of Zebra fish sample observation capability of Zebra fish microfluidic chip System in the prior art and the present invention

The existing zebra fish fixing technology participating in comparison comprises the following steps:

1. directly anaesthetizing the zebra fish (Tricain anaesthesia);

2. fixing the zebra fish by using agarose gel or other gel (manual agarose embedding);

ZEBRA technology;

VAST technique;

fish-trap technique;

6. the same inventor patent of patent No. 202010546028.0 (hereinafter referred to as the former patent).

The comparison items are the degree of automation, whether anesthesia is needed, the degree of flux and the observed quantity of the sample.

The results are shown in the following table.

Table 1 comparison of zebra fish sample observation capabilities of the prior art and zebra fish microfluidic chip systems of the present invention

Tricain anesthesia

Agarose/hydrogel embedding

ZEBRA

VAST

Fish-trap

The last patent

The invention

Degree of automation

Hand operated

Hand operated

Hand operated

Automatic

Automatic

Automatic

Automatic

Whether or not anesthesia is required

Is that

Is that

Whether or not

Is that

Whether or not

Whether or not

Whether or not

Degree of flux

Is low in

Is low in

Is low in

Is low in

Height of

Height of

Height of

Observed quantity of sample

Multiple purpose

Multiple purpose

Is single

Is single

Is single

Is single

Multiple purpose

As shown in the above table, the existing technologies for acquiring physiological information data of zebra fish are generally classified into two types, i.e. anesthesia and non-anesthesia: the device can play a role in rapidly fixing the zebra fish under the anesthesia condition, and although the data of a plurality of zebra fish can be simultaneously collected under one lens under the condition, the anesthesia time efficiency is very limited under the condition of not influencing the normal body health of the zebra fish, and the manual operation is difficult to avoid, so that the device has great instability and failure rate. Furthermore, it is also common to immobilize zebrafish after anesthesia using agarose or other gels, but such techniques often require manual embedding and are difficult to achieve with directional immobilization and difficult to further expand in throughput. The invention relates to a method for collecting information of zebra fish, which is characterized in that a micro-fluidic chip is arranged on a flow path of a zebra fish, a micro-fluidic chip is arranged on a flow path of the zebra fish, and a support structure is arranged on the flow path of the micro-fluidic chip, wherein the micro-fluidic chip is used for collecting information of a plurality of zebra fish. In summary, the invention has the advantage of simultaneously capturing the physiological information of a plurality of zebra fish under the condition of realizing single microscope visual field under the premise of no anesthesia and automatic directional fixation of the zebra fish, the high-flux and high-content zebra fish fry screening system greatly reduces the time cost, and contributes to further promoting large-scale drug screening, rapid detection and acquisition of various physiological information.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.