Drug concentration and water flow velocity gradient analysis micro-fluidic chip based on repeated capture and release of zebra fish and application of micro-fluidic chip

Abstract

The invention discloses a micro-fluidic chip for gradient analysis of drug concentration and water flow velocity based on repeated capture and release of zebra fish and application of the micro-fluidic chip. The chip integrates a multi-channel structure based on a glass substrate, and comprises a zebra fish inlet, a zebra fish channel, a detection channel and an outlet, a reagent channel and a liquid channel are respectively arranged on two sides of the middle section of the detection channel, a magnet groove is vertically arranged at the communication part, and a movable permanent magnet is arranged in the magnet groove. Through downward movement of a magnet groove permanent magnet in a detection channel, flexible space constraint is formed by a magnetic pole end and a protruding structure on the inner wall of the channel, and lossless fixation of the zebra fish is achieved. The flow speed of liquid is regulated and controlled by controlling the permanent magnet in the liquid channel; the reagent channel adopts a multi-layer bending structure and is combined with a magnetic control shunting technology, so that uniform distribution of drug concentration gradient is ensured. According to the invention, the whole-process closed-loop control of directional capture-release-analysis of zebra fish is realized through dynamic regulation and control of the magnet, and the reliability and efficiency of high-throughput drug screening are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic chips, and particularly relates to a microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish, and its application. Background Art

[0002] Due to the characteristics of high homology with human genes (similarity up to 87%), transparent embryos and rapid reproduction, zebrafish have become an important model organism for studying human disease mechanisms and drug screening. In zebrafish experiments, in vivo capture and release are key pretreatment steps for microinjection, behavioral analysis and drug exposure. The core lies in achieving rapid and directional fixation and non-destructive release of zebrafish. However, the existing technologies mainly rely on mechanical trap capture devices, which have the following significant defects: (1) Uncontrollable capture direction: Traditional traps rely on passive interception and cannot accurately control the body position of zebrafish (such as head / tail orientation), resulting in a positioning error rate of up to 30% - 40% in micromanipulation; (2) Irreversible release process: The mechanical buckle structure is prone to damage the mucus layer on the fish body surface, and reverse fluid flushing is required for release, causing stress response in zebrafish and affecting the reliability of subsequent experimental data; (3) Poor device reusability: The mechanical structure has problems of seal failure caused by wear, and the capture efficiency decreases by more than 60% after repeated use.

[0003] In recent years, magnetic manipulation technology has been introduced into the microfluidic field due to the advantages of non-contact control. However, existing solutions mainly focus on the manipulation of magnetic nanoparticles or simple liquid flow blockage. Although the magnetic control valve structure can achieve the on / off of pipelines, its single vertical movement mode of a magnet has the following limitations: (1) Single functionality: It can only achieve the on / off of liquid flow and cannot meet the multi-dimensional fixation requirements of zebrafish in vivo; (2) Insufficient spatial resolution: The magnetic field action range is difficult to match the millimeter-level body length of zebrafish, easily causing fish body slippage; (3) Irreversible operation: After the magnet is fixed, the chip needs to be disassembled to release zebrafish, which violates the requirements of high-throughput experiments.

[0004] In addition, most existing microfluidic chips adopt a single-layer flow channel design, and the zebrafish channel and reagent flow channel interfere with each other, resulting in uneven distribution of drug concentration gradient and seriously affecting the accuracy of toxicology experiments. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the present invention provides a microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish, and its application, and realizes the full-process closed-loop control of zebrafish directional capture - release - analysis through specific structural design.

[0006] The present invention is realized through the following technical solutions:

[0007] A microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish, comprising a glass substrate and a detection chip disposed thereon. The detection chip includes a zebrafish inlet, a zebrafish channel, and a detection channel that are sequentially connected. The end of the detection channel is connected to an outlet. On one side of the middle section of the detection channel, there is a reagent channel. The top of the reagent channel is provided with a reagent inlet, and the bottom extends into a curved pipe and communicates with the detection channel. On the other side of the middle section of the detection channel, there is a liquid channel. The head of the liquid channel is provided with a liquid inlet, and the end communicates with the detection channel.

[0008] A first magnet groove is vertically provided at the connection between the reagent channel and the detection channel. A third magnet groove is vertically provided at the connection between the liquid channel and the detection channel. A second magnet groove is provided on the detection channel. A movable permanent magnet is provided in each magnet groove.

[0009] The second magnet groove and the detection channel form a zebrafish fixing mechanism. When the permanent magnet in the second magnet groove moves down to the bottom, the magnetic end thereof forms a space constraint with the convex structure on the inner wall of the detection channel to jointly clamp and fix the zebrafish.

[0010] Preferably, the reagent channel extends downward from the reagent inlet to form a pyramid-shaped multi-layer curved pipe array. The top of the curved pipe close to the detection channel is connected to the bottom of the curved pipe far from the detection channel. The top opening of the topmost curved pipe forms the reagent inlet, and the bottom of the bottommost curved pipe communicates with the side wall of the detection channel through the first magnet groove.

[0011] Preferably, the liquid channel extends upward from the liquid inlet to form an inverted pyramid-shaped multi-layer vertical pipe array perpendicular to the detection channel. The top of the vertical pipe far from the detection channel is connected to the bottom of the vertical pipe close to the detection channel. The bottom opening of the bottommost vertical pipe forms the liquid inlet, and the top of the topmost vertical pipe communicates with the side wall of the detection channel through the third magnet groove.

[0012] Preferably, the bottom end of the reagent channel is provided with 3 curved pipes respectively corresponding to 3 first magnet grooves and communicating with the detection channel. The top end of the liquid channel is provided with 3 vertical pipes respectively corresponding to 3 third magnet grooves and communicating with the detection channel.

[0013] Preferably, the zebrafish channel is composed of two diagonal pipes. One extends obliquely from the lower zebrafish inlet to the upper left and then converges into the detection channel. The other extends obliquely from the upper zebrafish inlet to the lower right and then converges into the detection channel.

[0014] Preferably, the axis of the second magnet groove is set at an angle of 30° to 60° with the extension direction of the detection channel.

[0015] Preferably, the convex structure on the inner wall of the detection channel is a semi-circular limiting block, and the arc curvature radius thereof matches the body surface curvature of the zebrafish.

[0016] Preferably, anti-disengagement limiting flanges are provided at the tops of the trough bodies of the first magnet trough, the second magnet trough and the third magnet trough.

[0017] Preferably, the detection chip is made of polydimethylsiloxane.

[0018] The application of the above microfluidic chip in drug analysis.

[0019] The beneficial effects of the present invention are as follows:

[0020] (1) Non-destructive clamping structure: The second magnet trough and the semi-circular limiting block are integrated in the detection channel, and the flexible magnetic field generated by the downward pressure of the magnet is used to realize the wrapped fixation of the fish body.

[0021] (2) Liquid flow rate control: The liquid flow rate is regulated by combining the magnet trough in the liquid channel with the magnet lift, which can meet the requirements of different experimental conditions.

[0022] (3) Precise control of drug concentration: The pyramid-shaped multi-layer bending structure of the reagent channel is combined with magnetic control shunt, so that the drug concentration is controllable and the diffusion uniformity is effectively improved. Description of the Drawings

[0023] Figure 1 It is the overall structure diagram of the microfluidic chip for drug concentration and water flow velocity gradient analysis based on repeated capture and release of zebrafish;

[0024] Figure 2 It is the structural schematic diagram of the detection chip;

[0025] Figure 3 It is the cross-sectional structural schematic diagram of the second magnet trough and the detection channel;

[0026] Figure 4 It is the structural schematic diagram of the first magnet trough and the third magnet trough;

[0027] Figure 5 It is the sectional view of the permanent magnet position when the channel is closed;

[0028] Figure 6 It is the sectional view of the permanent magnet position when the channel is opened;

[0029] Figure 7 It is the photo of capturing the zebrafish;

[0030] Figure 8 It is the output flow rate diagram after cutting different numbers of channels under the same conditions in the reagent channel: A is for three channels, B is for two channels, and C is for single channel;

[0031] Figure 9 Output flow velocity diagrams after cutting different numbers of channels under the same conditions in the liquid channel: A is for three channels, B is for two channels, and C is for a single channel;

[0032] In the figure: 1. Zebrafish inlet; 2. Zebrafish channel; 3. First magnet slot; 4. Second magnet slot; 5. Reagent inlet; 6. Reagent channel; 7. Detection channel; 8. Outlet; 9. Third magnet slot; 10. Liquid channel; 11. Liquid inlet; 12. Glass substrate; 13. Polydimethylsiloxane layer; 14. Permanent magnet; 15. Protruding structure. Specific embodiments

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] In the description of the present invention, terms indicating orientation or positional relationships such as "top", "bottom", "head", "tail", "upper", "lower", "inner", "outer", etc. are only based on the orientation shown in the drawings for the convenience of describing the present invention, rather than indicating or implying that the device referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0035] Embodiment 1

[0036] A microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish, as Figure 1 、 2 shown, includes a glass substrate 12 and a polydimethylsiloxane layer 13 provided thereon. A detection chip is provided inside the polydimethylsiloxane layer 13. The detection chip includes a zebrafish inlet 1, a zebrafish channel 2, and a detection channel 7 that are connected in sequence. The end of the detection channel 7 is connected to an outlet 8; on one side of the middle section of the detection channel 7, there is a reagent channel 6. A reagent inlet 5 is provided at the top of the reagent channel 6, and the bottom extends as a curved pipe and communicates with the detection channel 7; on the other side of the middle section of the detection channel 7, there is a liquid channel 10. The head of the liquid channel 10 is provided with a liquid inlet 11, and the end communicates with the detection channel 7.

[0037] As Figures 2-4 shown, a first magnet slot 3 is vertically provided at the connection between the reagent channel 6 and the detection channel 7, a third magnet slot 9 is vertically provided at the connection between the liquid channel 10 and the detection channel 7, and a second magnet slot 4 is provided on the detection channel 7. A movable permanent magnet 14 is provided in each magnet slot.

[0038] As Figure 2 、 3As shown, the second magnet groove 4 and the detection channel 7 form a zebrafish fixing mechanism. When the permanent magnet 14 in the second magnet groove 4 moves down to the bottom, the magnetic pole end thereof and the protruding structure 15 on the inner wall of the detection channel 7 form a spatial constraint to jointly clamp and fix the zebrafish.

[0039] As Figure 2 shown, the reagent channel 6 extends downward from the reagent inlet 5 to form a pyramid-shaped multi-layer curved pipe array, wherein the top of the curved pipe on the side close to the detection channel 7 is connected to the bottom of the curved pipe on the side far from the detection channel 7, and the top opening of the topmost curved pipe forms the reagent inlet 5, and the bottom of the bottommost curved pipe communicates with the side wall of the detection channel 7 through the first magnet groove 3.

[0040] As Figure 2 shown, the liquid channel 10 extends upward from the liquid inlet 11 to form an inverted pyramid-shaped multi-layer vertical pipe array perpendicular to the detection channel 7, wherein the top of the vertical pipe on the side far from the detection channel 7 is connected to the bottom of the vertical pipe on the side close to the detection channel 7, the bottom opening of the bottommost vertical pipe forms the liquid inlet 11, and the top of the topmost vertical pipe communicates with the side wall of the detection channel 7 through the third magnet groove 9.

[0041] As Figure 2 shown, in this embodiment, the bottom end of the reagent channel 6 is provided with 3 curved pipes respectively corresponding to 3 first magnet grooves 3 to communicate with the detection channel 7, and the top end of the liquid channel 10 is provided with 3 vertical pipes respectively corresponding to 3 third magnet grooves 9 to communicate with the detection channel 7. By changing the array numbers (the numbers of the curved pipes and the vertical pipes) of the reagent channel 6 and the liquid channel 10, different experimental conditions can be satisfied.

[0042] As Figure 2 shown, the three curved pipes of the reagent channel 6 are arranged in sequence along the liquid flow direction of the detection channel 7, wherein one curved pipe close to the zebrafish channel 2 is arranged 9 mm in front of the protruding structure 15, and the distance between the three curved pipes is 18 mm each; the three vertical pipes of the liquid channel 10 are arranged in sequence along the liquid flow direction of the detection channel 7, wherein one vertical pipe close to the zebrafish channel 2 is arranged 0.2 mm behind the protruding structure 15, and the distance between the three vertical pipes is 18 mm each.

[0043] As Figure 2 shown, in this embodiment, the zebrafish channel 2 is composed of two oblique pipes, one extending obliquely from the lower zebrafish inlet 1 to the upper left and then converging into the detection channel 7; the other extending obliquely from the upper zebrafish inlet 1 to the lower right and then converging into the detection channel 7.

[0044] In a preferred solution, the axis of the second magnet groove 4 is arranged at an angle of 30° to 60° with the extending direction of the detection channel 7.

[0045] As Figure 2 、 3 shown, the convex structure 15 on the inner wall of the detection channel 7 is a semi-circular limiting block, and the arc curvature radius thereof matches the body surface curvature of the zebrafish.

[0046] In a preferred solution, anti-detachment limiting flanges are provided at the tops of the groove bodies of the first magnet groove 3, the second magnet groove 4, and the third magnet groove 9.

[0047] The permanent magnet 14 in each magnet groove has an S pole and an N pole. An electromagnet is arranged in the area corresponding to the permanent magnet 14 outside the microfluidic chip and is controlled by an electromagnetic drive circuit. By changing the current direction in the electromagnetic coil, repulsive and attractive forces are generated on the permanent magnet 14, so that the movement of the permanent magnet 14 can be manipulated. As Figure 4 shown, the permanent magnet 14 embedded in the first magnet groove 3 and the third magnet groove 9 moves perpendicular to the glass substrate 12. As Figure 5 shown, when the permanent magnet 14 is attracted and approaches the glass substrate 12, the channel is blocked and the liquid flow cannot pass through, thereby realizing an open circuit. As Figure 6 shown, when the permanent magnet 14 is repelled and moves away from the glass substrate 12, the channel is unblocked and the liquid flow can pass through. The permanent magnet 14 embedded in the second magnet groove 4 moves horizontally on the glass substrate 12. As Figure 3 shown, when the permanent magnet 14 is attracted and approaches the detection channel 7, it moves to a preset boundary and stops. At this time, the permanent magnet 14 and the convex structure 15 on the side wall of the detection channel 7 form a spatial constraint to clamp and fix the zebrafish. When the experiment is completed, the permanent magnet 14 is repelled and moves away from the detection channel 7 until it stops at the preset boundary. At this time, the permanent magnet 14 completely exits the detection channel 7, and the detection channel 7 is unblocked and allows the zebrafish to pass through.

[0048] The steps of the above microfluidic chip for analyzing the drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish in actual operation are specifically as follows:

[0049] (1) First, pump clear water from the zebrafish inlet 1 to apply pressure to the channel, move the permanent magnet 14 in the second magnet groove 4 to the bottom, and then pump a zebrafish from the zebrafish inlet 1. The zebrafish passes through the zebrafish channel 2 and the detection channel 7 and is captured at the second magnet groove 4, as Figure 7 shown.

[0050] (2) Pump the drug from the reagent inlet 5, and by controlling the permanent magnet 14 in the first magnet groove 3, adjust the number of on-off bends of the curved pipe of the reagent channel 6, and different concentration drug stimulation studies on the same zebrafish can be carried out.

[0051] The reagent inlet 5, the reagent channel 6, the first magnet slot 3 and the permanent magnet 14 therein form a drug concentration control area. By controlling the position of the permanent magnet 14 in the first magnet slot 3, the on-off of the reagent channel 6 can be controlled. Since the inlet flow rate remains unchanged, the drug concentration flowing into the reagent channel 6 can thus be controlled. As Figure 8 shown are the output flow rates after cutting off different numbers (1, 2, 3) of channels under the same conditions.

[0052] As Figure 8 shown in A, when all three channels are open, the flow rate of each channel is 0.4 m / s; as Figure 8 shown in B, when two channels are open, the flow rate of each channel is 0.55 m / s; as Figure 8 shown in C, when a single channel is open, the flow rate of the channel is 1.3 m / s.

[0053] (3) Pump liquid from the liquid inlet 11, and by controlling the number of vertical pipe on-offs of the liquid channel 10 by adjusting the permanent magnet 14 in the third magnet slot 9, research on different flow rate stimulations of the same zebrafish tail can be carried out.

[0054] The liquid inlet 11, the liquid channel 10, the third magnet slot 9 and the permanent magnet 14 therein form a water flow velocity gradient control area. By controlling the position of the permanent magnet 14 in the third magnet slot 9, the on-off of the liquid channel 10 can be controlled. Since the inlet flow rate remains unchanged, the liquid flow velocity flowing into the liquid channel 10 can thus be controlled. As Figure 9 shown are the output flow rates after cutting off different numbers (1, 2, 3) of channels under the same conditions.

[0055] As Figure 9 shown in A, when all three channels are open, the flow rate of each channel is 0.25 m / s; as Figure 9 shown in B, when two channels are open, the flow rate of each channel is 0.35 m / s; as Figure 9 shown in C, when a single channel is open, the flow rate of the channel is 0.65 m / s.

[0056] (4) Steps (2) and (3) above can be implemented simultaneously to conduct joint research on drug concentration stimulation and flow rate stimulation.

[0057] (5) After the experiment is completed, adjust the permanent magnet 14 in the second magnet slot 4 to a position far away from the detection channel 7 to release the zebrafish. The zebrafish is recovered through the detection channel 7 and the outlet 8.

[0058] The embodiments described above are only a part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope claimed in the claims. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish, characterized in that, It includes a glass substrate (12) and a detection chip disposed thereon. The detection chip includes a zebrafish inlet (1), a zebrafish channel (2), and a detection channel (7) that are sequentially connected. The end of the detection channel (7) is connected to an outlet (8). On one side of the middle section of the detection channel (7), there is a reagent channel (6). The top of the reagent channel (6) is provided with a reagent inlet (5), and the bottom extends into a curved pipe and communicates with the detection channel (7). On the other side of the middle section of the detection channel (7), there is a liquid channel (10). The head end of the liquid channel (10) is provided with a liquid inlet (11), and the end communicates with the detection channel (7). A first magnet groove (3) is vertically provided at the connection between the reagent channel (6) and the detection channel (7). A third magnet groove (9) is vertically provided at the connection between the liquid channel (10) and the detection channel (7). A second magnet groove (4) is provided on the detection channel (7). A movable permanent magnet (14) is provided in each magnet groove. The second magnet groove (4) and the detection channel (7) form a zebrafish fixing mechanism. When the permanent magnet (14) in the second magnet groove (4) moves down to the bottom, its magnetic pole end forms a spatial constraint with the protruding structure (15) on the inner wall of the detection channel (7) to jointly clamp and fix the zebrafish.

2. The microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish according to claim 1, wherein The reagent channel (6) extends downward from the reagent inlet (5) to form a pyramid-shaped multi-layer curved pipe array. The top of the curved pipe close to the detection channel (7) is connected to the bottom of the curved pipe far from the detection channel (7). The top opening of the topmost curved pipe forms the reagent inlet (5), and the bottom of the bottommost curved pipe communicates with the side wall of the detection channel (7) through the first magnet groove (3).

3. The microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish according to claim 1, wherein, The liquid channel (10) extends upward from the liquid inlet (11) to form an inverted pyramid-shaped multi-layer vertical pipe array perpendicular to the detection channel (7). The top of the vertical pipe far from the detection channel (7) is connected to the bottom of the vertical pipe close to the detection channel (7). The bottom opening of the bottommost vertical pipe forms the liquid inlet (11), and the top of the topmost vertical pipe communicates with the side wall of the detection channel (7) through the third magnet groove (9).

4. A microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish according to claim 1, characterized in that, The bottom end of the reagent channel (6) is provided with 3 curved pipes that respectively correspond to 3 first magnet grooves (3) and communicate with the detection channel (7). The top end of the liquid channel (10) is provided with 3 vertical pipes that respectively correspond to 3 third magnet grooves (9) and communicate with the detection channel (7).

5. A microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish according to claim 1, characterized in that, The zebrafish channel (2) is composed of two diagonal pipes. One extends diagonally from the lower zebrafish inlet (1) to the upper left and then converges into the detection channel (7). The other extends diagonally from the upper zebrafish inlet (1) to the lower right and then converges into the detection channel (7).

6. A microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish according to claim 1, characterized in that, The axis of the second magnet groove (4) is set at an angle of 30° to 60° with the extension direction of the detection channel (7).

7. A microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish according to claim 1, characterized in that, The protruding structure (15) on the inner wall of the detection channel (7) is a semi-circular limiting block, and the arc curvature radius thereof matches the body surface curvature of the zebrafish.

8. A microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish according to claim 1, characterized in that Anti-detachment limiting flanges are provided at the top of the groove bodies of the first magnet groove (3), the second magnet groove (4), and the third magnet groove (9).

9. A microfluidic chip for analyzing drug concentration and water flow velocity gradient based on repeated capture and release of zebrafish according to claim 1, characterized in that, The material of the detection chip is polydimethylsiloxane.

10. Use of the microfluidic chip according to any one of claims 1-9 in pharmaceutical analysis.

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