CN115537330A - Microfluidic chip for detecting multiple phenotypes of zebra fish and application thereof - Google Patents
Microfluidic chip for detecting multiple phenotypes of zebra fish and application thereof Download PDFInfo
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- CN115537330A CN115537330A CN202211269217.3A CN202211269217A CN115537330A CN 115537330 A CN115537330 A CN 115537330A CN 202211269217 A CN202211269217 A CN 202211269217A CN 115537330 A CN115537330 A CN 115537330A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
Abstract
The application discloses a micro-fluidic chip for detecting zebra fish multivariate phenotype includes: the microstructure units comprise a liquid input channel, a liquid output channel, and a zebra fish juvenile behavior phenotype test channel and a zebra fish juvenile brain phenotype test channel which are arranged between the liquid input channel and the liquid output channel; the zebra fish juvenile behavior phenotype test channel is configured to detect the behavior phenotype of a first zebra fish juvenile, the zebra fish juvenile brain phenotype test channel is configured to detect the brain phenotype of a second zebra fish juvenile, and the microfluidic chip is simple and convenient to operate, does not damage the zebra fish, is low in cost and high in throughput, and can simultaneously test the influence of a medicament on the behavioral phenotype and the brain phenotype of the zebra fish juvenile under a microscopic visual field.
Description
Technical Field
The application relates to the technical field of microfluidic chips, in particular to a microfluidic chip for detecting the multivariate phenotype of zebra fish and application thereof.
Background
Zebrafish have many advantages as a model organism, and the various advantages are as follows: high homology with human gene, easy reproduction, low cost, rapid development, transparency of zebra fish embryo and juvenile fish, and simple administration mode.
Compared with phenotypic drug screening based on animal models such as cells and mice, drug screening based on zebrafish phenotype has unique advantages. Live embryos or young fish exhibit diverse changes in biological characteristics and have an integrated vertebrate organ system, so the range of phenotypes that can be measured in zebrafish is much broader than in cells. For example, sedation, pain, tumor metastasis, intestinal motility, and vascular tone are some of the phenotypes associated with disease that are observable in zebrafish but difficult to monitor in cells. Furthermore, the small size of zebrafish embryos means that many thousands of live embryos can be screened for phenotypic effects. Although screening methods based on zebrafish phenotypes are not as high-throughput as cell, yeast or in vitro activity assays, the size of screening zebrafish embryos far exceeds the size available in all other vertebrate systems.
There are many devices and techniques currently available for screening zebrafish phenotypic drugs, including anesthetics, capillaries, agarose, and gelatin. Anesthetic is a common agent used to fix zebra fish in a desired position, and although it is easy to use, it may cause damage to the nervous system of zebra fish. The ultrafine capillary tube can be used for fixing and turning over the zebra fish juvenile fish, but the capillary tube has the defects of poor control and time and labor waste in operation. These time-consuming, irreversible and tedious manual processes can damage the fragile bodies of zebrafish embryos and juvenile fish, thereby affecting the validity of experimental results. Agarose and gelatin may also be used to embed zebrafish larvae, but may also cause damage to zebrafish.
The zebra fish microfluidic chip is a branch in the microfluidic chip technology and is a high-efficiency multifunctional device. The technology can simultaneously process a plurality of zebra fish samples in a high-flux mode, and accurately control the direction of the zebra fish and the corresponding physiological microenvironment of the zebra fish. Although the zebra fish microfluidic chip device has the advantages of automatic operation, rapid operation, time and labor saving and reduction of interaction among zebra fish, the zebra fish microfluidic chip device still has the defects of difficult realization technology, high cost, capability of detecting the phenotype of only a single organ part of the zebra fish and the like.
Therefore, the development of a microfluidic chip which is simple to operate, does not damage zebra fish, is low in cost and high in throughput, and can simultaneously test the influence of a drug on the behavioral phenotype and the brain phenotype of the zebra fish juvenile fish is urgently needed in the field.
Disclosure of Invention
The application aims to provide the microfluidic chip for detecting the multi-phenotype of the zebra fish, the microfluidic chip is simple and convenient to operate, does not damage the zebra fish, is low in cost and high in flux, and can be used for simultaneously testing the influence of a medicament on the behavioral phenotype and the brain phenotype of the zebra fish juvenile fish.
The application provides a micro-fluidic chip for detecting polynary phenotype of zebra fish, which is characterized by comprising: the micro-structure units comprise a liquid input channel, a liquid output channel, and a zebra fish juvenile behavior phenotype test channel and a zebra fish juvenile brain phenotype test channel which are arranged between the liquid input channel and the liquid output channel;
the zebra fish juvenile behavioral phenotype test channel is configured to detect a behavioral phenotype of a first zebra fish juvenile, and sequentially comprises a first fixed microchannel, a first limiting channel and a first fixed microchamber which are in fluid communication along an axial direction; the first stationary microchannel is configured to hold a head of the first zebra fish fry at the time of detection, the first confinement channel is configured to accommodate a body of the first zebra fish fry at the time of detection, the first stationary microchamber is configured to accommodate a tail of the first zebra fish fry at the time of detection, a width of the first confinement channel is smaller than widths of the first stationary microchannel and the first stationary microchamber, and the first stationary microchamber is designed to be a wide rectangle;
the zebra fish juvenile brain phenotype test channel is configured to detect a brain phenotype of a second zebra fish juvenile, and sequentially comprises a second fixed microchannel, a second limiting channel and a second fixed microchamber which are communicated with each other along the axial direction; the second fixed microchannel is configured to fix the head of the second zebra fish juvenile fish when detected, the second restriction channel is configured to accommodate the body of the second zebra fish juvenile fish when detected, the second fixed microchamber is configured to accommodate the tail of the second zebra fish juvenile fish when detected, the width of the second restriction channel is smaller than the width of the second fixed microchannel and the second fixed microchamber, and the second fixed microchamber is designed to be a narrow rectangle.
In another preferred embodiment, the first fixed micro-chamber has a length of 2500-3000 μm and a width of 2300-2800 μm, and the second fixed micro-chamber has a length of 3000-3800 μm and a width of 500-900 μm.
In another preferred embodiment, the first limiting channel has a length of 380 to 450 μm and a width of 250 to 350 μm.
In another preferred example, the length of the second restriction channel is 380-450 μm, and the width is 250-350 μm.
In another preferred example, the width of each of the first fixed microchannel and the second fixed microchannel is between 500 and 950 μm.
In another preferred embodiment, the length of each of the first fixed microchannel and the second fixed microchannel is between 6000 and 9000 μm.
In another preferred example, the width of the liquid input channel and the liquid output channel is between 750 and 950 μm.
In another preferred example, the liquid input channel, the first fixed micro-channel, the first limiting channel, the first fixed micro-chamber and the liquid output channel are all rounded at the joint.
In another preferred example, the liquid input channel, the first fixed micro-channel, the first limiting channel, the first fixed micro-chamber and the liquid output channel are all rounded at the joint.
In another preferred embodiment, the number of microstructure units is 1 to 10.
The application also provides application of the microfluidic chip in drug screening.
In another preferred example, the microfluidic chip comprises a microfluidic chip base and a cover plate connected in a non-permanent bonding manner.
In another preferred example, the material of the base of the microfluidic chip comprises glass, and the material of the cover plate of the microfluidic chip comprises a polydimethylsiloxane cover plate.
In another preferred example, the distance between the zebrafish juvenile behavioral phenotype test channel and the zebrafish juvenile brain phenotype test channel is in the range of 5-11 mm.
It is to be understood that within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described below (e.g. in the examples) can be combined with each other to constitute new or preferred technical solutions. Not to be reiterated herein, but to the extent of space.
The present specification describes a number of technical features distributed throughout the various technical aspects, and if all possible combinations of technical features (i.e. technical aspects) of the present specification are listed, the description is made excessively long. In order to avoid this problem, the respective technical features disclosed in the above summary of the invention of the present application, the respective technical features disclosed in the following embodiments and examples, and the respective technical features disclosed in the drawings may be freely combined with each other to constitute various new technical solutions (which are considered to have been described in the present specification) unless such a combination of the technical features is technically infeasible. For example, in one example, the feature a + B + C is disclosed, in another example, the feature a + B + D + E is disclosed, and the features C and D are equivalent technical means for the same purpose, and technically only one feature is used, but not simultaneously employed, and the feature E can be technically combined with the feature C, then the solution of a + B + C + D should not be considered as being described because the technology is not feasible, and the solution of a + B + C + E should be considered as being described.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is to be understood that the drawings in the following description are merely exemplary of the application, and that one of ordinary skill in the art, without any inventive faculty, may derive other embodiments from these drawings.
FIG. 1 is a schematic structural design diagram of a zebra fish microfluidic chip in the embodiment of the application;
FIG. 2 is a schematic diagram showing the distribution of a liquid input channel, a fixed microchannel, a restriction channel, a fixed microchamber and a liquid output channel of the zebra fish microfluidic chip according to the embodiment of the present application;
FIG. 3 is a schematic view of the hydrodynamic simulation of a behavioral phenotype test micro-channel and a brain phenotype test micro-channel of the zebra fish microfluidic chip according to the embodiment of the present application;
FIG. 4 is a diagram of a zebra fish microfluidic chip in an embodiment of the present application;
fig. 5 is a schematic diagram of loading zebra fish juvenile fish into a zebra fish microfluidic chip in the embodiment of the present application, which is respectively (a) a diagram of an effect of the zebra fish juvenile fish loaded into a test channel for detecting a behavioral phenotype of the zebra fish juvenile fish, (B) a diagram of an effect of the zebra fish juvenile fish loaded into a test channel for detecting a brain phenotype of the zebra fish juvenile fish, and (C) a diagram of a brain of the zebra fish juvenile fish under a fluorescence microscope;
FIG. 6 is a schematic view showing zebra fish larvae (A) both eyes of the zebra fish larvae, (B) hearts of the zebra fish larvae, (C) bodies of the zebra fish larvae, and (D) tails of the zebra fish larvae in the example of the present application;
FIG. 7 is a diagram of the zebrafish juvenile fish of the present application (A) olfactory epithelial nerves, olfactory bulb and subcortical, (B) cerebral cortex and anterior region, (C) mesoencephalic cap, (D) cerebellum, (E) five brain regions of medulla oblongata;
FIG. 8 is a graph showing the effect of organic solvent DMSO and various concentrations of methanol on the brain phenotype of zebra fish in the examples of the present application.
In the drawings, the designations are as follows:
10-micro-structural unit
1-zebra fish juvenile fish behavioral phenotype test channel
11-first stationary microchannel
12-first restricted passage
13-first fixed microchamber
2-zebra fish juvenile fish brain phenotype test channel
21-second stationary microchannel
22-second restriction channel
23-second fixed microchamber
3-liquid input channel
4-liquid outlet channel
Detailed Description
The inventor firstly discloses a micro-fluidic chip for detecting the multi-phenotype of the zebra fish through extensive and intensive research, the test channels of the micro-fluidic chip for detecting the phenotype of the zebra fish juvenile fish are mutually independent, and the design can avoid the interaction between the zebra fish juvenile fish, thereby reducing the experimental error; in addition, the micro-fluidic chip skillfully designs the structure of a fixed micro-chamber for accommodating the tail of the zebra fish juvenile based on the control of the movable range of each organ part of the zebra fish juvenile body, thereby realizing the simultaneous and better detection of behavioral phenotype characteristic change information and brain phenotype characteristic change information of more organ parts of the zebra fish juvenile.
Term(s) for
As used herein, the terms "chip" and "microfluidic chip" are used interchangeably;
as used herein, the terms "zebrafish juvenile behavioral phenotype test microchannel", "zebrafish behavioral phenotype test channel" and "zebrafish juvenile behavioral phenotype test channel" are used interchangeably;
as used herein, the terms | "zebrafish juvenile fish brain phenotype test microchannel", "zebrafish brain phenotype test channel", and "zebrafish juvenile fish brain phenotype test channel" are used interchangeably;
it is noted that, in the present patent application, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, the use of the verb "comprise a" to define an element does not exclude the presence of another, same element in a process, method, article, or apparatus that comprises the element. In the present patent application, if it is mentioned that a certain action is executed according to a certain element, it means that the action is executed according to at least the element, and two cases are included: performing the action based only on the element, and performing the action based on the element and other elements. The expression of a plurality of, a plurality of and the like includes 2, 2 and more than 2, more than 2 and more than 2.
In the present invention, all the directional indications (such as up, down, left, right, front, rear, etc.) are used only to explain the relative positional relationship between the respective members, the motion situation, etc. in a certain posture (as shown in the drawing), and if the certain posture is changed, the directional indication is changed accordingly.
In the following description, numerous technical details are set forth in order to provide a better understanding of the present application. However, it will be understood by those skilled in the art that the technical solutions claimed in the present application may be implemented without these technical details and with various changes and modifications based on the following embodiments.
Microfluidic chip for detecting multi-phenotype of zebra fish
The application aims to provide the microfluidic chip which is simple and convenient to operate, does not damage zebra fish, is low in cost and high in flux and can be used for detecting the multi-phenotype of the zebra fish;
another purpose of this application is to provide the application of above-mentioned zebra fish micro-fluidic chip in the medicine screening.
The application provides a zebra fish microfluidic chip which can be used for detecting behaviors of organ parts such as mouth, eyes, body, tail, heart and the like of the zebra fish juvenile fish and a change phenotype of cell activity of cerebral neurons, wherein fig. 6 shows the organ parts of the zebra fish juvenile fish.
The microfluidic chip includes: the device comprises a plurality of micro-structure units 10, a liquid inlet channel 3, a liquid outlet channel 4, a zebra fish juvenile behavior phenotype test channel 1 and a zebra fish juvenile brain phenotype test channel 2, wherein the zebra fish juvenile behavior phenotype test channel 1 and the zebra fish juvenile brain phenotype test channel 2 are arranged between the liquid inlet channel and the liquid outlet channel; the liquid input channel is configured to input zebra fish juvenile fish to be detected, and the liquid output channel is configured to output zebra fish juvenile fish after detection is completed;
the zebra fish juvenile behavior phenotype test channel 1 is configured to detect a behavior phenotype of a first zebra fish juvenile, and the zebra fish juvenile behavior phenotype test channel 1 sequentially comprises a first fixed micro-channel 11, a first limiting channel 12 and a first fixed micro-chamber 13 which are in fluid communication along an axial direction; the first fixed micro-channel 11 is configured to fix the head of the first zebra fish juvenile fish at the time of detection, the first limiting channel 12 is configured to accommodate the body of the first zebra fish juvenile fish at the time of detection, the first fixed micro-chamber 13 is configured to accommodate the tail of the first zebra fish juvenile fish at the time of detection, the width of the first limiting channel 12 is smaller than the widths of the first fixed micro-channel 11 and the first fixed micro-chamber 13, and the first fixed micro-chamber 13 is designed to be wide and rectangular, so that the movable range of each organ part of the zebra fish juvenile fish body is larger, and therefore the behavioral phenotype characteristic change information of more organ parts of the zebra fish juvenile fish is better detected; preferably, the first fixed microchamber has a length of 2500 to 3000 μm and a width of 2300 to 2800 μm.
The zebra fish juvenile brain phenotype test channel 2 is configured to detect a brain phenotype of a second zebra fish juvenile, and the zebra fish juvenile brain phenotype test channel 2 sequentially comprises a second fixed microchannel 21, a second limiting channel 22 and a second fixed microchannel 23 which are in fluid communication along the axial direction; the second fixing micro-channel 21 is configured to fix the head of the second zebra fish juvenile fish at the time of detection, the second limiting channel 22 is configured to accommodate the body of the second zebra fish juvenile fish at the time of detection, the second fixing micro-chamber 23 is configured to accommodate the tail of the second zebra fish juvenile fish at the time of detection, the width of the second limiting channel 22 is smaller than the widths of the second fixing micro-channel 21 and the second fixing micro-chamber 23, and the second fixing micro-chamber 23 is designed to be a narrow rectangle, so that the movable range of each organ part of the zebra fish juvenile fish body is smaller, and therefore the zebra fish juvenile fish is better fixed, and the brain phenotype characteristic change information of the brain of the zebra fish juvenile fish is detected. Preferably, the second fixed micro-chamber has a length of 3000 to 3800 μm and a width of 500 to 900 μm.
The width difference between the fixed micro-channel and the fixed micro-chamber ensures that the zebra fish juvenile fish is fixed in the fixed micro-channel, the limiting channel and the fixed micro-chamber under the continuous action of hydromechanics (the fixed micro-channel is used for fixing the head of the zebra fish juvenile fish, and the limiting channel and the fixed micro-chamber are used for fixing the body and the tail of the zebra fish juvenile fish).
The zebra fish juvenile fish behavioral phenotype test microchannel of the zebra fish microfluidic chip adopts a back or belly upward type to directionally fix the zebra fish juvenile fish, and can be used for collecting behavioral phenotype characteristic information of organs such as zebra fish juvenile fish eyes, mouths, bodies, tails, hearts and the like; the zebra fish juvenile fish brain phenotype test microchannel of the zebra fish microfluidic chip adopts a back-up type directional fixation zebra fish juvenile fish, and can be used for collecting the characteristic information of the brain part (neuron cell calcium flow change) of the zebra fish juvenile fish.
Preferably, the height of the liquid input channel, the fixed micro-channel, the limiting channel, the fixed micro-chamber and the liquid output channel of the zebra fish microfluidic chip is 450-1100 microns, and the width of the liquid input channel, the fixed micro-channel, the limiting channel, the fixed micro-chamber and the liquid output channel is 200-2800 microns. The height and the width can ensure that the zebra fish juvenile fish (5 to 9 days after fertilization) can pass through smoothly and unimpededly. And the joints of the liquid input channel, the fixed micro-chamber and the liquid output channel are all designed into circular arcs with the radius of 100-300 mu m, so that the zebra fish juvenile fish can smoothly enter each fixed micro-channel and each fixed micro-chamber, and the zebra fish juvenile fish is prevented from being damaged due to sharp turning.
Preferably, the fixing micro-channel and the limiting channel for detecting the behavioral phenotype and the brain phenotype of the zebra fish juvenile fish in the zebra fish micro-fluidic chip are integrally designed in a funnel shape, so that the fixing micro-channel and the limiting channel can better fix the head and the body of the zebra fish juvenile fish.
Preferably, the first and second fixed microchannels in the zebra fish microfluidic chip for detecting behavioral phenotype and brain phenotype of the zebra fish juvenile are 6000-9000 μm in length and 500-950 μm in width.
Preferably, the first confinement channel has a length of 380-450 μm and a width of 250-350 μm; the second confinement channel has a length of 380-450 μm and a width of 250-350 μm.
The preparation method of the zebra fish microfluidic chip can be prepared by adopting conventional technologies in the field such as high-precision numerical control processing or 3D printing.
In some embodiments, the zebrafish microfluidic chip comprises a microfluidic chip substrate and a cover plate, and is connected in a non-permanent bonding manner.
In some embodiments of the present application, the material of the substrate of the zebra fish microfluidic chip comprises glass, and the material of the cover plate of the zebra fish microfluidic chip comprises Polydimethylsiloxane (PDMS).
In some embodiments of the present application, the non-permanent bond is referred to as a "permanent bond" that is not separable after the Polydimethylsiloxane (PDMS) coverslip and glass substrate are bonded.
There are many devices and techniques currently available for screening zebrafish phenotypic drugs, including anesthetics, capillaries, agarose, and gelatin. Anesthetic is a common agent used to fix zebra fish in a desired position, and although it is easy to use, it may cause damage to the nervous system of zebra fish. The ultrafine capillary tube can be used for fixing and turning over the zebra fish juvenile fish, but the capillary tube has the defects of poor control and time and labor waste in operation. These time-consuming, irreversible and tedious manual processes can damage the fragile body of zebrafish embryos and juvenile fish, thereby affecting the validity of experimental results. Agarose and gelatin may also be used to embed zebrafish larvae, but may also cause damage to zebrafish. The limitations of the above tools and techniques have prompted the application of microfluidic technology as an ideal technology that has accuracy, repeatability, high throughput, and versatility, and can be used for high throughput-scale drug screening based on zebrafish and for research and analysis of zebrafish under controlled conditions. Although there are a number of microfluidic techniques and devices for manipulating zebrafish for specific organ viewing, their applicability is greatly limited by the high cost, complex external systems and the damage that can be inflicted on zebrafish. Moreover, the existing zebra fish microfluidic chip for testing the zebra fish juvenile fish has a single function, and can only be applied to the behavioral phenotype test of each part of the zebra fish juvenile fish or the brain phenotype test of the zebra fish. The zebra fish micro-fluidic chip provided by the application can solve the problems.
The main advantages of the present application
(a) The microfluidic chip for detecting the multivariate phenotype of the zebra fish is simple and convenient to operate, does not damage the zebra fish, is low in cost and high in flux, and can be repeatedly used;
(b) The zebra fish microfluidic chip solves the problem that the testing effect of the zebra fish microfluidic chip device is single, and can be used for testing the zebra fish multivariate phenotype of the influence of a medicament on the behavioral phenotype and the brain phenotype of the zebra fish juvenile fish in one chip;
(c) The first fixed micro-chamber of the zebra fish micro-fluidic chip is designed to be a wide rectangle and is used for observing the tail of the zebra fish, so that the influence of the generation of bubbles on the movement of the tail of the zebra fish is reduced, and the detection of a behavioral phenotype is facilitated;
(d) The second fixed micro-chamber of the zebra fish micro-fluidic chip is designed to be a narrow rectangle, and the tail movement of the zebra fish is limited, so that the brain area of the zebra fish can be better fixed, and the detection of the brain phenotype is facilitated.
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be understood that these are merely examples that the reader may take and are not intended to limit the scope of the invention.
Example 1:
zebra fish microfluidic chip and preparation method thereof
It should be noted that the experimental materials and reagents used are all consumable materials and reagents which are conventionally available from commercial sources unless otherwise specified.
The Polydimethylsiloxane (PDMS) used in this example was purchased from Mikupffer microfluidics, inc., and included a prepolymer and a crosslinker.
The zebra fish microfluidic chip in the embodiment 1 has the following specific structure:
referring to fig. 1-2, the zebra fish microfluidic chip has a plurality of microstructure units 10, each microstructure unit 10 includes a liquid input channel 3, a liquid output channel 4, and a zebra fish juvenile behavioral phenotype test channel 1 and a zebra fish juvenile brain phenotype test channel 2 disposed between the liquid input channel and the liquid output channel;
the zebra fish juvenile behavior phenotype test channel 1 is configured to detect a behavior phenotype of a first zebra fish juvenile, and the zebra fish juvenile behavior phenotype test channel 1 sequentially comprises a first fixed micro-channel 11, a first limiting channel 12 and a first fixed micro-chamber 13 which are in fluid communication along an axial direction; the first fixed micro-channel 11 is configured to fix the head of the first zebra fish juvenile fish at the time of detection, the first limiting channel 12 is configured to accommodate the body of the first zebra fish juvenile fish at the time of detection, the first fixed micro-chamber 13 is configured to accommodate the tail of the first zebra fish juvenile fish at the time of detection, the width of the first limiting channel 12 is smaller than the widths of the first fixed micro-channel 11 and the first fixed micro-chamber 13, and the first fixed micro-chamber 13 is designed to be a wide rectangle for releasing the tail of the zebra fish and observing the swing of the tail for behavioral shooting of the zebra fish;
the zebra fish juvenile brain phenotype test channel 2 is configured to detect a brain phenotype of a second zebra fish juvenile, and the zebra fish juvenile brain phenotype test channel 2 sequentially comprises a second fixed micro-channel 21, a second limiting channel 22 and a second fixed micro-chamber 23 which are in fluid communication along the axial direction; the second fixing micro-channel 21 is configured to fix the head of the second zebra fish juvenile fish at the time of detection, the second limiting channel 22 is configured to accommodate the body of the second zebra fish juvenile fish at the time of detection, the second fixing micro-chamber 23 is configured to accommodate the tail of the second zebra fish juvenile fish at the time of detection, the width of the second limiting channel 22 is smaller than the widths of the second fixing micro-channel 21 and the second fixing micro-chamber 23, and the second fixing micro-chamber 23 is designed to be a narrow rectangle for better fixing the tail of the zebra fish for shooting the brain of the zebra fish.
The widths of the fixed micro-channel and the limiting channel are smaller than the widths of the liquid input channel and the fixed micro-chamber, so that the zebra fish juvenile fish is fixed in the fixed micro-channel, the limiting channel and the fixed micro-chamber under the continuous action of the fluid (the fixed micro-channel is used for fixing the head of the zebra fish juvenile fish, and the limiting channel and the fixed micro-chamber are used for fixing the body and the tail of the zebra fish juvenile fish), and the technical effect of fixing the zebra fish juvenile fish is achieved.
A schematic structural design diagram of the zebra fish microfluidic chip in this embodiment 1 is shown in fig. 1, and the zebra fish microfluidic chip is provided with a plurality of micro-structural units 10, each micro-structural unit includes a liquid input channel 3, a liquid output channel 4, and a zebra fish juvenile behavior phenotype test channel 1 and a zebra fish juvenile brain phenotype test channel 2 which are arranged between the liquid input channel and the liquid output channel, and are shown in fig. 2;
referring to fig. 2, in the present embodiment, the widths of the liquid input channel 3 and the liquid output channel 4 of the zebra fish behavioral phenotype test channel 1 and the brain phenotype test channel 2 of the zebra fish microfluidic chip are both 860 μm.
The heights of the liquid input channel, the fixed micro-channel, the limiting channel, the fixed micro-chamber and the liquid output channel of the zebra fish behavioral phenotype test channel 1 and the brain phenotype test channel 2 of the zebra fish microfluidic chip are all 620 micrometers.
The zebra fish behavioral phenotype test channel of the zebra fish microfluidic chip and the fixed microchannel and the limiting channel of the zebra fish brain phenotype test channel are designed into an hourglass shape so as to better fix the head of the zebra fish juvenile fish, the height of the zebra fish behavioral phenotype test channel is 620 microns, the width of the zebra fish behavioral phenotype test channel is gradually narrowed from 860 microns to 260 microns along the flowing direction of fluid, and the length of the zebra fish behavioral phenotype test channel is 420 microns.
A first fixed micro-channel 11 of a zebra fish behavioral phenotype test micro-channel 1 of the zebra fish micro-fluidic chip is widened to 2860 mu m along a fluid flowing direction to form a first fixed micro-chamber, the width of the first fixed micro-chamber 13 is 2860 mu m, the first fixed micro-chamber is designed to be a wide rectangle to enable the movable range of each organ part of a zebra fish juvenile fish body to be larger, therefore behavioral phenotype characteristic change information of more organ parts of the zebra fish juvenile fish can be better detected, and the height of the first fixed micro-chamber is 620 mu m.
The second fixed micro-channel 21 of the zebra fish brain phenotype test channel 2 of the zebra fish microfluidic chip is widened to 860 microns through the second limiting channel along the fluid flowing direction to form a second fixed micro-chamber 23, the length of the second fixed micro-chamber is 3600 microns, the second fixed micro-chamber 23 is designed to be a narrow rectangle so that the movable range of each organ part of the zebra fish juvenile body is smaller, and therefore the zebra fish juvenile fish can be better fixed, the change information of the brain phenotype characteristics of the zebra fish juvenile fish brain can be detected conveniently, and the height of the second fixed micro-chamber is 620 microns.
The liquid input channel 3 and the liquid output channel 4 are in fluid communication with the zebra fish juvenile fish behavioral phenotype test channel 1 and the zebra fish juvenile fish brain phenotype test channel 2, the liquid input channel 3 is arranged at one end of the zebra fish microfluidic chip substrate, and the liquid output channel 4 is arranged at the other end of the zebra fish microfluidic chip substrate; the joints of the liquid input channel 3 and the liquid output channel 4 with the fixed micro-channel and the fixed micro-chamber are all round corners, and the radius of the round corners is 200 mu m.
Fig. 3 shows a schematic fluid dynamics simulation diagram of the zebra fish microfluidic chip in example 1, which is a schematic fluid dynamics simulation diagram of the behavioral phenotype test microchannel (a) and the brain phenotype test microchannel (B), respectively. Wherein the flow rate is most suitable for fixing the zebra fish juvenile fish within the range of 15-17 mL/h.
The zebra fish microfluidic chip in example 1 was prepared by copying a microfluidic hollow channel in the zebra fish microfluidic chip from a mold using Polydimethylsiloxane (PDMS).
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 lightly wipe the copper plate to remove stains at corners and gaps, then ultrasonically cleaning for 20min, and drying by using an air gun to finish the preparation of the zebra fish microfluidic chip template.
PDMS (prepolymer A) and PDMS (crosslinking agent B) were placed in the same container at a ratio of 10. The mixed PDMS was then poured onto a cleaned copper template and again evacuated to completely remove air bubbles. Putting into a baking oven at 80 ℃ and baking for 4-6 h. And after the PDMS is completely solidified, taking out and cooling to room temperature, then taking the PDMS off the mold, and punching by using a puncher according to the positions of the inlet and the outlet.
Preparing a glass microfluidic negative plate by adopting a conventional method in the field according to the size of the designed zebra fish microfluidic chip;
after the bonding surface of the substrate and the glass cover plate of the PDMS zebra fish microfluidic chip is treated by plasma, the substrate and the glass cover plate are bonded together to complete the preparation of the zebra fish microfluidic chip, as shown in FIG. 4.
The behavioral phenotype test microchannel of the zebra fish microfluidic chip prepared by the embodiment adopts a back or belly upward type directional fixation zebra fish juvenile fish, and can be used for collecting the behavioral phenotype characteristic information of the zebra fish juvenile fish eyes, mouth, body, tail, heart and other organ parts (as shown in fig. 6); the cerebral phenotype test micro-channel of the zebra fish micro-fluidic chip adopts a back-up type directionally fixed zebra fish juvenile fish and can be used for collecting the characteristic information of the cerebral part (neuron cell calcium flow change) of the zebra fish juvenile fish. The schematic diagram of the zebra fish juvenile fish loaded into the microchannel of the zebra fish microfluidic chip in the embodiment of the application is shown in fig. 5.
Example 2:
testing the Effect of organic solvent DMSO and different concentrations of methanol on brain phenotype of zebra fish juvenile fish
This example uses the zebra fish microfluidic chip of example 1 in this application to test the effect of organic solvents DMSO and different concentrations of methanol on the brain phenotype of zebra fish larvae.
First, a solution is prepared: control group solution 0.02% dmso solution (diluted with purified water), test group solutions 0.2% methanol, 0.4% methanol (diluted with purified water), respectively; secondly, loading the zebra fish juvenile fish by using the zebra fish microfluidic chip and the 30mL injector in the embodiment 1; subsequently, the chip loaded with the zebra fish juvenile fish and cleaned is fixed on a microscope stage, and whether each brain of the chip is normally active is observed, wherein each brain area of the brain is shown in fig. 7; and finally, testing: the control solution is firstly introduced to dissolve, and then the photo test is carried out for 10 minutes. The control solution was switched to the test solution at 10 minute intervals. The test groups were subjected to a photographing test for 10 minutes. The testing time of each zebra fish juvenile fish is 30 minutes in total, 5 zebra fish juvenile fish brain samples are contained, and the shooting is finished in 150 minutes in total. The fish needs to be kept still when photographed by a fluorescence microscope to reduce the influence of the outside on the juvenile fish. And respectively representing the activity change index of brain nerve cells of the zebra fish juvenile fish by adopting pulse counting, pulse average relative height and pulse average half-peak width. Counting the pulse number of times of large-scale nerve impulses in each brain area of the brain of the zebra fish juvenile fish; the average relative height of the pulse is the pulse intensity of large-scale nerve impulses appearing in each brain area of the brain of the zebra fish juvenile fish, the unit of the pulse intensity is a brightness unit when the zebra fish juvenile fish is shot by a microscope, the brightness is difficult to determine, and the brightness, namely the fluorescence intensity of different zebra fish juvenile fish samples is different, so that no unit exists; the average half-peak width of the pulse is one half of the pulse duration of large-scale nerve impulse in each brain area of the brain of the zebra fish juvenile fish, and the unit is a frame. As a result, as shown in FIG. 8, 0.2% methanol inhibited the activity of the brain of the zebrafish juvenile fish compared to 0.02% DMSO; compared with 0.2% methanol, 0.4% methanol promotes brain activity of young zebra fish.
The result of the embodiment 2 in the application shows that the microfluidic chip for detecting the multi-phenotype of the zebra fish provided by the application has feasibility and stability, has high expansibility (expanding to more kinds of drug testing and screening) and economy (low manufacturing cost), and provides powerful technical support for large-scale and high-flux drug screening.
In addition, the zebra fish microfluidic chip can be used for simultaneously shooting the brain and the ethology of zebra fish, and a test for the influence of an organic solvent DMSO and methanol with different concentrations on the behavioral phenotype of the zebra fish juvenile fish is not described in detail in the application, but the zebra fish can be shot after the dosing process is finished because a dosing process needs to be carried out in a shooting experiment scheme, and the zebra fish does not need to be observed in the dosing process, for example, the behavioral phenotype can be shot when the dosing is carried out in the brain phenotype shooting.
All documents mentioned in this application are to be considered as being integrally included in the disclosure of this application so as to be subject to modification as necessary. Further, it is understood that various changes or modifications may be made to the present application by those skilled in the art after reading the above disclosure of the present application, and such equivalents are also within the scope of the present application as claimed.
Claims (10)
1. A microfluidic chip for detecting a multivariate phenotype of zebrafish, comprising: the microstructure units comprise a liquid input channel, a liquid output channel, and a zebra fish juvenile behavior phenotype test channel and a zebra fish juvenile brain phenotype test channel which are arranged between the liquid input channel and the liquid output channel;
the zebra fish juvenile behavioral phenotype test channel is configured to detect a behavioral phenotype of a first zebra fish juvenile, and sequentially comprises a first fixed microchannel, a first limiting channel and a first fixed microchamber which are in fluid communication along an axial direction; the first fixed microchannel is configured to fix the head of the first zebra fish larva at the time of detection, the first limiting channel is configured to accommodate the body of the first zebra fish larva at the time of detection, the first fixed microchannel is configured to accommodate the tail of the first zebra fish larva at the time of detection, the width of the first limiting channel is smaller than the widths of the first fixed microchannel and the first fixed microchannel, and the first fixed microchannel is designed to be a wide rectangle;
the zebra fish juvenile brain phenotype test channel is configured to detect a brain phenotype of a second zebra fish juvenile, and sequentially comprises a second fixed microchannel, a second limiting channel and a second fixed microchamber which are communicated with each other along the axial direction; the second fixed microchannel is configured to fix the head of the second zebra fish juvenile fish when detected, the second restriction channel is configured to accommodate the body of the second zebra fish juvenile fish when detected, the second fixed microchamber is configured to accommodate the tail of the second zebra fish juvenile fish when detected, the width of the second restriction channel is smaller than the width of the second fixed microchannel and the second fixed microchamber, and the second fixed microchamber is designed to be a narrow rectangle.
2. The microfluidic chip of claim 1, wherein the first fixed microchamber has a length of 2500-3000 μm and a width of 2300-2800 μm, and the second fixed microchamber has a length of 3000-3800 μm and a width of 500-900 μm.
3. The microfluidic chip of claim 2, wherein the first confinement channel has a length of 380 to 450 μm and a width of 250 to 350 μm.
4. The microfluidic chip of claim 3, wherein the second confinement channel has a length of 380 to 450 μm and a width of 250 to 350 μm.
5. The microfluidic chip of claim 4, wherein the width of each of the first stationary microchannel and the second stationary microchannel is between 500 to 950 μm.
6. The microfluidic chip of claim 5, wherein the first stationary microchannel and the second stationary microchannel each have a length between 6000 and 9000 μm.
7. The microfluidic chip of claim 1, wherein the liquid input channel and the liquid output channel each have a width of between 750 μm and 950 μm.
8. The microfluidic chip of claim 1, wherein the liquid input channel, the first fixed microchannel, the first confinement channel, the first fixed microchamber, and the liquid output channel are all rounded at their junctions.
9. The microfluidic chip of claim 1, wherein the liquid input channel, the first fixed microchannel, the first confinement channel, the first fixed microchamber, and the liquid output channel are all rounded at their junctions.
10. Use of a microfluidic chip according to any of claims 1 to 9 in drug screening.
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| CN202211269217.3A CN115537330A (en) | 2022-10-17 | 2022-10-17 | Microfluidic chip for detecting multiple phenotypes of zebra fish and application thereof |
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