Marine fish microinjection needle and preparation method thereof

Abstract

The application belongs to the field of aquaculture, and specifically discloses a seawater fish micro-injection needle and a preparation method thereof. The method comprises the following steps: preparing a glass capillary tube and a needle drawing device; drawing the glass needle; breaking the needle; grinding the needle tip; testing the quality of the needle; methyl cellulose treatment; needle storage and use. The micro-injection needle prepared by the method can effectively solve the problems of hard seawater fish egg shell difficult to penetrate, easy breaking of the needle tip during injection, easy sticking of the needle, and great damage to the fertilized eggs, and is suitable for micro-injection research of various seawater fish species, has a wide application prospect, provides important support for gene editing research of seawater fish, and has good application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of aquaculture, and particularly relates to a seawater fish microinjection needle and a preparation method thereof. BACKGROUND

[0002] In recent years, the seawater aquaculture industry in China has developed rapidly, and the seawater fish aquaculture yield reached 192.56 million tons in 2022. However, problems such as slow growth and frequent disease outbreaks have seriously hindered the development of the seawater fish aquaculture industry. Microinjection technology, as an important tool for modern biomedical research, is widely used in gene editing, transgenic research, developmental biology, and drug research, and plays an important role in improving the growth rate, disease resistance, food conversion rate, and other traits of fish, constructing transgenic fish, and studying the differentiation, migration, and growth of cells during embryonic development.

[0003] Currently, microinjection technology for freshwater fish is relatively mature, but compared with freshwater fish, seawater fish eggs are usually wrapped in a hard eggshell. The injection needle prepared by the traditional needle grinding method cannot penetrate the eggshell and the needle tip is easily damaged. Therefore, special treatment is needed for the injection needle to adapt to the unique physiological and environmental conditions of seawater fish. SUMMARY

[0004] The purpose of the present application is to provide a seawater fish microinjection needle and a preparation method thereof to solve the above problems.

[0005] To achieve the above purpose, the present application provides the following technical solutions:

[0006] In a first aspect, the present application provides a preparation method of a seawater fish microinjection needle, comprising the following steps:

[0007] S1, preparing a glass capillary tube and a needle drawing device, the needle drawing device comprising a vertical needle drawing instrument, a needle forging instrument, a needle grinding instrument, and a stereomicroscope;

[0008] S2, drawing a glass needle: placing the glass capillary tube of step S1 into the needle drawing instrument, setting the parameters of the needle drawing instrument, and drawing;

[0009] S3, breaking the needle: using the needle forging instrument to process the injection needle, obtaining an injection needle with the required diameter according to the different characteristics of the seawater fish eggs, and obtaining a flat needle mouth;

[0010] S4, grinding the needle: using the needle grinding instrument to polish and modify the needle tip of the injection needle obtained in step S3;

[0011] S5, testing the quality of the needle: checking the injection needle obtained in step S4 under the stereomicroscope and performing a functional test, requiring that the injection needle can smoothly inject liquid and the needle tip can smoothly penetrate the cell membrane;

[0012] S6, a 2% methyl cellulose solution is prepared and sprayed on the surface of the micro-injection needle obtained in step S5.

[0013] Further, in step S1, the glass capillary tube has an outer diameter of 1.00 mm, an inner diameter of 0.60 mm, and is made of transparent borosilicate; the vertical needle drawing instrument is a program-controlled vertical needle drawing instrument, model PL-200, a glass tube forging needle instrument, model PF-22C, and a glass capillary tube needle grinding instrument, model PG-22C.

[0014] Further, in step S2, the needle drawing instrument is set to a temperature of 650°C, a pulling force of 30 N, a drawing speed of 30 mm / s, and a gas pressure of 50 kPa. The glass capillary tube is heated and gradually softened, and the middle part becomes thinner and thinner under the action of the pulling force, and finally breaks at the thinnest part to form two microneedles. After the glass needles are cooled, the upper part is removed.

[0015] Further, in step S3, the glass capillary tube of step S2 is placed in the clamp of the needle forging instrument, the platinum wire heating element and the injection needle are placed in the field of view of the microscope, the adjustment knob is operated to adjust the injection needle to the desired diameter (determined by the inner diameter of the needle tip opening required in step S4, which should not exceed this size) and parallel to the glass bead; the heater adjustment knob is set to a temperature of 600°C, and the foot switch is pressed to heat the glass bead on the platinum wire to orange red. Immediately after the injection needle tip is fused into the glass bead, the foot switch is released. At this time, the tip of the injection needle will be adhered to the glass bead. The platinum heating element is moved vertically to separate the injection needle from the platinum wire, and a flat needle opening is obtained.

[0016] Further, in step S4, the glass needle is installed on the micro-sanding machine of the needle grinding instrument, and the speed is controlled between 2000-5000 RPM. The needle tip is polished to a length of 9 mm, and the inner diameter of the needle tip opening is set to a liquid discharge volume of 0.5-1 nL. The needle tip is smooth, sharp, and free of burrs. After grinding, the needle tip is cleaned to remove glass debris and dust. If the needle is too long, it will affect the penetration ability of the micro-injection needle. If the liquid discharge volume is too high, it can easily cause the fish eggs to die. If the liquid discharge volume is too low, the gene editing efficiency will be too low. Fish eggs with different characteristics require a liquid discharge volume of 0.5-1 nL, and the inner diameter of the needle tip opening required is different. For example, for pompano eggs and large yellow croaker eggs, a needle tip opening inner diameter of 0.5 μm can meet the requirements of a liquid discharge volume of 0.5-1 nL, which can avoid the situation that injection causes the fish eggs to die under this inner diameter condition.

[0017] Further, in step S6, the 2% methyl cellulose solution is prepared by slowly adding methyl cellulose with a viscosity of 4000CPS into 1 / 4 of the total amount of ultrapure water (70 DEG C) and dispersing while stirring with a magnetic stirrer to obtain a hot gel, then slowly adding the remaining 3 / 4 of the ultrapure water (4 DEG C) and stirring on ice to obtain a 2% methyl cellulose solution; and the spraying method is to uniformly spray the surface of the microinjection needle obtained in step S5 using a spray bottle with a nozzle diameter of 0.30mm.

[0018] In a second aspect, the application provides a seawater fish microinjection needle prepared by the above preparation method.

[0019] The application has the following beneficial effects:

[0020] 1. The application uses high-quality borosilicate glass capillary tubes, precise control of the needle drawing instrument, and fine needle grinding treatment to make the size and shape of the microinjection needle highly consistent, and the needle tip smooth, sharp and not easily damaged, so as to penetrate the hard eggshell of seawater fish and accurately control the injection position and injection amount, reduce mechanical damage to cells or embryos, and improve the injection success rate.

[0021] 2. The use of a micro-sanding machine for needle tip grinding treatment makes the cross section smoother and causes less damage when piercing, and can be used in different biological samples (such as seawater fish embryos and seawater fish zygotes), and through reasonable speed and time control, the consistency of the needle tip quality is ensured. Adapt to its specific physiological and environmental conditions, improve the stability and effect of injection operation.

[0022] 3. On the basis of the foregoing process, the application strictly controls the inner diameter of the needle opening and the length of the needle to ensure that the microinjection needle can not only penetrate the eggshell of seawater fish, but also will not damage the zygote, and at the same time, liquid is injected into the egg, ensuring the injection effect. Too large needle opening and too long needle will significantly affect the injection effect.

[0023] 4. The needle tip surface is covered with a layer of methyl cellulose film, which can effectively prevent the zygote from adhering to the injection needle during the microinjection process of the zygote with adhesion, and significantly improve the injection efficiency.

[0024] 5. The preparation method of the microinjection needle is not only suitable for basic research fields such as gene editing, transgenic research and developmental biology, but also can be used for drug screening and cell physiology research, and has a wide application prospect.

[0025] In summary, the manufacturing method of the microinjection needle of the present application provides a high-precision, high-strength, high-success-rate, easy-to-operate and highly adaptable microinjection needle manufacturing method through a series of optimized steps. These advantages not only improve the reliability and efficiency of microinjection experiments on seawater fish, but also expand the application range of microinjection technology, providing an important tool for biomedical research and biotechnology development. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a schematic diagram of the needle breaking in the microinjection needle preparation method of Example 1 of the present application;

[0027] Figure 2 is a schematic diagram of polishing the needle tip in the microinjection needle preparation method of Example 1 of the present application;

[0028] Figure 3 is a schematic diagram of microinjection operation with the microinjection needle according to Example 1 of the present application;

[0029] Figure 4 is a schematic diagram of the microinjection needle of Example 1 of the present application injecting phenol red into the fertilized egg;

[0030] Figure 5 is a schematic diagram of the fertilized egg adhesion without spraying 2% methyl cellulose in Comparative Example 1 of the present application;

[0031] Figure 6 is a schematic diagram of the microinjection needle injecting into the fertilized egg when the needle tip opening is too large in Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0033] Example 1

[0034] The present embodiment provides a preparation method of a microinjection needle for seawater fish, comprising the following steps:

[0035] S1, prepare glass capillary and pull needle equipment, glass capillary parameters are length 90.00mm, outer diameter 1.00mm, inner diameter 0.60mm, material is high-quality transparent borosilicate which can keep stable in high salinity environment and is not easy to be corroded; the glass capillary is soaked in anhydrous ethanol for 24 hours, immersed in ultrapure water for 3 times, and then wiped dry with a dust-free cloth to ensure that the surface is free of dust and grease, and placed in an oven for drying. The pull needle equipment includes a vertical pull needle instrument (model PL-200), a forging needle instrument (model PF-22C), a grinding needle instrument (model PG-22C), and a stereomicroscope.

[0036] S2, the glass capillary of step S1 is placed in the clamp of the pull needle instrument, carefully installed into the heating groove, and the pull needle instrument parameters are set as temperature 650℃, pulling force 30N, and drawing speed 30mm / s. The glass capillary is heated and gradually softened, the middle part becomes thinner and thinner under the action of pulling force, and finally breaks at the thinnest part (as shown in Figure 1 ), forming two microneedles. After waiting for the glass needle to cool down, it is carefully taken out and the upper part is reserved.

[0037] S3, the glass capillary of step S2 is placed in the clamp of the broken needle instrument, the platinum wire heating element and the injection needle are placed in the field of view of the microscope, the adjusting knob is operated, and the injection needle is adjusted to a position parallel to the glass bead at an outer diameter of 1μm. The heater adjusting knob is set at a temperature of 600℃, and the foot switch is pressed. The glass bead on the platinum wire is heated to orange red, and the foot switch is immediately released after the injection needle tip is fused into the glass bead. At this time, the tip of the pipette will be adhered to the glass bead. The platinum heating element is moved vertically to separate the injection needle from the platinum wire, and a flat needle port is obtained.

[0038] S4, the tip of the injection needle obtained in step S2 is strengthened and polished by using the grinding needle instrument (as shown in Figure 2 ), the drawn glass needle is installed on the micro-sanding machine of the grinding needle instrument, the rotating speed is controlled between 2000-5000RPM, first slow and then fast, and the polishing time at the highest speed is not less than 30 seconds. The polishing is performed to a needle tip length of 9mm, a needle tip opening inner diameter of 0.5μm, and a smooth, sharp and burr-free needle tip. After grinding, the needle tip is immersed in anhydrous ethanol, cleaned with a dust-free cloth, and dried in an oven to remove residual glass debris and dust.

[0039] S5, the injection needle obtained in step S4 is checked under a stereomicroscope. The injection needle should be able to smoothly inject liquid and the needle tip should be able to smoothly penetrate the cell membrane. The qualified needle is carefully stored on the needle rack to avoid damage to the needle tip.

[0040] S6, a 2% methylcellulose aqueous solution was prepared, methylcellulose with a viscosity (CPS) of 4000 was slowly added to 1 / 4 of the total amount of ultrapure water (70°C) for dispersion while stirring with a magnetic stirrer to obtain a hot gel, and then the remaining 3 / 4 of the ultrapure water (4°C) was slowly added to obtain a 2% methylcellulose solution while stirring on ice; the surface of the microinjection needle obtained in step S5 was uniformly sprayed using a spray bottle with a nozzle diameter of 0.30 mm.

[0041] The microinjection needle prepared according to the method of this example was tested and shown to be able to successfully penetrate the egg shell of seawater fish and complete microinjection into the egg (as shown in Figures 3-4 ).

[0042] The microinjection needle of this example has been successfully applied to injection of pompano eggs and large yellow croaker eggs, and the specific operation is as follows:

[0043] Pompano egg injection: the injection needle was installed on the mechanical arm of the microinjection manipulator, and the microinjection manipulator was adjusted under the microscope to ensure that the needle tip was aligned with the pompano fertilized egg and gently pierced the fertilized egg membrane to slowly inject the injection solution. After injection, the microinjection needle was slowly withdrawn to avoid damaging the injection site.

[0044] Large yellow croaker egg injection: the injection needle was installed on the mechanical arm of the microinjection manipulator, and the microinjection manipulator was adjusted under the microscope to ensure that the needle tip was aligned with the large yellow croaker fertilized egg and gently pierced the fertilized egg membrane to inject the injection solution under the yolk membrane of the large yellow croaker egg, close to the cell nucleus. After injection, the microinjection needle was slowly withdrawn to avoid damaging the injection site.

[0045] Using the microinjection needle of this example, the injection solution can be successfully injected into the pompano eggs and large yellow croaker eggs through the above operation, the microinjection needle tip is not damaged and there is no fertilized egg adhesion, and the fertilized eggs are not damaged.

[0046] Comparative Example 1

[0047] This comparative example provides a method for preparing a microinjection needle for seawater fish, which is basically the same as Example 1, except that the spraying treatment of the 2% methylcellulose aqueous solution in step S6 is not performed, resulting in the problem of fertilized egg adhesion on the needle tip (as shown in Figure 5 ).

[0048] Comparative Example 2

[0049] This comparative example provides a method for preparing a microinjection needle for seawater fish, which is basically the same as Example 1, except that the inner diameter of the needle tip opening is 1 μm, resulting in the problem of excessive single injection volume (as shown in Figure 6 ).

[0050] The above merely describes the preferred embodiments of the present application and is not used to limit the present application. Although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of preparing a microinjection needle for marine fish, characterized by, The method comprises the following steps: S1, preparing a glass capillary and a needle drawing device, wherein the needle drawing device comprises a vertical needle drawing instrument, a needle forging instrument, a needle grinding instrument, and a stereomicroscope; S2, drawing a glass needle: placing the glass capillary of step S1 into the needle drawing instrument, setting parameters of the needle drawing instrument, and drawing; wherein the parameters of the needle drawing instrument are set as follows: temperature 650℃, pulling force 30N, drawing speed 30mm / s, and air pressure 50kPa; the glass capillary is heated and gradually softened, the middle part becomes thinner and thinner under the action of pulling force, and finally breaks at the thinnest part to form two microneedles; after the glass needle is cooled, it is taken out, and the upper part is reserved; S3, breaking the needle: processing the injection needle by using the needle forging instrument, obtaining the injection needle with a required diameter according to different characteristics of the fertilized eggs of the injection seawater fish, and obtaining a flat needle port; S4, grinding the needle: grinding and modifying the needle tip of the injection needle obtained in step S3 by using the needle grinding instrument; the glass needle is installed on a micro-sanding machine of the needle grinding instrument, the rotating speed is controlled to be between 2000-5000RPM, the grinding time at the highest rotating speed is not less than 30 seconds, the needle tip is ground and modified to a length of 9mm, the needle tip opening inner diameter is set to a size of liquid discharge amount of 0.5-1nL, the needle tip is smooth, sharp, and free of burrs; after the grinding is completed, the needle tip is cleaned to remove glass debris and dust; S5, testing the quality of the needle: checking the injection needle obtained in step S4 under the stereomicroscope and performing a function test; the injection needle is required to be able to smoothly inject liquid, and the needle tip is required to be able to smoothly penetrate the cell membrane; S6, preparing a 2% methyl cellulose solution and spraying it on the surface of the micro-injection needle obtained in step S5; wherein the preparation method of the 2% methyl cellulose solution is as follows: slowly adding methyl cellulose with a viscosity of 4000CPS into 1 / 4 of total water, which is ultrapure water with a temperature of 70℃, to disperse, while stirring by using a magnetic stirrer, to obtain a hot gel; then slowly adding the remaining 3 / 4 of ultrapure water with a temperature of 4℃, and stirring on ice to obtain the 2% methyl cellulose solution; the spraying method is to uniformly spray the micro-injection needle obtained in step S5 on the surface by using a spray bottle with a nozzle diameter of 0.30mm.

2. The method of claim 1, wherein the marine fish microinjection needle is prepared by the steps of: In step S1, the parameters of the glass capillary are as follows: outer diameter 1.00mm, inner diameter 0.60mm, and material transparent borosilicate; the vertical needle drawing instrument is a program-controlled vertical needle drawing instrument with a model number of PL-200, the glass tube needle forging instrument has a model number of PF-22C, and the glass capillary needle grinding instrument has a model number of PG-22C.

3. The method of claim 1, wherein the marine fish microinjection needle is prepared by the steps of: In step S3, the glass capillary of step S2 is placed into a clamp of the needle forging instrument, a platinum wire heating element and the injection needle are placed in the field of view of the microscope, the injection needle is adjusted to a position parallel to the glass bead at a required diameter by operating an adjusting knob, the heater adjusting knob is set to 600℃, and a foot switch is pressed to heat the glass bead on the platinum wire to orange red, the foot switch is immediately released after the injection needle tip is fused into the glass bead, at this time, the tip of the injection needle is adhered to the glass bead, the platinum heating element is vertically moved to separate the injection needle from the platinum wire, and a flat needle port is obtained.

4. The method of claim 1, wherein the marine fish microinjection needle is prepared by the steps of: When the needle tip opening inner diameter is 0.5μm, the liquid discharge amount applied to the eggs of the silver pomfret and the large yellow croaker is 0.5-1nL.

5. The sea water fish microinjection needle prepared by the method of any one of claims 1-4.

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