Sample application device and preparation method of alginate gel

Abstract

The invention discloses a sample application device and a preparation method of alginate gel. The sample application device comprises a sample application gun head and a liquid supplementing assembly, the sample application gun head is of a funnel structure with an upper opening and a lower opening, the upper opening is an inlet of the sample application gun head and is communicated with the liquid supplementing assembly, and the lower opening is an outlet gun tip of the sample application gun head; the inner diameter of the inlet is larger than that of the gun tip of the outlet; and the liquid supplementing assembly is configured to maintain the stability of the volume of a solution in the sample application gun head. The alginate gel is prepared by using a sample application device, the preparation method of the alginate gel is improved, and the mode of dropwise adding a sodium alginate sample solution into a metal salt solution by virtue of external conditions in the prior art is improved into the mode of enabling the sodium alginate sample solution to naturally enter the metal salt solution by using a siphon principle; the impact with the liquid level of the metal salt solution is avoided, so that the formed gel is uniform in shape, smooth and round, and the subsequent experiment requirements are better met.

Description

Technical Field

[0001] The present invention relates to the technical field of biological microcapsule preparation, and more specifically to a spotting device and a method for preparing alginate gel. Background Art

[0002] Microencapsulation technology, as it's commonly known, involves completely encapsulating solid particles, liquid droplets, gases, or living cells in an inert polymer material to form microcapsules. This technology has been further expanded to include the microencapsulation of pancreatic islet cells in alginate-polylysine membranes using an all-aqueous system. This has led to widespread application in biology, medicine, reproductive medicine, and other fields, including cell transplantation, drug delivery, and artificial insemination.

[0003] In the microcapsule preparation scheme, sodium alginate gel spheres need to be prepared. Currently, there are two main methods for preparing sodium alginate gel spheres: one is to inject a sodium alginate suspension into a saline solution from above through a needle, and an air jet is generally set around the liquid jet to assist the sodium alginate solution in forming into spheres; the other is to use high-voltage electrostatic method to prepare sodium alginate gel spheres. Both methods have a common feature, that is, both spray the sodium alginate solution through a certain type of needle, and the needle is placed 5-10cm above the saline solution. At the same time, other auxiliary conditions (such as air jet or voltage) are used. The alginate gel spheres prepared in this way have the following disadvantages and shortcomings:

[0004] (1) The impact of sodium alginate droplets dripping from a high altitude into the surface of the salt solution will cause the alginate gel spheres formed to have irregular shapes, mostly disc-shaped or irregular shapes. Uniform spherical alginate gels are necessary for subsequent experiments.

[0005] (2) Existing technology cannot achieve micro-scale preparation. The production lower limit of the above method is 2 mL, and it is impossible to microencapsulate single cells or very small amounts of samples, which has certain limitations.

[0006] (3) The size range of the prepared microcapsules is small, with a diameter range of 300-900 μm, and only spherical microcapsules can be prepared, with a narrow range of applications. Summary of the Invention

[0007] In view of the above problems, one object of the present invention is to provide a spotting device for preparing alginate gel.

[0008] An object of the present invention is to provide a method for preparing alginate gel.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] According to one aspect of the present invention, there is provided a spotting device comprising:

[0011] Spotting pipette tip and rehydration components;

[0012] The sample spotting gun head is a funnel structure with upper and lower openings, wherein the upper opening is the inlet of the sample spotting gun head, which is connected to the rehydration component, and the lower opening is the outlet tip of the sample spotting gun head;

[0013] The inner diameter of the inlet is larger than the inner diameter of the outlet gun tip;

[0014] The fluid replenishing component is configured to maintain a stable volume of the solution in the spotting pipette tip.

[0015] In addition, an optional solution is that the inlet is fixedly connected to a first equal-diameter extension tube, and the inner diameter of the first equal-diameter extension tube is equal to the inner diameter of the inlet;

[0016] and / or

[0017] The outlet gun tip is fixedly combined with a second equal-diameter extension tube, and the inner diameter of the second equal-diameter extension tube is equal to the inner diameter of the outlet gun tip.

[0018] In addition, an optional solution is that the fluid infusion component is one of a peristaltic pump, a continuous pipette or a quantitative flow pusher;

[0019] The fluid replenishment component is a continuous pipette or a quantitative flow pusher, and the fluid replenishment component is sealed and connected to the inlet of the sample spotting gun head.

[0020] In addition, an optional solution is that the inner diameter L1 of the outlet tip of the spotting gun head is in the range of 0.2-2 mm, the inner diameter L2 of the inlet of the spotting gun head is in the range of 2-20 mm, and the height H of the spotting gun head is in the range of 10-70 mm.

[0021] According to another aspect of the present invention, a method for preparing an alginate gel is provided, wherein the alginate gel is prepared by using a spotting device, comprising:

[0022] Step S10, mixing the sample solution and 5% sodium alginate solution to obtain a sodium alginate-sample mixed solution, wherein the concentration of sodium alginate in the sodium alginate-sample mixed solution is 0.5%-1.5%;

[0023] Step S20, injecting the sodium alginate-sample mixture into the sample spotting pipette tip, and configuring a rehydration component to maintain a stable volume of the sodium alginate-sample mixture in the sample spotting pipette tip;

[0024] Step S30, the outlet tip of the spotting gun is pointed downward and clicks a metal salt solution with a concentration of 1-3% (w / v) at a set frequency, and each click forms an alginate gel;

[0025] Step S40: collecting the prepared alginate gel every 5 minutes until the required amount of alginate gel is prepared.

[0026] In addition, an optional solution is that in step S30, the metal salt solution is placed in a container with an inner diameter of 30-60 mm, and the container moves horizontally or rotates around the Z axis as the outlet gun tip of the spotting gun head clicks.

[0027] In addition, an optional solution is that the alginate gel formed in step S30 is spherical, and step S30 includes:

[0028] Step S301: Each time the tip of the spotting gun head is clicked, the tip of the spotting gun head is immersed in the metal salt solution 0.5-5 mm below the liquid surface, and stays at this position for 20-100 ms before leaving the metal salt solution to form a spherical sodium alginate colloid.

[0029] In addition, an optional solution is that the alginate gel formed in step S30 is ellipsoidal, and step S30 includes:

[0030] Step S302: Each time the tip of the spotting gun head is clicked, the tip of the spotting gun head is immersed in the metal salt solution 0.5-5 mm below the liquid surface, and stays at this position for 20-500 ms before leaving the metal salt solution to form an ellipsoidal sodium alginate colloid.

[0031] In addition, an optional solution is that the alginate gel formed in step S30 is in the form of an elongated strip, and step S30 includes:

[0032] In step S303, with each click, the outlet tip of the spotting gun head is immersed 0.5-5 mm below the liquid surface of the metal salt solution, and the outlet tip is dragged in the direction of leaving the metal salt solution as the gel is formed. After the gel reaches the target length, the outlet tip is controlled to leave the metal salt solution.

[0033] In addition, an optional solution is to include before step S10:

[0034] Step S01, conducting a preliminary experiment based on the target diameter of the prepared gel to determine the size of the sample tip and the volume V of the sodium alginate-sample mixture in the sample tip during preparation;

[0035] The inner diameter L1 of the outlet tip of the spotting gun head is in the range of 0.2-2 mm, the inner diameter L2 of the inlet of the spotting gun head is in the range of 2-20 mm, and the height H of the spotting gun head is 10-70 mm.

[0036] During the preparation work, the volume V of the sodium alginate-sample mixture in the tip of the spotting pipette is in the range of 10-500 μL;

[0037] The inner diameter L1 of the outlet tip of the spotting gun head is smaller than the target diameter of the prepared gel, and the volume V of the sodium alginate-sample mixture in the spotting gun head is proportional to the diameter of the prepared gel.

[0038] The beneficial effects of the present invention are as follows:

[0039] To address the technical problems existing in the prior art, the present invention provides a sample spotting device and a method for preparing an alginate gel. The sample spotting device is used to prepare an alginate gel, and the method improves the alginate gel preparation method. The prior art method of dripping a sodium alginate sample solution into a metal salt solution under external conditions is improved to utilize a siphon principle to allow the sodium alginate sample solution to naturally enter the metal salt solution, avoiding collision with the metal salt solution surface. This results in a uniform, smooth, and rounded gel shape, better meeting the requirements of subsequent experiments. Compared to existing methods, this alginate gel preparation method is more convenient, low-cost, and has a wide range of applications.

[0040] The shape of the gel can be controlled by the spotting device to achieve the preparation of spherical, ellipsoidal and long strip gels. Spherical gels with a diameter range of 0.1-2.5mm, as well as ellipsoidal gels or long strip gels with a diameter range of 0.10-2mm and a length range of 0.1-100mm can be prepared.

[0041] By using a spotting device combined with this preparation method, single cells or small amounts of samples can be gelled, with a minimum sample volume of 20 μL being processed. The number of samples and the number of prepared gels can be precisely controlled, and the number of prepared gels can be accurately controlled and recorded.

[0042] In addition, the preparation method can realize the preparation of a gel with low sodium alginate concentration, and the minimum concentration of sodium alginate is 0.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0044] Figure 1 A schematic structural diagram of a spotting gun tip provided in an embodiment of the present invention is shown.

[0045] Figure 2 A schematic structural diagram of a spotting device provided by an embodiment of the present invention is shown.

[0046] Figure 3 The figure shows the effect of microencapsulation of the gel prepared by the prior art.

[0047] Figure 4The figure shows the effect of microencapsulation of the gel prepared by the preparation method provided in this application.

[0048] Figure 5 A schematic diagram showing a large amount of alginate gel prepared by the preparation method provided in this application. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and examples. It will be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention. It should also be noted that, for ease of description, the accompanying drawings only illustrate portions relevant to the present invention, not all structures.

[0050] In the description of the present invention, unless otherwise specified or limited, the terms "connected," "connected," and "fixed" should be understood broadly. For example, they may refer to fixed connections, detachable connections, or integration; they may refer to direct connections or connections through an intermediate gap; they may refer to internal communication between two components or interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0051] In the present invention, unless otherwise clearly stipulated and limited, the first feature being "on" or "under" the second feature may include the first and second features being in direct contact, or the first and second features not being in direct contact but being in contact through another feature between them.

[0052] In the description of this embodiment, terms such as "upper," "lower," "left," and "right" are used to refer to positions or locations based on those shown in the accompanying drawings. These terms are intended solely to facilitate description and simplify operation, and are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used solely for descriptive purposes and have no special meaning.

[0053] In view of the defects of the prior art, the present invention provides a spotting device, which combines Figure 1-2 As shown, the spotting device includes a spotting gun head 1 and a liquid replenishing component 2.

[0054] The sample spotting gun tip 1 is a funnel structure with upper and lower openings, wherein the upper opening is the inlet 11 of the sample spotting gun tip 1 , which is connected to the rehydration component 2 , and the mixed solution enters the sample spotting gun tip 1 through the rehydration component 2 .

[0055] The lower opening of the spotting gun head 1 is an outlet gun tip 12 of the spotting gun head 1 , and the mixed solution flows out through the outlet gun tip 12 .

[0056] The inner diameter of the inlet 11 is larger than the inner diameter of the outlet gun tip 12. By controlling the volume V of the mixed solution in the spotting gun tip 1 and the inner diameter of the outlet gun tip 12, the resultant force of the surface tension of the mixed solution in the spotting gun tip 1 is made greater than the gravity acting on the mixed solution, thereby preventing the mixed solution from flowing out of the spotting gun tip 1.

[0057] The spotting gun tip 1 immerses the outlet gun tip 12 into the metal salt solution, and uses the siphon effect to squeeze the mixed solution in the spotting gun tip 1 into the metal salt solution. The sodium alginate in the mixed solution contacts with the metal cations to complete ion exchange to form an alginate gel. During this process, the mixed solution needs to be replenished for the spotting gun tip 1 through the rehydration component 2 according to the consumption rate of the mixed solution in the spotting gun tip 1 to maintain the stability of the solution volume in the spotting gun tip 1.

[0058] In a specific embodiment, a first equal-diameter extension tube may be fixedly connected to the inlet 11 of the spotting gun head 1 , and the inner diameter of the first equal-diameter extension tube is equal to the inner diameter of the inlet.

[0059] In a specific embodiment, a second equal-diameter extension tube may be fixedly connected to the outlet gun tip 12 of the spotting gun head 1 , and the inner diameter of the second equal-diameter extension tube is equal to the inner diameter of the outlet gun tip 12 .

[0060] In a specific embodiment, the inner wall of the spotting gun tip 1 is a smooth surface; in another embodiment, the inner wall of the spotting gun tip 1 is processed with threads.

[0061] In one embodiment, the rehydration component 2 can be a hose with a diameter smaller than the inner diameter of the inlet 11. One end of the hose is non-sealedly connected to the inlet 11, and the other end is immersed in the mixed solution to maintain the volume of the mixed solution in the spotting gun tip 1 stable during operation.

[0062] In one embodiment, when the alginate gel is prepared by tapping the metal salt solution with the sample tip 1 at a fixed frequency, the consumption rate of the mixed solution in the sample tip 1 is stable. The volume of the mixed solution in the sample tip 1 can be controlled by a rate-controlled liquid feeding device (such as a peristaltic pump) to maintain a stable volume. Alternatively, if the uniformity of the alginate gel preparation is not required, manual liquid replenishment can be performed using a syringe.

[0063] In other embodiments, a continuous pipette or a quantitative flow injector can be sealed and connected to the inlet 11 of the spotting gun head 1 as a rehydration component 2 for rehydration. When the outlet gun tip 12 is immersed in the metal salt solution, the mixed solution is pushed into the metal salt solution by external force to prepare alginate gel.

[0064] In one embodiment, the spotting gun head 1 is further connected to an automatic driving assembly (not shown) to control the spotting gun head 1 to perform repetitive operations of immersing in and leaving the metal salt solution.

[0065] In this embodiment, the spotting gun tip 1 needs to be customized and is a non-standard part, and is made of metal material or polymer material.

[0066] In a specific example, the inner diameter L1 of the outlet tip of the spotting gun head 1 ranges from 0.2 to 2 mm, the inner diameter L2 of the inlet 11 of the spotting gun head 1 ranges from 2 to 20 mm, and the height H of the spotting gun head 1 ranges from 10 to 70 mm.

[0067] Another embodiment of the present invention provides a method for preparing an alginate gel, wherein the alginate gel is prepared using the spotting device provided in the above embodiment, and specifically comprises the following steps:

[0068] Step S10, mixing the sample solution and 5% sodium alginate solution to obtain a sodium alginate-sample mixed solution, wherein the concentration of sodium alginate in the sodium alginate-sample mixed solution is 0.5%-1.5%;

[0069] Step S20, injecting the sodium alginate-sample mixed solution into the spotting pipette tip 1, and configuring the rehydration component 2 to maintain the stability of the volume of the sodium alginate-sample mixed solution in the spotting pipette tip 1;

[0070] Step S30, the outlet tip 12 of the spotting gun head 1 points downward and hits the metal salt solution with a concentration of 1-3% (w / v) at a set frequency, and each click forms an alginate gel;

[0071] Step S40: collecting the prepared alginate gel every 5 minutes until the required amount of alginate gel is prepared.

[0072] In step S10, the sample solution is a cell-containing diluent or culture medium, such as oocyte culture medium, semen diluent, etc. The function of the sodium alginate solution is to exchange ions when the sodium alginate contacts the metal salt solution, and the reaction is rapid, and the alginate gel is solidified instantly upon contact.

[0073] The alginate gel prepared in this embodiment can be directly used for sustained release of drugs, nucleic acids, plasmids, single cells or multiple cells, etc., and can also be used for subsequent microencapsulation processing.

[0074] In step S20 , the sodium alginate-sample mixture is replenished to the sample spotting gun tip 1 through the liquid replenishing component 2 .

[0075] In one specific embodiment, in step S30, the metal salt solution is placed in a container with an inner diameter of 30-60 mm, such as a dish-shaped container or other container. During operation of the spotting tip 1, the container moves horizontally or rotates about the Z-axis in response to the tapping action of the outlet tip 12 of the spotting tip 1, thereby dispersing the individual alginate gels and preventing the aggregation of a large amount of gel at a single point, which can occur when the spotting tip 1 frequently taps the metal salt solution.

[0076] In a specific embodiment, in step S30, each time the spotting gun head 1 hits the metal salt solution, the outlet gun tip 12 of the spotting gun head 1 needs to be immersed 0.5-5 mm below the liquid surface of the metal salt solution. The sodium alginate-sample mixture in the spotting gun head 1 is naturally squeezed into the metal salt solution through the siphon principle. The sodium alginate in the sodium alginate-sample mixture and the metal salt solution exchange ions when they come into contact, and solidify instantly to form an alginate gel, and the sample is wrapped inside.

[0077] In one embodiment, the alginate gel formed in step S30 is spherical. Step S30 specifically includes step S301. With each click, the outlet tip 12 of the spotting gun head 1 is immersed 0.5-5 mm below the liquid surface of the metal salt solution, and stays at this position for 20-100 ms before leaving the metal salt solution to form a spherical sodium alginate colloid. In actual production, the specific position and residence time of the outlet tip 12 below the liquid surface of the metal salt solution can be adjusted according to the target diameter and shape of the prepared gel. Reasonable values are selected within the above-mentioned size range. Generally speaking, the deeper the outlet tip 12 of the spotting gun head 1 is immersed below the liquid surface of the metal salt solution, the faster the liquid is discharged and the shorter the residence time required to reach the target diameter. When the outlet tip 12 of the spotting gun head 1 is immersed below the liquid surface of the metal salt solution at a certain position, the longer the residence time at this position, the larger the diameter of the spherical gel formed.

[0078] In one embodiment, the alginate gel formed in step S30 is ellipsoidal. Step S30 specifically includes step S302, wherein, with each click, the outlet tip 12 of the spotting gun head 1 is immersed 0.5-5 mm below the liquid surface of the metal salt solution, and stays at this position for 20-500 ms before leaving the metal salt solution to form an ellipsoidal sodium alginate colloid. In actual production, the specific position and residence time of the outlet tip 12 below the liquid surface of the metal salt solution can be adjusted according to the target diameter and shape of the prepared gel, and reasonable values are selected within the above-mentioned size range. Generally speaking, the deeper the outlet tip 12 of the spotting gun head 1 is immersed below the liquid surface of the metal salt solution, the faster the liquid is discharged, and the shorter the residence time required to achieve the target diameter and target shape; when the outlet tip 12 of the spotting gun head 1 is immersed below the liquid surface of the metal salt solution, the ellipsoidal gel can be obtained as the residence time at this position increases (compared to the preparation of spherical gel).

[0079] In this embodiment, the speed at which the sodium alginate-sample mixture flows out of the spotting gun tip 1 is affected by the inner diameter of the outlet gun tip 12 and the volume of the sodium alginate-sample mixture within the spotting gun tip 1. When the inner diameter of the outlet gun tip 12 is small and the volume of the sodium alginate-sample mixture within the spotting gun tip 1 is large, the outlet gun tip 12 can form an ellipsoidal gel even after a short residence time (e.g., 20 ms) in the metal salt solution. Preferably, the residence time of the outlet gun tip 12 in the metal salt solution ranges from 50 to 500 ms.

[0080] In one embodiment, the alginate gel formed in step S30 is in the form of an elongated strip. Step S30 specifically includes step S303, wherein, with each click, the outlet tip 12 of the spotting gun tip 1 is immersed 0.5-5 mm below the liquid surface of the metal salt solution and, as the gel is formed, is dragged in a direction away from the metal salt solution until the gel reaches a target length, at which point the outlet tip 12 is controlled to exit the metal salt solution. In actual production, the specific position at which the outlet tip 12 is immersed below the liquid surface of the metal salt solution and the speed of movement can be adjusted based on the target diameter and shape of the prepared gel, with reasonable values selected within the aforementioned size range. Generally speaking, the deeper the outlet tip 12 of the spotting gun tip 1 is immersed below the liquid surface of the metal salt solution, the faster the liquid discharge, the shorter the residence time required to reach the target diameter, and the faster the upward movement of the spotting gun tip 1.

[0081] In one embodiment, step S01 is included before step S10 , in which a preliminary experiment is performed according to the target diameter of the prepared gel to determine the size of the spotting tip 1 and the volume V of the sodium alginate-sample mixture in the spotting tip 1 during the preparation work.

[0082] The inner diameter L1 of the outlet tip of the spotting gun head 1 is in the range of 0.2-2 mm, the inner diameter L2 of the inlet 11 of the spotting gun head 1 is in the range of 2-20 mm, and the height H of the spotting gun head 1 is in the range of 10-70 mm.

[0083] During the preparation work, the volume V of the sodium alginate-sample mixture in the sample spotting pipette tip 1 ranges from 10 to 500 μL.

[0084] During the preliminary experiment, the inner diameter L1 of the outlet tip of the spotting gun head needs to be controlled to be smaller than the target diameter of the prepared gel. The inner diameter L2 of the inlet 11 of the spotting gun head 1 and the height H of the spotting gun head 1 are adjusted according to the inner diameter L1 of the outlet tip of the spotting gun head and can be controlled within a reasonable range.

[0085] During preliminary experiments, the diameter of the prepared alginate gel can be fine-tuned by adjusting the volume V of the sodium alginate-sample mixture within the tip, with an adjustment accuracy of 0.05 mm. When the outlet tip 12 is immersed in the metal salt solution, the higher the liquid level of the sodium alginate-sample mixture within the tip 1, the greater the liquid pressure, and the faster the liquid discharge from the tip 1. Therefore, a larger volume V of the sodium alginate-sample mixture within the tip results in a larger diameter of the prepared gel.

[0086] In one embodiment, when preparing the alginate gel, the working temperature is 0-38°C.

[0087] In this embodiment, the diameter of the spherical gel body that can be prepared is in the range of 0.1-2.5 mm, the diameter of the ellipsoidal gel body or the long strip gel body is in the range of 0.1-2 mm, and the length is in the range of 0.1-100 mm. Figure 3 The figure shows the effect of microencapsulation of the gel prepared by the prior art. Figure 4 This is a diagram showing the effect of microencapsulation of the gel prepared by the preparation method provided in this application. Figure 5 Schematic diagram of a large amount of alginate gel prepared by the preparation method provided in this application. Figure 3 and Figure 4 The microcapsules obtained after microencapsulation of the gel prepared by the preparation method provided in this application are full and uniform, with a smooth surface, and are more in line with subsequent experimental standards.

[0088] In one embodiment, the cation of the metal salt solution used in step S30 is Ca 2+ 、Ba 2+ 、Cu 2+ 、Zn 2+ 、Sr 2+ 、Fe 2+ 、Fe 3+ 、Al3+ One of the above.

[0089] The preparation method of the alginate gel provided by the present invention will be described in detail below with reference to the examples.

[0090] Example 1: Preparation of alginate gel spheres using oocytes

[0091] A preliminary experiment was conducted based on the target diameter of the prepared gel. The material of the spotting gun tip 1 was polypropylene, the inner diameter L1 of the outlet tip 12 of the spotting gun tip 1 was set to 0.5 mm, the inner diameter L2 of the inlet 11 of the spotting gun tip 1 was set to 4.2 mm, and the height H of the spotting gun tip was set to 27.1 mm.

[0092] The oocyte culture medium and sodium alginate were mixed to form a sodium alginate-oocyte culture medium mixed solution (hereinafter referred to as the mixed solution), wherein the concentration of sodium alginate in the mixed solution was 1.2%.

[0093] Use a mouth pipette to inject the mixed solution into the spotting pipette tip 1. The injection volume V is determined to be 20 μL based on the results of the preliminary experiment, and the oocyte is slowly aspirated into the lower part of the spotting pipette tip 1 under a stereoscope.

[0094] The metal salt solution is a CaCl2 solution with a concentration of 1.5% (w / v), and the operating temperature is 37°C.

[0095] The outlet tip 12 of the spotting gun head 1 is immersed in the metal salt solution for 70 ms, and the sample is spotted once.

[0096] The obtained alginate gel ball contains one oocyte and can be used for subsequent operations.

[0097] Example 2: Preparation of semen sodium alginate gel balls

[0098] A preliminary experiment was conducted based on the target diameter of the prepared gel. The material of the sample spotting gun head 1 was 304 stainless steel. The inner diameter L1 of the outlet tip 12 of the sample spotting gun head 1 was set to 0.4 mm, the inner diameter L2 of the inlet 11 of the sample spotting gun head 1 was set to 4 mm, and the height H of the sample spotting gun head was set to 12.4 mm.

[0099] The semen was diluted with egg yolk-containing diluent to a final density of 5 × 10 7 / mL, and then add a 4% sodium alginate solution and slowly mix to form a sodium alginate-semen mixed solution (hereinafter referred to as the mixed solution), the concentration of sodium alginate in the mixed solution is 1%.

[0100] According to preliminary experiments, the volume V of the mixed solution in the spotting pipette tip 1 was set to 50 μL, 100 μL and 150 μL, respectively, and the gels were prepared respectively.

[0101] The metal salt solution is a CaCl2 solution with a concentration of 1.5% (w / v), and the operating temperature is 4°C.

[0102] The outlet tip 12 of the spotting gun head 1 is immersed in the metal salt solution for 70 ms, and the spotting frequency is 3 times / s.

[0103] The diameters of the semen gel balls prepared corresponding to the volume V of the mixed solution in the spotting gun tip 1 are 0.52 mm, 0.58 mm, and 0.65 mm, respectively.

[0104] Example 3: Preparation of semen sodium alginate gel balls

[0105] A preliminary experiment was conducted based on the target diameter of the prepared gel. The material of the spotting gun head 1 was 304 stainless steel. Compared with Example 2, the inner diameter L1 of the outlet tip 12 of the spotting gun head 1 was set to 0.5 mm, and other dimensions remained unchanged.

[0106] The semen was diluted with egg yolk-containing diluent to a final density of 5 × 10 7 / mL, and then add a 4% sodium alginate solution and slowly mix to form a sodium alginate-semen mixed solution (hereinafter referred to as the mixed solution), the concentration of sodium alginate in the mixed solution is 1%.

[0107] According to preliminary experiments, the volume V of the mixed solution in the spotting pipette tip 1 was set to 50 μL, 100 μL and 150 μL, respectively, and the gels were prepared respectively.

[0108] The metal salt solution is a CaCl2 solution with a concentration of 1.5% (w / v), and the operating temperature is 4°C.

[0109] The outlet tip 12 of the spotting gun head 1 is immersed in the metal salt solution for 70 ms, and the spotting frequency is 3 times / s.

[0110] The diameters of the semen gel balls prepared corresponding to the volume V of the mixed solution in the sample spotting pipette tip 1 are 0.58 mm, 0.66 mm, and 0.75 mm, respectively.

[0111] Example 4: Preparation of semen sodium alginate gel balls

[0112] A preliminary experiment was conducted based on the target diameter of the prepared gel. The material of the spotting gun head 1 was 304 stainless steel. Compared with Example 2, the inner diameter L1 of the outlet tip 12 of the spotting gun head 1 was set to 0.6 mm, and the other dimensions remained unchanged.

[0113] The semen was diluted with egg yolk-containing diluent to a final density of 5 × 10 7 / mL, and then add a 4% sodium alginate solution and slowly mix to form a sodium alginate-semen mixed solution (hereinafter referred to as the mixed solution), the concentration of sodium alginate in the mixed solution is 1%.

[0114] According to preliminary experiments, the volume V of the mixed solution in the spotting pipette tip 1 was set to 50 μL, 100 μL and 150 μL, respectively, and the gels were prepared respectively.

[0115] The metal salt solution is a CaCl2 solution with a concentration of 1.5% (w / v), and the operating temperature is 4°C.

[0116] The outlet tip 12 of the spotting gun head 1 is immersed in the metal salt solution for 70 ms, and the spotting frequency is 3 times / s.

[0117] The diameters of the semen gel balls prepared corresponding to the volume V of the mixed solution in the sample spotting pipette tip 1 are 0.65 mm, 0.75 mm, and 0.80 mm, respectively.

[0118] Example 5: Preparation of calcium alginate long gel

[0119] A preliminary experiment was conducted based on the target diameter of the prepared gel. The material of the spotting gun tip 1 was polypropylene, the inner diameter L1 of the outlet tip 12 of the spotting gun tip 1 was set to 0.9 mm, the inner diameter L2 of the inlet 11 of the spotting gun tip 1 was set to 4.7 mm, and the height H of the spotting gun tip was 34.2 mm.

[0120] The concentration of sodium alginate in the sample solution was 1%, the metal salt solution was a CaCl2 solution with a concentration of 1.5% (w / v), and the operating temperature was 4°C.

[0121] According to the preliminary experiment, the volume V of the sample solution in the tip 1 of the spotting pipette was set to 100 μL.

[0122] The outlet tip 12 of the spotting gun head 1 is immersed in the metal salt solution for 1000 ms.

[0123] The gel prepared by this solution is in the shape of a strip with a diameter of 1.3 mm and a length of 80-90 mm.

[0124] Example 6: Preparation of micro-sample alginate gel spheres

[0125] A preliminary experiment was conducted based on the target diameter of the prepared gel. The material of the spotting gun tip 1 was polypropylene, the inner diameter L1 of the outlet tip 12 of the spotting gun tip 1 was set to 0.5 mm, the inner diameter L2 of the inlet 11 of the spotting gun tip 1 was set to 4.2 mm, and the height H of the spotting gun tip was set to 27.1 mm.

[0126] The concentration of sodium alginate in the sample solution was 0.8%, the metal salt solution was a CaCl2 solution with a concentration of 1.5% (w / v), and the operating temperature was 4°C.

[0127] Place the sample solution in the sample tip 1, with a volume V of 10 μL.

[0128] The outlet tip 12 of the spotting gun head 1 is immersed in the metal salt solution for 70 ms, and the spotting is continued until the sample solution is completely immersed in the metal salt solution.

[0129] Since the total amount of sample solution is extremely small, the amount of solution in the sample tip cannot be maintained constant, and the diameter of the prepared gel balls gradually becomes smaller, with a diameter of about 0.5-0.7 mm, and the number of gel balls is 50-80.

[0130] By comparing Example 2, Example 3, and Example 4, it can be seen that, when other conditions remain unchanged during the preparation process, the larger the inner diameter L1 of the outlet tip 12 of the spotting gun head 1 is, the larger the diameter of the prepared gel ball is.

[0131] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A spotting device, characterized in that: include: Spotting pipette tip and rehydration components; The sample spotting gun head is a funnel structure with upper and lower openings, wherein the upper opening is the inlet of the sample spotting gun head, which is connected to the rehydration component, and the lower opening is the outlet tip of the sample spotting gun head; The inner diameter of the inlet is larger than the inner diameter of the outlet gun tip; The fluid replenishing component is configured to maintain a stable volume of the solution in the spotting pipette tip.

2. The spotting device according to claim 1, characterized in that The inlet is fixedly connected with a first equal-diameter extension tube, and the inner diameter of the first equal-diameter extension tube is equal to the inner diameter of the inlet; and / or The outlet gun tip is fixedly combined with a second equal-diameter extension tube, and the inner diameter of the second equal-diameter extension tube is equal to the inner diameter of the outlet gun tip.

3. The spotting device according to claim 1, characterized in that: The fluid infusion component is one of a peristaltic pump, a continuous pipette or a quantitative flow pusher; The fluid replenishment component is a continuous pipette or a quantitative flow pusher, and the fluid replenishment component is sealed and connected to the inlet of the sample spotting gun head.

4. The spotting device according to claim 1, characterized in that The inner diameter L1 of the outlet tip of the spotting gun head has a value range of 0.2-2 mm, the inner diameter L2 of the inlet of the spotting gun head has a value range of 2-20 mm, and the height H of the spotting gun head has a value range of 10-70 mm.

5. A method for preparing an alginate gel, comprising preparing the alginate gel by using the spotting device according to claim 1, wherein: include: Step S10, mixing the sample solution and 5% sodium alginate solution to obtain a sodium alginate-sample mixed solution, wherein the concentration of sodium alginate in the sodium alginate-sample mixed solution is 0.5%-1.5%; Step S20, injecting the sodium alginate-sample mixture into the sample spotting pipette tip, and configuring a rehydration component to maintain a stable volume of the sodium alginate-sample mixture in the sample spotting pipette tip; Step S30, the outlet tip of the spotting gun is pointed downward and clicks a metal salt solution with a concentration of 1-3% (w / v) at a set frequency, and each click forms an alginate gel; Step S40: collecting the prepared alginate gel every 5 minutes until the required amount of alginate gel is prepared.

6. The method for preparing alginate gel according to claim 5, characterized in that: In step S30 , the metal salt solution is placed in a dish-shaped container, and the container moves horizontally or rotates around the Z axis as the outlet gun tip of the spotting gun head clicks.

7. The method for preparing alginate gel according to claim 5, characterized in that: The alginate gel formed in step S30 is spherical. Step S30 includes: Step S301: Each time the tip of the spotting gun head is clicked, the tip of the spotting gun head is immersed in the metal salt solution 0.5-5 mm below the liquid surface, and stays at this position for 20-100 ms before leaving the metal salt solution to form a spherical sodium alginate colloid.

8. The method for preparing alginate gel according to claim 5, characterized in that: The alginate gel formed in step S30 is ellipsoidal. Step S30 includes: Step S302: Each time the tip of the spotting gun head is clicked, the tip of the spotting gun head is immersed in the metal salt solution 0.5-5 mm below the liquid surface, and stays at this position for 20-500 ms before leaving the metal salt solution to form an ellipsoidal sodium alginate colloid.

9. The method for preparing alginate gel according to claim 5, characterized in that: The alginate gel formed in step S30 is in the form of a long strip. Step S30 includes: In step S303, with each click, the outlet tip of the spotting gun head is immersed 0.5-5 mm below the liquid surface of the metal salt solution, and the outlet tip is dragged in the direction of leaving the metal salt solution as the gel is formed. After the gel reaches the target length, the outlet tip is controlled to leave the metal salt solution.

10. The method for preparing alginate gel according to claim 5, characterized in that: Before step S10, the following steps are included: Step S01, conducting a preliminary experiment based on the target diameter of the prepared gel to determine the size of the sample tip and the volume V of the sodium alginate-sample mixture in the sample tip during preparation; The inner diameter L1 of the outlet tip of the spotting gun head is in the range of 0.2-2 mm, the inner diameter L2 of the inlet of the spotting gun head is in the range of 2-20 mm, and the height H of the spotting gun head is in the range of 10-70 mm. During the preparation work, the volume V of the sodium alginate-sample mixture in the tip of the spotting pipette is in the range of 10-500 μL; The inner diameter L1 of the outlet tip of the spotting gun head is smaller than the target diameter of the prepared gel, and the volume V of the sodium alginate-sample mixture in the spotting gun head is proportional to the diameter of the prepared gel.

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