An embryo biopsy culture dish with microstructure positioning and its application

Abstract

The present invention relates to the technical fields of biomedical engineering and reproductive genetics, and particularly relates to an embryo biopsy culture dish with microstructure positioning and its application, including: a culture dish body; the culture dish body includes a bottom wall and a side wall extending along the periphery of the bottom wall, and an upper opening space suitable for accommodating a sample is formed between the bottom wall and the side wall. The bottom wall is sequentially provided with a central micro-column array region, a gradient hydrophobic region, and a superhydrophobic region from the center to the periphery; the central micro-column array region includes independent conical micro-columns; the contact angle of the surface of the gradient hydrophobic region gradually changes from 120° on the side close to the central micro-column array region to 150° on the side far from the central micro-column array region. The culture dish of the present invention, through the design of combining a micro-column array with a hydrophobic outer ring, does not require a mineral oil covering droplet during use, thereby significantly improving the clarity of microscopic imaging, avoiding contamination, and ensuring the accuracy of subsequent operations.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedical engineering and reproductive genetics, and particularly relates to an embryo biopsy culture dish with microstructural positioning and its application. Background Art

[0002] Embryo biopsy is a commonly used technical means in assisted reproductive technology (ART) and embryo research, mainly used to extract a small number of cells from early embryos for genetic testing or analysis. By embryo biopsy, the genetic status of embryos can be evaluated, and healthy embryos can be screened for transplantation or further research. Embryo biopsy usually takes place at an early stage of embryo development (usually 3 to 6 days after fertilization), and a small number of cells (usually 1 to 10 cells) are taken from the embryo by micromanipulation technology for genetic analysis, thereby detecting chromosomal abnormalities, single-gene diseases or other genetic problems of the embryo.

[0003] Currently, the methods of embryo biopsy include microblade cutting, microneedle aspiration and laser cutting, etc. As a micromanipulation technology, it needs to be carried out in a specific experimental environment. The culture dish is an indispensable tool in the process of embryo biopsy, which provides a stable growth environment and an operation platform for the embryo. Therefore, the design and structure of the culture dish have an important impact on the efficiency and accuracy of embryo biopsy.

[0004] Traditional culture dishes rely on mineral oil to cover and confine the liquid droplets, but the introduction of the oil phase leads to differences in optical refractive index, significantly reducing the resolution of microscopic imaging. Moreover, the use of mineral oil easily causes the residue of oil droplets in the mouth pipette, resulting in sample contamination and affecting the subsequent amplification and typing effects. In addition, manual labeling or fluorescence coding lacks the data chain of the operation process, easily leading to mismatches between the sample and the number, and unable to achieve the full-process traceability of "sample ID - operation log - gene data".

[0005] Therefore, there is an urgent need to develop a new type of culture dish that can simplify the operation process, does not require mineral oil coverage, has high imaging quality, low pollution possibility, is durable and can achieve full-process traceability, so as to assist in improving the efficiency and accuracy of embryo biopsy. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides an embryo biopsy culture dish with microstructural positioning and its application. Through the design of combining a microcolumn array with a hydrophobic outer ring, the culture dish of the present invention does not require a mineral oil covering droplet during use, thereby significantly improving the clarity of microscopic imaging, while avoiding the contamination of the embryo sample to be tested by mineral oil residues and ensuring the accuracy of subsequent operations; in addition, the combined traceability label system realizes the one-to-one correspondence between the culture dish and the embryo, avoids misalignment of sample coding, and records information such as operation time, making the operation process traceable; at the same time, the culture dish is prepared by using materials such as polydimethylsiloxane (PDMS) in combination with advanced processing techniques, further extending its service life.

[0007] In a first aspect of the present invention, the present invention proposes a culture dish. According to an embodiment of the present invention, it includes: a culture dish body; the culture dish body includes a bottom wall and a side wall extending along the outer periphery of the bottom wall, and an upper opening space suitable for accommodating a sample is formed between the bottom wall and the side wall. Among them, the bottom wall is sequentially provided with a central microcolumn array region, a gradient hydrophobic region, and a superhydrophobic region from the center to the outer periphery; the central microcolumn array region includes independent conical microcolumns; the contact angle of the surface of the gradient hydrophobic region gradually changes from 120° on the side close to the central microcolumn array region to 150° on the side far from the central microcolumn array region. The culture dish according to the present invention significantly improves the droplet stability and operation accuracy during the sample detection process through a unique structural design; among them, the conical microcolumn structure in the central microcolumn array region can fix the droplet through the droplet pinning effect, preventing it from spreading or moving, and ensuring the stability of embryo cells during the operation; at the same time, the contact angle of the gradient hydrophobic region gradually changes from 120° to 150°, thereby restricting the spread of the droplet, and further restricting the droplet spread through the superhydrophobic region. The central microcolumn array region, the gradient hydrophobic region, and the superhydrophobic region work together to ensure the stability of the droplet during the operation, reduce the contact between the droplet and the surface of the culture dish, reduce the risk of contamination, and further improve the operation accuracy and result reliability. This design does not require a mineral oil covering droplet, avoids the optical refractive index difference introduced by mineral oil, significantly improves the clarity of microscopic imaging, and at the same time reduces the contamination of the sample by mineral oil residues; in addition, the structural design of this culture dish can be applied to various application scenarios of embryo sample detection, has broad versatility and high durability, and further effectively improves the efficiency and accuracy of embryo sample detection.

[0008] According to an embodiment of the present invention, the above-mentioned culture dish may further have the following additional technical features:

[0009] According to an embodiment of the present invention, the water contact angle of the surface of the central microcolumn array region ≤ 30°.

[0010] According to an embodiment of the present invention, the water contact angle of the surface of the superhydrophobic region is ≥150°, and the rolling angle is ≤10°.

[0011] According to an embodiment of the present invention, a traceability label area is provided on the culture dish body.

[0012] According to an embodiment of the present invention, the traceability label area includes: one or more coding labels for uniquely identifying the culture dish.

[0013] According to an embodiment of the present invention, the coding label includes one or more of a high-frequency RFID label, a ultra-high-frequency RFID label, and a high-frequency NFC label.

[0014] According to an embodiment of the present invention, the material of the culture dish base structure includes one or more of polydimethylsiloxane, polymethyl methacrylate, polycarbonate, polystyrene, polytetrafluoroethylene, polylactic acid, polyglycolic acid, polyethylene, and polypropylene.

[0015] According to an embodiment of the present invention, a hydrophobic coating material is provided on the surface of the gradient hydrophobic region.

[0016] According to an embodiment of the present invention, the hydrophobic coating material includes one or more of perfluorooctyltriethoxysilane, polydimethylsiloxane, perfluoropolyether, and fluorinated polystyrene.

[0017] According to an embodiment of the present invention, a superhydrophobic coating material is provided on the surface of the superhydrophobic region.

[0018] According to an embodiment of the present invention, the superhydrophobic coating material includes one or more of fluorinated silica, perfluorooctyltriethoxysilane, perfluorododecyltriethoxysilane, fluorinated polystyrene, and fluorocarbon compounds.

[0019] According to an embodiment of the present invention, the culture dish further includes: an upper cover covering the culture dish body.

[0020] In a second aspect of the present invention, the present invention provides an embryo biopsy method. According to an embodiment of the present invention, the embryo biopsy method includes: placing a sample containing the embryo to be tested into the central microcolumn array region of the culture dish described in the first aspect and performing detection on it. The embryo biopsy method of the present invention significantly improves the accuracy of the detection operation and the reliability of the detection results by placing the sample containing the embryo to be tested in the central microcolumn array region of the culture dish. Specifically, the microcolumn structure of the culture dish can effectively fix the droplet, ensuring the stability of the embryo during the operation and avoiding embryo damage or operation errors caused by droplet movement or diffusion; at the same time, this method uses the gradient hydrophobic region and superhydrophobic region of the culture dish to reduce the contact area between the droplet and the surface of the culture dish, reducing the risk of contamination and avoiding imaging blur caused by mineral oil coverage, significantly improving the clarity of microscopic imaging; in addition, this detection method simplifies the steps of adding and removing mineral oil in traditional embryo biopsy, reduces the operation complexity, and improves the work efficiency; in addition, combined with the traceability label region of the culture dish, this detection method can also record the operation information during the embryo biopsy process, realize full-process traceability, and ensure the integrity and repeatability of experimental data; this method is applicable to different types of embryos, can meet different application requirements, and has a wide application prospect.

[0021] According to an embodiment of the present invention, the above embryo biopsy method may further have the following additional technical features:

[0022] According to an embodiment of the present invention, the embryo to be tested is a mammalian embryo.

[0023] In a third aspect of the present invention, the present invention provides the use of the culture dish described in the first aspect or the embryo biopsy method described in the second aspect in embryo genetic testing, livestock breeding, and basic biological research.

[0024] Those skilled in the art can understand that the features and advantages described above for the culture dish or the embryo biopsy method also apply to this use, and will not be repeated here.

[0025] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, where:

[0027] Figure 1Schematic diagram of the main structure of the embryo biopsy culture dish with micro-structure positioning in Embodiment 1 of the present invention. Among them, 1 is the central micro-column array area; 2 is the gradient hydrophobic area, 3 is the super-hydrophobic area, and 4 is the FM1108 high-frequency RFID tag;

[0028] Figure 2 Schematic diagram of the conical micro-column array area at the center of the embryo biopsy culture dish with micro-structure positioning in Embodiment 1 of the present invention. Among them, 5 is the conical micro-column, and the micro-column array formed by it can fix the operation droplet through the pinning effect;

[0029] Figure 3 Schematic diagram of the operation table collaborative system architecture of the embryo biopsy culture dish with micro-structure positioning in Embodiment 1 of the present invention. Among them, 6 is the chip reader installed on the operation table, and it is necessary to ensure that the FM1108 high-frequency RFID tag 4 is closely connected to the reader 6 during detection. Detailed implementation mode

[0030] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0031] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0032] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0033] In this article, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0034] In this article, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0035] Culture dish

[0036] The present invention provides a culture dish. According to an embodiment of the present invention, it includes: a culture dish body; the culture dish body includes a bottom wall and a side wall extending along the outer periphery of the bottom wall, and the bottom wall and the side wall form an upper opening space suitable for accommodating a sample. Wherein, the bottom wall is sequentially provided with a central micro-column array region, a gradient hydrophobic region, and a super-hydrophobic region from the center to the outer periphery; the central micro-column array region includes independent conical micro-columns; the contact angle of the surface of the gradient hydrophobic region gradually changes from 120° on the side close to the central micro-column array region to 150° on the side far from the central micro-column array region. The culture dish according to the present invention significantly improves the droplet stability and operation accuracy during the sample detection process through a unique structural design; among them, the conical micro-column structure in the central micro-column array region can fix the droplet through the droplet pinning effect, preventing it from spreading or moving, and ensuring the stability of embryonic cells during the operation; at the same time, the contact angle of the gradient hydrophobic region gradually changes from 120° to 150°, thereby restricting the spread of the droplet, and further restricting the droplet spread through the super-hydrophobic region. The central micro-column array region, the gradient hydrophobic region, and the super-hydrophobic region work together to ensure the stability of the droplet during the operation, reduce the contact between the droplet and the surface of the culture dish, reduce the pollution risk, and further improve the operation accuracy and result reliability. This design does not require mineral oil to cover the droplet, avoiding the optical refractive index difference introduced by mineral oil, significantly improving the clarity of microscopic imaging, and at the same time reducing the pollution of the sample by mineral oil residues; in addition, the structural design of this culture dish can be applied to various application scenarios of embryonic sample detection, with broad versatility and high durability, and further effectively improving the efficiency and accuracy of embryonic sample detection. Exemplarily, the process parameters of the central micro-column array region of the culture dish can be set as follows (diameter 2.5 mm), composed of conical micro-columns (height 20 μm, bottom diameter 5 μm, pitch 10 μm), and the water contact angle ≤ 30°; this region is processed by femtosecond laser, and the laser parameters are: wavelength 1030 ± 10 nm, single pulse energy 0.5 μJ, repetition frequency 100 kHz; the scanning path is a concentric spiral trajectory, with a pitch of 8 - 12 μm; after processing, the micro-column cone angle is 85° ± 2°, and the ratio of the pitch to the bottom diameter is 2:1; it should be noted that the parameter settings and preparation process of the central micro-column array region of the culture dish are not limited to the above method and can be adjusted and optimized according to actual needs.

[0037] According to an embodiment of the present invention, the water contact angle of the surface of the central micro-column array region ≤ 30°. Thus, the low contact angle design ensures the stability of the droplet in the central micro-column array region and the accuracy of the experimental operation during the sample detection process, while reducing the friction between the droplet and the surface, protecting the embryonic cells to be tested from mechanical damage; at the same time, this design also improves the spreading performance of the droplet, ensuring that the embryonic cells to be tested can be evenly distributed in the droplet, thereby further improving the accuracy of the detection result.

[0038] According to an embodiment of the present invention, the water contact angle of the surface of the superhydrophobic region is ≥150°, and the rolling angle is ≤10°. Thus, the superhydrophobic region with a high contact angle and a low rolling angle provides excellent hydrophobic performance for the culture dish, ensuring that the droplets during the sample detection process will not spread or remain during the operation, further reducing the risk of contamination and improving the reliability of the results.

[0039] According to an embodiment of the present invention, a traceability label area is provided on the culture dish body. Thus, the existence of the traceability label area enables the culture dish to achieve full-process traceability from sample placement to operation completion, avoiding sample mismatching, ensuring the accuracy and repeatability of experimental data, and further enhancing the reliability of experimental results. Exemplarily, the position of the traceability label area can be on the side wall of the culture dish.

[0040] According to an embodiment of the present invention, the traceability label area includes: one or more coding labels for uniquely identifying the culture dish. Thus, the coding label is used to quickly identify the culture dish, and the data storage unit is used to record operation information (such as operation time, operator, environmental conditions, etc.); the two work together to facilitate the management and analysis of experimental data, further enhancing the traceability of experimental operations and the integrity of data analysis.

[0041] According to an embodiment of the present invention, the coding label includes one or more of a high-frequency RFID tag, a ultra-high-frequency RFID tag, and a high-frequency NFC tag. Thus, the type of coding label can be selected according to different experimental environments, operation requirements, and cost requirements, further enhancing the versatility and flexibility of the culture dish; Exemplarily, the coding label can be an FM1108 high-frequency RFID tag, which is embedded in the middle section of the side wall of the culture dish, and its antenna is printed on the curved surface of the culture dish using a serpentine copper wire to ensure good signal transmission and adaptation to the curved surface structure of the culture dish; Further, the surface of the tag has a packaging layer, including a metal shielding mesh and a perfluoropolyether protective coating, for protecting the tag from the external environment and improving the stability and durability of the tag; In addition, for data reading of the tag, the tag data can be read through an external card reader system. The operating frequency of the card reader system is 860~960 MHz, and it supports the ISO / IEC 18000-63 communication protocol to ensure efficient data transmission and accurate reading; Among them, the card reader system includes a fixed bracket, which is installed on the operating table and used to keep a preset communication distance between the card reader and the tag on the side wall of the culture dish to ensure stable signal transmission; When reading data, the card reader system is connected to the computer of the operating laser through a data transmission interface to real-time transmit the embryo ID, operation time, and operator information stored in the tag, thereby realizing precise tracking and management of the culture dish.

[0042] According to an embodiment of the present invention, the material of the culture dish base structure includes one or more of polydimethylsiloxane, polymethyl methacrylate, polycarbonate, polystyrene, polytetrafluoroethylene, polylactic acid, polyglycolic acid, polyethylene, and polypropylene. Thus, different base materials can be selected according to different application scenarios and requirements; for example, polydimethylsiloxane (PDMS) has good optical transparency and biocompatibility and is very suitable for microscopy imaging and cell culture, while materials such as polycarbonate have higher mechanical strength and are suitable for working environments that require frequent operation.

[0043] According to an embodiment of the present invention, a hydrophobic coating material is provided on the surface of the gradient hydrophobic region. Thus, the droplet confinement ability is further optimized, the contact area between the droplet and the culture dish surface is reduced, the pollution risk is lowered, and the operation accuracy and imaging quality are improved. For example, the process parameters of the gradient hydrophobic region of the culture dish can be set as follows (width 0.5 mm), and a 1064 nm fiber laser is used for power gradient scanning (5W→15W); perfluorooctyltriethoxysilane is introduced into a vacuum chamber (10⁻³ Pa), and the deposition time is 30 - 60 min; the spacing between the contact angle test points in the gradient region is 0.1 mm, and the angle change rate is 60 ° / mm; it should be noted that the hydrophobic coating material provided on the surface of the gradient hydrophobic region of the culture dish and its preparation process are not limited to the above method and can be adjusted and optimized according to actual needs.

[0044] According to an embodiment of the present invention, the hydrophobic coating material includes one or more of perfluorooctyltriethoxysilane, polydimethylsiloxane, perfluoropolyether, and fluorinated polystyrene. Thus, different hydrophobic coating materials can be selected according to different application scenarios and requirements, and then the contact angle and rolling angle can be adjusted, thereby optimizing the stability and operation accuracy of the droplet during sample detection.

[0045] According to an embodiment of the present invention, a superhydrophobic coating material is provided on the surface of the superhydrophobic region. Thus, the droplet confinement ability is further optimized, the contact area between the droplet and the surface is reduced, the pollution risk is lowered, and the operation accuracy and imaging quality are improved. For example, the process parameters of the superhydrophobic region of the culture dish can be set as follows (a circular ring region of 3.5 - 10 mm), and fluorinated SiO2 nanoparticles (50 nm) are deposited by electrostatic spraying. The electrostatic spraying process parameters include: the concentration of the fluorinated SiO2 dispersion is 3 - 5 wt%, the spraying speed is 15 - 25 mm / s; the spraying voltage is 25 - 35 kV, and the distance between the nozzle and the dish surface is 10 ± 0.5 mm; the coating curing conditions: 70°C / 10 min, and the fluorine element content after curing is ≥45 at%; it should be noted that the superhydrophobic coating material provided on the surface of the superhydrophobic region of the culture dish and its preparation process are not limited to the above method and can be adjusted and optimized according to actual needs.

[0046] According to an embodiment of the present invention, the superhydrophobic coating material includes one or more of fluorinated silica, perfluorooctyltriethoxysilane, perfluorododecyltriethoxysilane, fluorinated polystyrene, and fluorocarbon compounds. Thus, different superhydrophobic coating materials can be selected according to different application scenarios and requirements, further improving the anti-pollution ability and droplet confinement ability of the culture dish.

[0047] According to an embodiment of the present invention, the culture dish further includes: an upper cover covering the culture dish body. Thus, by providing the upper cover, while protecting the sample to be tested in the dish, it reduces the fluctuations in temperature and humidity, provides a more stable growth environment for it, and avoids sample contamination.

[0048] Embryo biopsy method

[0049] The present invention provides an embryo biopsy method. According to an embodiment of the present invention, the embryo biopsy method includes: placing a sample containing the embryo to be tested into the central microcolumn array region of the aforementioned culture dish and performing detection on it. The embryo biopsy method of the present invention significantly improves the accuracy of the detection operation and the reliability of the detection results by placing the sample containing the embryo to be tested in the central microcolumn array region of the culture dish. Specifically, the microcolumn structure of the culture dish can effectively fix the droplet, ensuring that the embryo remains stable during the operation and avoiding embryo damage or operation errors caused by droplet movement or diffusion; at the same time, this method uses the gradient hydrophobic region and superhydrophobic region of the culture dish to reduce the contact area between the droplet and the surface of the culture dish, reducing the risk of contamination and avoiding imaging blurring caused by mineral oil coverage, significantly improving the clarity of microscopic imaging; in addition, this detection method simplifies the steps of adding and removing mineral oil in traditional embryo biopsy, reduces the operation complexity, and improves the work efficiency; in addition, combined with the traceability label region of the culture dish, this detection method can also record the operation information during embryo biopsy, realize full-process traceability, and ensure the integrity and repeatability of experimental data; this method is applicable to different types of embryos, can meet different application requirements, and has a wide application prospect.

[0050] According to an embodiment of the present invention, the embryo to be tested is a mammalian embryo. Exemplarily, the embryo to be tested can be livestock embryos such as bovine embryos, ovine embryos, porcine embryos, rabbit embryos, equine embryos, etc.; thus, the embryo biopsy is applicable to different types of embryos and has a wide application prospect.

[0051] Use in embryo genetic testing, livestock breeding, and basic biological research

[0052] The present invention provides the use of the aforementioned culture dish or the aforementioned embryo biopsy method in embryo genetic testing, livestock breeding, and basic biological research.

[0053] Those skilled in the art can understand that the features and advantages described above for the petri dish or embryo biopsy method also apply to this use and will not be elaborated here.

[0054] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchases.

[0055] Example 1: Embryo Biopsy Practice

[0056] 1. Preparation of an Embryo Biopsy Petri Dish with Microstructure Positioning

[0057] The petri dish of the present invention is designed based on the principles of "droplet pinning effect" and "gradient hydrophobic synergistic constraint" to solve the problems of blurred imaging and contamination caused by the traditional petri dish relying on mineral oil coverage. Its core design includes:

[0058] Central microcolumn array area: The droplet pinning effect is generated through the geometric structure of the conical microcolumns (height 20 μm, bottom diameter 5 μm, spacing 10 μm) to fix the embryo droplet and prevent the droplet from moving or spreading during the operation.

[0059] Gradient hydrophobic area: The contact angle gradually increases from 120° in the central area to 150° towards the periphery, forming a hydrophobic gradient to further restrain the creeping of the droplet edge and reduce the contact area between the droplet and the petri dish surface.

[0060] Superhydrophobic outer ring area: The contact angle ≥ 150° and the rolling angle ≤ 10°, ensuring that the droplet is completely restricted in the central operation area and avoiding contamination of the peripheral area.

[0061] Traceability label system: Integrate high-frequency RFID tags to achieve full-process traceability of embryo ID and operation information.

[0062] Among them, the basic structure of the petri dish is prepared from polydimethylsiloxane (PDMS).

[0063] The basic parameters and preparation conditions of the surface of the central microcolumn array area are set as follows:

[0064] The central micro-pillar array region (diameter 2.5 mm) consists of conical micro-pillars (height 20 μm, bottom diameter 5 μm, pitch 10 μm), with a water contact angle ≤ 30°; this region is processed by femtosecond laser, and the laser parameters are: wavelength 1030 ± 10 nm, single pulse energy 0.5 μJ, repetition frequency 100 kHz; the scanning path is a concentric spiral trajectory, with a pitch of 8 - 12 μm; after processing, the cone angle of the micro-pillars is 85° ± 2°, and the ratio of the pitch to the bottom diameter is 2:1.

[0065] The basic parameters and preparation conditions of the surface of the gradient hydrophobic region are set as follows:

[0066] The gradient hydrophobic region (width 0.5 mm), with the contact angle gradually changing from 120° to 150° (gradually changing from the region close to the central micro-pillar array region to the outer super-hydrophobic region); this region is subjected to a power gradient scan (5W → 15W) using a 1064 nm fiber laser; perfluorooctyltriethoxysilane is introduced into a vacuum chamber (10⁻³ Pa), and the deposition time is 30 - 60 min; the distance between the contact angle test points in the gradient region is 0.1 mm, and the angle change rate is 60 ° / mm.

[0067] The basic parameters and preparation conditions of the surface of the super-hydrophobic region are set as follows:

[0068] The super-hydrophobic region (a circular ring region of 3.5 - 10 mm), with a water contact angle ≥ 150° and a rolling angle ≤ 10°; this region deposits fluorinated SiO2 nanoparticles (50 nm) by electrostatic spraying, and the electrostatic spraying process parameters include: the concentration of the fluorinated SiO2 dispersion liquid is 3 - 5 wt%, the spraying speed is 15 - 25 mm / s; the spraying voltage is 25 - 35 kV, and the distance between the nozzle and the dish surface is 10 ± 0.5 mm; the coating curing conditions: 70℃ / 10min, and the fluorine element content after curing is ≥ 45 at%.

[0069] In the middle section of the side wall of the embryo biopsy culture dish with micro-structure positioning, an FM1108 high-frequency RFID tag is embedded. Its antenna is printed with a serpentine copper wire on the PDMS curved surface, and the tag encapsulation layer includes a metal shielding net and a perfluoropolyether protective coating; the FM1108 high-frequency RFID tag has dual functions: 1. Unique identification coding: accurate identification of the culture dish is achieved through RFID technology; 2. Data storage: records embryo ID, operation time, and operator information; the tag realizes data reading and writing through an external card reader system to complete the full-process information traceability.

[0070] The AES-256-GCM encryption protocol is adopted, and the storage area is divided into: read-only area: dish ID (64-bit unique coding); writable area: operation timestamp, operator ID; blockchain hash value: each operation record generates a Merkle Root and is stored in the chip.

[0071] Schematic diagram of the main structure of an embryo biopsy culture dish with micro-structure positioning is shown in Figure 1 , and the schematic diagram of the conical micro-column array area in the center of the embryo biopsy culture dish with micro-structure positioning is shown in Figure 2 , and the operation table collaborative system architecture of the embryo biopsy culture dish with micro-structure positioning is shown in Figure 3 .

[0072] Thus, an embryo biopsy culture dish with micro-structure positioning that can be used for embryo biopsy is prepared and obtained.

[0073] 2. Embryo biopsy practice

[0074] The inventor uses the embryo biopsy culture dish with micro-structure positioning prepared by the present invention to carry out embryo biopsy practice, and the specific steps are as follows:

[0075] Preparation of the operating fluid: The preparation of the embryo biopsy operating fluid needs to be carried out under strict aseptic conditions. Its core components include: electrolyte and buffer system (i.e., HEPES buffer; including sodium chloride 85 - 120 mM, potassium chloride 5 - 6 mM, phosphate 0.5 - 1 mM, sodium bicarbonate 25 mM; used to maintain osmotic pressure and pH = 7.2 - 7.4), energy substances (glucose 5.5 mM, sodium pyruvate 0.3 mM, sodium lactate 10 - 25 mM), amino acids and protective agents (alanine, proline, etc. 0.1 - 1 mM, human serum albumin 0.5 - 1% w / v), as well as antibiotics and chelating agents (gentamicin sulfate 50 μg / mL, EDTA 0.5 - 1 mM); during the specific preparation, first dissolve inorganic salts with ultrapure water, then add energy substances, amino acids and EDTA in sequence and stir until completely dissolved, and then adjust the pH = 7.2 - 7.4 with HEPES buffer, and control the osmotic pressure at 285 - 295 mOsm / kg; after preparation, filter and sterilize by positive pressure through a 0.22 μm filter membrane, sub-pack into sterile centrifuge tubes, store at -20°C (for long term), and preheat to 37°C before use.

[0076] Drop about 40 μL of the operating fluid onto the central micro-column array area of the culture dish prepared as described above; due to the pinning effect of the micro-column array area and the hydrophobic effect of the periphery, the operating droplet will stably exist in the center of the culture dish; then use a mouth pipette to transfer the embryo to be biopsied into the operating liquid groove to obtain a culture dish containing the embryo.

[0077] Install a chip reader on the biopsy operating table, and place the culture dish containing the embryo on the operating table to ensure that the FM1108 high-frequency RFID tag is in close contact with the chip reader; at this time, the number of the culture dish will be read, and the operation information will be stored in the supporting computer, and the operation record will start.

[0078] The biopsy operation is performed on the embryo using a laser biopsy system. After the biopsy is completed, the culture dish containing the embryo is separated from the chip reader. At this time, the operation record ends, and the operation information such as the biopsy time will be recorded.

[0079] The results show that: (1) The biopsy operation was successfully completed;

[0080] (2) The numbers of the biopsied embryos and the operation information such as the corresponding operation time were all recorded.

[0081] In the description of this specification, the descriptions with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0082] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A culture dish, characterized in that, Comprising: A culture dish body; The culture dish body includes a bottom wall and a side wall extending along the outer periphery of the bottom wall. The bottom wall and the side wall form an upper open space suitable for accommodating a sample. Wherein, The bottom wall is sequentially provided with a central micro-column array region, a gradient hydrophobic region, and a super-hydrophobic region from the center to the outer periphery; The central micro-column array region includes independent conical micro-columns; The water contact angle of the surface of the gradient hydrophobic region gradually changes from 120° on the side close to the central micro-column array region to 150° on the side far from the central micro-column array region; The water contact angle of the surface of the central micro-column array region ≤ 30°; The water contact angle of the surface of the super-hydrophobic region ≥ 150°, and the rolling angle ≤ 10°.

2. The petri dish according to claim 1, wherein A traceability label area is provided on the culture dish body.

3. The petri dish according to claim 2, wherein The traceability label area includes: One or more coding labels for uniquely identifying the culture dish.

4. The petri dish according to claim 3, wherein The coding label includes one or more of a high-frequency RFID label, a ultra-high-frequency RFID label, and a high-frequency NFC label.

5. The petri dish according to claim 1, characterized in that, The material of the culture dish basic structure includes one or more of polydimethylsiloxane, polymethyl methacrylate, polycarbonate, polystyrene, polytetrafluoroethylene, polylactic acid, polyglycolic acid, polyethylene, and polypropylene.

6. The petri dish according to claim 1, characterized in that, A hydrophobic coating material is provided on the surface of the gradient hydrophobic region.

7. The petri dish according to claim 6, wherein, The hydrophobic coating material includes one or more of perfluorooctyltriethoxysilane, polydimethylsiloxane, perfluoropolyether, and fluorinated polystyrene.

8. The petri dish according to claim 1, wherein, A super-hydrophobic coating material is provided on the surface of the super-hydrophobic region.

9. The petri dish according to claim 8, characterized in that, The super-hydrophobic coating material includes one or more of fluorinated silica, perfluorooctyltriethoxysilane, perfluorododecyltriethoxysilane, fluorinated polystyrene, and fluorocarbon compounds.

10. The petri dish according to claim 1, characterized in that, The culture dish further includes: An upper cover covering the culture dish body.

11. An embryo biopsy method, characterized in that, The embryo biopsy method includes: Placing a sample containing the embryo to be tested into the central micro-column array region of the culture dish according to any one of claims 1 to 10 and detecting it; Wherein, the embryo biopsy method is for non-diagnostic and non-therapeutic purposes; The embryo to be tested is a non-human mammalian embryo.

12. Use of the culture dish according to any one of claims 1 to 10 or the embryo biopsy method according to claim 11 in livestock breeding and basic biological research.

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