Embryo biopsy culture dish with microstructure positioning and application thereof

Abstract

The invention relates to the technical field of biomedical engineering and reproductive genetics, in particular to an embryo biopsy culture dish with microstructure positioning and application of the embryo biopsy culture dish. The culture dish body comprises a bottom wall and a side wall extending along the periphery of the bottom wall, the bottom wall and the side wall form an upper opening space suitable for containing a sample, and the bottom wall is sequentially provided with a central micro-column array area, a gradient hydrophobic area and a super-hydrophobic area from the center to the periphery; the central micro-column array area comprises independent conical micro-columns; the contact angle of the surface of the gradient hydrophobic area gradually changes from 120 degrees close to one side of the central micro-column array area to 150 degrees away from one side of the central micro-column array area. According to the culture dish disclosed by the invention, through the design of combining the micro-column array and the hydrophobic outer ring, liquid drops do not need to be covered by mineral oil when the culture dish is used, so that the definition of microscopic imaging is remarkably improved, pollution is avoided, and the accuracy of subsequent operation is ensured.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical engineering and reproductive genetics, and in particular to an embryo biopsy culture dish with microstructure positioning and application thereof. Background Art

[0002] Embryo biopsy is a commonly used technique in assisted reproductive technology (ART) and embryo research. It is mainly used to extract a small number of cells from early embryos for genetic testing or analysis. Embryo biopsy can evaluate the genetic status of the embryo and screen healthy embryos for transplantation or further research. Embryo biopsy usually uses micromanipulation technology to remove a small number of cells (usually 1 to 10 cells) from the embryo in the early stages of embryonic development (usually 3 to 6 days after fertilization) for genetic analysis, thereby detecting chromosomal abnormalities, single gene diseases or other genetic problems in the embryo.

[0003] Currently, methods of embryo biopsy include microknife cutting, microneedle puncture aspiration and laser cutting. As a micromanipulation technology, it needs to be performed in a specific experimental environment. The culture dish is an indispensable tool in the embryo biopsy process. It provides a stable growth environment and operating 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 constrain droplets, but the introduction of the oil phase leads to differences in optical refractive index, significantly reducing the resolution of microscopic imaging. The use of mineral oil can easily lead to residual oil droplets in the mouth pipette, causing sample contamination and affecting subsequent amplification and typing results. In addition, manual labeling or fluorescent coding lacks an operation process data link, which can easily lead to mismatches between samples and numbers, making it impossible to achieve full-process traceability of "sample ID-operation log-genetic 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 covering, has high imaging quality, is less likely to be contaminated, is durable and can achieve full process traceability, so as to help improve 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 a certain extent. To this end, the present invention provides an embryo biopsy culture dish with microstructure positioning and its application. The culture dish of the present invention is designed by combining a microcolumn array with a hydrophobic outer ring, so that no mineral oil covering droplets are required when in use, thereby significantly improving the clarity of microscopic imaging, while avoiding the contamination of the embryo sample to be tested by mineral oil residues, ensuring the accuracy of subsequent operations; in addition, the joint traceability label system realizes a one-to-one correspondence between the culture dish and the embryo, avoiding sample coding misalignment, and recording information such as operation time, so that the operation process can be traced; at the same time, the culture dish is made of materials such as polydimethylsiloxane (PDMS) combined with advanced processing technology, which further extends its service life.

[0007] In the 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 periphery of the bottom wall, 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-pillar array region, a gradient hydrophobic region and a super-hydrophobic region from the center to the periphery; the central micro-pillar array region includes separate conical micro-pillars; the contact angle of the surface of the gradient hydrophobic region gradually changes from 120° on the side close to the central micro-pillar array region to 150° on the side away from the central micro-pillar array region. The culture dish according to the present invention has a unique structural design, which significantly improves the droplet stability and operation accuracy during the sample detection process; wherein, the tapered micro-column structure in the central micro-column array area can fix the droplets through the droplet pinning effect, prevent them from diffusing or moving, and ensure that the embryonic cells remain stable during the operation; at the same time, the contact angle of the gradient hydrophobic area gradually changes from 120° to 150°, thereby constraining the diffusion of the droplets, and further constraining the diffusion of the droplets through the super-hydrophobic area, the central micro-column array area, the gradient hydrophobic area and the super-hydrophobic area work together to ensure that the droplets remain stable during the operation, reduce the contact between the droplets 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 mineral oil to cover the droplets, avoids the optical refractive index difference introduced by mineral oil, significantly improves the clarity of microscopic imaging, and reduces the contamination of the sample by mineral oil residues; in addition, the structural design of the culture dish can be applied to a variety of application scenarios for embryo sample detection, has a wide range of 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 also have the following additional technical features: According to an embodiment of the present invention, the water contact angle of the surface of the central micro-pillar array region is ≤30°.

[0009] According to an embodiment of the present invention, the water contact angle on the surface of the super-hydrophobic region is ≥150°, and the rolling angle is ≤10°.

[0010] According to an embodiment of the present invention, a traceability label area is provided on the culture dish body.

[0011] According to an embodiment of the present invention, the traceability label area includes: one or more coded labels for uniquely identifying the culture dish.

[0012] According to an embodiment of the present invention, the coding tag includes one or more of a high-frequency RFID tag, an ultra-high-frequency RFID tag, and a high-frequency NFC tag.

[0013] 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.

[0014] According to an embodiment of the present invention, a hydrophobic coating material is provided on the surface of the gradient hydrophobic region.

[0015] According to an embodiment of the present invention, the hydrophobic coating material includes one or more of perfluorooctyltriethoxysilane, polydimethylsiloxane, perfluoropolyether and fluorinated polystyrene.

[0016] According to an embodiment of the present invention, a super-hydrophobic coating material is disposed on the surface of the super-hydrophobic region.

[0017] According to an embodiment of the present invention, the super hydrophobic coating material includes one or more of fluorinated silicon dioxide, perfluorooctyltriethoxysilane, perfluorododecyltriethoxysilane, fluorinated polystyrene and fluorocarbons.

[0018] According to an embodiment of the present invention, the culture dish further comprises: an upper cover covering the culture dish body.

[0019] 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 comprises: placing a sample containing an embryo to be tested into the central micro-column array area of ​​the culture dish described in the first aspect, and testing it. The embryo biopsy method of the present invention significantly improves the accuracy of the detection operation and the reliability of the detection result by placing a sample containing the embryo to be tested in the micro-column array area in the center of the culture dish for detection. Specifically, the micro-column structure of the culture dish can effectively fix the droplets, ensure that the embryo remains stable during the operation, and avoid embryo damage or operational errors caused by the movement or diffusion of the droplets; at the same time, the method uses the gradient hydrophobic area and super-hydrophobic area of ​​the culture dish to reduce the contact area between the droplets and the surface of the culture dish, reducing the risk of contamination, while avoiding imaging blur caused by mineral oil coverage, and significantly improving the clarity of microscopic imaging; in addition, the detection method simplifies the steps of adding and removing mineral oil in traditional embryo biopsy, reduces the complexity of operation, and improves work efficiency; in addition, combined with the traceability label area of ​​the culture dish, the detection method can also record the operation information during the embryo biopsy process, realize full process traceability, and ensure the integrity and repeatability of the experimental data; the method is suitable for different types of embryos, can meet different application needs, and has broad application prospects.

[0020] According to an embodiment of the present invention, the above embryo biopsy method may also have the following additional technical features: According to an embodiment of the present invention, the embryo to be tested is a mammalian embryo.

[0021] In the third aspect of the present invention, the present invention proposes 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.

[0022] Those skilled in the art will appreciate that the features and advantages described above for the culture dish or embryo biopsy method are also applicable to this application and will not be described in detail here.

[0023] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 Schematic diagram of the main structure of the embryo biopsy culture dish with microstructure positioning in Example 1 of the present invention, wherein 1 is the central micro-pillar array area; 2 is the gradient hydrophobic area, 3 is the super hydrophobic area, and 4 is the FM1108 high-frequency RFID tag; Figure 2Schematic diagram of the conical micro-pillar array area in the center of the embryo biopsy culture dish with micro-structure positioning in Example 1 of the present invention, wherein 5 is a conical micro-pillar, and the micro-pillar array formed by the conical micro-pillar can fix the operating droplet through the pinning effect; Figure 3 This is a diagram of the collaborative system architecture of the operating table of the embryo biopsy culture dish with microstructure positioning in Example 1 of the present invention, wherein 6 is a chip reader installed on the operating table. During detection, it is necessary to ensure that the FM1108 high-frequency RFID tag 4 is tightly connected to the reader 6. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0027] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0028] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0029] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0030] Petri dish The present invention proposes a culture dish. According to an embodiment of the present invention, the culture dish comprises: a culture dish body; the culture dish body comprises a bottom wall and a side wall extending along the periphery of the bottom wall, the bottom wall and the side wall form an upper opening space suitable for accommodating a sample, wherein the bottom wall is provided with a central micro-pillar array region, a gradient hydrophobic region and a super-hydrophobic region in sequence from the center to the periphery; the central micro-pillar array region comprises independent conical micro-pillars; the contact angle of the surface of the gradient hydrophobic region gradually changes from 120° on the side close to the central micro-pillar array region to 150° on the side away from the central micro-pillar array region. The culture dish according to the present invention has a unique structural design, which significantly improves the droplet stability and operation accuracy during the sample detection process; wherein, the tapered micro-column structure in the central micro-column array area can fix the droplets through the droplet pinning effect, prevent them from diffusing or moving, and ensure that the embryonic cells remain stable during the operation; at the same time, the contact angle of the gradient hydrophobic area gradually changes from 120° to 150°, thereby constraining the diffusion of the droplets, and further constraining the diffusion of the droplets through the super-hydrophobic area, the central micro-column array area, the gradient hydrophobic area and the super-hydrophobic area work together to ensure that the droplets remain stable during the operation, reduce the contact between the droplets 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 mineral oil to cover the droplets, avoids the optical refractive index difference introduced by mineral oil, significantly improves the clarity of microscopic imaging, and reduces the contamination of the sample by mineral oil residues; in addition, the structural design of the culture dish can be applied to a variety of application scenarios for embryo sample detection, has a wide range of versatility and high durability, and further effectively improves the efficiency and accuracy of embryo sample detection. Exemplarily, the process parameters of the central microcolumn array area of ​​the culture dish can be set as follows (diameter 2.5 mm), composed of conical microcolumns (height 20 μm, bottom diameter 5 μm, spacing 10 μm), with a water contact angle ≤30°; this area 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; the cone angle of the microcolumns after processing is 85°±2°, and the ratio of spacing to bottom diameter is 2:1; it should be noted that the parameter setting of the central microcolumn array area 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.

[0031] According to an embodiment of the present invention, the water contact angle of the surface of the central micro-pillar array region is ≤30°. Thus, the low contact angle design ensures the stability of the droplets in the central micro-pillar array region and the accuracy of the experimental operation during the sample detection process, while reducing the friction between the droplets and the surface, protecting the embryonic cells to be tested from mechanical damage; at the same time, the design also improves the spreading performance of the droplets, ensuring that the embryonic cells to be tested can be evenly distributed in the droplets, thereby further improving the accuracy of the detection results.

[0032] According to an embodiment of the present invention, the water contact angle on the surface of the super-hydrophobic region is ≥150°, and the rolling angle is ≤10°. Thus, the super-hydrophobic region design with high contact angle and low rolling angle provides excellent hydrophobic performance for the culture dish, ensuring that the droplets in 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.

[0033] According to an embodiment of the present invention, a traceability label area is provided on the culture dish body. Thus, the presence of the traceability label area enables the culture dish to achieve full process traceability from sample placement to operation completion, avoids sample mismatching, ensures the accuracy and repeatability of experimental data, and further enhances the reliability of experimental results. Exemplarily, the traceability label area can be located on the side wall of the culture dish.

[0034] According to an embodiment of the present invention, the traceability label area includes: one or more coded labels for uniquely identifying the culture dish. Thus, the coded 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, and further enhance the traceability of experimental operations and the integrity of data analysis.

[0035] According to an embodiment of the present invention, the coding tag includes one or more of a high-frequency RFID tag, an ultra-high-frequency RFID tag, and a high-frequency NFC tag. As a result, the type of coding tag can be selected according to different experimental environments, operating requirements, and cost requirements, further enhancing the versatility and flexibility of the culture dish; illustratively, the coding tag 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 adapt to the curved surface structure of the culture dish; further, the surface of the tag has an encapsulation layer, including a metal shielding mesh and a perfluoropolyether protective coating, which is used to protect the tag from the external environment and improve the stability and durability of the tag; in addition, regarding the data reading of the tag, the tag data can be read through an external card reader system, and the operating frequency of the card reader system is 860~960 MHz, supporting ISO / IEC 18000-63 communication protocol ensures efficient data transmission and accurate reading; the card reader system includes a fixed bracket installed on the operating table, which is used to maintain a preset communication distance between the card reader and the label 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 operating the laser through a data transmission interface to transmit the embryo ID, operation time and operator information stored in the label in real time, thereby realizing accurate tracking and management of the culture dish.

[0036] According to an embodiment of the present invention, the material of the culture dish infrastructure includes one or more of polydimethylsiloxane, polymethyl methacrylate, polycarbonate, polystyrene, polytetrafluoroethylene, polylactic acid, polyglycolic acid, polyethylene and polypropylene. Thus, different basic 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 microscopic imaging and cell culture, while materials such as polycarbonate have higher mechanical strength and are suitable for working environments that require frequent operations.

[0037] According to an embodiment of the present invention, a hydrophobic coating material is provided on the surface of the gradient hydrophobic region. Thus, the restraining ability of the droplets is further optimized, the contact area between the droplets and the surface of the culture dish is reduced, the risk of contamination is reduced, and the operation accuracy and imaging quality are improved. Exemplarily, the process parameters of the gradient hydrophobic region of the culture dish can be set as follows (width 0.5 mm), using a 1064 nm fiber laser for power gradient scanning (5W→15W); passing perfluorooctyl triethoxysilane in a vacuum chamber (10⁻³ Pa), the deposition time is 30~60min; the contact angle test point spacing in the gradient area is 0.1 mm, and the angle change rate is 60 ° / mm; it should be noted that the hydrophobic coating material and its preparation process provided on the surface of the gradient hydrophobic region of the culture dish are not limited to the above method, and can be adjusted and optimized according to actual needs.

[0038] According to an embodiment of the present invention, the hydrophobic coating material includes one or more of perfluorooctyl triethoxysilane, polydimethylsiloxane, perfluoropolyether and fluorinated polystyrene. Therefore, different hydrophobic coating materials can be selected according to different application scenarios and requirements, and the contact angle and rolling angle can be adjusted to optimize the stability of the droplets and the operation accuracy during the sample detection process.

[0039] According to an embodiment of the present invention, a super-hydrophobic coating material is provided on the surface of the super-hydrophobic area. Thus, the restraining ability of the droplets is further optimized, the contact area between the droplets and the surface is reduced, the risk of contamination is reduced, and the accuracy of the operation and the imaging quality are improved. Exemplarily, the process parameters of the super-hydrophobic area of ​​the culture dish can be set as follows (3.5~10 mm ring area), and the fluorinated SiO 2 Nanoparticles (50 nm) were deposited. The electrostatic spraying process parameters included: fluorinated SiO 2The dispersion concentration 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 are 70°C / 10min, and the fluorine content after curing is ≥45 at%; it should be noted that the super-hydrophobic coating material and the preparation process thereof set on the surface of the super-hydrophobic area of ​​the culture dish are not limited to the above method, and can be adjusted and optimized according to actual needs.

[0040] According to an embodiment of the present invention, the super-hydrophobic coating material includes one or more of fluorinated silicon dioxide, perfluorooctyl triethoxysilane, perfluorododecyl triethoxysilane, fluorinated polystyrene and fluorocarbon compounds. Thus, different super-hydrophobic 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.

[0041] According to an embodiment of the present invention, the culture dish further comprises: an upper cover covering the culture dish body. Thus, by providing the upper cover, the sample to be tested in the dish is protected while reducing fluctuations in temperature and humidity, providing a more stable growth environment for the sample and avoiding sample contamination.

[0042] Embryo biopsy method The present invention provides an embryo biopsy method. According to an embodiment of the present invention, the embryo biopsy method comprises: placing a sample containing an embryo to be tested into the central micro-column array area of ​​the aforementioned culture dish, and testing it. The embryo biopsy method of the present invention significantly improves the accuracy of the detection operation and the reliability of the detection result by placing a sample containing the embryo to be tested in the micro-column array area in the center of the culture dish for detection. Specifically, the micro-column structure of the culture dish can effectively fix the droplets, ensure that the embryo remains stable during the operation, and avoid embryo damage or operational errors caused by the movement or diffusion of the droplets; at the same time, the method uses the gradient hydrophobic area and super-hydrophobic area of ​​the culture dish to reduce the contact area between the droplets and the surface of the culture dish, reducing the risk of contamination, while avoiding imaging blur caused by mineral oil coverage, and significantly improving the clarity of microscopic imaging; in addition, the detection method simplifies the steps of adding and removing mineral oil in traditional embryo biopsy, reduces the complexity of operation, and improves work efficiency; in addition, combined with the traceability label area of ​​the culture dish, the detection method can also record the operation information during the embryo biopsy process, realize full process traceability, and ensure the integrity and repeatability of the experimental data; the method is suitable for different types of embryos, can meet different application needs, and has broad application prospects.

[0043] According to an embodiment of the present invention, the embryo to be tested is a mammal embryo. For example, the embryo to be tested can be a livestock embryo such as a bovine embryo, a sheep embryo, a pig embryo, a rabbit embryo, a horse embryo, etc. Therefore, the embryo biopsy is applicable to different types of embryos and has a wide range of application prospects.

[0044] Uses in embryo genetic testing, livestock breeding, and basic biological research The present invention proposes the use of the aforementioned culture dish or the aforementioned embryo biopsy method in embryo genetic detection, livestock breeding and basic biological research.

[0045] Those skilled in the art will appreciate that the features and advantages described above for the culture dish or embryo biopsy method are also applicable to this application and will not be described in detail here.

[0046] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0047] Example 1: Embryo biopsy practice 1. Preparation of embryo biopsy dish with microstructure positioning The culture dish of the present invention is designed based on the principles of "droplet pinning effect" and "gradient hydrophobic cooperative constraint", aiming to solve the imaging blur and contamination problems caused by the traditional culture dish relying on mineral oil covering. Its core design includes: Central micro-pillar array area: The geometric structure of the conical micro-pillars (height 20 μm, bottom diameter 5 μm, and spacing 10 μm) produces a droplet pinning effect to fix the embryo droplets and prevent the droplets from moving or spreading during operation.

[0048] Gradient hydrophobic area: The contact angle gradually increases from 120° in the central area to 150° toward the periphery, forming a hydrophobic gradient, which further restricts the creeping of the droplet edge and reduces the contact area between the droplet and the culture dish surface.

[0049] Super hydrophobic outer ring area: contact angle ≥150°, rolling angle ≤10°, ensuring that the droplets are completely confined to the central operating area to avoid contaminating the peripheral area.

[0050] Traceability label system: Integrates high-frequency RFID tags to achieve full-process traceability of embryo ID and operation information.

[0051] Among them, the basic structure of the culture dish is made of polydimethylsiloxane (PDMS).

[0052] The basic parameters and preparation conditions of the surface of the central micropillar array area are set as follows: The central micropillar array area (diameter 2.5 mm) is composed of conical micropillars (height 20 μm, bottom diameter 5 μm, spacing 10 μm), with a water contact angle ≤30°; this area 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 8-12μm; the cone angle of the micropillar after processing is 85°±2°, and the ratio of spacing to bottom diameter is 2:1.

[0053] The basic parameters and preparation conditions of the gradient hydrophobic area surface are set as follows: Gradient hydrophobic area (width 0.5 mm), the contact angle gradually changes from 120° to 150° (gradually changes from the area close to the central micropillar array to the outer super-hydrophobic area); the area is scanned with a 1064 nm fiber laser for power gradient (5W→15W); perfluorooctyltriethoxysilane is introduced into the vacuum chamber (10⁻³ Pa) for a deposition time of 30~60 min; the contact angle test points in the gradient area are 0.1 mm apart, and the angle change rate is 60° / mm.

[0054] The basic parameters and preparation conditions of the super-hydrophobic surface are set as follows: The super-hydrophobic area (circular area of ​​3.5-10 mm) has a water contact angle of ≥150° and a rolling angle of ≤10°. The fluorinated SiO 2 Nanoparticles (50 nm) were deposited. The electrostatic spraying process parameters included: fluorinated SiO 2 The dispersion concentration 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 are: 70℃ / 10min, and the fluorine content after curing is ≥45 at%.

[0055] An FM1108 high-frequency RFID tag is embedded in the middle section of the side wall of the embryo biopsy culture dish with microstructure positioning. Its antenna is printed on the PDMS surface with a serpentine copper wire, and the tag packaging layer includes a metal shielding mesh 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: recording embryo ID, operation time and operator information; the tag realizes data reading and writing through an external card reader system to complete full-process information traceability.

[0056] Using AES-256-GCM encryption protocol, the storage area is divided into: read-only area: dish ID (64-bit unique code); writable area: operation timestamp, operator ID; blockchain hash value: each operation record generates a Merkle Root and stores it in the chip.

[0057] Schematic diagram of the main structure of the embryo biopsy dish with microstructure positioning Figure 1 , showing the tapered micropillar array area in the center of the embryo biopsy dish with microstructure positioning Figure 2 , the operation table collaborative system architecture of the embryo biopsy culture dish with microstructure positioning is shown in Figure 3 .

[0058] Thus, an embryo biopsy culture dish with microstructure positioning that can be used for embryo biopsy is prepared.

[0059] 2. Embryo biopsy practice The inventors used the embryo biopsy culture dish with microstructure positioning prepared by the present invention to perform embryo biopsy practice, and the specific steps are as follows: Preparation of operating fluid: The preparation of embryo biopsy operating fluid must be carried out under strict aseptic conditions. Its core components include: electrolytes 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), and antibiotics and chelating agents (gentamicin sulfate 50 μg / mL, EDTA 0.5~1 mM). In the specific preparation, first use ultrapure water to dissolve inorganic salts, then add energy substances, amino acids and EDTA in turn and stir until completely dissolved, then use HEPES buffer to adjust pH=7.2~7.4, and control the osmotic pressure at 285~295 mOsm / kg; after preparation, sterilize by positive pressure filtration through a 0.22 μm filter membrane, dispense into sterile centrifuge tubes, store at -20°C (long term), and preheat to 37°C before use.

[0060] Place a 40 μL drop of operating liquid in the central microcolumn array area of ​​the culture dish prepared above; due to the pinning effect of the microcolumn array area and the hydrophobic effect of the periphery, the operating drop will stably exist in the center of the culture dish; then use a mouth pipette to transfer the embryo to be biopsied to the operating liquid tank to obtain a culture dish containing the embryo.

[0061] Install the 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 culture dish number will be read, the operation information will be stored in the matching computer, and the operation record will begin.

[0062] Use the laser biopsy system to perform a biopsy on the embryo. 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 biopsy time and other operation information will be recorded.

[0063] The results showed that: (1) the biopsy operation was completed smoothly; (2) The biopsied embryo number, corresponding operation time and other operation information are recorded.

[0064] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0065] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A culture dish, characterized in that: include: Petri dish body; The culture dish body comprises a bottom wall and a side wall extending along the periphery of the bottom wall, wherein the bottom wall and the side wall form an upper opening space suitable for accommodating the sample. in, The bottom wall is provided with a central micro-pillar array region, a gradient hydrophobic region and a super hydrophobic region in sequence from the center to the periphery; The central micro-pillar array region includes separate conical micro-pillars; The water contact angle of the surface of the gradient hydrophobic region gradually changes from 120° on the side close to the central micro-pillar array region to 150° on the side away from the central micro-pillar array region.

2. The culture dish according to claim 1, characterized in that The water contact angle on the surface of the central micro-pillar array region is ≤30°.

3. The culture dish according to claim 1, characterized in that The water contact angle on the surface of the super-hydrophobic region is ≥150°, and the rolling angle is ≤10°.

4. The culture dish according to claim 1, characterized in that A traceability label area is provided on the culture dish body.

5. The culture dish according to claim 4, characterized in that: The traceability label area includes: One or more coded tags for uniquely identifying the culture dish.

6. The culture dish according to claim 5, characterized in that: The coding tag includes one or more of a high-frequency RFID tag, an ultra-high-frequency RFID tag, and a high-frequency NFC tag.

7. The culture dish according to claim 1, characterized in that: 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.

8. The culture dish according to claim 1, characterized in that: A hydrophobic coating material is disposed on the surface of the gradient hydrophobic region.

9. The culture dish according to claim 8, characterized in that The hydrophobic coating material includes one or more of perfluorooctyltriethoxysilane, polydimethylsiloxane, perfluoropolyether and fluorinated polystyrene.

10. The culture dish according to claim 1, characterized in that: A super-hydrophobic coating material is disposed on the surface of the super-hydrophobic region.

11. The culture dish according to claim 10, characterized in that: The super hydrophobic coating material includes one or more of fluorinated silicon dioxide, perfluorooctyltriethoxysilane, perfluorododecyltriethoxysilane, fluorinated polystyrene and fluorocarbons.

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

13. An embryo biopsy method, characterized in that: The embryo biopsy method comprises: A sample containing the embryo to be tested is placed in the central micro-column array area of ​​the culture dish according to any one of claims 1 to 12, and tested.

14. The embryo biopsy method according to claim 13, characterized in that: The embryo to be tested is a mammalian embryo.

15. Use of the culture dish according to any one of claims 1 to 12 or the embryo biopsy method according to claim 13 or 14 in embryo genetic testing, livestock breeding and basic biological research.

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