Single sperm capturing device based on micromanipulation needle and sperm pool PCR (Polymerase Chain Reaction) tube

Abstract

The utility model relates to a single sperm capturing device based on a micromanipulation needle and a sperm pool PCR (Polymerase Chain Reaction) tube, and belongs to the technical field of assisted reproduction and single cell operation. The device is composed of a micromanipulation module, a PCR tube mechanism and a composite operation dish, the micromanipulation module integrates a micromanipulation needle and a micromanipulation system, and single sperm grabbing and three-dimensional accurate positioning are achieved; the PCR tube mechanism forms a sperm pool through 2 [mu] LPBS liquid drops on the tube wall, a 3 [mu] LPBS buffer solution is preset at the bottom, and a gradient liquid film is constructed to optimize sperm positioning; the composite operation dish is of a one-pool double-area structure, and the whole process integration of sperm braking, cleaning and transferring is achieved. According to the device, a traditional droplet transfer step is eliminated through a micromanipulation needle direct sperm discharging and pool entering technology, the single-time operation time is effectively shortened, the capture success rate is effectively increased, and an efficient and stable technical platform is provided for single sperm genome analysis.

Description

Technical Field

[0001] This utility model relates to the field of assisted reproduction and single-cell manipulation technology, specifically a single sperm capture device based on a micromanipulation needle and a sperm pool PCR tube. Background Technology

[0002] Gene sequencing and transcriptome sequencing of single sperm cells are of great significance for research on andrological genetic diseases. Effective collection and preservation of single sperm cells are prerequisites for in-depth research. Currently, existing techniques involve collecting single sperm cells under a microscope using a pipette or micropipette. Specifically, the single sperm cell is immobilized using a micromanipulation device, grasped with a micromanipulation needle, and placed in a drop of PBS. Under a microscope, it is then aspirated using a pipette or micropipette and transferred to a PCR tube containing 5 μL of PBS. However, pipettes may introduce contamination from bacteria, fungi, viruses, or exogenous DNA. Furthermore, this method requires a high level of operator skill, and the thick PBS layer in the PCR tube makes it prone to loss during transfer.

[0003] Furthermore, this process, due to the use of pipettes or manual operation of micropipettes, may result in excessive PBS being injected into the PCR tube, which could negatively interfere with the subsequent intracytoplasmic sperm amplification system and its success rate. Utility Model Content

[0004] To address the problems of the prior art, this invention provides a single sperm capture device based on a micromanipulation needle and a sperm pool PCR tube.

[0005] To solve the above-mentioned technical problems, this utility model is achieved through the following technical solution: a single sperm capture device based on a micromanipulation needle and a sperm pool PCR tube, comprising:

[0006] The micromanipulation module includes the micromanipulation needle and the micromanipulation system;

[0007] The PCR tube mechanism has a sperm pool on its tube wall for holding sperm, and a PBS buffer droplet is placed at the bottom of the tube wall.

[0008] The composite operating dish has a rectangular sperm swimming pool, a PVP immobilization zone, and a PBS washing zone.

[0009] This application describes a method for collecting single sperm cells using a micromanipulation needle. Two microliters of PBS are then aspirated from a PCR tube using a micropipette and placed in the sperm pool. The PCR tube is then held horizontally, and under a microscope, the micromanipulation needle is used to transfer the single sperm cell into the sperm pool on the PCR tube wall. After centrifugation, the sample is stored. This device is simple to operate, highly efficient, and can be effectively used for subsequent research.

[0010] Preferably, the sperm pool is composed of 2 μL of PBS droplets, and the volume of the PBS buffer droplets is set to 3 μL.

[0011] Preferably, the number of rectangular sperm swimming pools is set to one, and the number of PVP braking zones and PBS washing zones are set to two each.

[0012] Preferably, the surfaces of the rectangular sperm swimming pool, PVP braking area, and PBS cleaning area of ​​the composite operating dish are hydrophobically treated to form annular hydrophobic boundaries, and the center of each area is a hydrophilic contact surface.

[0013] The beneficial effects of this utility model are as follows:

[0014] This invention overcomes the technical bottleneck of traditional single sperm capture, which requires switching between multiple devices, through an integrated operation system design of a micromanipulation module and a PCR tube mechanism. Specifically, it features: a simplified operation path: utilizing the integrated "grab-stop-discharge" action of the micromanipulation needle eliminates redundant steps such as droplet transfer and microscope repositioning in traditional methods, reducing the single operation time to 1±0.2 minutes (compared to 1.5±0.2 minutes for conventional methods); and a quantitatively improved success rate: the synergistic effect of the "1+2+2" functional partitions of the composite operation dish (1 swimming pool + 2 stopping zones + 2 washing zones) and the sperm pool in the PCR tube wall effectively improves the success rate of single sperm capture. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the overall structure of the composite operating dish of this utility model.

[0016] Figure 2 This is a schematic diagram of the PCR tube mechanism operation structure of this utility model.

[0017] Figure 3 This is a schematic diagram of clinical validation experimental data for this utility model.

[0018] Figures 1 to 3 In the middle: 1. Micromanipulation module; 11. Micromanipulation needle; 12. Micromanipulation system; 2. PCR tube mechanism; 21. Sperm pool; 22. PBS buffer droplet; 3. Composite operation dish; 31. Rectangular sperm swimming pool; 32. PVP immobilization area; 33. PBS washing area. Detailed Implementation

[0019] The technical solutions of this utility model will be clearly and completely described below with reference to the embodiments of this utility model. Obviously, the described embodiments are only some embodiments of this utility model, and not all embodiments. Based on the embodiments of this utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of this utility model.

[0020] like Figures 1 to 3 The single sperm capture device based on a micromanipulation needle and a sperm pool PCR tube is shown, comprising: a micromanipulation module 1, including the micromanipulation needle 11 and a micromanipulation system 12; a PCR tube mechanism 2, the tube wall of which is provided with a sperm pool 21 for accommodating sperm, and a PBS buffer droplet 22 is provided at the bottom of the tube wall of the PCR tube mechanism 2; and a composite operation dish 3, which has a rectangular sperm swimming pool 31, a PVP immobilization area 32 and a PBS washing area 33.

[0021] The sperm pool consists of 2 μL of PBS droplets, and the PBS buffer droplets are set at 3 μL. There is one rectangular sperm motility pool 31, and two PVP immobilization zones 32 and two PBS washing zones 33.

[0022] The surfaces of the rectangular sperm swimming pool 31, PVP braking zone 32, and PBS cleaning zone 33 of the composite operating dish 3 are hydrophobically treated to form annular hydrophobic boundaries, and the center of each region is a hydrophilic contact surface.

[0023] Workflow of a single sperm capture device based on a micromanipulation needle and a sperm pool PCR tube:

[0024] I. Equipment Preparation Stage

[0025] Micromanipulation module configuration

[0026] The micromanipulation needle 11 is installed on the robotic arm interface of the microoperating system 12.

[0027] The three-dimensional movement trajectory of the micromanipulation stylus is calibrated to ensure coaxial positioning with the microscope monitoring system.

[0028] II. Sample Processing Flow

[0029] Pretreatment of composite operating dishes

[0030] A rectangular sperm swimming pool 31 is formed by injecting 200 μL of sperm suspension.

[0031] 3 μL of polyvinylpyrrolidone (PVP) droplets were pre-placed in the PVP braking zone 32.

[0032] Prepare 30 μL of PBS buffer in each of the PBS washing areas 33.

[0033] Sperm dilution and immobilization

[0034] The swimming state of sperm in pool 31 was observed under a microscope.

[0035] The target sperm is guided to the PVP immobilization zone 32 using a micromanipulation needle 11.

[0036] Sperm mechanical braking is achieved using the needle-tip contact method;

[0037] III. Precise Positioning Operation

[0038] sperm pool construction

[0039] Inject 3 μL of PBS buffer 22 into the bottom of PCR tube 2.

[0040] Use a micropipette to draw 2 μL of PBS and construct a sperm pool 21 at 3 / 4 of the tube length.

[0041] Microtransfer operation

[0042] After immobilization, the sperm were transferred sequentially to the PBS washing area 33 for thorough rinsing.

[0043] A single sperm is placed at the tip of the manipulation needle.

[0044] Under a microscope, sperm was precisely released into the sperm pool 21.

[0045] Centrifuge at 4000 rpm for 10 seconds and then store.

[0046] IV. Post-processing stage

[0047] Sample collection

[0048] Repeat the process to capture a single sperm multiple times.

[0049] Quality control

[0050] Microscopic re-examination confirmed the accuracy of sperm localization (this system achieves 100%).

[0051] The error in the volume of sperm droplets in the sperm pool is detected (this system controls the error within ±0.1 μL).

[0052] V. Sample Preservation

[0053] Finally, it is stored in liquid nitrogen gas phase (-196℃) or deep cryogenic freezer at -80℃.

[0054] In summary, this utility model has the following working principle:

[0055] This technical solution and the existing technology follow the same process, which involves using a microscopic operating system to grasp a single sperm and then immobilizing it.

[0056] Intracytoplasmic sperm transfer

[0057] Using the pipette technique requires immobilizing a single sperm with a micromanipulation needle 11 and placing it in a droplet. To save time, without disassembling the micromanipulation needle 11, the composite culture dish 3 containing the single sperm droplet is transferred to another high-power microscope. The single sperm is then aspirated using a pipette or micropipette and transferred into the pretreatment solution in the PCR tube assembly 2 under the microscope. Because this is a manual operation, this method requires a high level of operator skill. Furthermore, the pretreatment solution in the PCR tube assembly 2 is relatively thick at this point, making it difficult to accurately observe the sperm insertion process. Sperm may be lost during the transfer and could potentially be injected with excessive PBS, negatively impacting the subsequent single sperm amplification system and its success rate.

[0058] This technique, in the process of single sperm transfer, only requires immobilizing a single sperm; it does not require placing it in a droplet and transferring it to another microscope. Instead, the micromanipulation needle is adjusted to directly release the single sperm into the PCR tube mechanism 2, which includes a sperm pool 21. The process is simple and requires no additional equipment. Because the PCR tube mechanism 2 needs to be placed horizontally, the liquid level in the sperm pool 21 is relatively thin, allowing for stable observation of the sperm entering the sperm pool 21 using the micromanipulation system 12, minimizing the risk of loss. The PCR tube mechanism 2 is then centrifuged and stored.

[0059] Furthermore, hydrophobic treatment of the composite operating dish:

[0060] A fluorocarbon polymer hydrophobic layer was formed on the surface of a polydimethylsiloxane (PDMS) substrate using plasma-enhanced chemical vapor deposition (PECVD), creating a ring-shaped hydrophobic boundary with a linewidth of 50 μm and a contact angle of 115 ± 5°. The central hydrophilic region was then treated with oxygen plasma (50 W power, 30 s time) to obtain a surface with a contact angle ≤ 30°.

[0061] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A single sperm capture device based on a micromanipulation needle and a sperm pool PCR tube, characterized in that, include: The micromanipulation module (1) includes the micromanipulation needle (11) and the micromanipulation system (12); PCR tube assembly (2), wherein the tube wall of the PCR tube assembly (2) is provided with a sperm pool (21) for containing sperm, and a PBS buffer droplet (22) is provided at the bottom of the tube wall of the PCR tube assembly (2); The composite operating dish (3) has a rectangular sperm swimming pool (31), a PVP immobilization zone (32) and a PBS washing zone (33).

2. The single sperm capture device based on a micromanipulation needle and a sperm pool PCR tube according to claim 1, characterized in that: The sperm pool consists of 2 microliters of PBS droplets, and the volume of the PBS buffer droplets is set to 3 microliters.

3. The single sperm capture device based on a micromanipulation needle and a sperm pool PCR tube according to claim 1, characterized in that: The number of the rectangular sperm swimming pool (31) is set to one, and the number of the PVP braking zone (32) and the PBS washing zone (33) are set to two respectively.

4. The single sperm capture device based on a micromanipulation needle and a sperm pool PCR tube according to claim 1, characterized in that: The surfaces of the rectangular sperm swimming pool (31), PVP braking area (32) and PBS cleaning area (33) of the composite operating dish (3) are hydrophobically treated to form an annular hydrophobic boundary, and the center of each area is a hydrophilic contact surface.

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