A chip for screening highly motile sperm based on microfluidics technology

Abstract

The present invention relates to a chip for realizing high-motility sperm screening based on microfluidic technology, comprising a substrate and a slide, wherein the substrate is used to support the slide; a loading pool is provided on the slide, the loading pool comprises a sample inlet, and a grading pool and a collecting pool are sequentially provided on the periphery of the loading pool; the loading pool, the grading pool and the collecting pool are sequentially connected; a sampling port is also provided on the slide, and the sampling port is connected to the collecting pool. The present invention solves the potential adverse effects caused by various operations such as sperm centrifugation and washing in vitro in the existing assisted reproductive technology process. It utilizes sperm chemotaxis and swimming characteristics along the interface to effectively screen high-motility sperm, obtain high-motility sperm required for assisted reproductive technology, and greatly improve the success rate of in vitro fertilization. It has a wide range of applications, and the device can be used in human reproduction and animal breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a chip for realizing high-motility sperm screening based on microfluidic technology. Background Art

[0002] The increasing incidence of infertility worldwide has greatly promoted the development of assisted reproductive technology. However, many operations in the assisted reproductive technology process may cause sperm damage, which ultimately leads to embryo maldevelopment or miscarriage. This is also one of the important reasons why the success rate of in vitro fertilization technology is difficult to exceed 50%. These damaging operations include: sperm centrifugation, density gradient centrifugation, and the influence of certain components in the separation fluid. To reduce the impact of these operations, centrifugation should be avoided as much as possible for the screening of highly active sperm. Therefore, it is necessary to study a sperm screening platform that is easy to operate and has little sample damage. Summary of the invention

[0003] The present invention proposes a chip for screening high-motility sperm based on microfluidic technology, which solves the potential adverse effects of various operations such as sperm in vitro centrifugation and washing in the existing assisted reproductive technology process. It utilizes sperm chemotaxis and swimming characteristics along the interface to effectively screen high-motility sperm, and can obtain high-motility sperm required for assisted reproductive technology, which can greatly improve the success rate of in vitro fertilization. It has a wide range of applications and can be used in human reproduction and animal breeding.

[0004] The technical solution of the present invention to solve the above problem is: a chip for screening high-motility sperm based on microfluidic technology, which is special in that:

[0005] It comprises a substrate and a slide, wherein the substrate is used to support the slide;

[0006] The slide is provided with a loading pool, the loading pool includes an injection port, and the periphery of the loading pool is provided with a classification pool and a collection pool in sequence; the loading pool, the classification pool and the collection pool are connected in sequence;

[0007] The slide is also provided with a sampling port, which is connected to the collection pool.

[0008] Furthermore, the above-mentioned classification pool is an annular pool, and the annular pool surrounds the loading pool.

[0009] Furthermore, the above-mentioned classification tank includes a first classification tank, a second classification tank and a third classification tank, and the first classification tank, the second classification tank and the third classification tank are concentrically arranged from inside to inside.

[0010] Furthermore, the first classification tank, the second classification tank and the third classification tank have different internal heights.

[0011] Furthermore, the heights of the first classification tank, the second classification tank and the third classification tank are successively reduced.

[0012] Furthermore, the first classification pool has a height of about 50um and a width of about 5000um, the second classification pool has a height of about 20um and a width of about 2500um, and the third classification pool has a height of about 5um and a width of about 2500um.

[0013] Furthermore, the grading pool and the collection pool are evenly distributed with flow-blocking columns, with a diameter of about 100 um. The flow-blocking columns are used to prevent collapse (prevent the chip from collapsing) and reduce the impact of liquid disturbance on sperm movement.

[0014] Furthermore, the number of the above-mentioned sampling ports is four, which are evenly distributed around the circumference of the collection pool.

[0015] Furthermore, the material of the above-mentioned carrier sheet is polydimethylsiloxane (PDMS) with good biocompatibility, and other microfluidic chip materials such as PC, PMMA, pure glass and the like may also be used.

[0016] Furthermore, the substrate is made of glass.

[0017] Advantages of the present invention:

[0018] 1) The present invention designs the chip as a concentric annular pool structure, with a loading pool, three-layer grading pools at different heights, and a collecting pool as the basic structure, and uses graded screening to achieve the screening of high-motility sperm, reduce the damage to sperm caused by centrifugation, and quickly complete sperm screening.

[0019] 2) The present invention designs a small circular baffle column at the corresponding position of each pool to prevent collapse and reduce the impact of liquid disturbance on sperm movement.

[0020] 3) The chip of the present invention uses the third classification pool as the sperm characteristic observation and evaluation area, and can observe sperm movement in real time with the assistance of a microscope, providing more possibilities for sperm characteristic evaluation for practical use.

[0021] 4) When the present invention is used, no other viscous substances are introduced, which is conducive to the later separation, and the screened sperm can be directly used for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a structural diagram of a chip that achieves high-motility sperm screening based on microfluidic technology;

[0023] Figure 2 is a longitudinal section view of the chip of the present invention;

[0024] Figure 3 It is a flow chart of the use of the present invention;

[0025] Figure 4 It is a cross-sectional schematic diagram of the present invention for real-time observation of sperm movement and speed measurement under a microscope;

[0026] Figure 5 It is a result diagram after conducting experiments using the present invention;

[0027] Figure 6 yes Figure 1 A partial enlarged view of the .

[0028] Among them: 1. substrate, 2. carrier film, 3. loading pool, 4. injection port, 5. collection pool, 6. sampling port, 7. first classification pool, 8. second classification pool, 9. third classification pool, 10. baffle column. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0030] Microfluidics refers to the technology that integrates or basically integrates the basic operating units such as sample preparation, reaction, separation, and detection involved in the fields of biological, chemical, and medical analysis processes onto a chip of a few square centimeters (or even smaller), forms a network of microchannels, and runs through the entire system with controllable fluids to replace various functions of conventional biological or chemical laboratories. Its basic characteristics and greatest advantages are the flexible combination and large-scale integration of multiple unit technologies on a tiny platform.

[0031] In view of the shortcomings of the traditional sperm screening method, the present invention combines the application of the new generation of high-throughput microfluidic technology to design a high-motility sperm screening chip, which can achieve non-destructive screening and collection of sperm under normal growth conditions, which is conducive to obtaining high-motility sperm required for assisted reproductive technology, so as to greatly improve the success rate of in vitro fertilization, and has a wide range of applications, not only for human reproduction (such as reproductive centers of various medical institutions), but also for animal breeding. It has great application prospects in assisted reproductive technology.

[0032] A chip for realizing high-motility sperm screening based on microfluidic technology comprises a substrate 1 and a slide 2, wherein the substrate 1 is used to support the slide 2; a loading pool 3 is provided on the slide 2, the loading pool 3 comprises a sampling port 4, and a classification pool and a collection pool 5 are sequentially provided around the loading pool 3; the loading pool 3, the classification pool and the collection pool 5 are sequentially connected; a sampling port 6 is also provided on the slide 2, and the sampling port 6 is connected to the collection pool 5. The classification pool is an annular pool, which surrounds the loading pool 3 as the center.

[0033] As a preferred embodiment of the present application, the classification pool is divided into three levels, including a first classification pool 7, a second classification pool 8 and a third classification pool 9. The first classification pool 7, the second classification pool 8 and the third classification pool 9 are concentrically arranged from the inside to the outside.

[0034] As a preferred embodiment of the present application, the first classification pool 7, the second classification pool 8 and the third classification pool 9 have different internal heights, and the heights of the first classification pool 7, the second classification pool 8 and the third classification pool 9 decrease in sequence.

[0035] As a preferred embodiment of the present application, the height inside the first classification pool 7 is about 50um and the width is about 5000um, the height inside the second classification pool 8 is about 20um and the width is about 2500um, and the height inside the third classification pool 9 is about 5um and the width is about 2500um.

[0036] See also Figure 6 As a preferred embodiment of the present application, baffle columns 10 are evenly arranged in each annular pool. The baffle columns are dispersed, and the density is not fixed but adjustable. The diameter is about 100um, which prevents the chip from collapsing and reduces the influence of liquid disturbance on sperm movement.

[0037] As a preferred embodiment of the present application, there are multiple sampling ports 6 , and the multiple sampling ports 6 are evenly distributed around the circumference of the collection pool 5 .

[0038] As a preferred embodiment of the present application, the material of the carrier sheet 2 is polydimethylsiloxane (PDMS) with good biocompatibility, or PC, PMMA or glass material. The substrate 1 is made of glass.

[0039] The present invention discloses a chip for screening high-activity sperm based on microfluidic technology. The height of the classification pool is gradually reduced and the channel width is used to classify and screen sperm. When the temperature is suitable, the sperm sample is passed through the middle injection port 4 and enters the loading pool 3. Since sperm has a tendency to the outermost collection pool 5 storing an inducer, the sperm will swim and pass through the first classification pool 7, the second classification pool 8 and the third classification pool 9 with decreasing heights in sequence, and finally reach collection in the outermost collection pool 5. The screening of high-activity sperm is achieved and the damage to sperm is avoided. At the same time, because of the transparency of PDMS and the design of the third classification pool 9 that only allows a single layer of sperm to pass through, a sperm feature evaluation area can be provided after the use of a microscope, so that more sperm motility features (such as movement speed, movement direction, etc.) can be evaluated in real time, providing a reference for the next step of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). It is conducive to its application in assisted reproductive technology.

[0040] Example

[0041] See also Figure 1 and Figure 2 , a chip for screening high-motility sperm based on microfluidic technology, comprising a substrate 1 and a carrier 2 arranged on the substrate 1. A multi-layer annular pool structure is concentrically arranged on the carrier 2. The innermost loading pool 3 and the outermost collecting pool 5 of the chip are both about 50um in height. Before adding the sample, liquid is first passed into the entire internal structure of the chip to avoid pressure difference, and the width of the two pools is wider than that of other pools, which is suitable for sampling, loading and collecting sperm. The chip has three annular classification pools: the height of the first classification pool 7 is about 50um and the width is about 5000um, the height of the second classification pool 8 is about 20um and the width is about 2500um, and the height of the third classification pool 9 is about 5um and the width is about 2500um. Four sampling ports 6 are evenly distributed on the outer circumference of the collection pool 5.

[0042] Since the height and width of each grading pool are different, they play different grading and screening roles. The first grading pool 7 performs coarse screening, the second grading pool 8 performs fine screening, and the third grading pool 9 performs single-layer sperm screening.

[0043] Due to the chemotaxis of sperm and the swimming characteristics along the interface, after the sperm is added to the innermost loading pool 3 through the injection port 4, it will move to the outermost collection pool 5 filled with inducers. After the initial screening in the first classification pool 7, the sperm with lower motility remains in the loading pool, and the sperm with higher motility swims into the second classification pool 8. The sperm with higher motility can pass through the second classification pool 8 and enter the channel of the third classification pool 9 connected to the collection pool. The design height of the channel of the third classification pool 9 can only allow a single layer of sperm to pass through. Finally, the sperm with high motility reaches the collection pool through the third classification pool 9.

[0044] A small circular baffle column 10 with a diameter of about 100um is designed at the corresponding position of each pool. The purpose is to prevent collapse and reduce the influence of liquid disturbance on sperm movement. The screened sample containing high-motility sperm can be sucked out at the sample outlet.

[0045] The present invention uses a loading pool, three-layer grading pools of different heights, and a collection pool as the basic structure. The high-activity sperm is screened and collected in the collection pool by grading screening to avoid damage to the sperm caused by excessive operations. The material used to make the chip is polydimethylsiloxane (PDMS) with good biocompatibility. The chip channel structure is at the micron level, which meets the conditions for sperm swimming. At the same time, the transparent characteristics of the chip can meet the conditions for observation under a microscope, which provides the possibility for the evaluation of other sperm characteristics in the later stage.

[0046] The present invention designs a small circular baffle column at the position corresponding to each annular pool, which greatly reduces the interference of liquid on the screening process in actual use and improves the accuracy; due to the transparency of PDMS and the channel design that only allows a single layer of sperm to pass through, a sperm characteristic evaluation area is provided, so that more sperm vitality characteristics can be observed and evaluated in real time, meeting the needs of actual operation. Because no substances such as agar or gel are introduced, the screened sperm can be directly used for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

[0047] Use examples of the present invention:

[0048] See also Figure 3 The chip for high-motility sperm screening based on microfluidic technology of the present invention is vacuumed, the nutrient solution is filled in the internal structural channels of the carrier, and then the inducer is added to the four sampling ports 6, and 1.5 ml of sperm sample is added to the injection port 4 of the loading pool 3. The mixture is allowed to stand at room temperature and observed in real time under a microscope. After 1 hour, the sample in the sampling port 6 is collected.

[0049] See also Figure 4 During the screening process, sperm characteristics (including sperm morphology and movement, etc.) were observed and evaluated in area 9 of the third classification pool using a microscope. The chip design meets the requirements of bright field / phase contrast incident light illumination imaging. The incident light passes through the microfluidic chip and enters the objective lens to achieve illumination imaging. In a liquid environment, a video with a resolution of 0.65μm, 2048×2048, a duration of 60s, and a frame rate of 10fps was captured using a phase contrast microscope with a 10x objective lens. The movement trajectory of each sperm is automatically tracked through a computer program, and the swimming speed of the sperm is calculated. The speed distribution map of the sperm is obtained through statistical analysis. See Figure 5 It can be concluded that the proportion of high-speed sperm after screening increases, and the average speed also increases. The chip has a significant effect on sperm motility screening.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art can still adjust the technical solutions described in the above embodiments or replace some of the technical features by equivalents. Therefore, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A chip for screening high-motility sperm based on microfluidics technology. Features: It comprises a substrate (1) and a carrier sheet (2), wherein the substrate (1) is used to support the carrier sheet (2); The carrier sheet (2) is provided with a loading pool (3), the loading pool (3) comprises an injection port (4), and a classification pool and a collection pool (5) are sequentially provided around the loading pool (3); the loading pool (3), the classification pool and the collection pool (5) are sequentially connected; The slide (2) is also provided with a sampling port (6), and the sampling port (6) is connected to the collection pool (5); The classification pool is an annular pool, and the annular pool surrounds the loading pool (3); The classification pool comprises a first classification pool (7), a second classification pool (8) and a third classification pool (9), and the first classification pool (7), the second classification pool (8) and the third classification pool (9) are concentrically arranged from the inside to the outside; The first classification tank (7), the second classification tank (8) and the third classification tank (9) have different internal heights; The heights of the first classification tank (7), the second classification tank (8) and the third classification tank (9) decrease in sequence; The first classification pool (7) has an interior height of about 50 um and a width of about 5000 um, the second classification pool (8) has an interior height of about 20 um and a width of about 2500 um, and the third classification pool (9) has an interior height of about 5 um and a width of about 2500 um; Flow blocking columns (10) are evenly distributed in the classification pool and the collection pool (5), and the diameter of the flow blocking columns (10) is 100 um.

2. A chip for screening high-motility sperm based on microfluidic technology according to claim 1, Features: The number of the sampling ports (6) is multiple, and the multiple sampling ports (6) are evenly distributed around the outer circumference of the collection pool (5).

3. A chip for screening high-motility sperm based on microfluidic technology according to claim 2, Features: The material of the carrier sheet (2) is polydimethylsiloxane, PC, PMMA or glass material.

4. A chip for selecting high-motility sperm based on microfluidic technology according to claim 3, Features: The substrate (1) is made of glass.

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