CN118109296B - Sperm fixation microstructure platform and device - Google Patents

Abstract

The present invention provides a sperm fixing microstructure platform and device, which can be applied to the fields of biomedical engineering technology, assisted reproductive technology and microfluidic technology. The sperm fixing device includes an analysis chamber, in which a sperm fixing microstructure platform is provided, and the sperm fixing microstructure platform is used to capture and fix sperm; wherein the sperm fixing microstructure platform includes a card slot and a guide slot; the width of the card slot is 4 to 6 μm, which is used to accommodate and fix the sperm head; the guide slot is arranged in a trumpet shape, and the trumpet-shaped small end is connected to the card slot, which is used to guide the sperm head into the card slot and allow the sperm flagellum to have space for movement. The sperm fixing microstructure platform and device provided by the present invention have the advantages of no damage, simple operation, orderly distribution, etc., and the key is to ensure the free swing of the sperm flagellum while fixing the sperm.

Description

Sperm fixation microstructure platform and device

Technical Field

The present invention relates to the fields of biomedical engineering technology, assisted reproductive technology and microfluidic technology, and more specifically, to a sperm fixation microstructure platform and device.

Background Art

Capturing and fixing individual sperm from a group of swimming sperm for research, analysis and manipulation at the single sperm level is of great value in biomedical engineering technology, assisted reproductive technology and other life science and medical technology fields. For example, after fixing the sperm, the flagella swing is analyzed and the swing waveform parameters are extracted, which can be used for sperm quality assessment.

However, existing sperm capture and fixation technologies have problems such as flagella constraint, sperm damage, difficult operation, and disordered distribution, and therefore have many limitations in practical applications.

Summary of the invention

In view of the above problems, the main purpose of the present invention is to provide a sperm fixation microstructure platform and device. Through the structural design of the slot and the guide groove, the sperm fixation device can not only effectively capture and fix sperm, but also ensure the free swing of the flagellum. In addition, the technical effects of no damage, easy operation, orderly distribution, etc. are also achieved at the same time.

To achieve the above object, the technical solution of the present invention is as follows:

The first aspect of the present invention provides a sperm fixation device, which includes an analysis chamber, in which a sperm fixation microstructure platform is provided, and the sperm fixation microstructure platform is used to capture and fix sperm; wherein the sperm fixation microstructure platform includes a card slot and a guide slot; the width of the card slot is 4 to 6 μm, and is used to accommodate and fix the sperm head; the guide slot is arranged in a trumpet shape, and the trumpet-shaped small end is connected to the card slot, and is used to guide the sperm head into the card slot and allow the sperm flagella to have activity space.

According to an embodiment of the present invention, the length of the above-mentioned slot is 4-6 μm.

According to an embodiment of the present invention, the width of the opening of the guide groove is 10-20 μm.

According to an embodiment of the present invention, the total length of the latch groove and the guide groove is 8 to 15 μm.

According to an embodiment of the present invention, the width of the above-mentioned slot is 5 μm, and the length is 5 μm.

According to an embodiment of the present invention, the width of the guide groove opening is 15 μm.

According to an embodiment of the present invention, the total length of the latch groove and the guide groove is 12 μm.

According to an embodiment of the present invention, the depths of the clamping groove and the guiding groove are the same, both of which are 2-5 μm.

According to an embodiment of the present invention, the sperm fixation microstructure platform is a column or a table with a height of 10 to 200 μm.

According to an embodiment of the present invention, the sperm fixation microstructure platform comprises a plurality of the above-mentioned slots and the above-mentioned guide slots, and ends of the plurality of the above-mentioned slots are connected via a connecting channel.

According to an embodiment of the present invention, the width of the connecting channel is 0.5-2 μm, and the depth is 2-5 μm.

According to an embodiment of the present invention, a plurality of the sperm fixation microstructure platforms are disposed in the analysis chamber, and the plurality of the sperm fixation microstructure platforms are arranged in an array.

According to an embodiment of the present invention, the analysis chamber is provided with an injection port, and sperm enters the analysis chamber through the injection port.

According to an embodiment of the present invention, the analysis chamber is provided with a panel made of a transparent material, and sperms are observed and analyzed through the panel made of the transparent material.

According to an embodiment of the present invention, the material of the cavity of the above-mentioned analysis cavity and the above-mentioned sperm fixation microstructure platform are both polydimethylsiloxane.

Another aspect of the present invention provides a sperm fixation microstructure platform for use in the sperm fixation device.

According to an embodiment of the present invention, the sperm fixing device provided by the present invention includes an analysis chamber, and a sperm fixing microstructure platform is provided in the analysis chamber, and the sperm fixing microstructure platform is used to capture and fix sperm. A groove is provided on the sperm fixing microstructure platform, and each groove includes a card slot and a guide slot. By using microfluidic technology, the width of the card slot is limited so that it can just accommodate a sperm head. Since the swimming mode of sperm can only move forward but not backward, once the sperm hits such a card slot, it is not easy to escape, thereby achieving the capture and fixation of sperm. By setting the shape of the guide slot to a trumpet shape, it is conducive to guiding sperm into the card slot and improving the efficiency of capturing and fixing sperm; at the same time, the trumpet-shaped design avoids the guide slot from binding the sperm flagellum, so that the sperm flagellum can maintain a swing similar to a free swimming state. Under the guidance of the guide slot, the sperm head falls into the card slot, but the sperm flagellum is located outside the card slot, that is, the guide slot and the area outside the guide slot, which has a relatively spacious activity space, so the flagellum can swing freely without constraints. Moreover, since the device only relies on the structural steric hindrance of the card slot to fix the sperm, the device does not damage the sperm at all, that is, it is non-destructive. At the same time, the device is easy to operate. You only need to add the sample and wait for a while. After capturing and fixing a sufficient number of sperm, you can start observation and analysis. In addition, since the microstructure platform is used as the capture site, the position of the captured and fixed sperm is more orderly and controllable, avoiding the interference between sperm caused by uncontrolled scattered disorder and staggered overlapping distribution, ensuring that the actual effective data volume is not reduced.

According to the embodiments of the present invention, the sperm fixation device provided by the present invention has the advantages of being damage-free, easy to operate, and orderly distributed, and the key is that while capturing and fixing sperm, it can ensure that the flagellum can swing freely without constraints.

The outstanding features and advantages of the embodiments of the present invention are that the free swinging of sperm flagella can be ensured while the sperm is fixed. On the one hand, this can make the flagella imaging not be disturbed by the fixed structure, which is conducive to image recognition and automated analysis, and can automatically identify most of the flagella and extract the corresponding parameters. On the other hand, since the swing of the flagella is not affected by the structure, the results obtained by such analysis can more realistically reflect the flagellar behavior of sperm in a free swimming state, such as the flagellar state of sperm when swimming in the body, which has a certain reference value in assisted reproduction.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the present invention will become more apparent through the following description of the embodiments of the present invention with reference to the accompanying drawings, in which:

FIG1 is a schematic diagram of a sperm fixation device provided in an embodiment of the present invention, wherein (a) is a schematic diagram of the sperm fixation device, (b) is a schematic diagram of the sperm fixation microstructure platform array after sperm fixation, and (c) is a schematic diagram of the sperm fixation microstructure platform after sperm fixation;

FIG2 is a top view of a sperm fixation microstructure platform provided in an embodiment of the present invention;

FIG3 is a transient photograph of a sperm with its head fixed provided in Experimental Example 1 of the present invention (the portion of the flagellum successfully identified by the program is superimposed with a white line); and

FIG4 is the sperm flagella fluctuation data provided in Experimental Example 2 of the present invention, wherein (a) is the superposition of flagella shapes at different times, and (b) is the change of curvature of flagella at different positions over time;

The markings in the accompanying drawings are as follows: 100 is the analysis chamber; 210 is the first injection port; 220 is the second injection port; 300 is the sperm fixation microstructure platform array; 310 is the sperm fixation microstructure platform; 311 is the card slot; 312 is the guide slot; 313 is the connecting channel; 400 is the panel of the analysis chamber.

DETAILED DESCRIPTION

In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

Capturing and fixing individual sperm from a group of swimming sperm for research, analysis and manipulation at the single sperm level is of great value in the fields of biomedical engineering, assisted reproductive technology, etc. For example, after fixing the sperm, the flagellar swing is analyzed and the swing waveform parameters are extracted, which can be used for sperm quality assessment.

In related examples, micropipette technology can be used to fix single sperm, but the operation is complicated and the efficiency is low. Sperm can be fixed on the surface using the surface adhesion method, but the distribution of sperm is disordered, and multiple sperm are often crowded and interfered, which is not conducive to analysis and operation. Micro-imprinting can be used to obtain a patterned adhesion array to control the distribution of adhered sperm, but the chemical treatment is complicated and time-consuming, and is incompatible with some sperm biochemical analysis methods. Single sperm can also be captured using optical tweezers and high-frequency electric field devices, but the focused light beam and high-intensity electric field both have potential damage to sperm, and the device is complex, costly, and inefficient. The above problems are not conducive to its application in reproductive medicine and other fields.

Microfluidic technology has many advantages, such as low sample and reagent consumption, fast high throughput, and high integration. The development of microfluidic technology has opened up broad space for life sciences, medicine and other fields. Using microfluidic chips, single sperm can be captured. For example, the pressure difference generated by the channel flow field can be used to squeeze sperm into a special small hole "trap" to bind the sperm. In addition, by applying an external flow field and taking advantage of the characteristics of sperm swimming against the current, sperm can actively swim into the "trap" array and cannot escape, thereby achieving large-scale single sperm capture. However, the above two microfluidic technologies have a common defect, that is, this type of "trap" structure not only binds sperm, but also limits the swing of sperm flagella. Especially when sperm is capacitated and in a super-activated state, the flagella swing is large and is more constrained by this type of "trap" structure. On the one hand, because the flagella will contact the vertical wall of the structure, resulting in image overlap, it becomes difficult to automatically analyze the flagellar parameters and it is difficult to fully extract the parameters of the entire flagellum. On the other hand, even if the flagellar parameters are successfully extracted, the authenticity and validity of the analysis results cannot be guaranteed because the oscillation of the flagella is limited by the structure and there is a gap between the oscillation of the flagella and the natural swimming state. In addition, the above two microfluidic technologies require precise control of the flow field, which is difficult to operate.

In summary, the above-mentioned related examples have problems such as flagella constraint, sperm damage, operational difficulties, and disordered distribution in the capture and fixation of sperm, which have many limitations in practical applications in the fields of biomedical engineering, assisted reproductive technology, etc.

In the process of realizing the present invention, it was found that by utilizing microfluidic technology and through the structural design of the card slot and the guide groove, the sperm fixation device solved the above-mentioned problems, and has the advantages of being damage-free, easy to operate, and orderly distributed. The key is that while effectively capturing and fixing sperm, it can also ensure that the flagella can swing freely without constraints.

Specifically, according to an embodiment of the present invention, the present invention provides a sperm fixation device, which includes an analysis chamber, in which a sperm fixation microstructure platform is provided, and the sperm fixation microstructure platform is used to capture and fix sperm; wherein the sperm fixation microstructure platform includes a card slot and a guide slot; the width of the card slot is 4 to 6 μm, and is used to accommodate and fix the sperm head; the guide slot is arranged in a trumpet shape, and the small end of the trumpet shape is connected to the card slot, and is used to guide the sperm head into the card slot and allow the sperm flagella to have activity space.

According to an embodiment of the present invention, the number of sperm fixation microstructure platforms in the analysis chamber may be one or more, and the present application does not limit the number of sperm fixation microstructure platforms in the analysis chamber. When the number of sperm fixation microstructure platforms in the analysis chamber is more than one, the multiple sperm fixation microstructure platforms may be arranged in an array.

According to an embodiment of the present invention, the number of the card slot and the guide slot can be one or more. The number of the card slot and the guide slot is the same, and the two are one-to-one corresponding. At the connecting part of the card slot and the guide slot, the width of the card slot and the guide slot is the same, and one card slot and one guide slot constitute a set of sperm fixation microstructures.

According to an embodiment of the present invention, the width of the card slot may be fixed, or may gradually increase from the inside to the outside, and the present invention does not limit the shape of the card slot.

According to the embodiment of the present invention, after the sperm is injected into the analysis chamber, the sperm head first enters the guide groove. Since the sperm can only swim forward but not backward, under the guidance of the guide groove, the sperm head enters the card slot. By setting the shape of the guide groove to be trumpet-shaped, it is not only conducive to guiding the sperm into the card slot, but also reduces the restraint on the sperm flagellum, so that the sperm flagellum can maintain a swing similar to the free swimming state.

According to an embodiment of the present invention, microfluidic technology is used to limit the width of the slot so that it can just accommodate a sperm head, and the shapes of the slot and the sperm head are closely matched. Since the swimming mode of sperm can only move forward but not backward, once the sperm hits such a slot, it is not easy to escape, thereby achieving the capture and fixation of the sperm.

According to an embodiment of the present invention, under the guidance of the guide groove, the sperm head sinks into the slot, but the sperm flagellum is located outside the slot, that is, the guide groove and the area outside the guide groove have a relatively spacious activity space, so the flagellum can swing freely without constraints. On the one hand, this can make the flagellum imaging not be interfered by the fixed structure, which is conducive to image recognition and automated analysis, and can realize automatic identification of most of the flagellum and extract the corresponding parameters. On the other hand, since the swing of the flagellum is not affected by the structure, the results obtained by such analysis can more realistically reflect the flagellar behavior of sperm in a free swimming state, such as the flagellar state of sperm when swimming in the body, which has a certain reference value in assisted reproduction.

According to the embodiments of the present invention, the sperm fixation device only relies on the structural steric hindrance of the slot to fix the sperm, which does not damage the sperm at all and is non-destructive; it only needs to add the sample and wait for a moment, and after a sufficient number of sperm are captured and fixed, observation and analysis can begin, and the operation is relatively convenient; the captured and fixed sperm are all located on the microstructure platform, and there will be no overlap or disorder between the sperm, thus avoiding mutual interference and affecting the effective data volume, and the device is orderly.

According to an embodiment of the present invention, the length of the card slot is 4-6 μm. Specifically, the length of the card slot may be 4 μm, 4.5 μm, 5 μm, 5.5 μm or 6 μm. Preferably, the length of the card slot is 5-6 μm.

According to an embodiment of the present invention, the width of the guide groove opening position is 10-20 μm. Specifically, the width of the guide groove opening position may be 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm or 20 μm. Preferably, the width of the guide groove opening position is 13-17 μm.

According to an embodiment of the present invention, the length of the card slot and the guide slot is 8 to 15 μm in total. Specifically, the length of the card slot and the guide slot is 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm or 15 μm in total. Preferably, the length of the card slot and the guide slot is 10 to 13 μm in total.

According to an embodiment of the present invention, the width of the card slot is 5 μm, and the length is 5 μm, and the width of the guide groove opening is 15 μm. The total length of the card slot and the guide groove is 12 μm.

According to an embodiment of the present invention, the depths of the card slot and the guide slot are the same, both 2-5 μm. Specifically, the depths of the card slot and the guide slot can be 2 μm, 3 μm, 4 μm or 5 μm. Preferably, the depths of the card slot and the guide slot are both 4-5 μm.

According to the embodiment of the present invention, by limiting the size of the card slot, it is beneficial for the card slot to capture and fix sperm; by limiting the size of the guide groove, the activity space of the sperm flagellum is guaranteed.

According to an embodiment of the present invention, the sperm fixation microstructure platform is a column or a platform with a height of 10 to 200 μm. Specifically, the height of the sperm fixation microstructure platform can be 10 μm, 20 μm, 30 μm, 50 μm, 70 μm, 100 μm, 120 μm, 150 μm, 180 μm, 200 μm. Preferably, the height of the sperm fixation microstructure platform can be 40 to 90 μm.

According to an embodiment of the present invention, the sperm fixation microstructure platform may be a cylinder, a prism, a frustum, a prism, or the like.

According to an embodiment of the present invention, the sperm fixation microstructure platform includes a plurality of slots and guide slots, and the ends of the plurality of slots are connected through a connecting channel; the connecting channel has a width of 0.5 to 2 μm and a depth of 2 to 5 μm.

According to an embodiment of the present invention, the width of the connection channel may be 0.5 μm, 0.7 μm, 1.0 μm, 1.3 μm, 1.5 μm, 1.8 μm or 2.0 μm, and the depth of the connection channel may be 2 μm, 2.5 μm, 3 μm, 4 μm, 4.5 μm or 5 μm.

According to an embodiment of the present invention, a plurality of clamping grooves and a plurality of guiding grooves are evenly distributed on the sperm fixing microstructure platform.

According to an embodiment of the present invention, the ends of the card slot can be connected through a connecting channel, which is a capillary channel, which can prevent the liquid from not being able to fill and residual bubbles; at the same time, the width of the channel is smaller than the sperm head, so that only liquid is allowed to pass through, and the sperm will not be able to drill out and escape.

For example, the sperm fixation microstructure platform may include 4 slots and 4 guide slots, forming 4 groups of sperm fixation microstructures, and the 4 groups of sperm fixation microstructures may form a quadruple platform orthogonally surrounded by the ends of the slots. In this case, each sperm fixation microstructure platform can capture and fix 4 sperms.

According to an embodiment of the present invention, a plurality of sperm fixation microstructure platforms are provided in the analysis chamber, and the plurality of sperm fixation microstructure platforms are arranged in an array.

According to the embodiments of the present invention, the present invention can fix sperm in a high-throughput and orderly manner. The array-arranged sperm fixation microstructure platform can make the structure of the fixed sperm arranged in a compact array, so as to capture sperm in large quantities, and the operation is simple, and high-throughput single sperm analysis and operation can be achieved. For example, the sperm fixation device provided by the present invention can be used for routine rapid screening of sperm in the medical field, etc.

According to the embodiments of the present invention, the microstructure array is arranged in an orderly and reasonable manner, and the sperms do not affect each other. Therefore, it has many advantages such as strong experimental controllability, large amount of effective data, and high analysis efficiency.

According to an embodiment of the present invention, the analysis chamber is provided with an injection port, and sperm enters the analysis chamber through the injection port.

According to an embodiment of the present invention, the analysis chamber is provided with a panel made of a transparent material, through which the morphology of sperm can be seen.

According to an embodiment of the present invention, one end of the sperm fixing microstructure platform where the clamping groove and the guiding groove are located may be in contact with the panel.

According to an embodiment of the present invention, after the sperm is injected into the analysis cavity through the injection port, it enters the card slot through the guidance of the guide groove. At this time, the state of the sperm can be observed through a panel made of transparent material, which is convenient for sperm quality assessment, etc.

According to an embodiment of the present invention, the materials of the cavity of the analysis chamber and the sperm fixation microstructure platform are both polydimethylsiloxane.

According to an embodiment of the present invention, the sperm immobilization device provided by the present invention can be prepared based on soft lithography technology.

According to an embodiment of the present invention, a method for preparing a sperm immobilization device may include:

S1. Draw the channel pattern in two layers, wherein the card slot, guide groove and connection channel area are the first layer, and the other areas are the second layer.

S2. Using a silicon wafer as a substrate and a photoresist as a substrate for attaching patterns, the photolithography process is performed twice to obtain a positive mold.

According to the embodiment of the present invention, each photolithography process includes the steps of coating, pre-baking, exposure, post-baking, developing, hard baking, etc. The two photolithography processes are respectively for the first layer and the second layer of the pattern.

S3. Mix the polydimethylsiloxane prepolymer and curing agent thoroughly, pour them onto the positive mold obtained by photolithography, perform thermal curing in an oven, cool them, remove the polydimethylsiloxane analysis cavity from the positive mold, then punch a hole at the injection port, and then attach the analysis cavity to a panel made of transparent material to obtain a sperm fixation device.

According to an embodiment of the present invention, the polydimethylsiloxane prepolymer and the curing agent can be fully mixed in a mass ratio of 10:1, and after the bubbles are removed by vacuum, they are poured onto the positive mold obtained by photolithography, and thermally cured in an oven at 80°C for 2 hours. After being taken out of the oven and cooled to room temperature, the polydimethylsiloxane prepolymer analysis cavity is peeled off from the positive mold, and a hole is punched at the injection port position of the polydimethylsiloxane prepolymer analysis cavity with a flat-headed needle, and then the channel is attached to the cover glass to complete the device.

According to an embodiment of the present invention, the present invention also provides a sperm fixation microstructure platform for the above-mentioned sperm fixation device.

According to the embodiments of the present invention, the present invention can ensure the free swinging of sperm flagella while fixing sperm. On the one hand, this can make the flagella imaging not be disturbed by the fixed structure, which is conducive to image recognition and automated analysis, and can realize automatic recognition of most of the flagella and extract the corresponding parameters. On the other hand, since the swing of flagella is not affected by the structure, the results obtained by such analysis can more realistically reflect the flagellar behavior of sperm in a free swimming state, such as the flagellar state of sperm when swimming in the body, which has a certain reference value in assisted reproduction.

According to the embodiments of the present invention, the sperm fixing device provided by the present invention can achieve non-destructiveness to sperm and is simple and quick to operate. Experiments show that the various motility parameters of sperm fixed by the sperm fixing device of the present invention do not change significantly within 2 hours, and the sperm can maintain a high activity for a long time. The advantage of this non-destructive fixation is more obvious in medical-related applications such as assisted reproduction. At the same time, compared with other microfluidic devices related to sperm fixation, the operation of this device is also simpler and more convenient. For example, there is no need for external flow field control, and only a simple injection of liquid is required for microscopic observation and analysis. In addition, the site controllability of sperm capture is also achieved, which can ensure the orderly distribution of sperm and avoid the situation where sperm is randomly distributed and interferes with each other.

According to the embodiments of the present invention, in general, the sperm fixation microstructure platform and device provided by the present invention have the advantages of being damage-free, easy to operate, orderly distributed, and high-throughput, and the key is to ensure the free swinging of sperm flagella while fixing sperm.

The following will describe the technical solutions in the embodiments of the present invention in detail in conjunction with the accompanying drawings in the embodiments of the present invention. It should be noted that the specific embodiments below are only for illustration and are not intended to limit the present invention.

As shown in FIG1 , the sperm fixation device includes an analysis chamber 100, an injection port 210 and an injection port 220 provided on the analysis chamber, a sperm fixation microstructure platform array 300 in the analysis chamber, and a panel 400 provided on the analysis chamber, wherein the sperm fixation microstructure platform array 300 includes a sperm fixation microstructure platform 310, and the sperm fixation microstructure platform 310 is a square column. Among them, the material of the analysis chamber 100 and the sperm fixation microstructure platform 300 is polydimethylsiloxane, and the panel 400 is a cover glass. During operation, the sperm sample is injected into the analysis chamber 100 from any injection port, and the sperm fixation microstructure platform array 300 captures and fixes the sperm. At this time, the sperm can be observed and analyzed through the panel made of transparent material, which is convenient for sperm quality assessment, etc.

As shown in FIG. 2 , the sperm fixing microstructure platform 310 includes 4 groups of sperm fixing microstructures, each of which includes a slot 311 and a guide slot 312. The length and width of the sperm fixing microstructure platform 310 are both 33 μm, and the height is 60 μm; the slot is used to accommodate and fix the sperm head, the width of the slot is 5 μm, and the length of the slot is 5 μm; the guide slot is arranged in a trumpet shape, and the trumpet-shaped small end is connected to the slot, which is used to guide the sperm head into the slot and allow the sperm tail to have sufficient activity space. The width of the opening position of the guide slot is 15 μm, the length of the slot and the guide slot is 12 μm in total, and the depth of the slot and the guide slot is 5 μm. The ends of the four slots are connected through a connecting channel 313, and the four groups of sperm fixing microstructures are orthogonally arranged to form a sperm fixing microstructure platform. At this time, the sperm fixing microstructure platform 310 can capture and fix four sperm. Among them, the width of the connecting channel 313 is 1 μm, and the depth of the connecting channel 313 is 5 μm.

Test Example 1

Sperm quality assessment

Sperm quality assessment is of great significance in the fields of reproductive medicine and animal husbandry. The traditional computer-aided sperm analysis system (CASA) extracts various parameters from the swimming trajectory of the sperm group to evaluate the quality of the sperm sample. However, since sperm swimming essentially depends on the swing of the flagella, directly analyzing the swing of the sperm flagella can more deeply reflect the movement characteristics of sperm, compared with CASA extracting downstream parameters from sperm swimming, and thus more truly and effectively evaluate sperm quality.

The sperm fixation device provided by the present invention can be used to analyze the flagellar movement of sperm in a high-throughput manner to evaluate the quality of sperm samples, as exemplified below:

Bovine sperm is easy to obtain and is similar to human sperm in morphology, size, and motility. It is a classic and universal model sperm in both basic experimental research and application technology development. Therefore, this test example uses bovine sperm for operation. Mix a bovine sperm sample thoroughly, draw 5μL, and inject it into any injection port of the device. If the sperm density of the sample is low, the density of live sperm can be increased by density gradient centrifugation and other treatment methods, and then the sample is injected into the device. After the device is filled, seal the two injection ports with grease to prevent the flow field from being generated in the subsequent main chamber, which affects the observation and analysis. Place the device on the 37℃ heated stage of the microscope, wait for enough sperm to fall into the sperm fixation microstructure (about 5 minutes), and then shoot and analyze them one by one. Each device has tens of thousands of sperm fixation microstructures, so in the end almost all sperm will be fixed, and there will be almost no free-swimming sperm to interfere with the shooting and analysis. Try to randomly select sperm in different areas of the chamber for shooting. Taking into account the compromise operation time, data quantity and data size, a frame rate of 300 frames per second was used, each sperm was photographed for 20-30 seconds, and a total of 50 sperm were photographed. Figure 3 is a transient photograph of a sperm with its head fixed (the identified sperm flagellum is printed in white).

The application of the sperm fixation device provided by the present invention is not limited to bovine sperm, and sperm of other species can also be used according to the above process, such as human sperm, other common livestock sperm (sheep sperm, pig sperm, etc.), mouse sperm, etc.

Test Example 2

Experimental study on sperm chemotaxis and other characteristics

In mammals, the encounter process between sperm and egg is extremely complex, and the reproductive tract needs to provide a series of guidance mechanisms to help high-quality sperm successfully find the egg and ultimately achieve fertilization. Chemotaxis is an important short-range guidance mechanism, where sperm senses the gradient of specific chemicals to determine the direction of the egg. However, the research on the chemotaxis response mechanism of mammalian sperm in related technologies is still insufficient, especially the research on the input and output response characteristics of the chemotaxis system is relatively rare.

By using the sperm fixation device provided by the present invention, the input and output response characteristics of sperm to chemical substances can be studied, for example, basic responses such as impulse response and step response.

Here are some examples:

As in Experimental Example 1, this Experimental Example also uses bovine sperm. Inject the sperm into the sperm fixture and seal it according to the method of Experimental Example 1. Use ultraviolet convergent laser to irradiate the light-released progesterone complex (1 μM). By adjusting the light intensity and irradiation duration, the concentration of progesterone in the environment near the irradiation point is regulated, and the corresponding changes in the sperm flagellar waveform are photographed and analyzed. Each sperm was tested 5 times for response, and a total of 100 sperm were tested. Figure 4 is the sperm fluctuation data. The changes in various parameters of the sperm flagellar waveform in the frequency domain are statistically analyzed, and finally the input-output characteristics of the sperm to the progesterone concentration are obtained.

The sperm fixation device provided by the present invention is not limited to the study of the chemotactic response characteristics of bovine sperm, but can also be used for other response characteristics of sperm of other species (as described in Experimental Example 1), such as thermotactic response, super-activation response, etc.

The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims (9)

1. A sperm fixation device, characterized in that the sperm fixation device comprises an analysis chamber, wherein a sperm fixation microstructure platform is provided in the analysis chamber, and the sperm fixation microstructure platform is used to capture and fix sperm; Wherein, the sperm fixation microstructure platform includes a card slot and a guide slot; The width of the card slot is 4-6 μm, and the length of the card slot is 4-6 μm, which is used to accommodate and fix the sperm head; The guide groove is arranged in a trumpet shape, and the trumpet-shaped small end is connected to the clamping groove, so as to guide the sperm head into the clamping groove and allow the sperm flagellum to have a moving space; The width of the guide groove opening is 13-17 μm; The total length of the card groove and the guide groove is 8-15 μm. 2. The sperm fixing device according to claim 1, characterized in that the width of the slot is 5 μm and the length is 5 μm; The width of the guide groove opening is 15 μm; The total length of the clamping groove and the guide groove is 12 μm. 3. The sperm fixing device according to claim 1 is characterized in that the depths of the clamping groove and the guide groove are the same, both of which are 2-5 μm. 4. The sperm fixation device according to claim 1, characterized in that the sperm fixation microstructure platform is a column or a table with a height of 10 to 200 μm. 5. The sperm fixation device according to claim 1, characterized in that the sperm fixation microstructure platform comprises a plurality of the slots and the guide slots, and the ends of the plurality of slots are connected through a connecting channel; The connecting channel has a width of 0.5-2 μm and a depth of 2-5 μm. 6. The sperm fixation device according to claim 1 is characterized in that a plurality of the sperm fixation microstructure platforms are provided in the analysis chamber, and the plurality of the sperm fixation microstructure platforms are arranged in an array. 7. The sperm fixation device according to claim 1, characterized in that the analysis chamber is provided with an injection port, and sperm enters the analysis chamber through the injection port; The analysis chamber is provided with a panel made of transparent material, through which sperm is observed and analyzed. 8. The sperm fixation device according to any one of claims 1 to 7, characterized in that the material of the cavity of the analysis cavity and the sperm fixation microstructure platform are both polydimethylsiloxane. 9. A sperm fixation microstructure platform used in the sperm fixation device according to any one of claims 1 to 8.