CN118109296B - Sperm fixing microstructure platform and device - Google Patents
Sperm fixing microstructure platform and device Download PDFInfo
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- CN118109296B CN118109296B CN202410337787.4A CN202410337787A CN118109296B CN 118109296 B CN118109296 B CN 118109296B CN 202410337787 A CN202410337787 A CN 202410337787A CN 118109296 B CN118109296 B CN 118109296B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/46—Means for fastening
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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Abstract
The invention provides a sperm fixing microstructure platform and a sperm fixing microstructure device, which can be applied to the technical fields of biomedical engineering, assisted reproduction and microfluidic. The sperm fixing device comprises an analysis cavity, wherein a sperm fixing microstructure platform is arranged in the analysis cavity and is used for capturing and fixing sperm; wherein the sperm fixing microstructure platform comprises a clamping groove and a guide groove; the width of the clamping groove is 4-6 mu m, and the clamping groove is used for accommodating and fixing the head of the sperm; the guiding groove is arranged in a horn shape, and the small end of the guiding groove is communicated with the clamping groove and is used for guiding the head of the sperm to enter the clamping groove and enabling the sperm flagellum to have a movable space. The sperm fixing microstructure platform and the sperm fixing microstructure device provided by the invention have the advantages of no damage, simplicity and convenience in operation, ordered distribution and the like, and are critical in fixing sperm and ensuring free swinging of sperm flagellum.
Description
Technical Field
The invention relates to the technical fields of biomedical engineering, assisted reproduction and microfluidics, in particular to a sperm fixing microstructure platform and a sperm fixing microstructure device.
Background
The method captures and fixes single sperms from swimming group sperms so as to study, analyze and operate at the single sperm level, and has important value in the fields of biomedical engineering technology, assisted reproduction technology and other life medicine science and technology. For example, after sperm are fixed, flagella oscillation is analyzed, and oscillation waveform parameters are extracted and can be used for sperm quality assessment.
However, the existing sperm capturing and fixing technology has the problems of flagella restraint, sperm injury, difficult operation, disordered distribution and the like, so that a plurality of limitations exist in practical application.
Disclosure of Invention
In view of the foregoing, it is a primary object of the present invention to provide a sperm immobilization microstructural platform and apparatus. Through the structural design of the clamping groove and the guide groove, the sperm fixing device not only can effectively capture and fix sperm, but also can ensure the free swing of flagellum. In addition, the technical effects of no damage, simple and convenient operation, ordered distribution and the like are achieved.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
In a first aspect, the present invention provides a sperm immobilization apparatus, said sperm immobilization apparatus comprising an analysis chamber having a sperm immobilization microstructure platform disposed therein, said sperm immobilization microstructure platform being configured to capture and immobilize sperm; wherein, the sperm fixing microstructure platform comprises a clamping groove and a guiding groove; the width of the clamping groove is 4-6 mu m, and the clamping groove is used for accommodating and fixing the head of the sperm; the guiding groove is arranged in a horn shape, and the small end of the guiding groove is communicated with the clamping groove and used for guiding the head of the sperm to enter the clamping groove and enabling the sperm flagellum to have a movable space.
According to an embodiment of the present invention, the length of the clamping groove is 4 to 6 μm.
According to an embodiment of the present invention, the width of the opening position of the guide groove is 10 to 20 μm.
According to an embodiment of the present invention, the lengths of the card slot and the guide slot are 8 to 15 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.
According to an embodiment of the present invention, the width of the opening position of the guide groove is 15 μm.
According to an embodiment of the present invention, the lengths of the card slot and the guide slot are 12 μm in total.
According to the embodiment of the invention, the depth of the clamping groove and the guide groove is the same and is 2-5 μm.
According to the embodiment of the invention, the sperm fixing microstructure platform is a cylinder or a platform body, and the height is 10-200 mu m.
According to an embodiment of the present invention, the sperm fixing microstructure platform includes a plurality of the clamping grooves and the guiding grooves, and ends of the clamping grooves are communicated through a connecting channel.
According to an embodiment of the present invention, the connecting channel has a width of 0.5 to 2 μm and a depth of 2 to 5 μm.
According to the embodiment of the invention, a plurality of sperm fixing microstructure platforms are arranged in the analysis cavity, and the sperm fixing microstructure platforms are arranged in an array.
According to an embodiment of the invention, the analysis chamber is provided with a sample inlet through which sperm enter the analysis chamber.
According to an embodiment of the invention, the analysis chamber is provided with a panel made of transparent material through which sperm are observed and analyzed.
According to the embodiment of the invention, the cavity of the analysis cavity and the material of the sperm fixing microstructure platform are both polydimethylsiloxane.
In another aspect, the invention provides a sperm immobilization microstructure platform for use in a sperm immobilization apparatus as described above.
According to the embodiment of the invention, the sperm fixing device comprises an analysis cavity, wherein a sperm fixing microstructure platform is arranged in the analysis cavity and is used for capturing and fixing sperm. The sperm fixing microstructure platform is provided with grooves, each groove comprises a clamping groove and a guide groove, the width of each clamping groove is limited by the microfluidic technology, so that the sperm fixing microstructure platform can just accommodate one sperm head, and the sperm can only move forward and not retract once being bumped into the clamping groove, so that the sperm is not easy to separate, and the capturing and fixing of the sperm are realized. The shape of the guide groove is horn-shaped, so that sperm can be guided into the clamping groove, and the efficiency of capturing and fixing sperm is improved; meanwhile, the horn-shaped design avoids the constraint of the guide groove on the sperm flagellum, so that the sperm flagellum can keep swinging similar to the swinging in a free swimming state. Under the guidance of the guide groove, the sperm head is sunk into the clamping groove, but sperm flagellum is positioned outside the clamping groove, namely the guide groove and the area outside the guide groove, and has a relatively wide movable space, so that the flagellum can freely swing without constraint. Furthermore, the device is completely harmless to sperm, i.e. is non-destructive, since only the structural steric hindrance of the clamping groove is relied upon to immobilize the sperm. Meanwhile, the device is convenient to operate, and can start observation and analysis only by waiting for a moment after sample addition and capturing and fixing enough sperms. In addition, the microstructure platform is used as a capturing site, so that the position of capturing the fixed sperms is orderly and controllable, the inter-sperm interference caused by uncontrolled scattering and disorder and staggered overlapping distribution is avoided, and the fact that the effective data volume is not broken is ensured.
According to the embodiment of the invention, the sperm fixing device provided by the invention has the advantages of no damage, simple operation, ordered distribution and the like, and has the key that the free swinging of the flagellum without restraint can be ensured while the sperm is captured and fixed.
A salient feature and advantage of embodiments of the present invention is the ability to secure free swinging of sperm flagella while immobilizing sperm. On one hand, the method can enable the flagellum imaging not to be interfered by a fixed structure, is favorable for image recognition and automatic analysis, and can realize automatic recognition of most of flagellum and extraction of corresponding parameters. On the other hand, since the swinging of the flagella is not influenced by the structure, the result obtained by analysis can reflect the flagella behavior of the sperm in a free swimming state, such as the flagella state of the sperm in the swimming state in vivo, which has a certain reference value in auxiliary reproduction.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent from the following description of embodiments of the present invention with reference to the accompanying drawings, in which:
FIG. 1 is a schematic diagram of a sperm immobilization device according to an embodiment of the present invention, wherein (a) is a schematic diagram of the sperm immobilization device, (b) is a schematic diagram of a sperm immobilization micro-structure platform array after immobilizing sperm, and (c) is a schematic diagram of a sperm immobilization micro-structure platform after immobilizing sperm;
FIG. 2 is a top view of a sperm immobilization microstructured platform provided in accordance with an embodiment of the present invention;
FIG. 3 is a photograph of sperm transient with fixed sperm head (white lines superimposed on the portion of flagella successfully identified by the procedure) provided in test example 1 of the present invention; and
FIG. 4 is sperm flagella fluctuation data provided in test example 2 of the present invention, wherein (a) is a superposition of flagella shapes at different times, and (b) is a change of curvature of different positions of flagella with time;
The figures are marked as follows: 100 is an analysis chamber; 210 is a first sample inlet; 220 is a second sample inlet; 300 is a sperm immobilization microstructural platform array; 310 is a sperm immobilization microstructural platform; 311 is a clamping groove; 312 is a guide groove; 313 is a connection channel; 400 is a faceplate of the analysis chamber.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent.
The method captures and fixes single sperms from the swimming group sperms so as to study, analyze and operate at the single sperm level, and has important value in the fields of biomedical engineering, assisted reproduction technology and the like. For example, after sperm are fixed, flagella oscillation is analyzed, and oscillation waveform parameters are extracted and can be used for sperm quality assessment.
In related examples, micropipette technology may be used for single sperm immobilization, but is complex to operate and less efficient. The surface adhesion method can be used for fixing the sperms on the surface, but the sperms are randomly distributed, so that the condition that a plurality of sperms are crowded and disturbed is common, and the analysis and the operation are not facilitated. The micro-imprinting method can be used for obtaining a patterned adhesion array and controlling the distribution of adhesion sperms, however, the chemical treatment is complex, time-consuming and incompatible with some sperm biochemical analysis methods. The optical tweezers and the high-frequency electric field device can be used for capturing single sperms, however, the focused light beam and the high-intensity electric field have potential damage to the sperms, the device is complex, the cost is high, the efficiency is low, and the problems are unfavorable for the application of the device in the fields of reproductive medicine and the like.
The microfluidic technology has the advantages of low consumption of sample reagents, high speed, high throughput, high integration level and the like. The development of microfluidic technology opens up a wide space in the fields of life sciences, medicine and the like. The capture of single sperm can be achieved by using a microfluidic chip. For example, sperm can be trapped by squeezing them into a specially-made small orifice "trap" using the pressure differential created by the channel flow field. In addition, by using the characteristic that the sperms tend to flow in a countercurrent way through an externally-applied flow field, the sperms can actively flow into a trap array and cannot escape, so that a large quantity of single sperms can be captured. However, the two microfluidic techniques described above have the common disadvantage that such "trap" structures, while confining sperm, also limit sperm flagellum oscillation. Especially when sperm is in a super-activated state after capacitation, flagella swing is larger and is more constrained by the 'trap' structure. On the one hand, since the flagella can contact with the vertical wall surface of the structure, the images are overlapped, so that the automatic analysis of the parameters of the flagella becomes difficult, and the parameters of the whole flagella are difficult to be completely extracted. On the other hand, even if the flagella parameters are successfully extracted, since the flagella swing is structurally limited, there is a gap from the swing in the natural swimming state, and the authenticity and the validity of the analysis result are difficult to be ensured. In addition, the two micro-fluidic technologies need to accurately control the flow field, and the operation difficulty is high.
In summary, the above related examples have the problems of flagellum restriction, sperm injury, difficult operation, disordered distribution and the like for capturing and fixing sperms, and have many limitations in practical application in the fields of biomedical engineering, assisted reproduction technology and the like.
In the process of realizing the invention, the microfluidic technology is utilized, and through the structural design of the clamping groove and the guide groove, the sperm fixing device solves the problems, has the advantages of no damage, simple operation and ordered distribution, and has the key that the flagellum can be ensured to freely swing without restraint while effectively capturing and fixing the sperm.
Specifically, according to an embodiment of the present invention, the present invention provides a sperm fixing apparatus, including an analysis chamber, in which a sperm fixing microstructure platform is disposed, the sperm fixing microstructure platform being used for capturing and fixing sperm; wherein the sperm fixing microstructure platform comprises a clamping groove and a guide groove; the width of the clamping groove is 4-6 mu m, and the clamping groove is used for accommodating and fixing the head of the sperm; the guiding groove is arranged in a horn shape, and the small end of the guiding groove is communicated with the clamping groove and is used for guiding the head of the sperm to enter the clamping groove and enabling the sperm flagellum to have a movable space.
According to the embodiment of the application, the number of the sperm fixing microstructure platforms in the analysis cavity can be one or a plurality of sperm fixing microstructure platforms, and the number of the sperm fixing microstructure platforms in the analysis cavity is not limited. When the number of the sperm fixing micro-structure platforms in the analysis cavity is a plurality, the sperm fixing micro-structure platforms can be arranged in an array.
According to the embodiment of the invention, the number of the clamping grooves and the guide grooves can be one or a plurality of. The number of the clamping grooves and the guide grooves is the same, the two are in one-to-one correspondence. At the communicating part of the clamping groove and the guiding groove, the widths of the clamping groove and the guiding groove are the same, and one clamping groove and one guiding groove form a group of sperm fixing microstructures.
According to the embodiment of the invention, the width of the clamping groove can be fixed or gradually increased from inside to outside, and the shape of the clamping groove is not limited.
According to the embodiment of the invention, after the sperm is injected into the analysis cavity, the sperm head firstly enters the guide groove, and the sperm head enters the clamping groove under the guidance of the guide groove because the sperm moves forward only and cannot move backward. By arranging the shape of the guide groove in a horn shape, the sperm can be guided into the clamping groove, and meanwhile, the constraint on sperm flagella is reduced, so that the sperm flagella can keep swinging similar to a free swimming state.
According to the embodiment of the invention, by utilizing the microfluidic technology, the width of the clamping groove is limited, so that the clamping groove can just accommodate one sperm head, and the clamping groove is relatively fit with the sperm head in shape, and as the swimming mode of the sperm can only advance and can not retreat, once the sperm collides into the clamping groove, the sperm is not easy to separate, thereby realizing the capturing and fixing of the sperm.
According to the embodiment of the invention, under the guidance of the guide groove, the sperm head is sunk into the clamping groove, but sperm flagellum is positioned outside the clamping groove, namely the guide groove and the area outside the guide groove, so that a wider movable space is provided, and the flagellum can freely swing without constraint. On one hand, the method can enable the flagellum imaging not to be interfered by a fixed structure, is favorable for image recognition and automatic analysis, and can realize automatic recognition of most of flagellum and extraction of corresponding parameters. On the other hand, since the swinging of the flagella is not influenced by the structure, the result obtained by analysis can reflect the flagella behavior of the sperm in a free swimming state, such as the flagella state of the sperm in the swimming state in vivo, which has a certain reference value in auxiliary reproduction.
According to the embodiment of the invention, the sperm fixing device is used for fixing sperm only by virtue of the structural steric hindrance of the clamping groove, so that the sperm fixing device has no damage to the sperm and no damage; after sample addition and waiting for a moment, the sufficient number of sperms can be captured and immobilized, so that observation and analysis can be started, and the operation is more convenient; the captured and fixed sperms are all positioned on the microstructure platform, so that overlapping and disordered sperms can not occur, the condition that effective data quantity is influenced due to mutual interference is avoided, and the method has order.
According to an embodiment of the invention, the length of the clamping groove 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 clamping groove is 5-6 μm.
According to an embodiment of the present invention, the width of the opening position of the guide groove is 10 to 20 μm. Specifically, the width of the opening position of the guide groove 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 opening position of the guide groove is 13 to 17 μm.
According to an embodiment of the present invention, the lengths of the card slot and the guide slot are 8 to 15 μm in total. Specifically, the lengths of the card slot and the guide slot are 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm or 15 μm in total. Preferably, the length of the clamping groove and the guide groove is 10-13 μm in total.
According to the embodiment of the invention, the width of the clamping groove is 5 mu m, and the length is 5 mu m; the width of the opening position of the guide groove was 15 μm. The length of the clamping groove and the guide groove is 12 μm in total.
According to the embodiment of the invention, the depth of the clamping groove and the depth of the guide groove are the same and are 2-5 mu m. Specifically, the depth of the card slot and the guide slot may be 2 μm, 3 μm, 4 μm or 5 μm. Preferably, the depth of the clamping groove and the guide groove is 4-5 μm.
According to the embodiment of the invention, the size of the clamping groove is limited, so that the clamping groove is beneficial to capturing and fixing sperms; by limiting the dimensions of the guide slot, the active space of sperm flagellum is ensured.
According to the embodiment of the invention, the sperm fixing microstructure platform is a cylinder or a platform body, and the height is 10-200 mu m. In particular, the sperm immobilization microstructure platform may have a height of 10 μm, 20 μm, 30 μm, 50 μm, 70 μm, 100 μm, 120 μm, 150 μm, 180 μm, 200 μm. Preferably, the sperm immobilization microstructure platform may have a height of 40 to 90 μm.
According to embodiments of the present invention, the sperm immobilization microstructure platform may be a cylinder, prism, truncated cone, pyramid, or the like.
According to the embodiment of the invention, the sperm fixing microstructure platform comprises a plurality of clamping grooves and guide grooves, and the ends of the clamping grooves are communicated through a connecting channel; the width of the connecting channel is 0.5-2 μm, and the depth is 2-5 μm.
According to embodiments 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. The depth of the connecting channel may be 2 μm, 2.5 μm, 3 μm, 4 μm, 4.5 μm or 5 μm.
According to the embodiment of the invention, the plurality of clamping grooves and the plurality of guide grooves are uniformly distributed on the sperm fixing microstructure platform.
According to the embodiment of the invention, the end parts of the clamping grooves can be communicated through the connecting channels, and the connecting channels are capillary channels, so that residual bubbles caused by the fact that liquid cannot be filled can be prevented; at the same time the width of the channel is smaller than the sperm head, thus allowing only liquid to pass through without allowing sperm to escape.
For example, the sperm immobilization microstructure platform may comprise 4 clamping grooves and 4 guiding grooves, forming 4 sets of sperm immobilization microstructures, and the 4 sets of sperm immobilization microstructures may form a quadruple platform orthogonally surrounded by the ends of the clamping grooves. At this point, each sperm immobilization microstructure platform may capture and immobilize 4 sperm.
According to the embodiment of the invention, a plurality of sperm fixing microstructure platforms are arranged in the analysis cavity and are arranged in an array.
According to the embodiment of the invention, the invention can fix sperms in a high-flux and orderly manner. The sperm fixing microstructure platform arranged in an array can enable the structure of the fixed sperm to be arranged in a compact array, so that the sperm can be captured in a large quantity, the operation is simple, and the high-flux single sperm analysis and operation are realized. For example, the sperm fixing device provided by the invention can be used for conventional and rapid screening of sperms in the medical field.
According to the embodiment of the invention, the microstructure array is orderly arranged and reasonably distributed, and sperms are not mutually influenced, so that the method has the advantages of strong experimental controllability, large effective data volume, higher analysis efficiency and the like.
According to an embodiment of the invention, the analysis chamber is provided with a sample inlet through which sperm enter the analysis chamber.
According to an embodiment of the invention, the analysis chamber is provided with a panel made of transparent material through which the morphology of the sperm is seen.
According to embodiments of the present invention, the end of the sperm immobilization microstructural platform where the clamping groove and the guide groove are located may be in contact with the faceplate.
According to the embodiment of the invention, after the sperms are injected into the analysis cavity through the sample inlet, the sperms enter the clamping groove through the guide of the guide groove, and at the moment, the state of the sperms can be observed through the panel made of the transparent material, so that the quality assessment of the sperms and the like can be conveniently carried out.
According to the embodiment of the invention, the cavity of the analysis cavity and the sperm fixing microstructure platform are made of polydimethylsiloxane.
According to embodiments of the present invention, sperm fixation devices provided herein may be prepared based on soft lithography techniques.
According to an embodiment of the present invention, a method of preparing a sperm immobilization device may include:
S1, drawing a channel pattern in two layers, wherein the clamping groove, the guide groove and the connecting channel area are the first layer, and the other areas are the second layer.
S2, using a silicon wafer as a substrate, using photoresist as an adhesion pattern substrate, and executing a photoetching process twice to obtain a male die.
According to the embodiment of the invention, each photoetching process comprises the steps of spin coating, pre-baking, exposure, post-baking, development, hard baking and the like. The two times of photoetching are respectively aimed at the first layer and the second layer of the pattern.
S3, fully mixing the polydimethylsiloxane prepolymer and the curing agent, pouring the mixture on a male die obtained by photoetching, performing thermal curing in an oven, cooling, taking down a polydimethylsiloxane analysis cavity from the male die, punching at a sample inlet position, and attaching the analysis cavity on a panel made of transparent materials to obtain the sperm fixing device.
According to an embodiment of the present invention, the polydimethylsiloxane prepolymer and the curing agent may be combined in an amount of 10:1, after removing bubbles in vacuum, pouring the mixture on a male die obtained by photoetching, and performing thermal curing in an oven at 80 ℃ for 2 hours. Taking out from the oven, cooling to room temperature, removing the polydimethylsiloxane prepolymer analysis cavity from the male mold, punching holes at the position of a sample inlet of the polydimethylsiloxane prepolymer analysis cavity by using a flat head needle, and attaching a channel to a cover glass to finish the manufacturing of the device.
The invention also provides a sperm immobilization microstructure platform for the sperm immobilization device according to the embodiment of the invention.
According to the embodiment of the invention, the sperm flagellum can be ensured to swing freely while fixing sperm. On one hand, the method can enable the flagellum imaging not to be interfered by a fixed structure, is favorable for image recognition and automatic analysis, and can realize automatic recognition of most of flagellum and extraction of corresponding parameters. On the other hand, since the swinging of the flagella is not influenced by the structure, the result obtained by analysis can reflect the flagella behavior of the sperm in a free swimming state, such as the flagella state of the sperm in the swimming state in vivo, which has a certain reference value in auxiliary reproduction.
According to the embodiment of the invention, the sperm fixing device provided by the invention can realize the damage-free sperm and is simple and quick to operate. Experiments show that the sperm fixed by the sperm fixing device has no significant change of various motility parameters within 2 hours, and the sperm can keep higher activity for a long time. The advantage of non-invasive fixation is more obvious in medical related applications such as assisted reproduction. Meanwhile, compared with other microfluidic devices related to sperm fixation, the device is simpler and more convenient to operate. For example, no additional flow field control is needed, and the analysis can be observed by a mirror by simply injecting liquid once. In addition, the site controllability of sperm capturing is realized, the ordered distribution of sperms can be ensured, and the conditions of disordered distribution and mutual interference of sperms are avoided.
According to the embodiment of the invention, in general, the sperm fixing microstructure platform and the sperm fixing microstructure platform device have the advantages of no damage, simple operation, ordered distribution, high flux and the like, and are critical in fixing sperm and ensuring free swinging of sperm flagellum.
The following describes the technical solution in the embodiment of the present invention in detail with reference to the drawings in the embodiment of the present invention. It should be noted that the following specific embodiments are only examples and are not intended to limit the present invention.
As shown in fig. 1, the sperm fixing apparatus includes an analysis chamber 100, a sample inlet 210 and a sample inlet 220 provided on the analysis chamber, a sperm fixing microstructure platform array 300 in the analysis chamber, and a panel 400 provided on the analysis chamber, wherein the sperm fixing microstructure platform array 300 includes a sperm fixing microstructure platform 310, and the sperm fixing microstructure platform 310 is a square column. Wherein, the analysis chamber 100 and the sperm fixing microstructure platform 300 are made of polydimethylsiloxane, and the panel 400 is a cover glass. In operation, a sperm sample is injected into the analysis chamber 100 from any sample inlet, the sperm is captured and immobilized by the sperm immobilization microstructure platform array 300, and at this time, the sperm can be observed and analyzed through a panel made of a transparent material, so that the sperm quality assessment and the like are facilitated.
As shown in fig. 2, sperm immobilization microstructure platform 310 comprises 4 sets of sperm immobilization microstructures, each set comprising a card slot 311, a guide slot 312. The sperm immobilization micro-structural platform 310 had a length and width of 33 μm and a height of 60 μm; the clamping groove is used for accommodating and fixing the head of the sperm, the width of the clamping groove is 5 mu m, and the length of the clamping groove is 5 mu m; the guide groove is horn-shaped, and the small end of the horn-shaped guide groove is communicated with the clamping groove and used for guiding the head of the sperm to enter the clamping groove and enabling the tail of the sperm to have sufficient movable space, the width of the opening position of the guide groove is 15 mu m, the sum of the lengths of the clamping groove and the guide groove is 12 mu m, and the depth of the clamping groove and the guide groove is 5 mu m. The ends of the 4 clamping grooves are communicated through the connecting channel 313, and the sperm fixing microstructure of 4 groups is orthogonally arranged to form a sperm fixing microstructure platform, at this time, 4 sperm can be captured and fixed by the sperm fixing microstructure platform 310. Wherein the width of the connection channel 313 is 1 μm and the depth of the connection channel 313 is 5 μm.
Test example 1
Sperm quality assessment
Sperm quality assessment is of great importance in the field of reproductive medicine and animal breeding. A conventional Computer-aided sperm analysis system (Computer-AIDED SPERM ANALYSIS, CASA) extracts parameters from the swimming track of a sperm population to assess sperm sample quality. However, since sperm motility is essentially dependent on flagella oscillation, sperm oscillation is directly analyzed, and the sperm motility is more deeply reflected in the motility characteristics of sperm than in the case of extracting downstream parameters from sperm motility by CASA, thereby more truly and effectively evaluating sperm quality.
The sperm immobilizing device provided by the invention can be used for high-throughput analysis of flagella swing of sperm, and is used for evaluating sperm sample quality, and specific examples are as follows:
Bovine sperm is easy to obtain, is similar to human sperm in morphology, size and exercise behavior, and is a classical and universal mode sperm in basic experimental research and application technology development, so that the experimental example adopts bovine sperm for operation. A sample of bovine sperm is thoroughly mixed, 5 μl is aspirated, and injected into any sample inlet of the device. If the density of the sperm sample is low, the density of the viable sperm can be increased by a density gradient centrifugation treatment method and the like, and then the sample is sampled and injected into the device. After the device is full, the two sample inlets are sealed by grease so as to prevent flow fields from being generated in the subsequent main cavity and influence the observation and analysis. The device was placed on a heated stage of a microscope at 37 ℃ and after enough sperm were trapped in the sperm immobilization microstructure (about 5 minutes), the analysis was taken one by one. Each device has a microstructure of tens of thousands of sperm immobilized, so that almost all sperm are immobilized at last, and almost no free swimming sperm interfere with shooting analysis. Sperm shots were randomly selected as much as possible in different areas of the chamber. Combining the compromise time, data amount and data size, using a frame rate of 300 frames/second, a total of 50 sperm were photographed for each sperm, and figure 3 is a photograph of sperm transient with the sperm head immobilized (identified sperm flagella printed as white).
The application of the sperm fixing device provided by the invention is not limited to bovine sperm, but other species of sperm can be applied according to the flow, such as human sperm, other common livestock sperm (sheep sperm, pig sperm and the like), mouse sperm and the like.
Test example 2
Sperm chemotaxis and other characteristic experimental study
In mammals, the process of meeting sperm and eggs is extremely complex, requiring the genital tract to provide a series of guidance mechanisms to help the high quality sperm successfully find the egg and ultimately achieve fertilization. Chemotactic mechanism is an important short-range guiding mechanism, and sperm can sense the direction of an ovum by sensing a specific chemical gradient. However, the chemotactic response mechanism of mammalian sperm has been under-studied in the related art, and particularly the input-output response characteristics of chemotactic systems have been relatively rarely studied.
By using the sperm fixing device provided by the invention, the input-output response characteristics of sperms to chemical substances, such as basic responses of impulse response, step response and the like, can be studied.
The following is illustrative:
In the same manner as in test example 1, bovine sperm was used in this test example. Sperm cells were injected and sealed into the sperm cell apparatus as described in test example 1. The ultraviolet converging laser is used for irradiating the light release type progesterone complex (1 mu M), the concentration of the progesterone in the environment near an irradiation point is regulated and controlled by regulating the light intensity and the irradiation time length, and the corresponding change of the sperm flagellum waveform is photographed and analyzed. Each sperm was tested 5 responses, a total of 100 sperm, and figure 4 is sperm fluctuation data. And (5) counting the change of each parameter of the sperm flagella waveform in a frequency domain, and finally obtaining the input-output characteristic of the sperm to the concentration of the progesterone.
The sperm fixing apparatus provided by the invention is not limited to the study of chemotactic response characteristics of bovine sperm, but can also be used for other response characteristics of sperm of other species (as described in test example 1), such as a chemotactic response, a super-activation response and the like.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (9)
1. The sperm fixing device is characterized by comprising an analysis cavity, wherein a sperm fixing microstructure platform is arranged in the analysis cavity and used for capturing and fixing sperm;
Wherein the sperm fixing microstructure platform comprises a clamping groove and a guide groove;
the width of the clamping groove is 4-6 mu m, and the length of the clamping groove is 4-6 mu m and is used for accommodating and fixing the head of a sperm;
the guiding groove is arranged in a horn shape, and the small end of the guiding groove in a horn shape is communicated with the clamping groove and is used for guiding the head of the sperm to enter the clamping groove and enabling the sperm flagellum to have a movable space;
The width of the opening position of the guide groove is 13-17 mu m;
The total length of the clamping groove and the guide groove is 8-15 mu m.
2. The sperm immobilization apparatus of claim 1, wherein said card slot has a width of 5 μm and a length of 5 μm;
the width of the opening position of the guide groove is 15 mu m;
the length of the clamping groove and the guide groove is 12 μm in total.
3. The sperm fixing apparatus as claimed in claim 1, wherein said clamping grooves and said guiding grooves are of the same depth of 2-5 μm.
4. The sperm immobilization apparatus of claim 1, wherein the sperm immobilization microstructure platform is a cylinder or a platform having a height of 10-200 μm.
5. The sperm immobilization apparatus of claim 1, wherein said sperm immobilization microstructural platform comprises a plurality of said card slots and said guide slots, ends of said plurality of card slots communicating through a connecting channel;
the width of the connecting channel is 0.5-2 mu m, and the depth is 2-5 mu m.
6. The sperm immobilization apparatus of claim 1, wherein a plurality of said sperm immobilization microstructural platforms are disposed within said analysis chamber, said plurality of sperm immobilization microstructural platforms being arranged in an array.
7. The sperm immobilization apparatus of claim 1, wherein said analysis chamber is provided with a sample inlet through which sperm enters said analysis chamber;
The analysis chamber is provided with a panel made of transparent material through which sperm are observed and analyzed.
8. The sperm immobilization apparatus of any one of claims 1 to 7, wherein the cavity of the analysis chamber and the sperm immobilization microstructure platform are both polydimethylsiloxane.
9. A sperm immobilization microstructural platform for use in a sperm immobilization device as defined in any one of claims 1 to 8.
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| US9340762B2 (en) * | 2010-08-20 | 2016-05-17 | Yu Sun | Method for automated sperm manipulation and device for holding sperm and oocytes |
| AU2014353050B2 (en) * | 2013-11-20 | 2019-10-03 | Brigham And Women's Hospital, Inc. | System and method for sperm sorting |
| US11491485B2 (en) * | 2018-04-09 | 2022-11-08 | Cornell University | Rheotaxis-based separation of motile sperm and bacteria using a microfluidic corral system |
| CN111019805B (en) * | 2019-12-20 | 2023-01-24 | 南通大学 | Microfluidic chip device for fixing single cell and performing medical analysis in situ and application thereof |
| US20230321659A1 (en) * | 2022-04-11 | 2023-10-12 | Massachusetts Institute Of Technology | Microfluidic chip for high-throughput cell pairing and fusion |
| CN117470842A (en) * | 2023-10-23 | 2024-01-30 | 中国科学院遗传与发育生物学研究所 | Sperm posture and movement state control method |
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