CN110551618B - A sperm sorting device - Google Patents
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- CN110551618B CN110551618B CN201910923820.0A CN201910923820A CN110551618B CN 110551618 B CN110551618 B CN 110551618B CN 201910923820 A CN201910923820 A CN 201910923820A CN 110551618 B CN110551618 B CN 110551618B
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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
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- Bioinformatics & Cheminformatics (AREA)
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Abstract
本发明属于微纳加工和生命科学相结合的交叉领域，尤其涉及一种精子分选装置。本发明提供的精子分选装置包括：精液腔，所述精液腔被水平设置的PDMS通孔膜分隔为上下两个腔室，下腔室为精液储存腔室，上腔室为精液回收腔室；与所述精液储存腔室相连通的精液流入通道；所述PDMS通孔膜采用微纳加工制成。本发明提供的精子分选装置所采用的过滤膜为利用微纳加工工艺制成的PDMS通孔膜，该方法制成的PDMS通孔膜的厚度、孔径、均一性与孔隙率都高度可控，从而使本发明提供的精子分选装置可通过提升其所设置的PDMS通孔膜孔隙率的方式，实现精子回收效率的大幅增加。
The invention belongs to the cross field of combining micro-nano processing and life science, and particularly relates to a sperm sorting device. The sperm sorting device provided by the present invention comprises: a semen cavity, the semen cavity is divided into two upper and lower chambers by a PDMS through-hole membrane arranged horizontally, the lower chamber is a semen storage chamber, and the upper chamber is a semen recovery chamber a semen inflow channel communicated with the semen storage chamber; the PDMS through-hole film is made by micro-nano processing. The filter membrane used in the sperm sorting device provided by the present invention is a PDMS through-hole membrane made by using a micro-nano processing technology. The thickness, pore size, uniformity and porosity of the PDMS through-hole membrane prepared by this method are highly controllable. , so that the sperm sorting device provided by the present invention can achieve a substantial increase in sperm recovery efficiency by increasing the porosity of the PDMS through-hole membrane set therein.
Description
Technical Field
The invention belongs to the crossing field of micro-nano processing and life science, and particularly relates to a sperm sorting device.
Background
According to the 'Chinese infertility status report' in 2010, the incidence rates of infertility of both men and women in one year, two years and more than ten years are respectively 10%, 15% and 25%, which are close to the average infertility rate of 15-20% in developed countries, and the couple who can not be bred is in a trend of youngization, the diagnosis age is 23 years, and the maximum age is 40 years. The main cause of male infertility is low sperm count, often associated with low sperm motility and impaired sperm function, resulting in an inability to naturally fertilize oocytes. Of all available Assisted Reproductive Technologies (ARTs), intracytoplasmic sperm injection (ICSI) has become a powerful tool for managing male infertility. One of the major challenges in successfully performing ICSI is the selection of highly motile sperm, as sperm quality directly determines whether it will fertilize the oocyte and ultimately lead to a viable delivery of the fertilized ovum. Therefore, techniques that help identify and select sperm with high motility will increase ART success rates.
Traditional sperm selection techniques, such as the upstream method and Percoll gradient centrifugation, have been widely used to separate highly motile sperm from the rest of the sample. However, none of these techniques is optimal: the upstream methods do not provide high yields of motile sperm and the centrifugation process can be detrimental to sperm. Conventional techniques separate highly motile sperm from semen samples with low sperm count (oligospermia), low sperm motility (oligospermia), which is the most common cause of male infertility, and therefore semen samples used for ICSI procedures are likely to be oligospermia, or both. Therefore, there is a need to develop new techniques that can be used in a clinical setting to improve the efficiency of motile sperm sorting.
For a sperm sorting device, the selection of the middle layer filtering membrane is crucial, and the size and the porosity of the membrane pores of the filtering membrane directly influence various key indexes such as the activity and the recovery rate of sorted sperm. However, the commercial polycarbonate filtering membranes adopted by the traditional sperm sorting device have poor preparation controllability, non-uniform pore size, non-uniform pore distribution and low porosity, and the sorting effect of the sperm sorting device is seriously influenced.
Disclosure of Invention
In view of the above, the invention aims to provide a sperm sorting device, wherein a filtering membrane arranged in the sperm sorting device is a polydimethylsiloxane through-hole membrane prepared by adopting a micro-nano processing mode, the membrane has good uniformity, and the pore diameter and the porosity can be randomly regulated and controlled according to needs; the sperm sorting device provided by the invention can greatly increase the sperm recovery efficiency by improving the porosity of the polydimethylsiloxane through-hole membrane arranged on the sperm sorting device.
The invention provides a sperm sorting device, comprising:
the semen cavity is divided into an upper cavity and a lower cavity by a polydimethylsiloxane through hole membrane which is horizontally arranged, the lower cavity is a semen storage cavity, and the upper cavity is a semen recovery cavity;
a semen inflow channel communicating with the semen storage chamber;
the polydimethylsiloxane through hole membrane is prepared by micro-nano processing.
Preferably, the pore diameter of the polydimethylsiloxane through-hole membrane is 5-20 μm; the hole pitch of the polydimethylsiloxane through hole film is 7-20 microns; the thickness of the polydimethylsiloxane through-hole film is 4-10 mu m.
Preferably, the method for preparing the polydimethylsiloxane through-hole membrane by adopting micro-nano processing comprises the following specific steps:
a) coating photoresist on a substrate, curing the photoresist layer, and performing flood exposure to obtain a broken-chain photoresist layer;
b) coating polydimethylsiloxane on the broken-chain photoresist layer, and curing to obtain a polydimethylsiloxane film layer;
c) plating metal on the polydimethylsiloxane film layer to obtain a metal film layer;
d) coating photoresist on the metal film layer, and carrying out exposure and development after the adhesive layer is cured to enable the adhesive layer to be patterned into a circular hole array;
e) and etching the metal film layer and the polydimethylsiloxane film layer in sequence, and then removing the metal film layer which is not etched to obtain the polydimethylsiloxane through-hole film.
Preferably, in step a), the photoresist comprises one or more of S1813 photoresist, AZ6112 photoresist and SPR220 photoresist.
Preferably, in the step b), the thickness of the coating is 4-10 μm.
Preferably, in step c), the metal film layer is an aluminum film layer; the thickness of the metal film layer is 150-500 nm.
Preferably, in step d), the photoresist comprises one or more of S1813 photoresist, AZ6112 photoresist and SPR220 photoresist.
Preferably, in the step e), the etching of the metal film layer is performed in an inductively coupled plasma enhanced reactive etching device; the polydimethylsiloxane film layer is etched in reactive ion etching equipment; the removing mode of the unetched metal film layer is wet etching.
Preferably, the device further comprises a chemotaxis sorting first channel and a chemotaxis sorting second channel which are respectively communicated with the semen recovery chamber;
cumulus cells are cultured in the chemotaxis sorting first channel; the chemotaxis sort second channel served as a control.
Preferably, the semen recovery device further comprises a temperature-gradient sorting first channel and a temperature-gradient sorting second channel which are respectively communicated with the semen recovery chamber, and a heating module;
the heating module is used for generating a temperature difference between the temperature-sensitive sorting first channel and the temperature-sensitive sorting second channel.
Compared with the prior art, the invention provides a sperm sorting device. The sperm sorting apparatus provided by the present invention comprises: the semen cavity is divided into an upper cavity and a lower cavity by a polydimethylsiloxane through hole membrane which is horizontally arranged, the lower cavity is a semen storage cavity, and the upper cavity is a semen recovery cavity; a semen inflow channel communicating with the semen storage chamber; the polydimethylsiloxane through hole membrane is prepared by micro-nano processing. In the invention, semen is firstly added from a semen inlet of a semen inflow channel, flows through the semen inflow channel and then enters a semen storage chamber, wherein the sperm with higher activity swims upwards against the gravity and passes through a polydimethylsiloxane through-hole membrane to enter a semen recovery chamber, thereby realizing the separation of the sperm with high activity. The filtering membrane adopted by the sperm sorting device provided by the invention is a Polydimethylsiloxane (PDMS) through-hole membrane prepared by a micro-nano processing technology, and the thickness, the aperture, the uniformity and the porosity of the PDMS through-hole membrane prepared by the method are highly controllable, so that more active sperms can be obtained in the sperm sorting process, and the sorting efficiency is improved. In addition, in the preferred technical scheme provided by the invention, the sperm sorting device is also integrated with a chemotaxis or thermotropic channel, so that the chemotaxis or chemotaxis optimization can be continuously carried out after the sperm is subjected to filtration membrane sorting, and the sperm with the highest quality can be obtained.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic view of a sperm cell sorting apparatus according to an embodiment of the present invention;
FIG. 2 is a schematic side view of a chemotactic sperm sorting device according to an embodiment of the present invention;
FIG. 3 is a schematic top view of a chemotactic sperm sorting device according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of a side view of a thermotropic sperm sorting device according to an embodiment of the present invention;
FIG. 5 is a schematic top view of a heat-tactic sperm sorting apparatus according to an embodiment of the present invention;
FIG. 6 is an SEM image of a PDMS film provided in example 1 of the present invention;
fig. 7 is a digital photograph of the semen inflow channel mold provided in example 2 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a sperm sorting device, comprising:
the semen cavity is divided into an upper cavity and a lower cavity by a polydimethylsiloxane through hole membrane which is horizontally arranged, the lower cavity is a semen storage cavity, and the upper cavity is a semen recovery cavity;
a semen inflow channel communicating with the semen storage chamber;
the polydimethylsiloxane through hole membrane is prepared by micro-nano processing.
Referring to fig. 1, fig. 1 is a schematic structural view of a sperm sorting apparatus according to an embodiment of the present invention. In fig. 1, 1 denotes a semen inlet, 2 denotes a semen inflow channel, 3 denotes a semen storage chamber, 4 denotes a polydimethylsiloxane through-hole membrane, 5 denotes a semen recovery chamber, 1-1 denotes a substrate, 1-2 denotes a first polymer layer, and 1-3 denotes a second polymer layer.
The sperm sorting device provided by the invention comprises a sperm cavity, wherein the sperm cavity is divided into an upper cavity and a lower cavity by a polydimethylsiloxane through-hole membrane 4 which is horizontally arranged, the lower cavity is a sperm storage cavity 3, and the upper cavity is a sperm recovery cavity 5. The polydimethylsiloxane through-hole membrane 4 is prepared by micro-nano processing, and the aperture, porosity and thickness of the polydimethylsiloxane through-hole membrane can be freely regulated and controlled according to needs. In one embodiment of the present invention, the pore size of the polydimethylsiloxane through-hole membrane 4 is preferably 5 to 20 μm, and specifically may be 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm. In one embodiment of the present invention, the hole pitch of the polydimethylsiloxane through hole film 4 is preferably 7 to 20 μm, and specifically may be 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm. In one embodiment of the present invention, the thickness of the polydimethylsiloxane through-hole film 4 is preferably 4 to 10 μm, and specifically may be 4 μm, 4.2 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, or 10 μm.
In the invention, the polydimethylsiloxane through-hole membrane 4 is prepared by micro-nano processing, and the specific preparation steps preferably comprise:
a) coating photoresist on a substrate, curing the photoresist layer, and performing flood exposure to obtain a broken-chain photoresist layer;
b) coating polydimethylsiloxane on the broken-chain photoresist layer, and curing to obtain a polydimethylsiloxane film layer;
c) plating metal on the polydimethylsiloxane film layer to obtain a metal film layer;
d) coating photoresist on the metal film layer, and carrying out exposure and development after the adhesive layer is cured to enable the adhesive layer to be patterned into a circular hole array;
e) and etching the metal film layer and the polydimethylsiloxane film layer in sequence, and then removing the metal film layer which is not etched to obtain the polydimethylsiloxane through-hole film.
In the preparation step of the polydimethylsiloxane through-hole film 4 provided by the invention, in the step a), the photoresist comprises one or more of but not limited to S1813 photoresist, AZ6112 photoresist and SPR220 photoresist; the coating mode is preferably spin coating, the rotation speed of the spin coating is preferably 3000-5000 rpm, specifically 3000rpm, 3500rpm, 4000rpm, 4500rpm or 5000rpm, the spin coating time is preferably 20-60 s, specifically 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s or 60 s; the thickness of the coating is preferably 0.5-2 μm, and specifically may be 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm or 2 μm; the curing mode is preferably baking, the baking temperature is preferably 105-130 ℃, specifically 105 ℃, 110 ℃, 115 ℃, 120 ℃, 125 ℃ or 130 ℃, and the baking time is preferably 60-120 s, specifically 60s, 70s, 80s, 90s or 100 s; the flood exposure is preferably carried out in an ultraviolet lithography machine of the type SUSS MA 6; the time of the flood exposure is preferably 7-12 s, and specifically may be 7s, 8s, 9s, 10s, 11s or 12 s.
In the preparation step of the polydimethylsiloxane through-hole membrane 4 provided by the invention, in the step b), the coated polydimethylsiloxane consists of a polydimethylsiloxane prepolymer and a curing agent, specifically SYLGARD 184 produced by Dow Corning can be selected, the product is a suit product consisting of a PDMS prepolymer and a curing agent, the PDMS prepolymer and the curing agent are uniformly mixed according to a proportion when the polydimethylsiloxane through-hole membrane is used, and the mass ratio of the PDMS prepolymer to the curing agent is preferably (5-15): 1, specifically 10: 1; the coating mode is preferably spin coating; the coating thickness is preferably 4-10 μm, and specifically can be 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm or 10 μm; the curing mode is preferably baking, the baking temperature is preferably 110-130 ℃, specifically 110 ℃, 115 ℃, 120 ℃, 125 ℃ or 130 ℃, and the baking time is preferably 15-40 min, specifically 15min, 20min, 25min, 30min, 35min or 40 min.
In the step of preparing the polydimethylsiloxane through-hole membrane 4 provided by the invention, in the step b), in order to improve the hydrophilicity of the surface of the glue layer and facilitate the coating of polydimethylsiloxane, preferably, before the polydimethylsiloxane is coated, the surface O is carried out on the broken-chain photoresist layer2And (4) carrying out plasma treatment. During the treatment, O2The flow rate is preferably 20-40 sccm, and specifically 30 sccm; the radio frequency power is preferably 20-40 w, and specifically can be 30 w; the treatment time is preferably 1-4 min, and specifically 2 min.
In the preparation step of the polydimethylsiloxane through-hole film 4 provided by the invention, in the step c), the plating mode is preferably electron beam evaporation, and the deposition rate of the electron beam evaporation is preferably 45-80A/s, and specifically can be 45A/s, 50A/s, 55A/s, 60A/s, 65A/s, 70A/s, 75A/s or 80A/s; the metal film layer is preferably an aluminum film layer; the thickness of the metal film layer is preferably 150-500 nm, and specifically can be 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm or 500 nm.
In the step of preparing the polydimethylsiloxane through-hole membrane 4 provided by the invention, in the step c), in order to improve the hydrophilicity of the surface of the polydimethylsiloxane membrane layer and increase the adhesion between the polydimethylsiloxane membrane layer and the metal coating, preferably, before plating, the polydimethylsiloxane membrane layer is subjected to surface O2And (4) carrying out plasma treatment. During the treatment, O2The flow rate is preferably 20-40 sccm, and specifically 30 sccm; the radio frequency power is preferably 100-300 w, and specifically can be 200 w; the treatment time is preferably 1-4 min, and specifically 2 min.
In the preparation step of the polydimethylsiloxane through-hole film 4 provided by the invention, in the step d), the photoresist comprises one or more of S1813 photoresist, AZ6112 photoresist and SPR220 photoresist; the coating mode is preferably spin coating, the rotation speed of the spin coating is preferably 2000-4000 rpm, specifically 2000rpm, 2500rpm, 3000rpm, 3500rpm or 4000rpm, the spin coating time is preferably 20-60 s, specifically 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s or 60 s; the thickness of the coating is preferably 0.5-2 μm, and specifically may be 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm or 2 μm; the curing mode is preferably baking, the baking temperature is preferably 50-80 ℃, specifically 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ or 80 ℃, and the baking time is preferably 60-120 s, specifically 60s, 70s, 80s, 90s, 100s, 110 s or 120 s; the exposure is preferably carried out in a uv lithography machine of the type SUSS MA 6; the exposure time is preferably 6-9 s, and specifically can be 6s, 6.5s, 7s, 7.5s, 8s, 8.5s or 9 s; the grade of the developing solution used for developing is preferably AZ-MIF-300; the developing time is preferably 40-60 s, and specifically can be 40s, 45s, 50s, 55s or 60 s.
In the preparation step of the polydimethylsiloxane through-hole membrane 4 provided by the invention, in the step e), the etching of the metal membrane layer is preferably carried out in an inductively coupled plasma enhanced reaction etching device, and the model of the device is preferably ICP 180; in the etching process, the used process gas is preferably Cl2HBr and BCl3Said Cl2The flow rate is preferably 5-20 sccm, specifically 5sccm, 10sccm, 15sccm or 20sccm, the flow rate of HBr is preferably 5-20 sccm, specifically 5sccm, 10sccm, 15sccm or 20sccm, and the BCl3The flow rate is preferably 20-40 sccm, and specifically can be 20sccm, 25sccm, 30sccm, 35 sccm or 40 sccm; the etching rate is preferably 4-5 nm/s, and specifically can be 4.45 mn/s.
In the step of preparing the polydimethylsiloxane through-hole membrane 4 provided by the invention, in the step e), the polydimethylsiloxane membrane layer is preferably etched in reactive ion etching equipment, and in the etching process, the used process gas is preferably O2And CF4Said O is2The flow rate is preferably 15-20 sccm, specifically 15sccm, 16sccm, 17sccm, 18sccm, 19sccm or 20sccm, and the CF4The flow rate of (C) is preferably 40 to 60sccm, which can be 40sccm, 45sccm, 50sccm, 55sccm or 60 sccm; the etching rate is preferably 0.2-0.3 mu m/min, and specifically can be 0.25 mu m/min.
In the preparation step of the polydimethylsiloxane through-hole film 4 provided by the invention, in the step e), the removing mode of the unetched metal film layer is preferably wet etching; the etching solution for wet etching is preferably a mixed solution of phosphoric acid, nitric acid and acetic acid.
In the invention, the sperm sorting device also comprises a semen inflow channel 2, wherein one end of the semen inflow channel 2 is opened to be used as a semen inlet 1, and the other end of the semen inflow channel is communicated with a semen storage chamber 3. When the sperms are sorted, the sperms are added from the semen inlet 1 of the semen inflow channel 2, flow through the semen inflow channel 2 and then enter the semen storage chamber 3.
In the present invention, the sperm sorting device is preferably further provided with a chemotaxis sorting channel, as shown in fig. 2 and 3, fig. 2 is a schematic side view of the chemotaxis sperm sorting device provided in the embodiment of the present invention, and fig. 3 is a schematic top view of the chemotaxis sperm sorting device provided in the embodiment of the present invention. In fig. 2 and 3, 1 denotes a semen inlet, 2 denotes a semen inflow channel, 3 denotes a semen storage chamber, 4 denotes a polydimethylsiloxane through-hole membrane, 5 denotes a semen recovery chamber, 6 denotes a chemotactic sorting channel outlet, 6-1 denotes a chemotactic sorting first channel, 6-2 denotes a chemotactic sorting second channel, 1-1 denotes a substrate, 1-2 denotes a first polymer layer, and 1-3 denotes a second polymer layer. 1-4 represent a third polymer layer. In the invention, the sperm sorting device provided with the chemotaxis sorting channel comprises a first chemotaxis sorting channel 6-1 and a second chemotaxis sorting channel 6-2, wherein one end openings of the first chemotaxis sorting channel 6-1 and the second chemotaxis sorting channel 6-2 are communicated with a semen recovery chamber 5, and the other end opening is used as an outlet 6 of the chemotaxis sorting channel; cumulus cells were cultured in the first channel 6-1 of chemotactic sorting, and the second channel 6-2 of chemotactic sorting was used as a control.
In the present invention, the sperm sorting device is preferably further provided with a thermotropic sorting channel, as shown in fig. 4 and 5, fig. 4 is a schematic side view of the thermotropic sperm sorting device according to the embodiment of the present invention, and fig. 5 is a schematic top view of the thermotropic sperm sorting device according to the embodiment of the present invention. In FIGS. 4 and 5, 1 denotes a semen inlet, 2 denotes a semen inflow channel, 3 denotes a semen storage chamber, 4 denotes a polydimethylsiloxane through-hole membrane, 5 denotes a semen recovery chamber, 7 denotes a thermotropic sorting channel outlet, 7-1 denotes a thermotropic sorting first channel, 7-2 denotes a thermotropic sorting second channel, 8 denotes a heating module, 1-1 denotes a substrate, 1-2 denotes a first polymer layer, and 1-3 denotes a second polymer layer. 1-4 represent a third polymer layer. In the present invention, the sperm sorting apparatus provided with the chemotaxis sorting channel includes a chemotaxis sorting first channel 7-1, a chemotaxis sorting second channel 7-2, and a heating module 8. One end opening of the first heat-taxiing sorting channel 7-1 and the second heat-taxiing sorting channel 7-2 is communicated with the semen recovery chamber 5, and the other end opening is used as an outlet 7 of the heat-taxiing sorting channel; the heating module 8 is used for generating a temperature difference between the temperature-sensitive sorting first channel 7-1 and the temperature-sensitive sorting second channel 7-2.
In the invention, the sperm sorting device can be prepared by a micro-nano processing technology, and by taking the sperm sorting device with the structure shown in fig. 1 as an example, the sperm sorting device can be specifically prepared according to the following steps:
firstly, preparing a polydimethylsiloxane through-hole membrane 4 by adopting a micro-nano processing technology;
then, processing the first polymerization layer 1-2 by adopting a micro-nano processing technology to form a semen inlet 1, a semen inflow channel 2 and a semen storage chamber 3; the material of the first polymeric layer 1-2 is preferably polydimethylsiloxane;
then, processing the second polymerization layer 1-3 by adopting a micro-nano processing technology to form a semen recovery chamber 5; the material of the second polymeric layers 1-3 is preferably polydimethylsiloxane;
next, bonding the first polymeric layer 1-2 formed with the semen inlet 1, the semen inflow channel 2 and the semen storage chamber 3 to the substrate 1-1, the substrate 1-1 preferably being made of glass;
and finally, bonding a polydimethylsiloxane through-hole membrane 4 to an opening right above the semen storage chamber 3 of the first polymerization layer 1-2, and aligning and bonding the semen recovery chamber 5 of the second polymerization layer 1-3 and the semen storage chamber 3 of the first polymerization layer 1-2 to obtain the sperm sorting device with the structure shown in the figure 1.
The filtering membrane adopted by the sperm sorting device provided by the invention is a Polydimethylsiloxane (PDMS) through-hole membrane prepared by a micro-nano processing technology, and the thickness, the aperture, the uniformity and the porosity of the PDMS through-hole membrane prepared by the method are highly controllable, so that more active sperms can be obtained in the sperm sorting process, and the sorting efficiency is improved. In addition, in the preferred technical scheme provided by the invention, the sperm sorting device is also integrated with a chemotaxis or thermotropic channel, so that the chemotaxis or chemotaxis optimization can be continuously carried out after the sperm is subjected to filtration membrane sorting, and the sperm with the highest quality can be obtained.
For the sake of clarity, the following examples are given in detail.
Example 1
Preparation of Polydimethylsiloxane (PDMS) via films comprising the following steps:
firstly, spin-coating S1813 photoresist on a silicon wafer substrate, wherein the spin-coating speed is 3000rpm, the spin-coating time is 40S, and the spin-coating thickness is 1 μm. Then, it was baked and cured at 115 ℃ for 90 seconds. And then, performing flood exposure by using an ultraviolet lithography machine (SUSS MA6) for 9s to obtain the photoresist layer with fully broken chains.
The prepared photoresist layer is subjected to surface O treatment on a photoresist remover2Plasma treatment, O2The flow rate is 30sccm, the RF power is 30w, and the processing time is 2 min. And then, spin-coating a PDMS film on the surface of the treated photoresist layer, wherein the spin-coated PDMS glue solution is SYLGARD 184 produced by Dow Corning, the product is a packaged product consisting of a PDMS prepolymer and a curing agent, the PDMS prepolymer and the curing agent are uniformly mixed according to the mass ratio of 10:1 when the spin-coating device is used, and the spin-coating thickness is 4.2-10 μm. And (3) after the PDMS is subjected to spin coating, baking and curing at 120 ℃ for 40 min.
The prepared PDMS film is subjected to surface O in Reactive Ion Etching (RIE) equipment2Plasma treatment, O2The flow rate is 30sccm, andthe frequency power is 200w, and the processing time is 2 min. And then, depositing an aluminum film layer on the surface of the treated PDMS film layer by adopting an electron beam evaporation mode, wherein the deposition rate is 60A/s, and the deposition thickness is 300 nm.
And spin-coating the surface of the aluminum film layer with the photoresist S1813 again, wherein the spin-coating speed is 3000rpm, the spin-coating time is 40S, and the spin-coating thickness is 1 μm. Then, it was baked at 65 ℃ for 90 seconds. And photoetching and patterning the photoresist layer to form a circular hole array, wherein the exposure time is 7.5s, the developing solution is AZ-MIF-300, and the developing time is 50 s.
Patterning a round hole array on the photoresist layer, and etching an aluminum film layer in inductively coupled plasma (ICP180) enhanced reactive etching (ICP180) with Cl as process gas2:10sccm，HBr:10sccm， BCl330 sccm; the etching rate was 4.45 nm/s. Then, it was placed in a Reactive Ion Etching (RIE) apparatus to etch the PDMS film with O as the process gas2:18sccm，CF450 sccm; the etching rate was 0.25 μm/min. Thereafter, the unetched aluminum film layer was removed using an etching solution containing 85mL of phosphoric acid (concentration 98 wt%), 25mL of nitric acid (concentration 98 wt%), and 10mL of acetic acid (concentration 100 wt%). And after the etching is finished, obtaining the PDMS film with the through hole structure.
Scanning Electron Microscope (SEM) observation of the PDMS film prepared in this example was performed, and as a result, fig. 6 is shown in fig. 6, and fig. 6 is an SEM image of the PDMS film provided in example 1 of the present invention. As can be seen from FIG. 6, the PDMS membrane prepared by this example has uniform pore size and pore distribution, 10 μm pore diameter, and 20 μm pore-to-pore distance.
Example 2
Sperm sorting device
This embodiment provides a sperm sorting apparatus having the structure shown in fig. 1, comprising: semen flows into the channel 2 and the semen lumen. Wherein the semen cavity is divided into an upper cavity and a lower cavity by a polydimethylsiloxane through-hole membrane 4 (prepared in embodiment 1) which is horizontally arranged, the lower cavity is a semen storage cavity 3, and the upper cavity is a semen recovery cavity 5; the semen inflow channel 2 has one end opening as a semen inlet 1 and the other end opening communicating with a semen storage chamber 3.
In the embodiment, the semen inlet 1, the semen inflow channel 2 and the semen storage chamber 3 are positioned in the first polymeric layer 1-2, and the material of the first polymeric layer 1-2 is polydimethylsiloxane; the semen recovery chamber 5 is positioned in the second polymeric layer 1-3, the material of the second polymeric layer 1-3 is polydimethylsiloxane; the first polymeric layer 1-2 is located on the substrate 1-1, and the substrate 1-1 is made of glass.
Preparation method of sperm sorting device
Preparing a semen inflow channel mold: spin-coating SU-8 photoresist on a four-inch silicon wafer substrate, wherein the photoresist is SU-83035, the spin-coating thickness is about 165 μm, the spin-coating rotation speed is 1300rpm, and the spin-coating time is 40 s; then baking at 65 deg.C for 5min, baking at 95 deg.C for 20min, and co-spin coating twice. And then carrying out exposure for 36s, baking at 95 ℃ for 4min after exposure, then developing for 12min, and finally hardening at 150 ℃ for 10min to obtain the semen inflow channel mold shown in fig. 7, wherein fig. 7 is a digital photo diagram of the semen inflow channel mold provided in embodiment 2 of the invention.
And pouring PDMS on the semen inflow channel mould, and demoulding to obtain the first polymerization layer 1-2 with the semen inflow channel 2. Then, holes are respectively drilled at the inlet end and the outlet end of the semen inflow channel 2 to respectively form a semen inlet 1 and a semen storage chamber 3; wherein, the inlet end has a punching aperture of 2mm, and the outlet end has a punching aperture of 10 mm.
PDMS is poured on the rectangular mould, and demoulding is carried out to obtain the second polymeric layer 1-3. Then, the second polymeric layer 1-3 is perforated to form a semen recovery chamber 5 with a perforation diameter of 10 mm.
The first polymeric layer 1-2 formed with the semen inlet 1, the semen inflow channel 2 and the semen storage chamber 3 is bonded to the substrate 1-1. Then, the PDMS film prepared in example 1 was bonded to the opening just above the semen storage chamber 3 of the first polymeric layer 1-2. Finally, the semen recovery chamber 5 of the second polymeric layer 1-3 is bonded in alignment with the semen storage chamber 3 of the first polymeric layer 1-2, resulting in the sperm sorting device of the structure shown in fig. 1.
In this embodiment, the bonding manner is oxygen plasma treatment of the bonding surface for 20s, and the bonding equipment is a plasma cleaning machine.
Example 3
Chemotaxis sperm sorting unit
This embodiment provides a sperm sorting apparatus having the structure shown in fig. 2 and 3, comprising: semen flows into channel 2, the semen lumen, chemotaxis sort first channel 6-1 and chemotaxis sort second channel 6-2. Wherein the semen cavity is divided into an upper cavity and a lower cavity by a polydimethylsiloxane through-hole membrane 4 (prepared in embodiment 1) which is horizontally arranged, the lower cavity is a semen storage cavity 3, and the upper cavity is a semen recovery cavity 5; an opening at one end of the semen inflow channel 2 is used as a semen inlet 1, and an opening at the other end is communicated with a semen storage chamber 3; one end openings of the first chemotaxis sorting channel 6-1 and the second chemotaxis sorting channel 6-2 are communicated with the semen recovery chamber 5, and the other end opening is used as an outlet 6 of the chemotaxis sorting channel; cumulus cells are cultured in the chemotaxis sorting first channel 6-1; chemotaxis sorting second lane 6-2 served as a control.
In the embodiment, the semen inlet 1, the semen inflow channel 2 and the semen storage chamber 3 are positioned in the first polymeric layer 1-2, and the material of the first polymeric layer 1-2 is polydimethylsiloxane; the semen recovery chamber 5, the chemotaxis sorting first channel 6-1 and the chemotaxis sorting second channel 6-2 are positioned in the second polymer layer 1-3, and the material of the second polymer layer 1-3 is polydimethylsiloxane; the third polymer layer 1-4 covers the opening right above the semen recovery chamber 5 and is used for sealing the semen recovery chamber 5, and the third polymer layer 1-4 is made of polydimethylsiloxane; the first polymeric layer 1-2 is located on the substrate 1-1, and the substrate 1-1 is made of glass.
Example 4
Chemotactic sperm sorting device
This embodiment provides a sperm sorting apparatus having the structure shown in fig. 4 and 5, comprising: the semen flows into the channel 2, the semen cavity, the first channel 7-1 for heat-taxing sorting, the second channel 7-2 for heat-taxing sorting and the heating module 8. Wherein the semen cavity is divided into an upper cavity and a lower cavity by a polydimethylsiloxane through-hole membrane 4 (prepared in embodiment 1) which is horizontally arranged, the lower cavity is a semen storage cavity 3, and the upper cavity is a semen recovery cavity 5; an opening at one end of the semen inflow channel 2 is used as a semen inlet 1, and an opening at the other end is communicated with a semen storage chamber 3; one end openings of the first heat-taxiing sorting channel 7-1 and the second heat-taxiing sorting channel 7-2 are communicated with the semen recovery chamber 5, and the other end openings are used as heat-taxiing sorting channel outlets 7; the heating module 8 is used for generating a temperature difference between the temperature-sensitive sorting first channel 7-1 and the temperature-sensitive sorting second channel 7-2.
In the embodiment, the semen inlet 1, the semen inflow channel 2 and the semen storage chamber 3 are positioned in the first polymeric layer 1-2, and the material of the first polymeric layer 1-2 is polydimethylsiloxane; the semen recovery chamber 5, the heat taxing sorting first channel 7-1, the heat taxing sorting second channel 7-2 and the heating module 8 are positioned in the second polymer layer 1-3, and the material of the second polymer layer 1-3 is polydimethylsiloxane; the third polymer layer 1-4 covers the opening right above the semen recovery chamber 5 and is used for sealing the semen recovery chamber 5, and the third polymer layer 1-4 is made of polydimethylsiloxane; the first polymeric layer 1-2 is located on the substrate 1-1, and the substrate 1-1 is made of glass.
Sperm sorting experiments
1) The experimental process comprises the following steps: an experiment was performed using the sperm sorting device prepared in example 2, which was first subjected to ultraviolet sterilization for half an hour, then 200 μ L of liquefied original sperm (activity rate 15%) was injected into the semen inlet, 200 μ L of HTF (Human Tube Fluid, shanghai constant distance biology) was injected from above the semen collection chamber, and then stored for 15min at 37 ℃, and then 2-5 μ L of semen was taken from above the semen collection chamber and placed into a disposable sperm counting cell (shanghai north-ang medical science and technology, ltd.), and sperm activity was detected using a fully automatic sperm analyzer (SAS-II, SAS).
2) The experimental results are as follows: the activity rate of the sorted sperms is improved from the original 15% to 87%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. A sperm sorting apparatus comprising:
the semen cavity is divided into an upper cavity and a lower cavity by a polydimethylsiloxane through hole membrane which is horizontally arranged, the lower cavity is a semen storage cavity, and the upper cavity is a semen recovery cavity;
a semen inflow channel communicating with the semen storage chamber;
the polydimethylsiloxane through-hole membrane has the aperture of 5-20 microns, the hole spacing of 7-20 microns and the thickness of 4-10 microns;
the polydimethylsiloxane through-hole membrane is prepared by micro-nano processing, and the specific preparation steps comprise:
a) coating photoresist on a substrate, curing the photoresist layer, and performing flood exposure to obtain a broken-chain photoresist layer;
b) coating polydimethylsiloxane on the broken-chain photoresist layer, and curing to obtain a polydimethylsiloxane film layer;
c) plating metal on the polydimethylsiloxane film layer to obtain a metal film layer;
d) coating photoresist on the metal film layer, and carrying out exposure and development after the adhesive layer is cured to enable the adhesive layer to be patterned into a circular hole array;
e) sequentially etching the metal film layer and the polydimethylsiloxane film layer, and then removing the metal film layer which is not etched to obtain the polydimethylsiloxane through-hole film;
in the step a), the photoresist comprises one or more of S1813 photoresist, AZ6112 photoresist and SPR220 photoresist;
in the step b), the thickness of the coating is 4-10 μm;
in the step c), the metal film layer is an aluminum film layer; the thickness of the metal film layer is 150-500 nm;
in the step d), the photoresist comprises one or more of S1813 photoresist, AZ6112 photoresist and SPR220 photoresist;
in the step e), etching the metal film layer is carried out in inductively coupled plasma enhanced reactive etching equipment; the polydimethylsiloxane film layer is etched in reactive ion etching equipment; the removing mode of the unetched metal film layer is wet etching.
2. The sperm sorting device of claim 1, further comprising a chemotaxis sorting first channel and a chemotaxis sorting second channel in communication with the semen recovery chamber, respectively;
cumulus cells are cultured in the chemotaxis sorting first channel; the chemotaxis sort second channel served as a control.
3. The sperm sorting device of claim 1, further comprising a temperature-sensitive sorting first channel and a temperature-sensitive sorting second channel in communication with the semen recovery chamber, respectively, and a heating module;
the heating module is used for generating a temperature difference between the temperature-sensitive sorting first channel and the temperature-sensitive sorting second channel.
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| WO2024007019A2 (en) * | 2022-07-01 | 2024-01-04 | DxNow, Inc. | Multi-well systems and methods for sorting sperm |
| WO2025137104A1 (en) * | 2023-12-22 | 2025-06-26 | Coopersurgical, Inc. | Multi-chamber systems and methods for sorting sperm |
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