Rapid sperm screening device based on electroosmotic flow and micro-column array manufacturing method

Abstract

The microfluidic device comprises a shell, a micro-column screening area, a positive electrode, a negative electrode and a cover plate, the positive electrode and the negative electrode are installed on the left side and the right side of the interior of the shell, the micro-column screening area is arranged between the positive electrode and the negative electrode, and the shell is covered with the cover plate; the micro-column screening area is composed of a micro-column array, and micro-channels for sperms to pass through are formed among micro-columns. The invention further discloses a manufacturing method of the micro-column array. The rapid sperm screening device is simple to operate. According to the invention, a micro-nano processing means is adopted to prepare a micro-channel environment for simulating sperm swimming of a microstructure, a high-viscosity buffer solution is adopted to simulate a body fluid environment for sperm swimming, and an electroosmotic flow method is adopted, so that a liquid sample moves from a positive electrode direction to a negative electrode direction, a sperm flowing environment is created in situ, and active sperm screening is accelerated.

Description

Technical Field

[0001] The present invention relates to the field of assisted reproduction, and in particular to a microfluidic device and a microcolumn array manufacturing method that can be used for sperm screening. Background Art

[0002] In the field of assisted reproduction, density gradient centrifugation or direct swim-up method is generally used to treat semen. Although both methods can obtain some sperm with high vitality, this method sometimes damages the integrity of sperm genes. For example, the sperm screening device disclosed in Chinese Patent Publication No. CN211445700U includes a box body, a box cover is provided at the upper end of the box body, a vertically slidable support plate is provided in the box body, and vertical plates are provided on the left and right sides of the support plate. A plurality of semen tanks are provided on the support plate, a semen outlet is provided on the side wall of the support plate, and a sealing ring is provided at the semen outlet. The bottom ends of the semen tanks are connected to the semen outlets through pipes; an opening with the same diameter as the semen outlet is provided on the side wall of the box body, and when the support plate moves to the bottom inner side of the box body, the semen outlet is aligned with the opening; the opening is connected to an inclined sperm swimming tube, and the top of the sperm swimming tube is connected to a collection box, and the upper and lower ends of the collection box are respectively provided with an inlet and an outlet, and a valve I is provided at the outlet. It simulates the sperm swimming environment, but there is still the possibility of sperm being damaged during the activity process. Summary of the invention

[0003] The technical problem to be solved by the embodiments of the present invention is to provide a microfluidic device and a microcolumn array manufacturing method that can be used to screen sperm, which can integrate the screening process while ensuring high DNA integrity, and effectively prevent semen from being contaminated during the entire assisted reproductive process.

[0004] In order to solve the above technical problems, an embodiment of the present invention provides a microfluidic device that can be used for screening sperm, including a shell, a microcolumn screening area, a positive electrode, a negative electrode, and a cover plate, wherein the positive electrode and the negative electrode are installed on the left and right sides of the inside of the shell, the microcolumn screening area is arranged between the positive electrode and the negative electrode, and the cover plate is covered on the shell; the microcolumn screening area is composed of a microcolumn array, and microchannels for sperm to pass through are formed between the microcolumns, the spacing between the microcolumns is 65-90μm, the height is 90-110μm, and the diameter of the microcolumns is 90-110μm.

[0005] Furthermore, the microcolumn is a straight columnar microcolumn or an umbrella columnar microcolumn.

[0006] Furthermore, the minimum circumscribed cylindrical diameter of the straight cylindrical microcolumn is 100 μm, the height is 100 μm, and the spacing between single microcolumns is 75 μm.

[0007] Furthermore, the umbrella-column-shaped microcolumn is composed of three or four columns whose tops are connected and whose bottoms are connected to the bottoms of adjacent umbrella-column-shaped microcolumns.

[0008] Furthermore, the inclination angle of the column of the umbrella-shaped microcolumn is 15°-75°.

[0009] Furthermore, the cover plate has a filling port.

[0010] Furthermore, the cover plate is fitted to the top of the microcolumn.

[0011] Furthermore, it also includes a heat spreader and a liquid cooling layer, wherein the liquid cooling layer is arranged at the bottom of the shell, and the heat spreader and the micro-column screening area are stacked in sequence from bottom to top.

[0012] The present invention also provides a method for manufacturing the above-mentioned micro-column array, including the steps of manufacturing the micro-column array into straight columnar micro-columns, wherein the steps include: S1: Design a mask according to array parameters, wherein the mask and the top view of the microcolumn array Figure 1 To; S2: using the mask to perform photolithography, and cutting the finished product after photolithography to obtain the desired micro-column array.

[0013] Further, the step of manufacturing the microcolumn array into umbrella-shaped microcolumns includes: S1: Design the mask according to array parameters; S2: The upper edge of a box made of target photoresist is tightly fitted under the mask, the interior of the box is filled with liquid photoresist, and the box is placed under a parallel ultraviolet light source. Reflectors are placed around the box and the mask according to the required angle, and a baffle is set directly above the box, so that the light of the parallel ultraviolet light source is reflected by the reflector into the preset window of the mask, and finally the umbrella-shaped microcolumn is formed.

[0014] The implementation of the embodiments of the present invention has the following beneficial effects: the rapid sperm screening device of the present invention is easy to operate. The present invention adopts micro-nano processing to prepare a microstructure to simulate the microchannel environment of sperm swimming, and adopts a high viscosity buffer to simulate the body fluid environment of sperm swimming. By using the electroosmotic flow method, the liquid sample is moved from the positive electrode direction to the negative electrode direction, and the flow environment of sperm is created in situ, which accelerates the screening of active sperm. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic structural diagram of the rapid sperm screening device manufactured in the first embodiment of the present invention during use; Figure 2A schematic diagram of the assembly of a microfluidic chip of a rapid sperm screening device made by the present invention; the assembly sequence is from top to bottom; Figure 3 Schematic diagram of the liquid flow channel of the liquid cooling layer in the first embodiment of the present invention; Figure 4 is a schematic diagram of the top view of the micro-column screening area; Figure 5 Schematic diagram of making umbrella-shaped micro-columns in the second embodiment of the invention; Figure 6 is a schematic diagram of an umbrella-shaped microcolumn. DETAILED DESCRIPTION

[0016] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0017] Embodiment 1: Reference Figure 1 The structural diagram shown.

[0018] A microfluidic device for screening sperms according to an embodiment of the present invention comprises a microcolumn screening area 1, a positive electrode 2, a negative electrode 3, a heat spreader 4, a cover plate 5, a liquid cooling layer 6, and a housing 7.

[0019] Combination Figure 2 As shown, the liquid cooling layer 6, the heat spreader 4, and the micro-column screening area 1 are installed in the outer shell 7 from bottom to top in sequence. The height of the outer shell should be exactly flush with the cover plate 5, and the outer shell should just cover all the accessories, namely the micro-column screening area 1, the positive electrode 2, the negative electrode 3, the heat spreader 4, the cover plate 5, and the liquid cooling layer 6.

[0020] The positive electrode 2 and the negative electrode 3 are installed on the left and right sides of the shell, and the micro-column screening area 1 is set between the positive electrode and the negative electrode. The positive electrode 2 and the negative electrode 3 are respectively inserted from both ends of the device using platinum wire electrodes. After the device is filled with liquid, a closed circuit is formed between the positive and negative electrodes and the liquid, and electroosmosis can be formed after power is turned on.

[0021] like Figure 3 As shown, the liquid cooling layer 6 has a circulation pipe inside, and the heat is evenly conducted with the micro-column screening area 1 through the heat spreader 4, so as to keep the samples in the micro-column screening area 1 at a constant temperature.

[0022] The cover plate 5 is located above the micro-column screening area 1, and the cover plate fits the micro-columns of the micro-column screening area 1. The cover plate should cover the entire area of ​​the micro-column screening area 1. A sample filling port is provided in the middle of the cover plate.

[0023] The size of the microcolumn screening area is 25 mm × 75 mm.

[0024] like Figure 4 As shown, the micro-column screening area is composed of a micro-column array. In this embodiment, the micro-columns are straight columns, and the minimum circumscribed cylinder diameter of a single micro-column is 100 μm and the height is 100 μm. The spacing between single micro-columns is 75 μm, and micro-channels are formed between the micro-column arrays to simulate the micro-channel environment of sperm swimming.

[0025] The embodiment of the present invention also provides a method for making the above-mentioned microcolumn, which is implemented by the following steps.

[0026] S1: The mask used in photolithography should pay attention to whether the photoresist used is positive or negative. First, the mask should be designed using computer-aided design (CAD) software. The pattern and its distribution on the mask should be consistent with the top view of the micropillar array. Figure 1 To.

[0027] S2: Micro-pillar array fabrication: Based on the design result of the straight columnar (8) array pattern in the previous step, photolithography is performed using a mask, and the finished product after photolithography is cut to obtain the desired micro-pillar array.

[0028] In this embodiment, the shape of the microcolumn screening area 1 is designed and the size is determined according to different sample loading requirements. Note that the height of the shell should be exactly flush with the cover plate 5, and then the transparent resin shell is prepared using 3D printing technology.

[0029] Embodiment 2: like Figure 5 , Figure 6 As shown, based on Example 1, the microcolumns in the microcolumn screening area in this embodiment are umbrella-column-shaped microcolumns, which are composed of three or four columns connected at the top and connected at the bottom to the bottom of the adjacent umbrella-column-shaped microcolumns.

[0030] The inclination angle of the column of the umbrella-shaped microcolumn is 15°-75°, such as 45°, 60°.

[0031] 3. Microchannels are formed between the umbrella-shaped microcolumns to simulate the microchannel environment of sperm swimming.

[0032] The embodiment of the present invention provides a method for making the above-mentioned microcolumn, which is implemented by the following steps.

[0033] S1: When using a mask for photolithography, you should pay attention to whether the photoresist used is positive or negative. When designing the mask using computer-aided design software, pay attention to the spacing between the staggered pillars and the inclination angle of the pillars.

[0034] S2: Use the mask designed for the umbrella-shaped micro-columns in the previous step. All the following equipment are placed under the parallel UV light source. The upper edge of a box made of the target photoresist fits tightly under the mask. The box is filled with liquid photoresist. Depending on the required angle, mirrors with specific angles are placed around the box and mask, such as 15°- 75°. A baffle is placed above the box and mask. The height of the baffle should be set reasonably to avoid blocking the UV reflected by the mirror and ensure that the reflected UV light can cover all positions on the mask.

[0035] Through the above method, the light from the parallel ultraviolet light source is reflected by the reflector into the preset window of the mask, and finally the umbrella-shaped micro-column is formed.

[0036] Embodiment 3: A microfluidic device for screening sperm of the present invention is used by the following method.

[0037] S1. Pre-treatment chip: Add high viscosity buffer to the sample outlet area to simulate the cervical mucus in the sperm swimming environment. Wait for a while to allow the high viscosity buffer to fill the micro-column array area.

[0038] S2. Loading samples onto the chip: Add sperm sample into the microcolumn screening area (1).

[0039] S3. Turn on the electroosmotic flow: Turn on the power of the positive electrode (2) and the negative electrode (3), and wait for about 30 to 60 minutes. The screening method adopted by the present invention uses the electroosmotic flow method, which can promote the flow of liquid and accelerate the sperm screening. S4. Sample collection: Recover the buffer solution near the positive electrode (2), which contains sperm with higher activity.

[0040] The above disclosure is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A microfluidic device for sperm screening, characterized in that: It includes an outer shell, a micro-column screening area, a positive electrode, a negative electrode, and a cover plate. The positive electrode and the negative electrode are installed on the left and right sides of the inner part of the outer shell. The micro-column screening area is arranged between the positive electrode and the negative electrode. The cover plate covers the outer shell. The micro-column screening area is composed of a micro-column array, and micro-channels for sperm to pass through are formed between the micro-columns. The spacing between the micro-columns is 65-90μm, the height is 90-110μm, and the diameter of the micro-columns is 90-110μm.

2. The microfluidic device for sperm screening according to claim 1, characterized in that: The microcolumn is a straight columnar microcolumn or an umbrella columnar microcolumn.

3. The microfluidic device for sperm screening according to claim 2, characterized in that: The minimum circumscribed cylindrical diameter of the straight cylindrical microcolumn is 100 μm, the height is 100 μm, and the spacing between individual microcolumns is 75 μm.

4. The microfluidic device for sperm screening according to claim 2, characterized in that: The umbrella-column-shaped microcolumn is composed of three or four columns whose tops are connected and whose bottoms are connected to the bottoms of the adjacent umbrella-column-shaped microcolumns.

5. The microfluidic device for sperm screening according to claim 4, characterized in that: The inclination angle of the column of the umbrella-shaped microcolumn is 15°-75°.

6. The microfluidic device for sperm screening according to claim 1, characterized in that: The cover plate has a filling port.

7. The microfluidic device for sperm screening according to claim 6, characterized in that: The cover plate is fitted to the top of the microcolumn.

8. The microfluidic device for sperm screening according to any one of claims 1 to 7, characterized in that: It also includes a heat spreader and a liquid cooling layer. The liquid cooling layer is arranged at the bottom of the shell. The heat spreader and the micro-column screening area are stacked in sequence from bottom to top.

9. A method for making the micropillar array of claim 1, characterized in that: The method comprises the steps of manufacturing the micro-column array into straight columnar micro-columns, the steps comprising: S1: designing a mask according to array parameters, wherein the mask is consistent with a top view of the microcolumn array; S2: using the mask to perform photolithography, and cutting the finished product after photolithography to obtain the desired micro-column array.

10. A method for making the micropillar array of claim 1, characterized in that: The method comprises the steps of manufacturing the microcolumn array into umbrella-shaped microcolumns, the steps comprising: S1: Design the mask according to array parameters; S2: The upper edge of a box made of target photoresist is tightly fitted under the mask, the interior of the box is filled with liquid photoresist, and the box is placed under a parallel ultraviolet light source. Reflectors are placed around the box and the mask according to the required angle, and a baffle is set directly above the box, so that the light of the parallel ultraviolet light source is reflected by the reflector into the preset window of the mask, and finally the umbrella-shaped microcolumn is formed.

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