A method for selecting high-motility sperm, its selection platform and preparation method

Abstract

This invention discloses a method for selecting high-moisture sperm, along with its selection platform and preparation method. By combining interdigital transducers and microfluidic channels, an acoustic microfluidic platform is constructed. Surface acoustic waves provided by the interdigital transducers are applied to the microfluidic channels. The acoustic capture force provided by two stages of interdigital transducers is used to capture and filter low-moisture sperm and temporarily store high-moisture sperm, respectively. Finally, the signal input to the second-stage interdigital transducer is turned off to collect high-moisture sperm. The selection method of this invention can capture and store sperm non-destructively, avoiding problems such as DNA peroxidation damage, breakage, and reduced sperm quality caused by conventional sperm selection methods. It achieves high-throughput, automated, and non-destructive screening and collection of high-moisture sperm. The selection platform improves upon the long sorting time and cumbersome manual operation required by traditional methods and enables the reuse of the acoustic microfluidic platform, further enhancing its clinical applicability.

Description

Technical Field

[0001] This invention belongs to the field of assisted reproductive technology, specifically relating to a method for selecting high-motility sperm, its selection platform, and preparation method. Background Technology

[0002] Microfluidics is a rapidly evolving tool that enables researchers to exert unprecedented control over microscale environments. It is being used in numerous fields, including biology, materials science, medicine, chemistry, and physical sciences. Working within microchannels, researchers can manipulate tiny objects of interest at high resolution. The small size and volume of these systems also offer many other key advantages, including portability, low cost, rapid prototyping, easier automation, and the ability to use limited sample sizes and reagents. Consequently, microfluidics research and applications have seen tremendous growth over the past 20 years.

[0003] Microfluidics refers to a novel technology that actively or passively manipulates one or more fluids at the micrometer scale. This technology can shrink the basic functions of chemical and biological laboratories to a chip of a few square centimeters, hence the name "lab-on-a-chip." This technology has also shown great potential in drug discovery, tissue engineering, and nanotechnology, enabling the rapid analysis and processing of thousands of drug, gene, or chemical samples on a lab-on-a-chip platform.

[0004] The application of surface acoustic wave (SAW) technology in microfluidics is a promising area of ​​development. One method involves fabricating interdigital transducers on piezoelectric substrates to manipulate minute particles. In microchannel fluids, particles experience forces primarily composed of gravity, buoyancy, acoustic radiation force, and Stokes force. The dominant force—acoustic radiation force—is mainly influenced by the SAW frequency and the particle size. Therefore, as the SAW frequency increases, the acoustic radiation force on the particles also increases.

[0005] Male infertility accounts for 40% of all infertility cases, mainly manifesting as azoospermia, oligospermia, asthenospermia, and teratospermia. Therefore, identifying high-motility, high-quality sperm from male semen samples is crucial for improving and resolving male infertility.

[0006] Currently, the main clinical methods for sperm selection are the "sperm swim-up method" and the "density gradient centrifugation method." The former uses the difference in sperm's ability to swim to the upper layer of the culture medium to separate and screen high-quality sperm; the latter uses the differences in motility, trajectory, and density between normal and abnormal sperm to select high-quality sperm through centrifugation. Although these two methods can screen for sperm with excellent morphology and motility, they have drawbacks such as long sorting time and multiple centrifugation operations. Furthermore, density gradient centrifugation is prone to causing oxidative damage and breakage of sperm DNA, reducing sperm quality and affecting fertilization success rate. Although there are staining assessment methods that can accurately evaluate sperm damage (such as sperm chromatin structure analysis and comet assays), the staining process also means that the sperm can no longer be used for fertilization with egg cells.

[0007] Therefore, there is an urgent clinical need to develop non-invasive assessment and screening techniques for high-quality sperm, thereby improving the success rate of male-factor assisted reproductive technologies. In recent years, researchers have been conducting in-depth research to address the shortcomings of existing high-quality sperm screening methods, focusing on developing new methods that can non-invasively and objectively screen for highly motile and high-quality sperm. Summary of the Invention

[0008] This invention discloses a method for selecting high-motility sperm, as well as its selection platform and preparation method. By combining interdigital transducers and microfluidic channels, an acoustic microfluidic platform is built. The surface acoustic waves provided by the interdigital transducers are applied to the inside of the microfluidic channels. The acoustic capture force provided by the two-stage interdigital transducers is used to capture and filter low-motility sperm and temporarily store high-motility sperm, respectively. Finally, the high-motility sperm are collected by shutting off the signal input of the second-stage interdigital transducer.

[0009] The technical solution of the present invention is as follows:

[0010] One objective of this invention is to provide a method for selecting high-moisture sperm. A signal generator is used to apply a radio frequency signal to two-stage interdigital transducers of a selection platform to generate two parallel surface acoustic wave fields acting inside a microfluidic channel. The sperm sample is introduced from the inlet of the microfluidic channel. By controlling the input radio frequency signal, an acoustic trapping force is generated to filter low-moisture sperm, temporarily store high-moisture sperm, and collect high-moisture sperm by turning off the radio frequency signal of the second-stage interdigital transducer.

[0011] Furthermore, the input signal frequency of the first-stage interdigital transducer in the radio frequency signal is 228-365MHz, and the generated surface acoustic wave wavelength is 10-16μm; the input signal frequency of the second-stage interdigital transducer is 456-912MHz, and the generated surface acoustic wave wavelength is 4-8μm.

[0012] The second objective of this invention is to provide a platform for selecting high-moisture sperm.

[0013] Furthermore, the preferred platform is composed of interdigital transducers combined with microfluidic channels. The interdigital transducers are arranged in a two-stage parallel structure, with a spacing of 10-20 mm between the two stages of interdigital transducers.

[0014] Furthermore, in the preferred platform, both the first-stage interdigital transducer and the second-stage interdigital transducer have 15-25 electrode pairs.

[0015] Furthermore, the electrode width and spacing of the first-stage interdigital transducer are equal, ranging from 2.5 to 4 μm; the electrode width and spacing of the second-stage interdigital transducer are equal, ranging from 1 to 2 μm.

[0016] Furthermore, the acoustic aperture of the electrode pair is 2-3 mm.

[0017] The third objective of this invention is to provide a method for preparing a high-motility sperm selection platform, specifically including the following steps:

[0018] S1. Fabrication of interdigital transducers: Chromium and gold layers are deposited on a lithium niobate substrate using photolithography and electron beam thermal evaporation. Then, the photoresist pattern is stripped to prepare an interdigital electrode pattern composed of chromium and gold. A SiO2 layer is deposited on the surface of the interdigital electrode using chemical vapor deposition.

[0019] S2. Fabrication of microfluidic channels: A silicon wafer mold for microfluidic channels is fabricated using maskless photolithography. The microfluidic channels are then fabricated using a casting process. The microfluidic channels are perforated, and the structural surfaces are activated by oxygen plasma.

[0020] S3. Combination of interdigital transducer and microfluidic channel: The microfluidic channel is fixed on the surface of the SiO2 layer of the interdigital transducer and placed within the acoustic aperture range of the interdigital transducer.

[0021] Furthermore, the lithium niobate substrate in S1 is 0.5 mm thick, double-sided polished, and 128° Y / X tangential.

[0022] Furthermore, in S1, the chromium layer has a thickness of 5 nm, the gold layer has a thickness of 50 nm, and the SiO2 layer has a thickness of 200 nm.

[0023] Furthermore, the microfluidic channel in S2 is made of PDMS, and the cross-sectional dimensions of the internal channel are 50 × 30 μm. 2 The channel length is 30mm.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. The high-motility sperm selection method provided by the present invention utilizes the acoustic capture force provided by the surface acoustic wave field generated by the interdigital transducer, which can non-destructively screen high-quality sperm, avoiding problems such as sperm DNA peroxidation damage, breakage, and reduced sperm quality caused by conventional selection methods.

[0026] 2. The high-moisture sperm selection method provided by this invention has the characteristics of high throughput and automation. By adjusting the input radio frequency signals of the first-stage interdigital transducer and the second-stage interdigital transducer, a large-scale, one-time collection of high-moisture sperm can be completed, reducing the long sorting time and cumbersome manual operation required in traditional methods.

[0027] 3. The high-motility sperm optimization platform provided by this invention combines interdigital transducers and microfluidic channels. Driven by the resonance of their respective input signals, the two interdigital transducers can generate two parallel, non-interfering surface acoustic wave fields with different acoustic trapping forces that propagate along the surface of the lithium niobate substrate and act within the microfluidic channels in their respective regions. This optimization platform is simple to prepare, has good results, and can be reused by cleaning the microfluidic channels with a cleaning solution. Using this optimization platform can effectively reduce costs and increase efficiency, and has high clinical practical value.

[0028] Figure Labels

[0029] Figure 1 This is an experimental flowchart of the high-motility sperm selection method of the present invention;

[0030] Figure 2 This is a schematic diagram of the interdigital transducer of the high-motility sperm selection platform of the present invention;

[0031] Figure 3 This is a diagram showing the arrangement of the two-stage interdigital transducers in the high-motility sperm selection platform of the present invention.

[0032] Figure 4 This is a schematic diagram of the microfluidic channel structure of the high-motility sperm selection platform of the present invention;

[0033] Figure 5 This is a side view of the structure of the high-motility sperm selection platform of the present invention. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0037] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0038] Example 1

[0039] This embodiment provides a method for selecting high-motility sperm, such as... Figure 1 As shown, the specific steps include the following:

[0040] First, a signal generator is used to apply a radio frequency signal to the two-stage interdigital transducers of the preferred platform to generate two parallel surface acoustic wave fields that act inside the microfluidic channel.

[0041] Then, the sperm sample is introduced through the microfluidic channel inlet. By adjusting the input radio frequency signal, an acoustic trapping force is generated to filter low-motility sperm and temporarily store high-motility sperm. The input signal frequency of the first-stage interdigital transducer is 228MHz, which resonates with its interdigital electrode to generate surface acoustic waves with a wavelength of 10μm. The input signal frequency of the second-stage interdigital transducer is 456MHz, which resonates with its interdigital electrode to generate surface acoustic waves with a wavelength of 4μm.

[0042] Finally, highly motile sperm were collected by turning off the radio frequency signal of the second-stage interdigital transducer.

[0043] Example 2

[0044] This embodiment provides a method for selecting high-motility sperm, such as... Figure 1 As shown, the specific steps include the following:

[0045] First, a signal generator is used to apply a radio frequency signal to the two-stage interdigital transducers of the preferred platform to generate two parallel surface acoustic wave fields that act inside the microfluidic channel.

[0046] Then, the sperm sample is introduced through the microfluidic channel inlet. By adjusting the input radio frequency signal, an acoustic trapping force is generated to filter low-motility sperm and temporarily store high-motility sperm. The input signal frequency of the first-stage interdigital transducer is 240MHz, which resonates with its interdigital electrode to generate surface acoustic waves with a wavelength of 13μm. The input signal frequency of the second-stage interdigital transducer is 685MHz, which resonates with its interdigital electrode to generate surface acoustic waves with a wavelength of 6μm.

[0047] Finally, highly motile sperm were collected by turning off the radio frequency signal of the second-stage interdigital transducer.

[0048] Example 3

[0049] This embodiment provides a method for selecting high-motility sperm, such as... Figure 1 As shown, the specific steps include the following:

[0050] First, a signal generator is used to apply a radio frequency signal to the two-stage interdigital transducers of the preferred platform to generate two parallel surface acoustic wave fields that act inside the microfluidic channel.

[0051] Then, the sperm sample is introduced through the microfluidic channel inlet. By adjusting the input radio frequency signal, an acoustic trapping force is generated to filter low-motility sperm and temporarily store high-motility sperm. The input signal frequency of the first-stage interdigital transducer is 365MHz, which resonates with its interdigital electrode to generate surface acoustic waves with a wavelength of 16μm. The input signal frequency of the second-stage interdigital transducer is 912MHz, which resonates with its interdigital electrode to generate surface acoustic waves with a wavelength of 8μm.

[0052] Finally, highly motile sperm were collected by turning off the radio frequency signal of the second-stage interdigital transducer.

[0053] Example 4

[0054] This embodiment provides a high-motility sperm selection platform, such as... Figure 5 As shown, the specific steps include the following:

[0055] S1. Fabrication of interdigitated transducers: Chromium and gold layers were deposited on a 0.5 mm thick, double-sided polished lithium niobate substrate with a 128° Y / X tangent using photolithography and electron beam thermal evaporation. Then, the photoresist pattern was stripped to fabricate a transducer composed of 5 nm chromium and 50 nm gold. Figure 2 The interdigitated electrode pattern shown is formed by depositing a 200nm SiO2 layer on the surface of the interdigitated electrode using chemical vapor deposition technology.

[0056] S2. Arrangement of interdigital transducers: Two stages of parallel interdigital transducers are arranged, as shown in the attached diagram. Figure 3 As shown, the two-stage interdigital transducers are spaced 10 mm apart. The first-stage interdigital transducer has 15 electrode pairs with an equal electrode width and spacing of 2.5 μm and an acoustic aperture of 2 mm. The second-stage interdigital transducer has 15 electrode pairs with an electrode width and spacing of 1 μm and an acoustic aperture of 2 mm.

[0057] S3. Fabrication of Microfluidic Channels: PDMS was selected as the microfluidic channel material. A silicon wafer mold for the microfluidic channels was fabricated using maskless photolithography. The PDMS microfluidic channels were then obtained through a casting process. Figure 4 As shown, the cross-sectional dimensions of its internal channel are 50 × 30 μm. 2 The channel length is 30mm. The microfluidic channel is perforated and the structural surface is activated by oxygen plasma.

[0058] S4. Combination of interdigital transducer and microfluidic channel: Fix the microfluidic channel on the surface of the SiO2 layer of the interdigital transducer and place it within the acoustic aperture range of the interdigital transducer.

[0059] Example 5

[0060] This embodiment provides a method for preparing a high-motility sperm selection platform, which specifically includes the following steps:

[0061] S1. Fabrication of interdigitated transducers: Chromium and gold layers were deposited on a 0.5 mm thick, double-sided polished lithium niobate substrate with a 128° Y / X tangent using photolithography and electron beam thermal evaporation. Then, the photoresist pattern was stripped to fabricate a transducer composed of 5 nm chromium and 50 nm gold. Figure 2 The interdigitated electrode pattern shown is formed by depositing a 200nm SiO2 layer on the surface of the interdigitated electrode using chemical vapor deposition technology.

[0062] S2. Arrangement of interdigital transducers: Arrange two stages of parallel interdigital transducers, such as... Figure 3 As shown, the two-stage interdigital transducers are spaced 15 mm apart. The first-stage interdigital transducer has 20 electrode pairs with an equal electrode width and spacing of 3.2 μm and an acoustic aperture of 2.5 mm. The second-stage interdigital transducer has 20 electrode pairs with an electrode width and spacing of 1.5 μm and an acoustic aperture of 2.5 mm.

[0063] S3. Fabrication of Microfluidic Channels: PDMS was selected as the microfluidic channel material. A silicon wafer mold for the microfluidic channels was fabricated using maskless photolithography. The PDMS microfluidic channels were then obtained through a casting process. Figure 4 As shown, the cross-sectional dimensions of its internal channel are 50 × 30 μm. 2 The channel length is 30mm. The microfluidic channel is perforated and the structural surface is activated by oxygen plasma.

[0064] S4. Combination of interdigital transducer and microfluidic channel: Fix the microfluidic channel on the surface of the SiO2 layer of the interdigital transducer and place it within the acoustic aperture range of the interdigital transducer.

[0065] Example 6

[0066] This embodiment provides a method for preparing a high-motility sperm selection platform, which specifically includes the following steps:

[0067] S1. Fabrication of interdigitated transducers: Chromium and gold layers were deposited on a 0.5 mm thick, double-sided polished lithium niobate substrate with a 128° Y / X tangent using photolithography and electron beam thermal evaporation. Then, the photoresist pattern was stripped to fabricate a transducer composed of 5 nm chromium and 50 nm gold. Figure 2 The interdigitated electrode pattern shown is formed by depositing a 200nm SiO2 layer on the surface of the interdigitated electrode using chemical vapor deposition technology.

[0068] S2. Arrangement of interdigital transducers: Two stages of parallel interdigital transducers are arranged, as shown in the attached diagram. Figure 3 As shown, the two-stage interdigital transducers are spaced 20 mm apart. The first-stage interdigital transducer has 25 electrode pairs with an equal electrode width and spacing of 4 μm and an acoustic aperture of 3 mm. The second-stage interdigital transducer has 25 electrode pairs with an electrode width and spacing of 2 μm and an acoustic aperture of 3 mm.

[0069] S3. Fabrication of Microfluidic Channels: PDMS was selected as the microfluidic channel material. A silicon wafer mold for the microfluidic channels was fabricated using maskless photolithography. The PDMS microfluidic channels were then obtained through a casting process. Figure 4 As shown, the cross-sectional dimensions of its internal channel are 50 × 30 μm. 2 The channel length is 30mm. The microfluidic channel is perforated and the structural surface is activated by oxygen plasma.

[0070] S4. Combination of interdigital transducer and microfluidic channel: Fix the microfluidic channel on the surface of the SiO2 layer of the interdigital transducer and place it within the acoustic aperture range of the interdigital transducer.

[0071] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for selecting high-motility sperm in a non-disease diagnosis and treatment method, characterized in that: A signal generator is used to apply radio frequency signals to the two-stage interdigital transducers of the preferred platform to generate two parallel surface acoustic wave fields that act inside the microfluidic channel. The sperm sample is introduced from the inlet of the microfluidic channel. By adjusting the input radio frequency signal, an acoustic trapping force is generated to filter low-motility sperm and temporarily store high-motility sperm. The high-motility sperm is collected by turning off the radio frequency signal of the second-stage interdigital transducer. The input signal frequency of the first-stage interdigital transducer in the radio frequency signal is 228-365 MHz, and the generated surface acoustic wave wavelength is 10-16 μm; the input signal frequency of the second-stage interdigital transducer is 456-912 MHz, and the generated surface acoustic wave wavelength is 4-8 μm.

2. A high-motility sperm selection platform, characterized in that, The preferred platform is composed of interdigital transducers combined with microfluidic channels. The interdigital transducers are arranged in a two-stage parallel structure, with a spacing of 10-20 mm between the two stages. Both the first-stage and second-stage interdigital transducers in the preferred platform have 15-25 electrode pairs. The electrode width and spacing of the first-stage interdigital transducers are equal, ranging from 2.5-4 μm. The electrode width and spacing of the second-stage interdigital transducers are equal, ranging from 1-2 μm. The acoustic aperture of each electrode pair is 2-3 mm.

3. A method for preparing a high-motility sperm selection platform, characterized in that, Specifically, the following steps are included: S1. Fabrication of interdigital transducers: Chromium and gold layers are deposited on a lithium niobate substrate using photolithography and electron beam thermal evaporation. Then, the photoresist pattern is stripped to prepare an interdigital electrode pattern composed of chromium and gold. A SiO2 layer is deposited on the surface of the interdigital electrode using chemical vapor deposition. S2. Fabrication of microfluidic channels: A silicon wafer mold for microfluidic channels is fabricated using maskless photolithography. The microfluidic channels are then fabricated using a casting process. The microfluidic channels are perforated, and the structural surfaces are activated by oxygen plasma. S3. Integration of the interdigital transducer and the microfluidic channel: The microfluidic channel is fixed on the surface of the SiO2 layer of the interdigital transducer and placed within the acoustic aperture range of the interdigital transducer; the material of the microfluidic channel in S2 is PDMS, and the cross-sectional dimensions of the internal channel are 50×30 μm. 2 The channel length is 30 mm.

4. The method for preparing a high-motility sperm selection platform according to claim 3, characterized in that, The lithium niobate substrate in S1 is 0.5 mm thick, double-sided polished, and 128° Y / X tangential.

5. The method for preparing a high-motility sperm selection platform as described in claim 3, characterized in that, The thickness of the chromium layer in S1 is 5 nm, the thickness of the gold layer is 50 nm, and the thickness of the SiO2 layer is 200 nm.

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