Sperm quality detection sensor and preparation method thereof

Abstract

The invention discloses a sperm quality detection sensor and a preparation method thereof, and belongs to the technical field of detection sensing, the sperm quality detection sensor comprises an electrode, the surface of the electrode is modified with an HA functional layer, and the HA functional layer has amino active sites; the redox probe and the electrode generate an electric signal; and the conductive layer is arranged between the electrode and the redox probe and is used for conducting between the electrode and the redox probe. According to the application, the HA functional layer is specifically combined with the healthy sperm surface receptor in the to-be-detected sperm, and the current between the electrode and the redox probe is influenced by the specific combination of the HA functional layer and the receptor by adopting a micro-current signal processing technology; the number of healthy sperms in the to-be-detected semen is represented through the current change of the sperm quality detection sensor, the sperm quality detection sensor breaks through the limitation of morphological interpretation in a traditional detection technology, the size of sperm detection equipment can be greatly reduced, and the sperm quality detection precision is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection sensors, and in particular relates to a sperm quality detection sensor and a preparation method thereof. Background Art

[0002] The innovation of reproductive health monitoring technology is of great significance to the assessment of human fertility and the development of assisted reproduction. As a core assessment method, sperm quality testing has an extremely important significance for couples preparing for pregnancy, assisted reproduction and the diagnosis of male reproductive diseases due to its high accuracy.

[0003] Traditional semen analysis primarily uses optical microscopy or computer-assisted semen analysis systems to detect and analyze sperm in semen. Optical microscopy requires multiple steps, including semen liquefaction, staining, fixation, and manual microscopic examination, which is time-consuming. Manual microscopic examination relies on the examiner's experience, and key indicators such as sperm acrosome integrity and neck curvature are visually inspected, resulting in poor consistency in sperm quality assessment. Computer-assisted semen analysis, on the other hand, uses image recognition algorithms for automated sperm analysis. However, this equipment is expensive and requires a phase-contrast microscope and high-speed camera system. Furthermore, when sperm density is low or uneven, the system can easily misidentify sperm-like particles (such as cell debris and white blood cells) as sperm, leading to significant detection errors. Summary of the Invention In response to one or more of the above-mentioned defects or improvement needs of the prior art, the present invention provides a sperm quality detection sensor to solve the problems of cumbersome detection process and large detection error in the existing sperm quality detection.

[0004] To achieve the above object, the present invention provides a sperm quality detection sensor, which includes: An electrode, wherein the electrode is a glassy carbon / gold electrode, and the surface of the electrode is modified with a HA functional layer, wherein the HA functional layer has amino active sites; A redox probe, wherein the redox probe undergoes a redox reaction with the electrode and generates an electrical signal, and the HA functional layer can bind to a sperm surface receptor in the semen to be tested to hinder electron exchange between the electrode and the redox probe; A conductive layer is provided between the electrode and the redox probe and is used for conducting electricity between the electrode and the redox probe.

[0005] As a further improvement of the present invention, the conductive layer includes a gold nanodendrite array and a graphene / Nafion composite matrix layer.

[0006] As a further improvement of the present invention, it also includes an isolation membrane, which is coated on the outer surface of the multilayer structure formed by the electrode, the conductive layer and the redox probe; the isolation membrane includes a film-forming structure formed by gradient volatilization of Nafion solution by ethanol, and the isolation membrane has a negatively charged ion channel.

[0007] As a further improvement of the present invention, the redox probe is formed by depositing Prussian blue using a mixed solution containing FeCl3, K3[Fe(CN)6], KCl and HCl.

[0008] As a further improvement of the present invention, the HA functional layer is formed by activating the carboxyl group with EDC / NHS and cross-linking with ADH through amide bonds, and epoxy ring-opening grafting of the hydroxyl group with PEG-DGE.

[0009] The present application also includes a method for preparing a sperm quality detection sensor, which comprises the following steps: Base treatment to form a base layer; preparing electrodes on the substrate layer and pre-treating the electrodes; providing a conductive layer on the surface of the electrode; Setting a HA functional layer at the end of the electrode; Electrochemical deposition of Prussian blue was used to form a redox probe; Blocking nonspecific sites on the surface of electrodes, conductive layers, and redox probes; Nafion selective membranes were prepared on the outer surfaces of the electrodes, conductive layer and redox probe.

[0010] As a further improvement of the present invention, preparing the electrode on the substrate layer includes: Preparation of working electrode: A gold electrode was prepared on the substrate layer using a photolithography process to serve as the working electrode. Counter electrode preparation: Platinum slurry is spray-printed on the substrate using a microelectronic printer to form a circular electrode; Preparation of reference electrode: Ag / AgCl slurry was printed on the substrate layer, and a stable reference electrode was formed by constant potential oxidation method.

[0011] As a further improvement of the present invention, pre-treating the electrode includes: Mechanical polishing: The working electrode is polished step by step using aluminum oxide suspensions of different particle sizes. Ultrasonic cleaning is used to remove aluminum oxide after each polishing step. Chemical activation: The working electrode was ultrasonically treated with a HCl:H2O2 mixture to remove oxides on the working electrode surface, and the oxygen-containing functional groups on the working electrode surface were activated by cyclic voltammetry in a H2SO4 solution.

[0012] As a further improvement of the present invention, the preparation of a conductive layer on the electrode surface comprises: Dendritic gold nanostructures were formed by constant potential pulse deposition of a 50 mM HAuCl4 solution containing 0.1 M KCl.

[0013] As a further improvement of the present invention, providing an HA functional layer at the end of the electrode includes: HA carboxyl groups were activated using EDC / NHS and then coupled with ADH at room temperature for 12 h.

[0014] The above-mentioned improved technical features can be combined with each other as long as they do not conflict with each other.

[0015] In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art: (1) The sperm quality detection sensor of the present invention adopts biological molecule directional recognition technology to specifically bind the HA functional layer to the surface receptors of healthy sperm in the semen to be tested, and adopts microcurrent signal processing technology to use the specific binding of the HA functional layer to the sperm surface receptor to affect the current between the electrode and the redox probe, and the current change of the sperm quality detection sensor is used to characterize the number of healthy sperm in the semen to be tested. The sperm quality detection sensor in this application can directly react to the integrity and fertilization ability of the sperm membrane in the semen through the electrochemical signal difference, breaking through the limitations of morphological interpretation in traditional detection technology, and can greatly increase the size of sperm detection equipment and improve the accuracy of sperm quality detection.

[0016] (2) The sperm quality detection sensor of the present invention has a three-dimensional gold dendrite structure that can effectively increase the specific surface area of ​​the conductive layer, allowing sperm in the semen to pass through the conductive layer to quickly bind to the HA functional layer on the electrode surface. Secondly, the graphene / Nafion composite matrix layer has good electrical conductivity, and the Nafion composite matrix can isolate other substances in the semen, preventing semen from contacting the electrode and affecting the normal operation of the electrode, thereby ensuring the accuracy of the electrical signal generated between the electrode and the redox probe, and improving the accuracy of the detection of healthy sperm count.

[0017] (3) The sperm quality detection sensor of the present invention is provided with an isolation membrane, which includes a membrane structure formed by volatilizing a Nafion solution through an ethanol gradient. After the Nafion solution is volatilized through an ethanol gradient, negatively charged ion channels are formed on the surface of the membrane structure. The negatively charged ion channels can synergistically exclude proteins larger than 5 kDa and positively charged cell fragments in semen, thereby avoiding the exclusion of damaged sperm and non-sperm proteins, allowing sperm with specific receptors to pass through the isolation membrane and then combine with the HA functional layer on the electrode, thereby avoiding interference from other non-sperm substances in semen and improving the detection accuracy of the sperm quality detection sensor for healthy sperm. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 2 is a schematic structural diagram of a sperm quality detection sensor according to an embodiment of the present invention; Figure 2 (a) is a schematic diagram of the model of the sperm quality detection sensor; Figure 2 Middle (b) is a schematic diagram of the model of sperm binding to hyaluronic acid in the sperm quality detection sensor; Figure 3 (a) is the it curve of the reaction between different concentrations of semen and HA on the working electrode surface of the sensor for 300s; Figure 3 Middle (b) is the it curve of the reaction between different concentrations of semen and HA on the working electrode surface of the sensor for 600s; Figure 4 Schematic diagram of the process of preparing a sperm quality detection sensor in an embodiment of the present invention. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only intended to illustrate the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0020] In the description of the present invention, it should be understood that, unless otherwise specified, the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be understood as limiting the present invention.

[0021] Furthermore, unless otherwise specified, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Thus, a feature specified as "first" or "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specified.

[0022] In the present invention, unless otherwise specified or limited, the terms "installed," "connected," "connect," "fixed," etc. should be understood in a broad sense. For example, they can refer to fixed connection, detachable connection, or integration; mechanical connection, electrical connection; direct connection, or indirect connection through an intermediate medium; internal communication between two components, or interaction between two components, unless otherwise specified. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0023] In the present invention, unless otherwise expressly specified or limited, when a first feature is "above" or "below" a second feature, it may mean that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediary. Furthermore, when a first feature is "above," "above," or "above" a second feature, it may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. When a first feature is "below," "below," or "below" a second feature, it may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.

[0024] See also Figures 1 to 4 The sperm quality detection sensor in the preferred embodiment of the present invention includes an electrode, which is a glassy carbon / gold electrode structure, and is modified with an HA (hyaluronic acid) functional layer on the electrode surface, and the HA functional layer has an amino active site; and a redox probe, which can generate an electrical signal between the electrode and the redox probe; a conductive layer, which is arranged between the electrode and the redox probe and is used for electrical conduction between the electrode and the redox probe. Specifically, the HA functional layer on the surface of the electrode in the present application can specifically bind to the receptor on the surface of healthy sperm in the semen to be tested. After the two are combined, the electron exchange between the electrode and the redox probe will be hindered. By detecting the current strength between the electrode and the redox probe, the number of healthy sperm in the semen that are bound to the HA functional layer can be obtained, thereby obtaining the number of healthy sperm in the semen to be tested.

[0025] The sperm quality detection sensor in the present application adopts biomolecular directional recognition technology to specifically bind the HA functional layer to the surface receptors of healthy sperm in the semen to be tested, and adopts microcurrent signal processing technology to utilize the specific binding of the HA functional layer to the receptor to affect the current between the electrode and the redox probe. The current change of the sperm quality detection sensor is used to characterize the number of healthy sperm in the semen to be tested. The sperm quality detection sensor in the present application can directly react to the integrity and fertilization ability of the sperm membrane in the semen through the electrochemical signal difference, breaking through the limitations of morphological interpretation in traditional detection technology, and can greatly increase the size of sperm detection equipment and improve the accuracy of sperm quality detection.

[0026] It is worth noting that the conductive layer in this application is arranged on the surface of the electrode layer, and although the HA functional layer is modified on the surface of the electrode, the HA functional layer is also embedded in the conductive layer, and the redox probe is arranged as a whole on the conductive layer to form a multilayer structure of electrode, conductive layer and redox probe.

[0027] Furthermore, as an optional embodiment of the present invention, the conductive layer in this application includes a gold nanodendrite array and a graphene / Nafion (perfluorosulfonic acid resin) composite matrix layer. The three-dimensional gold dendrite structure can effectively increase the specific surface area of ​​the conductive layer, allowing sperm in the semen to be tested that pass through the conductive layer to quickly bind to the HA functional layer on the electrode surface. Secondly, the graphene / Nafion composite matrix layer has excellent electrical conductivity, and the Nafion composite matrix can isolate other substances in the semen, preventing semen from contacting the electrode and affecting the normal operation of the electrode, thereby ensuring the accuracy of the electrical signal generated between the electrode and the redox probe, and improving the accuracy of the detection of healthy sperm count.

[0028] Furthermore, as an optional embodiment of the present invention, the sperm quality detection sensor in the present application also includes an isolation membrane, which is coated on the outer surface of the multi-layer structure of the electrode, the conductive layer and the redox probe, and the isolation membrane includes a film-forming structure formed by the gradient volatilization of the Nafion solution by ethanol. After the Nafion solution is volatilized by the gradient ethanol, negatively charged ion channels will be formed on the surface of the membrane structure. The negatively charged ion channels can synergistically repel proteins larger than 5kDa and positively charged cell fragments in the semen to avoid the exclusion of damaged sperm and non-sperm proteins, so that sperm with receptors pass through the isolation membrane and then combine with the HA functional layer on the electrode, avoiding interference from other non-sperm substances in the semen, and improving the detection accuracy of the sperm quality detection sensor for healthy sperm.

[0029] Furthermore, as an optional embodiment of the present invention, prior to providing the isolation membrane, the present application further includes blocking nonspecific sites on the electrode, conductive layer, and redox probe. This is accomplished by soaking the electrode, conductive layer, and redox probe in a BSA (bovine serum albumin) buffer solution, followed by a 30-minute soaking treatment in a 0.1M glycine solution. This double-blocks the nonspecific sites and reduces nonspecific adsorption to less than 5%. Specifically, nonspecific site blocking refers to the fact that BSA binds to the hydrophobic surface (hydrophobic regions of carbon-based materials and gold electrodes) on the electrode or conductive layer to form a physical barrier, preventing other hydrophobic molecules from nonspecifically adsorbing on the metal electrode surface, thereby protecting the electrode. Furthermore, glycine can coordinate with metal ions through its carboxyl group, thereby reducing the adsorption of foreign proteins on the metal active sites. The combination of BSA and glycine reacts with activated groups (EDC / NHS-activated carboxyl groups and epoxy groups) on the electrode surface to form covalent bonds or hydrogen bonds, thereby blocking the active groups.

[0030] Furthermore, as an optional embodiment of the present invention, the redox probe in this application is formed by depositing Prussian blue using a mixed solution containing FeCl3 (ferric chloride), K3[Fe(CN)6] (potassium ferrocyanide), KCl (potassium chloride), and HCl (hydrochloric acid). The redox probe can react with a gold electrode to produce an oxidation-reduction reaction, generating an electrical signal between the redox probe and the gold electrode.

[0031] Furthermore, as an optional embodiment of the present invention, the HA functional layer in this application is formed by activating the carboxyl groups with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) / NHS (N-hydroxysuccinimide) and cross-linking them with ADH (adipic acid dihydrazide) through amide bonds, and then epoxy-ring-opening grafting of the hydroxyl groups with PEG-DGE (polyethylene glycol diglycidyl ether). By activating the carboxyl groups with EDC / NHS and cross-linking them with ADH through amide bonds, and then epoxy-ring-opening grafting of the hydroxyl groups with PEG-DGE, a double-cross-linked spatial network is formed, which enables the fixed density of HA to reach 2.3~3.1μg / mm 2 , improve the binding density and stability between the HA functional layer and the electrode, improve the binding efficiency between HA and the receptors in sperm, and increase the capture efficiency of sperm.

[0032] Furthermore, as an optional embodiment of the present invention, the electrodes in this application include a working electrode, a counter electrode, and a reference electrode. The working electrode is a gold electrode, the counter electrode is a circular electrode sheet printed with platinum slurry, and the reference electrode is a stable reference layer formed by constant potential oxidation of Ag / AgCl slurry.

[0033] It is worth noting that the sperm quality detection sensor in the present invention also includes a base layer, and the above-mentioned electrodes, redox probes and conductive layer are all arranged on the base layer. The base layer cooperates with the isolation membrane to form a closed sensor structure. The healthy sperm in the semen to be tested can enter the interior of the sensor through the ion channel on the isolation membrane, and specifically bind to the HA functional layer on the electrode surface to hinder the transmission of electrical signals between the electrode and the redox probe, change the detection current of the sensor, and thus characterize the number of healthy sperm in the semen to be tested.

[0034] Furthermore, with respect to the sperm quality detection sensor in the present application, it also includes a method for preparing the sperm quality detection sensor, which comprises the following steps: Base treatment to form a base layer; preparing electrodes on the substrate layer and pre-treating the electrodes; providing a conductive layer on the surface of the electrode; providing a HA functional layer on the electrode; Electrochemical deposition of Prussian blue was used to form a redox probe; Blocking nonspecific sites on the surface of electrodes, conductive layers, and redox probes; Nafion selective membranes were prepared on the outer surfaces of the electrodes, conductive layer and redox probe.

[0035] Furthermore, as an optional embodiment of the present invention, the substrate treatment in this application to form the substrate layer specifically includes: cutting a PET film to a predetermined size, ultrasonically cleaning it sequentially with acetone, ethanol, and deionized water, and drying it with nitrogen to form the substrate layer. PET film itself has good flexibility and chemical stability, making it suitable as a substrate material for flexible sensors. A gradient solvent cleaning process consisting of acetone, ethanol, and deionized water can remove grease (acetone), polar contaminants (ethanol), and residual ions (water), respectively. Nitrogen purging can prevent residual fibers on the surface of the substrate layer, thereby obtaining a clean and dry substrate layer.

[0036] Furthermore, as an optional embodiment of the present invention, the substrate processing to form the substrate layer in the present application includes: A silicon wafer is used as the substrate. It is cleaned sequentially with acetone, isopropyl alcohol, and deionized water. After being purged with nitrogen, it is treated with oxygen plasma. Silicon wafers, with their high flatness and compatibility with photolithography processes, are also suitable for use as the substrate layer. Oxygen plasma treatment enhances the hydrophilicity of the silicon wafer's surface.

[0037] Furthermore, as an optional embodiment of the present invention, the preparation of the electrode on the substrate layer of the present application specifically includes: Preparation of working electrode: A gold electrode was prepared on the substrate layer using a photolithography process to serve as the working electrode. Counter electrode preparation: Platinum slurry is spray-printed on the substrate using a microelectronic printer to form a circular electrode; Preparation of reference electrode: Ag / AgCl slurry was printed on the substrate layer, and a stable reference electrode was formed by constant potential oxidation method.

[0038] Furthermore, as an optional embodiment of the present invention, the preparation of the electrode of the present application includes: Gold electrode photolithography preparation: Spin-coat photoresist on the surface of the silicon wafer, expose and develop, and then perform metal deposition to form a gold electrode pattern on the surface of the silicon wafer; External integration of the reference electrode: Use Ag / AgCl electrode and fix it to the edge of the silicon wafer with conductive silver glue. The distance between the Ag / AgCl electrode and the gold electrode should be 1~1.5mm to avoid the electrolyte causing a short circuit between the gold electrode and the reference electrode.

[0039] Furthermore, as an optional embodiment of the present invention, the pretreatment of the electrode in this application specifically includes: Mechanical polishing: The working electrode is polished step by step using aluminum oxide suspensions of different particle sizes. Ultrasonic cleaning is used to remove aluminum oxide after each polishing step. Chemical activation: The working electrode was ultrasonically treated with a HCl:H2O2 mixture to remove oxides on the working electrode surface. The oxygen-containing functional groups (carboxyl or hydroxyl) on the working electrode surface were activated by cyclic voltammetry in a H2SO4 solution to enhance the hydrophilicity and electrochemical activity of the working electrode.

[0040] Furthermore, as an optional embodiment of the present invention, the configuration of the conductive layer in this application specifically includes: Dendritic gold nanostructures were formed by constant potential pulse deposition of a 50 mM HAuCl4 solution containing 0.1 M KCl.

[0041] Furthermore, as an optional embodiment of the present invention, the configuration of the conductive layer in this application includes: Porous gold nanostructures were formed in a solution containing 0.1 M H2SO4 and 1 mM HAuCl4 using a square wave pulse method.

[0042] Furthermore, as an optional embodiment of the present invention, the HA functional layer is provided at the end of the electrode in the present application and specifically includes: Activation of HA carboxyl groups with EDC / NHS and subsequent coupling with ADH at room temperature for 12 hours can form stable amide bonds and avoid conformational destruction of HA caused by excessive cross-linking.

[0043] Furthermore, as an optional embodiment of the present invention, the HA functional layer is provided on the electrode in the present application, including: The electrode was immersed in a 1 mM ethanol solution for 12–14 h to form a self-assembled monolayer to thiolate the gold surface; 10 mg / mL HA containing 1 mM EDC and 0.5 mM NHS was used to react with the thiol groups on the electrode surface.

[0044] Furthermore, as an optional embodiment of the present invention, the formation of the redox probe in the present application specifically includes: Prussian blue nanoparticle layers were formed by cyclic voltammetry in a 0.1 M KCl solution containing 2 mM FeCl3, 2 mM K3[Fe(CN)6], and 0.1 M HCl.

[0045] Furthermore, as an optional embodiment of the present invention, the non-specific site blocking in the present application specifically includes: The substrate, electrode, conductive layer, and Prussian blue structure were blocked with 1% BSA at room temperature and then treated with 0.1 M glycine solution for 30 min to double-block the unreacted sites, so that the relative ratio of the background signal to the positive signal after blocking was <5%.

[0046] Furthermore, as an optional embodiment of the present invention, the preparation of the Nafion selective membrane in this application specifically includes: A Nafion solution diluted with ethanol was drop-coated and dried to form a cation exchange membrane with a pore size of 1~2nm.

[0047] The sperm quality detection sensor is prepared using the above-mentioned preparation method of the sperm quality detection sensor.

[0048] Example 1: Sensor substrate processing and electrode preparation: Substrate treatment: Cut a 1 cm × 1 cm PET film with a thickness of 0.18 mm and ultrasonically clean it with acetone (40°C), ethanol (30°C), and deionized water for 15 minutes each (ultrasonic power 100 W). The PET film was then blown dry with 0.3 MPa pressure nitrogen.

[0049] Preparation of working electrode: A 0.5 mm gold electrode with a thickness of 100 nm was prepared on a PET film using a photolithography process. The gold electrode was used as the working electrode with a diameter of 3 mm.

[0050] Counter electrode preparation: Platinum slurry was spray-printed on a PET film using a microelectronic printer to form a circular electrode with a diameter of 3 mm and an electrode thickness of 20±2 μm.

[0051] Reference electrode preparation: Ag / AgCl slurry was printed on a PET film, and a stable reference layer was formed by constant potential oxidation. Specifically, in the constant potential oxidation method, Ag / AgCl was used as the reference electrode benchmark and oxidation was continued at a potential of +0.5 V for 600 s.

[0052] Working electrode pretreatment: Mechanical polishing: Use 1.0 μm, 0.3 μm, and 0.05 μm alumina suspensions in stages. The alumina powder and ultrapure water in the alumina suspension are prepared in a mass ratio of 1:1. Polish step by step. After each polishing step, ultrasonic cleaning with deionized water and anhydrous ethanol for 5 minutes each is required to avoid uneven surface roughness caused by residual particles. Chemical activation: Surface oxides were removed by ultrasonic treatment in a mixture of HCl:H₂O₂ (volume ratio 3:1) for 10 minutes. Surface oxygen-containing functional groups were then activated by cyclic voltammetry in 0.1 M H₂SO₄. Cyclic voltammetry was performed with a scan rate of 50 mV / s between -0.2 V and +1.2 V using Ag / AgCl as the reference, for 20 cycles.

[0053] Conductive layer modification: Gold Nanostructure Modification: Potentiostatic pulse deposition was performed using a mixture of 0.1 M KCl and 50 mM HAuCl₄ to form uniform, dendritic Au nanostructures (SEM-verified particle size of 20 ± 5 nm), enhancing conductivity and surface area. The pH of the 0.1 M KCl and 50 mM HAuCl₄ solution was 1.5. Potentiostatic pulse deposition consisted of deposition at -0.2 V for 0.5 s, followed by deposition at 0.5 V for 1 s, for a total of 100 cycles.

[0054] HA functional layer fixed: HA cross-linking: HA carboxyl groups were activated using EDC / NHS at a pH of 5.5 for 2 hours at 4°C. Subsequently, ADH was coupled at room temperature for 12 hours. The molar ratio of EDC to NHS was 1:2, the ADH concentration was 10 mM, and the pH was 7.4.

[0055] Verification of HA immobilization: Atomic force microscopy (AFM) was used to characterize the thickness of the HA layer (target 50-100 nm) and surface roughness (Ra < 5 nm) to ensure uniform coverage; and XPS (X-ray photoelectron spectroscopy) was used to detect the nitrogen content to verify the amino cross-linking efficiency.

[0056] Prussian Blue Electrochemical Deposition: Prussian blue (PB) nanoparticle layers were formed using cyclic voltammetry in a 0.1 M KCl solution containing 2 mM FeCl₃, 2 mM K₃[Fe(CN)₆]₆, and 0.1 M HCl. Cyclic voltammetry was performed between -0.2 V and +0.6 V for 10 cycles at a scan rate of 50 mV / s.

[0057] Containment and selective barrier construction: Nonspecific site blocking: Block with 1% BSA at room temperature for 2 hours, followed by treatment with 0.1 M glycine for 30 minutes to double-block unreacted sites. Specifically, the BSA solution was PBS buffer containing 0.05% Tween-20, and the glycine solution was at a pH of 8.5.

[0058] Preparation of Nafion selective membrane: After drop-coating 0.5% Nafion, the membrane was dried at 40% humidity and 25°C for 24 hours to form a cation exchange membrane with a pore size of 1-2 nm. Electrochemical impedance spectroscopy (EIS) was used to verify membrane integrity and ensure that the low-frequency impedance of the Nafion selective membrane was >1 kΩ·cm. 2 Preferably, the Nafiong solution is obtained by diluting with ethanol.

[0059] Example 2: Sensor substrate processing and electrode preparation: Substrate treatment: A 1.5 cm × 1.5 cm silicon wafer with a thickness of 500 μm was used as the substrate. The wafer was ultrasonically cleaned in acetone (50°C), isopropyl alcohol (40°C), and deionized water for 20 minutes each at an ultrasonic power of 120 W. After a nitrogen purge at a pressure of 0.4 MPa, the wafer was treated with a 100 W oxygen plasma for 5 minutes to enhance the hydrophilicity of the silicon wafer surface. Gold electrode photolithography preparation: Spin-coating photoresist: AZ 5214 photoresist was used for spin coating at a speed of 3000 rpm to a thickness of 1.5 μm, and pre-baked at 90°C for 90 s.

[0060] Exposure and development: The working electrode and counter electrode were designed using a mask, and UV exposure was performed using AZ 300 MIF developer for 30 seconds. The working electrode was a 2mm diameter circular electrode, and the counter electrode was a ring electrode with an outer diameter of 4mm and an inner diameter of 3mm. The UV exposure parameters were 365nm and 100 mJ / cm 2 .

[0061] Metal deposition: Electron beam evaporation of a gold layer with a thickness of 150 nm and an evaporation rate of 0.5 Å / s was used to form the electrode pattern after lift-off.

[0062] External integrated reference electrode: Ag / AgCl reference electrode: An Ag / AgCl electrode with a diameter of 1 mm was selected and fixed to the edge of the silicon substrate using conductive silver glue. The distance between the Ag / AgCl reference electrode and the working electrode was 1 mm.

[0063] Working electrode pretreatment: Mechanical polishing: 1.0 μm, 0.3 μm, and 0.05 μm alumina suspensions were used in stages. Alumina powder and ultrapure water were prepared in a 1:1 mass ratio. Polishing was performed step by step. After each polishing step, ultrasonic cleaning was performed with deionized water and anhydrous ethanol for 5 minutes each to avoid uneven surface roughness caused by residual particles.

[0064] Chemical activation: Ultrasonic treatment with an HCl:H₂O₂ mixture for 10 minutes removed surface oxides. Surface oxygen-containing functional groups (such as carboxyl and hydroxyl groups) were then activated by cyclic voltammetry in 0.1 M H₂SO₄, enhancing the electrode's hydrophilicity and electrochemical activity. The volume ratio of HCl to H₂O₂ was 3:1, and the cyclic voltammetry voltage was -0.2 V to +1.2 V. The reference electrode was Ag / AgCl, with a scan rate of 50 mV / s and 20 cycles.

[0065] Modification of the electrode conductive layer: A porous gold nanostructure (SEM verified that the pore size of the gold nanostructure was 50±10 nm) was formed in a mixed solution of 0.1 M H2SO4 and 1 mM HAuCl4 using a square wave pulse method; the voltage in the square wave pulse method was -0.1 V to +0.3 V, the duty cycle was 50%, the frequency was 10 Hz, and the deposition time was 300 s.

[0066] HA covalent immobilization: Thiolation of gold surface: The gold electrode was immersed in 1 mM ethanol solution for 12 h to form a self-assembled monolayer.

[0067] HA coupling: 10 mg / mL HA was reacted with thiol groups on the electrode surface. XPS was used to detect the S2p peak to verify coupling efficiency. The 10 mg / mL HA solution contained 1 mM EDC and 0.5 mM NHS. The reaction pH was 7.0 and the reaction was carried out at room temperature for 6 hours.

[0068] Prussian blue layer fixation: The electrolyte was adjusted to a mixed solution of 3 mM FeCl3, 3 mM K3[Fe(CN)6] and 0.2 M KCl. The constant potential method was used with a voltage of +0.4 V and a deposition time of 120 s to control the PB layer thickness to 80±10 nm (the PB layer thickness was verified using AFM).

[0069] Anti-pollution barrier construction: Nafion membrane optimization: spin coating 1% Nafion solution at 3000 rpm for 30 s, annealing at 80°C for 1 hour to form a dense film. The EIS low-frequency impedance of the dense film is >2 kΩ·cm. 2 .

[0070] Sperm quality detection sensor performance test: Linear range: tested by standard sperm suspension, the calibration curve covers 10 4 ~10 7 cells / mL, and the limit of detection (LOD) was ≤200 cells / mL.

[0071] Anti-interference test: Add 10 times the concentration of protein (BSA), glucose and metal ions (Na + , K + ), the detection signal change was <5%, proving that the Nafion membrane effectively shielded the interference of large molecules.

[0072] Stability and reproducibility: The sperm quality detection sensor was stored at 4°C for 30 days and then tested again. The response signal attenuation was <8%; and the relative standard deviation (RSD) of the same batch of electrodes (n>5) was <4%, meeting clinical testing requirements.

[0073] At the same time, sperm quality detection sensors were used to test semen of different concentrations. Figure 3 (a) represents the IT curve of the reaction between sperm and HA on the working electrode surface of the sensor for 300s. Figure 3 (b) represents the it curve of the reaction between sperm and HA on the working electrode surface of the sensor for 600s. It can be seen that the sperm concentration is 37×10 6 The signal of the sample with sperm concentration of 10×10 6 The sample signal of sperm / mL was in line with expectations; and the signal showed a downward trend over time. The signal of the sample after 600s was generally lower than that of the sample after 300s, indicating that the longer the time, the more sperm bound to HA on the electrode surface and the smaller the signal.

[0074] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A sperm quality detection sensor, characterized in that: include: An electrode, wherein the electrode is a glassy carbon / gold electrode, and the surface of the electrode is modified with a HA functional layer, wherein the HA functional layer has amino active sites; A redox probe, wherein the redox probe generates an electrical signal with the electrode, and the HA functional layer can bind to a sperm surface receptor in the semen to be tested to hinder electron exchange between the electrode and the redox probe; A conductive layer is provided between the electrode and the redox probe and is used for conducting electricity between the electrode and the redox probe.

2. The sperm quality detection sensor according to claim 1, characterized in that: The conductive layer includes a gold nanodendrite array and a graphene / Nafion composite matrix layer.

3. The sperm quality detection sensor according to claim 1, characterized in that: It also includes an isolation membrane, which is coated on the outer surface of the multilayer structure formed by the electrode, the conductive layer and the redox probe; the isolation membrane includes a film-forming structure formed by gradient volatilization of Nafion solution by ethanol, and the isolation membrane has a negatively charged ion channel.

4. The sperm quality detection sensor according to claim 1, characterized in that: The redox probe is formed by depositing Prussian blue using a mixed solution containing FeCl3, K3[Fe(CN)6], KCl and HCl.

5. The sperm quality detection sensor according to claim 1, characterized in that: The HA functional layer is formed by activating the carboxyl group with EDC / NHS and cross-linking with ADH through amide bonds, and epoxy ring-opening grafting of the hydroxyl group with PEG-DGE.

6. A method for preparing a sperm quality detection sensor, for preparing the sperm quality detection sensor as claimed in any one of claims 1 to 5, characterized in that: The steps include: Base treatment to form a base layer; preparing electrodes on the substrate layer and pre-treating the electrodes; providing a conductive layer on the surface of the electrode; providing a HA functional layer on the electrode; Electrochemical deposition of Prussian blue was used to form a redox probe; Blocking nonspecific sites on the surface of electrodes, conductive layers, and redox probes; Nafion selective membranes were prepared on the outer surfaces of the electrodes, conductive layer and redox probe.

7. The method for preparing a sperm quality detection sensor according to claim 6, wherein: The electrode is prepared on the substrate layer, comprising: Preparation of working electrode: A gold electrode was prepared on the substrate layer using a photolithography process to serve as the working electrode. Counter electrode preparation: Platinum slurry is spray-printed on the substrate using a microelectronic printer to form a circular electrode; Preparation of reference electrode: Ag / AgCl slurry was printed on the substrate layer, and a stable reference electrode was formed by constant potential oxidation method.

8. The method for preparing a sperm quality detection sensor according to claim 6, wherein: Pre-treatment of the electrode includes: Mechanical polishing: The working electrode is polished step by step using aluminum oxide suspensions of different particle sizes. Ultrasonic cleaning is used to remove aluminum oxide after each polishing step. Chemical activation: The working electrode was ultrasonically treated with a HCl:H2O2 mixture to remove oxides on the working electrode surface, and the oxygen-containing functional groups on the working electrode surface were activated by cyclic voltammetry in a H2SO4 solution.

9. The method for preparing a sperm quality detection sensor according to claim 6, wherein: The preparation of the conductive layer on the electrode surface comprises: Dendritic gold nanostructures were formed by constant potential pulse deposition of a 50 mM HAuCl4 solution containing 0.1 M KCl.

10. The method for preparing a sperm quality detection sensor according to claim 6, wherein: Providing a HA functional layer at the end of the electrode includes: HA carboxyl groups were activated using EDC / NHS and then coupled with ADH at room temperature for 12 h.

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