A sperm quality detection sensor and a preparation method thereof

Abstract

This invention discloses a sperm quality detection sensor and its preparation method, belonging to the field of detection and sensing technology. It includes an electrode with an HA functional layer modified on its surface, the HA functional layer having amino active sites; a redox probe that generates an electrical signal with the electrode; and a conductive layer disposed between the electrode and the redox probe for conduction between them. This application specifically binds the HA functional layer to receptors on the surface of healthy sperm in the test semen and employs microcurrent signal processing technology. The specific binding of the HA functional layer to the receptors affects the current between the electrode and the redox probe. The change in current of the sperm quality detection sensor characterizes the number of healthy sperm in the test semen. This sperm quality detection sensor overcomes the limitations of morphological interpretation in traditional detection technologies, significantly reducing the size of sperm detection equipment and improving the accuracy of sperm quality detection.

Description

Technical Field

[0001] This invention belongs to the field of detection sensor technology, specifically relating to a sperm quality detection sensor and its preparation method. Background Technology

[0002] Innovations in reproductive health monitoring technology are of great significance to the development of human fertility assessment and assisted reproduction. Sperm quality testing, as a core assessment method, is of great importance to couples preparing for pregnancy, assisted reproduction, and the diagnosis of male reproductive diseases due to its accuracy.

[0003] Traditional semen analysis primarily employs optical microscopy or computer-aided semen analysis systems to detect and analyze sperm in semen. Optical microscopy requires multiple steps, including semen liquefaction, staining and fixation, and manual microscopic examination, which is time-consuming. Furthermore, manual microscopic examination relies on the experience of the examiner, and key indicators such as sperm acrosome integrity and sperm neck curvature are assessed visually, leading to inconsistent sperm quality assessments. Computer-aided semen analysis systems use image recognition algorithms for automated sperm detection; however, these systems are expensive, requiring phase-contrast microscopes and high-speed imaging systems. Additionally, when sperm density is too low or uneven, the system can easily misidentify similar particles (such as cell debris and white blood cells) as sperm, resulting in significant detection errors. Summary of the Invention

[0004] In view of 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.

[0005] To achieve the above objectives, the present invention provides a sperm quality detection sensor, comprising:

[0006] The electrode is a glassy carbon / gold electrode, and the surface of the electrode is modified with an HA functional layer, the HA functional layer having amino active sites;

[0007] A redox probe that undergoes a redox reaction with the electrode and generates an electrical signal; the HA functional layer can bind to sperm surface receptors in the semen to prevent electron exchange between the electrode and the redox probe.

[0008] A conductive layer is disposed between the electrode and the redox probe for conducting electricity between the electrode and the redox probe.

[0009] As a further improvement of the present invention, the conductive layer comprises a gold nanodendritic array and a graphene / Nafion composite matrix layer.

[0010] As a further improvement of the present invention, it also includes an isolation membrane, which covers 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 the evaporation of Nafion solution through an ethanol gradient, and the isolation membrane has negatively charged ion channels.

[0011] 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.

[0012] As a further improvement of the present invention, the HA functional layer is formed by activating the carboxyl groups with EDC / NHS and crosslinking them with amide bonds with ADH, and by using PEG-DGE to perform epoxy ring-opening grafting on the hydroxyl groups.

[0013] This application also includes a method for preparing a sperm quality detection sensor, which includes the following steps:

[0014] Substrate treatment to form a base layer;

[0015] Electrodes are fabricated on a substrate and pretreated.

[0016] A conductive layer is formed on the electrode surface;

[0017] An HA functional layer is provided at the electrode tip;

[0018] A redox probe was formed by electrochemical deposition of Prussian blue.

[0019] Non-specific sites on the surfaces of electrodes, conductive layers, and redox probes are sealed.

[0020] Nafion selective films were prepared on the outer surfaces of electrodes, conductive layers, and redox probes.

[0021] As a further improvement of the present invention, the electrode fabrication on the substrate layer includes:

[0022] Working electrode fabrication: A gold electrode is fabricated on the substrate layer using photolithography to serve as the working electrode;

[0023] Electrode fabrication: A microelectronic printer is used to spray platinum paste onto the substrate to form a circular electrode;

[0024] Reference electrode preparation: Ag / AgCl slurry is printed on the substrate layer, and a stable reference electrode is formed by constant potential oxidation.

[0025] As a further improvement of the present invention, the pretreatment of the electrodes includes:

[0026] Mechanical polishing: The working electrode is polished step by step using alumina suspensions of different particle sizes in stages. After each polishing stage, ultrasonic cleaning is used to remove the alumina.

[0027] Chemical activation: The working electrode was ultrasonically treated with a mixture of HCl:H2O2 to remove oxides from its surface. The oxygen-containing functional groups on the surface of the working electrode were then activated by cyclic voltammetry in H2SO4 solution.

[0028] As a further improvement of the present invention, the preparation of the conductive layer on the electrode surface includes:

[0029] Dendritic gold nanostructures were formed by constant potential pulse deposition of a 50 mM HAuCl4 solution containing 0.1 M KCl.

[0030] As a further improvement of the present invention, the HA functional layer is provided at the electrode end, including:

[0031] The HA carboxyl group was activated using EDC / NHS, and then coupled with ADH at room temperature for 12 hours.

[0032] The aforementioned improved technical features can be combined with each other as long as they do not conflict with each other.

[0033] In summary, the beneficial effects of the above-described technical solutions conceived by this invention compared with the prior art include:

[0034] (1) The sperm quality detection sensor of the present invention uses biomolecular directional recognition technology to specifically bind the HA functional layer to the surface receptors of healthy sperm in the test semen, and uses microcurrent signal processing technology to utilize the specific binding of the HA functional layer to the sperm surface receptors to affect the current between the electrode and the redox probe. The change in current of the sperm quality detection sensor characterizes the number of healthy sperm in the test semen. The sperm quality detection sensor of the present application can directly reflect the integrity of the sperm membrane and fertilization capacity in the semen through electrochemical signal difference, breaking through the limitations of morphological interpretation in traditional detection technology, which can greatly improve the size of sperm detection equipment and improve the accuracy of sperm quality detection.

[0035] (2) The sperm quality detection sensor of the present invention has a three-dimensional dendritic gold structure that can effectively increase the specific surface area of ​​the conductive layer, allowing sperm in the test semen that has passed 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 conductivity, and the Nafion composite matrix can isolate other substances in the semen, preventing the 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 healthy sperm count detection.

[0036] (3) The sperm quality detection sensor of the present invention is provided with an isolation membrane, which includes a film-forming structure formed by evaporating Nafion solution with ethanol gradient. After the Nafion solution is evaporated with ethanol gradient, negatively charged ion channels will be formed on the surface of the membrane structure. The negatively charged ion channels can synergistically repel proteins with a value greater than 5kDa and positively charged cell debris in the semen, so as to avoid the rejection of damaged sperm and non-sperm proteins. This allows sperm with specific receptors to pass through the isolation membrane and bind to 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. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the structure of the sperm quality detection sensor in an embodiment of the present invention;

[0038] Figure 2 (a) is a schematic diagram of a sperm quality detection sensor model; Figure 2 (b) is a schematic diagram of the model of sperm binding to hyaluronic acid in a sperm quality detection sensor;

[0039] Figure 3 (a) shows the it curves of the reaction between different concentrations of the test semen and the HA on the working electrode surface of the sensor for 300 s; Figure 3 (b) shows the it curves of the reaction between different concentrations of the test semen and the HA on the working electrode surface of the sensor for 600 s;

[0040] Figure 4 This is a schematic flowchart of the sperm quality detection sensor preparation method in an embodiment of the present invention. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0042] In the description of this invention, it should be understood that, unless otherwise stated, the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0043] Furthermore, unless otherwise stated, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0044] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0045] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0046] Please see Figures 1-4The sperm quality detection sensor in a preferred embodiment of the present invention includes an electrode, which is a glassy carbon / gold electrode structure, and has an HA (hyaluronic acid) functional layer modified on its surface. The HA functional layer has amino active sites. It also includes a redox probe that generates an electrical signal with the electrode, and a conductive layer disposed between the electrode and the redox probe for conducting electricity between them. Specifically, the HA functional layer on the electrode surface can specifically bind to receptors on the surface of healthy sperm in the test semen. After binding, it hinders electron exchange between the electrode and the redox probe. By detecting the current strength between the electrode and the redox probe, the number of healthy sperm in the semen bound to the HA functional layer can be obtained, thus determining the number of healthy sperm in the test semen.

[0047] The sperm quality detection sensor in this application employs biomolecular directional recognition technology to specifically bind the HA functional layer to receptors on the surface of healthy sperm in the test semen. It also utilizes microcurrent signal processing technology to leverage the specific binding of the HA functional layer to the receptors to influence the current between the electrode and the redox probe. The change in current from the sperm quality detection sensor characterizes the number of healthy sperm in the test semen. This sperm quality detection sensor can directly reflect the integrity of the sperm membrane and fertilization capacity in semen through electrochemical signal difference, overcoming the limitations of morphological interpretation in traditional detection technologies. This significantly improves the size of sperm detection equipment and enhances the accuracy of sperm quality detection.

[0048] It is worth noting that the conductive layer in this application is disposed 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 on the conductive layer as a whole, forming a multilayer structure of electrode, conductive layer and redox probe.

[0049] Furthermore, as an optional embodiment of the present invention, the conductive layer in this application comprises a gold nanodendritic array and a graphene / Nafion (perfluorosulfonic acid resin) composite matrix layer. The three-dimensional dendritic gold structure can effectively increase the specific surface area of ​​the conductive layer, allowing sperm in the test semen to quickly bind to the HA functional layer on the electrode surface. Secondly, the graphene / Nafion composite matrix layer has good conductivity, and the Nafion composite matrix can isolate other substances in the semen, preventing the semen from contacting the electrode and affecting its normal operation, thus ensuring the accuracy of the electrical signal generated between the electrode and the redox probe and improving the accuracy of healthy sperm count detection.

[0050] Furthermore, as an optional embodiment of the present invention, the sperm quality detection sensor in this application also includes an isolation membrane. The isolation membrane covers the outer surface of the multilayer structure of electrodes, conductive layer and redox probe. The isolation membrane includes a film-forming structure formed by gradient evaporation of Nafion solution with ethanol. After the Nafion solution is evaporated by gradient ethanol, negatively charged ion channels are formed on the surface of the membrane structure. The negatively charged ion channels can synergistically repel proteins greater than 5kDa and positively charged cell debris in semen, so as to avoid the rejection of damaged sperm and non-sperm proteins. This allows sperm with receptors to pass through the isolation membrane and bind to the HA functional layer on the electrode, avoiding interference from other non-sperm substances in semen and improving the detection accuracy of the sperm quality detection sensor for healthy sperm.

[0051] Furthermore, as an optional embodiment of the present invention, before setting the isolation membrane, this application further includes sealing non-specific sites on the electrode, conductive layer, and redox probe. This is mainly achieved by immersing the electrode, conductive layer, and redox probe in a BSA (bovine serum albumin) buffer solution, followed by immersion in a 0.1M glycine solution for 30 minutes, thus doubly sealing the non-specific sites and reducing non-specific adsorption to below 5%. Specifically, non-specific site sealing here refers to BSA binding 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 non-specifically adsorbing onto the surface of the metal electrode, thereby protecting the electrode. Secondly, glycine can coordinate with metal ions through its carboxyl groups, thereby reducing the adsorption of impurities by the metal active sites. Through the combination of BSA and glycine, it can react with the active groups (carboxyl groups and epoxy groups activated by EDC / NHS) on the electrode surface to form covalent bonds or hydrogen bonds, achieving the sealing of active groups.

[0052] 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 comprising FeCl3 (ferric chloride), K3[Fe(CN)6] (potassium ferricyanide), KCl (potassium chloride), and HCl (hydrochloric acid). The redox probe can react with a gold electrode to undergo a redox reaction, generating an electrical signal between the redox probe and the gold electrode.

[0053] 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) via amide bonds, and then performing epoxy ring-opening grafting on the hydroxyl groups using PEG-DGE (polyethylene glycol diglycidyl ether). By activating the carboxyl groups with EDC / NHS and cross-linking them with ADH via amide bonds, and then performing epoxy ring-opening grafting on the hydroxyl groups using PEG-DGE, a double-crosslinked spatial network can be formed, resulting in a HA fixation density of 2.3~3.1 μg / mm². 2 This improves the binding density and stability of the HA functional layer to the electrode, increases the binding efficiency of HA to receptors in sperm, and enhances the sperm capture efficiency.

[0054] Furthermore, as an optional embodiment of the present invention, the electrode in this application includes 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 formed by platinum paste printing, and the reference electrode is a stable reference layer formed by constant potential oxidation of Ag / AgCl paste.

[0055] It is worth noting that the sperm quality detection sensor in this invention also includes a base layer. The electrodes, redox probes, and conductive layers are all disposed on the base layer. The base layer and the isolation membrane cooperate to form a closed sensor structure. Healthy sperm in the semen to be tested can enter the sensor through the ion channels on the isolation membrane and specifically bind to the HA functional layer on the electrode surface to block the transmission of electrical signals between the electrode and the redox probe, thereby changing the detection current of the sensor to characterize the number of healthy sperm in the semen to be tested.

[0056] Furthermore, regarding the sperm quality detection sensor in this application, it also includes a method for preparing the sperm quality detection sensor, which includes the following steps:

[0057] Substrate treatment to form a base layer;

[0058] Electrodes are fabricated on a substrate and pretreated.

[0059] A conductive layer is formed on the electrode surface;

[0060] An HA functional layer is formed on the electrode;

[0061] A redox probe was formed by electrochemical deposition of Prussian blue.

[0062] Non-specific sites on the surfaces of electrodes, conductive layers, and redox probes are sealed.

[0063] Nafion selective films were prepared on the outer surfaces of electrodes, conductive layers, and redox probes.

[0064] Furthermore, as an optional embodiment of the present invention, the substrate treatment to form the substrate layer specifically includes: cutting a PET film to a predetermined size, sequentially ultrasonically cleaning it with acetone, ethanol, and deionized water, and then drying it with nitrogen gas to form the substrate layer. PET film itself has good flexibility and chemical stability, making it suitable as a substrate material for flexible sensors. The gradient solvent cleaning consisting of acetone, ethanol, and deionized water can remove grease (acetone), polar contaminants (ethanol), and ionic residues (water), respectively. Nitrogen purging can prevent fiber residue on the substrate layer surface, thereby obtaining a clean and dry substrate layer.

[0065] Furthermore, as an optional embodiment of the present invention, the substrate treatment to form the substrate layer in this application includes:

[0066] Silicon wafers were selected as the substrate and were sequentially cleaned with acetone, isopropanol, and deionized water. After purging with nitrogen, the silicon wafers were treated with oxygen plasma. Silicon wafers possess high flatness and photolithography compatibility, making them suitable as a substrate layer. Oxygen plasma treatment enhances the surface hydrophilicity of the silicon wafers.

[0067] Furthermore, as an optional embodiment of the present invention, the fabrication of the electrode on the substrate layer specifically includes:

[0068] Working electrode fabrication: A gold electrode is fabricated on the substrate layer using photolithography to serve as the working electrode;

[0069] Electrode fabrication: A microelectronic printer is used to spray platinum paste onto the substrate to form a circular electrode;

[0070] Reference electrode preparation: Ag / AgCl slurry is printed on the substrate layer, and a stable reference electrode is formed by constant potential oxidation.

[0071] Furthermore, as an optional embodiment of the present invention, the preparation of the electrode of this application includes:

[0072] Gold electrode photolithography fabrication: Photoresist is spin-coated onto the surface of a silicon wafer, exposed and developed, and then metal deposition is performed to form a gold electrode pattern on the surface of the silicon wafer;

[0073] Externally integrated reference electrode: An Ag / AgCl electrode is selected and fixed to the edge of the silicon wafer using conductive silver paste. The Ag / AgCl electrode is spaced 1~1.5mm from the gold electrode to avoid short circuit between the gold electrode and the reference electrode caused by the electrolyte.

[0074] Furthermore, as an optional embodiment of the present invention, the pretreatment of the electrodes in this application specifically includes:

[0075] Mechanical polishing: The working electrode is polished step by step using alumina suspensions of different particle sizes in stages. After each polishing stage, ultrasonic cleaning is used to remove the alumina.

[0076] Chemical activation: The working electrode is ultrasonically treated with a mixture of HCl:H2O2 to remove oxides on the surface of the working electrode. The oxygen-containing functional groups (carboxyl or hydroxyl groups) on the surface of the working electrode are activated by cyclic voltammetry in H2SO4 solution to enhance the hydrophilicity and electrochemical activity of the working electrode.

[0077] Furthermore, as an optional embodiment of the present invention, the provision of the conductive layer in this application specifically includes:

[0078] Dendritic gold nanostructures were formed by constant potential pulse deposition of a 50 mM HAuCl4 solution containing 0.1 M KCl.

[0079] Furthermore, as an optional embodiment of the present invention, the conductive layer in this application includes:

[0080] Porous gold nanostructures were formed in a solution containing 0.1 M H2SO4 and 1 mM HAuCl4 using a square wave pulse method.

[0081] Furthermore, as an optional embodiment of the present invention, the provision of an HA functional layer at the electrode end in this application specifically includes:

[0082] By activating the HA carboxyl group with EDC / NHS and then coupling it with ADH at room temperature for 12 hours, a stable amide bond can be formed, and the conformational destruction of HA caused by excessive cross-linking can be avoided.

[0083] Furthermore, as an optional embodiment of the present invention, the provision of an HA functional layer on the electrode in this application includes:

[0084] The electrode was immersed in a 1 mM ethanol solution for 12-14 hours to form a self-assembled monolayer, thereby thiolizing the gold surface.

[0085] The 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.

[0086] Furthermore, as an optional embodiment of the present invention, the formation of the redox probe in this application specifically includes:

[0087] Prussian blue nanoparticle layers were formed in a 0.1 M KCl solution containing 2 mM FeCl3, 2 mM K3[Fe(CN)6], and 0.1 M HCl using cyclic voltammetry.

[0088] Furthermore, as an optional embodiment of the present invention, the non-specific site blocking in this application specifically includes:

[0089] The substrate, electrodes, conductive layer, and Prussian blue structure were sealed at room temperature using 1% BSA, and then treated with 0.1M glycine solution for 30 min to double-seal unreacted sites, so that the relative ratio of background signal to positive signal after sealing was <5%.

[0090] Furthermore, as an optional embodiment of the present invention, the preparation of the Nafion selective membrane in this application specifically includes:

[0091] A Nafion solution diluted with ethanol was drop-coated and dried to form a cation exchange membrane with a pore size of 1-2 nm.

[0092] The sperm quality detection sensor was prepared using the method described above.

[0093] Example 1:

[0094] Sensor substrate preparation and electrode fabrication:

[0095] Substrate treatment: Cut 1 cm × 1 cm PET film with a thickness of 0.18 mm. Clean the film with acetone (40℃), ethanol (30℃), and deionized water for 15 minutes each (ultrasonic power 100 W). Dry the PET film by blowing nitrogen gas at a pressure of 0.3 MPa.

[0096] Working electrode fabrication: A 0.5 mm gold electrode with a thickness of 100 nm was fabricated on a PET film using photolithography. The gold electrode was used as the working electrode with a diameter of 3 mm.

[0097] Electrode preparation: Platinum paste was sprayed onto a PET film using a microelectronic printer to form a circular electrode with a diameter of 3 mm and a thickness of 20 ± 2 μm.

[0098] Reference electrode preparation: Ag / AgCl slurry is printed on PET film, and a stable reference layer is formed by constant potential oxidation method; specifically, in constant potential oxidation method, Ag / AgCl is used as the reference electrode reference, and oxidation is carried out continuously at +0.5V potential for 600s.

[0099] Working electrode pretreatment:

[0100] Mechanical polishing: Alumina suspensions of 1.0 μm, 0.3 μm, and 0.05 μm are used in stages. The alumina powder in the alumina suspension is mixed with ultrapure water at a mass ratio of 1:1. Polishing is carried out step by step. After each polishing stage, ultrasonic cleaning with deionized water and anhydrous ethanol is required for 5 minutes each to avoid particle residue that may cause uneven surface roughness.

[0101] Chemical activation: The surface oxides were removed by ultrasonic treatment with a HCl:H2O2 (volume ratio 3:1) mixture for 10 minutes. Subsequently, the oxygen-containing functional groups on the surface were activated by cyclic voltammetry in 0.1 M H2SO4. Specifically, in the cyclic voltammetry, Ag / AgCl was used as the reference, and cyclic scanning was performed between -0.2V and +1.2V for 20 cycles at a scan rate of 50mV / s.

[0102] Conductive layer modification:

[0103] Gold nanostructure modification: A potentiostatic pulse deposition process was performed using a mixed solution containing 0.1 M KCl and 50 mM HAuCl4 to form uniform dendritic Au nanostructures (SEM verified particle size 20 ± 5 nm), improving conductivity and specific surface area. The pH of the 0.1 M KCl and 50 mM HAuCl4 mixed solution was 1.5. In the potentiostatic pulse deposition, deposition was performed at -0.2 V for 0.5 s, followed by 1 s at 0.5 V, forming one cycle, for a total of 100 cycles.

[0104] HA functional layer fixed:

[0105] HA crosslinking: The HA carboxyl groups were activated using EDC / NHS at pH 5.5 for 2 hours at 4°C; subsequently, it was coupled with ADH at room temperature for 12 hours. The molar ratio of EDC to NHS was 1:2, the concentration of ADH was 10 mM, and the pH was 7.4.

[0106] HA immobilization verification: The thickness (target 50-100 nm) and surface roughness (Ra<5 nm) of the HA layer were characterized by atomic force microscopy (AFM) to ensure uniform coverage; and the amino crosslinking efficiency was verified by detecting the nitrogen content by XPS (X-ray photoelectron spectroscopy).

[0107] Prussian blue electrochemical deposition: Prussian blue (PB) nanoparticle layers were formed in a 0.1 M KCl solution containing 2 mM FeCl3, 2 mM K3[Fe(CN)6], and 0.1 M HCl using cyclic voltammetry. Specifically, the cyclic voltammetry was performed at -0.2 V to +0.6 V for 10 cycles at a scan rate of 50 mV / s.

[0108] Construction of closed and selective barriers:

[0109] Non-specific site blocking: Unreacted sites were blocked for 2 hours at room temperature with 1% BSA solution, followed by treatment with 0.1 M glycine solution for 30 minutes. Specifically, the BSA solution was PBS buffer containing 0.05% Tween-20, and the glycine solution was at pH 8.5.

[0110] Preparation of Nafion-selective membranes: After drop-coating with 0.5% Nafion, the membranes were dried at 40% humidity and 25°C for 24 hours to form cation exchange membranes with pore sizes of 1-2 nm. The integrity of the membranes was verified by electrochemical impedance spectroscopy (EIS), ensuring that the low-frequency impedance of the Nafion-selective membrane was >1 kΩ·cm. 2 Preferably, the Nafiong solution here is obtained by diluting it with ethanol.

[0111] Example 2:

[0112] Sensor substrate preparation and electrode fabrication:

[0113] Substrate treatment: A 1.5 cm × 1.5 cm silicon wafer with a thickness of 500 μm was used as the substrate; it was ultrasonically cleaned for 20 minutes each with acetone (50℃), isopropanol (40℃) and deionized water, with an ultrasonic power of 120 W. After purging with nitrogen at a pressure of 0.4 MPa, it was treated with 100 W oxygen plasma for 5 minutes to enhance the hydrophilicity of the silicon wafer surface.

[0114] Gold electrode photolithography fabrication:

[0115] Spin coating of photoresist: AZ 5214 photoresist was used for spin coating at a speed of 3000 rpm, with a coating thickness of 1.5 μm, and pre-baked at 90℃ for 90 s.

[0116] Exposure and Development: The mask design includes a working electrode and a counter electrode. UV exposure is performed using AZ 300 MIF developer for 30 seconds. The working electrode is a 2mm diameter circular electrode, and the counter electrode is a ring electrode with an outer diameter of 4mm and an inner diameter of 3mm. The UV exposure parameters are 365nm and 100 mJ / cm². 2 .

[0117] Metal deposition: Gold layer is evaporated by electron beam to a thickness of 150 nm at an evaporation rate of 0.5 Å / s, and electrode patterns are formed after peeling.

[0118] Externally integrated reference electrode:

[0119] Ag / AgCl reference electrode: An Ag / AgCl electrode with a diameter of 1 mm is selected and fixed to the edge of the silicon substrate with conductive silver paste. The distance between the Ag / AgCl reference electrode and the working electrode is 1 mm.

[0120] Working electrode pretreatment:

[0121] Mechanical polishing: Alumina suspensions of 1.0 μm, 0.3 μm, and 0.05 μm were used in stages. Alumina powder and ultrapure water were mixed at a mass ratio of 1:1 and polished step by step. After each polishing stage, the surface was ultrasonically cleaned for 5 minutes each with deionized water and anhydrous ethanol to avoid uneven surface roughness caused by particle residue.

[0122] Chemical activation: Surface oxides were removed by ultrasonic treatment with a HCl:H₂O₂ mixture for 10 minutes. Subsequently, oxygen-containing functional groups (such as carboxyl and hydroxyl groups) on the surface were activated by cyclic voltammetry in 0.1 MH₂SO₄ to enhance the hydrophilicity and electrochemical activity of the electrode. The volume ratio of HCl to H₂O₂ was 3:1, the cyclic voltammetry voltage was -0.2 V to +1.2 V, the reference electrode was Ag / AgCl, the scan rate was 50 mV / s, and the cycle was 20 times.

[0123] Electrode conductive layer modification: Porous gold nanostructures were formed in a mixed solution of 0.1 M H2SO4 and 1 mM HAuCl4 using a square wave pulse method (SEM verification showed that the pore size of the gold nanostructures was 50 ± 10 nm); 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.

[0124] HA is covalently fixed:

[0125] Thiolization of gold surface: The gold electrode was immersed in 1 mM ethanol solution for 12 hours to form a self-assembled monolayer.

[0126] HA Coupling: 10 mg / mL HA was reacted with thiol groups on the electrode surface, and the coupling efficiency was verified by detecting the S2p peak using XPS. The 10 mg / mL HA contained 1 mM EDC and 0.5 mM NHS. The pH of the HA reaction with the electrode surface was 7.0, and the reaction was carried out at room temperature for 6 hours.

[0127] 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. A 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 at 80±10 nm (the PB layer thickness was verified by AFM).

[0128] Construction of anti-pollution barriers:

[0129] Nafion membrane optimization: A 1% Nafion solution was spin-coated at 3000 rpm for 30 seconds, followed by annealing at 80°C for 1 hour to form a dense film. The EIS low-frequency impedance of the dense film was >2 kΩ·cm. 2 .

[0130] Sperm quality detection sensor performance test:

[0131] Linear range: The calibration curve covers 10 using standard sperm suspension testing. 4 ~10 7 cells / mL, detection limit (LOD) ≤200 cells / mL.

[0132] Anti-interference test: Add 10 times the concentration of protein (BSA), glucose, and metal ions (Na+). + K + The detection signal change was less than 5%, proving that the Nafion membrane effectively shields macromolecular interference.

[0133] Stability and reproducibility:

[0134] After storing the sperm quality detection sensor at 4°C for 30 days, it was tested again, and the response signal attenuation was <8%; and the relative standard deviation (RSD) of the same batch of electrodes (n>5) was <4%, which meets the requirements for clinical testing.

[0135] Simultaneously, a sperm quality detection sensor was used to test semen samples of different concentrations. Figure 3 In the middle (a), the it curve of the reaction between sperm and HA on the working electrode surface of the sensor after 300 s is shown. Figure 3 (b) represents the it curve of the reaction between sperm and HA on the working electrode surface of the sensor after 600 s. It can be seen that the sperm concentration is 37 × 10⁻⁶. 6 The sample signal per 1000 sperm / mL was lower than that of a sperm concentration of 10 × 10⁻⁶. 6 The sample signal was as expected, with the number of sperm bound to HA on the electrode surface increasing over time. The signal of the sample after 600 seconds of resting was generally lower than that after 300 seconds of resting, indicating that the longer the time, the more sperm bound to HA on the electrode surface, and the smaller the signal.

[0136] Those skilled in the art will readily understand 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 within the scope of protection of the present invention.

Claims

1. A sperm quality detection sensor, characterized in that, include: The electrode is a glassy carbon / gold electrode, and the surface of the electrode is modified with an HA functional layer, the HA functional layer having amino active sites; A redox probe that generates an electrical signal with the electrode; the HA functional layer can bind to sperm surface receptors in the semen to block electron exchange between the electrode and the redox probe. A conductive layer is disposed between the electrode and the redox probe 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 comprises a gold nanodendritic 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 covers 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 the evaporation of Nafion solution through an ethanol gradient, and the isolation membrane has negatively charged ion channels.

4. The sperm quality detection sensor according to claim 1, characterized in that, The redox probe was 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 groups with EDC / NHS and crosslinking them with amide bonds with ADH, and then using PEG-DGE to perform epoxy ring-opening grafting on the hydroxyl groups.

6. A method for preparing a sperm quality detection sensor, used to prepare the sperm quality detection sensor as described in any one of claims 1 to 5, characterized in that, Includes the following steps: Substrate treatment to form a base layer; Electrodes are fabricated on a substrate and pretreated. A conductive layer is formed on the electrode surface; An HA functional layer is formed on the electrode; A redox probe was formed by electrochemical deposition of Prussian blue. Non-specific sites on the surfaces of electrodes, conductive layers, and redox probes are sealed. Nafion selective films were prepared on the outer surfaces of electrodes, conductive layers, and redox probes.

7. The method for preparing the sperm quality detection sensor according to claim 6, characterized in that, The electrode is fabricated on the substrate layer including: Working electrode fabrication: A gold electrode is fabricated on the substrate layer using photolithography to serve as the working electrode; Electrode fabrication: A microelectronic printer is used to spray platinum paste onto the substrate to form a circular electrode; Reference electrode preparation: Ag / AgCl slurry is printed on the substrate layer, and a stable reference electrode is formed by constant potential oxidation.

8. The method for preparing the sperm quality detection sensor according to claim 6, characterized in that, Electrode pretreatment includes: Mechanical polishing: The working electrode is polished step by step using alumina suspensions of different particle sizes in stages. After each polishing stage, ultrasonic cleaning is used to remove the alumina. Chemical activation: The working electrode was ultrasonically treated with a mixture of HCl:H2O2 to remove oxides from its surface. The oxygen-containing functional groups on the surface of the working electrode were then activated by cyclic voltammetry in H2SO4 solution.

9. The method for preparing the sperm quality detection sensor according to claim 6, characterized in that, The conductive layer disposed on the electrode surface includes: 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 the sperm quality detection sensor according to claim 6, characterized in that, The HA functional layer at the electrode tip includes: The HA carboxyl group was activated using EDC / NHS, and then coupled with ADH at room temperature for 12 hours.

Images