Working electrode for hormone in-vivo detection, preparation method of working electrode and electrochemical sensor

Abstract

The invention discloses a working electrode for hormone in-vivo detection, a preparation method of the working electrode and an electrochemical sensor, and relates to the technical field of hormone detection, the working electrode comprises an electrode substrate and a sensing layer located on the surface of the electrode substrate; the sensing layer comprises an inductor layer, the inductor layer comprises an enzyme or a receptor, the enzyme directly or indirectly takes female reproductive hormone as a catalytic substrate, and the receptor is a female reproductive hormone receptor. According to the invention, the enzyme or the receptor is used as a material of the inductor layer, the enzyme directly or indirectly takes the female reproductive hormone as a catalytic substrate, the receptor is a female reproductive hormone receptor, and the enzyme or the receptor is not consumed in the process of reacting with the female reproductive hormone. Therefore, when the working electrode is used for detecting the female reproductive hormone, the working electrode can be repeatedly used, continuous monitoring can be achieved, the working electrode is high in anti-interference performance, and the detection range of the concentration of the female reproductive hormone can reach 10-1000 pM.

Description

Technical Field

[0001] The present invention relates to the technical field of hormone detection, and in particular to a working electrode for in-vivo hormone detection, a preparation method thereof, and an electrochemical sensor. Background Art

[0002] Female reproductive hormones have a significant impact on female health and are primarily categorized into two main groups: pituitary hormones and ovarian hormones. Pituitary hormones include follicle-stimulating hormone (FSH) and luteinizing hormone (LH), while ovarian hormones include estradiol (E2) and progesterone (P4). These hormones play a key role in female ovulation, menstrual cycles, pregnancy, ovarian syndrome, and postpartum depression. Specifically, follicle-stimulating hormone promotes follicular development, regulates follicle selection and closure, and synergizes with estrogen on ovulation and corpus luteum formation. Luteinizing hormone stimulates theca cells to synthesize androgens, providing a substrate for estradiol synthesis, promoting oocyte maturation prior to ovulation, and promoting the synthesis and secretion of progesterone and estrogen. Estradiol primarily promotes the development of female reproductive organs, the appearance of secondary sexual characteristics, and regulates follicle growth and endometrial repair. Progesterone primarily acts late in the menstrual cycle by promoting endometrial thickening in preparation for embryo implantation. Monitoring the levels of these hormones allows for an accurate assessment of a woman's health. For example, ovulation typically occurs mid-cycle, and changes in the levels of follicle-stimulating hormone, luteinizing hormone, and estradiol can help determine the exact time of ovulation. Furthermore, women's health issues such as polycystic ovary syndrome (PCOS) are also associated with abnormal levels of these hormones, making hormone level monitoring an important tool for diagnosis and treatment.

[0003] Existing hormone detection methods are mainly carried out through blood sampling, which requires special medical equipment and cannot achieve continuous and real-time monitoring. Electrochemical sensors show obvious advantages in real-time monitoring. They can not only provide test results in real time, but also have high sensitivity and a high degree of automation. At present, some electrochemical sensors have been used to detect hormones in environmental samples (such as food) or in vitro samples (such as saliva and urine), such as monitoring estradiol residues in food, but they still face some challenges, such as being able to only be used once and not being able to achieve continuous monitoring. Specifically, this is because the substances in the electrodes of electrochemical sensors that react with hormones, such as antibodies, aptamers, etc., change their structure after binding with hormones and can no longer be used.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] Based on the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a working electrode for in vivo hormone detection, a preparation method thereof, and an electrochemical sensor, aiming to solve the problem that hormone electrodes or sensors can only be used once and cannot achieve continuous monitoring.

[0006] The technical solutions of the present invention are as follows:

[0007] A first aspect of the present invention provides a working electrode, wherein the working electrode comprises an electrode substrate and a sensing layer located on a surface of the electrode substrate;

[0008] The sensing layer includes a sensor layer, and the sensor layer includes an enzyme or a receptor. The enzyme directly or indirectly uses female reproductive hormone as a catalytic substrate, and the receptor is a female reproductive hormone receptor.

[0009] Optionally, the working electrode further includes a polymer protective layer, which is located on the surface of the sensing layer, and the polymer protective layer includes at least one of chitosan, polyurethane, alginate, agarose, collagen, silk fibroin, polytetravinylpyridine-polystyrene block copolymer, polyvinyl alcohol and polyethylene glycol.

[0010] Optionally, the sensing layer further includes a conductive polymer layer, which is located between the electrode substrate and the sensing layer; the conductive polymer layer includes at least one of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid, polyaniline, poly(p-phenylene glycol) and polypyrrole.

[0011] Optionally, the sensing layer further comprises a metal nanoparticle layer, wherein the metal nanoparticle layer is located between the conductive polymer layer and the sensor layer, and the metal nanoparticles in the metal nanoparticle layer are chemically bonded to the enzyme or receptor in the sensor layer.

[0012] Optionally, the electrode substrate includes a metal electrode or a conductive non-metal electrode.

[0013] Optionally, the female reproductive hormone includes one of progesterone, follicle-stimulating hormone, luteinizing hormone and estradiol.

[0014] Optionally, the enzyme includes one of an enzyme that uses progesterone as a direct or indirect catalytic substrate and an enzyme that uses estradiol as a direct or indirect catalytic substrate; the receptor includes one of a follicle-stimulating hormone receptor, a luteinizing hormone receptor, and an estradiol receptor;

[0015] The enzyme that directly or indirectly catalyzes progesterone as a substrate includes one of cytochrome P450 17A1, 3β-hydroxysteroid dehydrogenase, cytochrome P450 3A4, and 20β-hydroxysteroid dehydrogenase; the enzyme that directly or indirectly catalyzes estradiol as a substrate includes one of cytochrome P450 1A1, cytochrome P450 1B1, 17β-hydroxysteroid dehydrogenase, sulfotransferase, and uridine diphosphate glucuronyltransferase; and the estradiol receptor includes estrogen receptor α or estrogen receptor β.

[0016] A second aspect of the present invention provides a method for preparing a working electrode as described in any one of the above aspects of the present invention, comprising the following steps:

[0017] providing an electrode substrate;

[0018] A sensing layer is formed on the surface of the electrode substrate to obtain the working electrode.

[0019] Optionally, the electrode substrate is prepared by etching combined with physical vapor deposition;

[0020] The sensing layer is formed on the surface of the electrode substrate by one of drop coating, spin coating, spray coating, doctor blade coating, printing and dip coating.

[0021] In a third aspect, the present invention provides an electrochemical sensor, wherein the electrochemical sensor comprises a counter electrode, a reference electrode, and at least one working electrode as described above.

[0022] Beneficial Effects: The present invention utilizes an enzyme or receptor as the sensing layer material. The enzyme directly or indirectly uses female reproductive hormone as a catalytic substrate, and the receptor is a female reproductive hormone receptor. The enzyme or receptor is not consumed during its interaction with female reproductive hormone. Therefore, the working electrode provided by the present invention is reusable for female reproductive hormone detection, enabling continuous monitoring. The working electrode also exhibits strong anti-interference properties and can detect female reproductive hormone concentrations within a range of 10 to 1000 pM. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the structure of the working electrode in an embodiment of the present invention.

[0024] Figure 2 Schematic diagram of the preparation process of the working electrode in an embodiment of the present invention.

[0025] Figure 3 This is the cyclic voltammetry stability test result of the first working electrode in Example 5.

[0026] Figure 4This is a current-voltage curve obtained by continuously monitoring estradiol solutions of different concentrations using an electrochemical sensor in Example 7.

[0027] Figure 5 This is a graph showing the change in current versus estradiol concentration obtained by using an electrochemical sensor to test estradiol solutions of different concentrations in Example 7.

[0028] Figure 6 This is the anti-interference test result diagram in Example 7.

[0029] Figure 7 This is a graph showing the change in current over time obtained by continuous monitoring of female rats for 6 hours using an electrochemical sensor in Example 7.

[0030] Figure 8 This is a graph showing the change in current over time obtained by continuous monitoring of female rats for 28 days using an electrochemical sensor in Example 7. DETAILED DESCRIPTION

[0031] The present invention provides a working electrode for in vivo hormone detection, a preparation method thereof, and an electrochemical sensor. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0032] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] In existing electrochemical sensors for hormone detection, substances in the electrodes that react with hormones, such as antibodies and aptamers, change their structure after binding with hormones and become unusable. Therefore, existing electrochemical sensors for hormone detection can only be used once and cannot achieve continuous monitoring. Based on this, an embodiment of the present invention provides a working electrode, wherein, as Figure 1 As shown, the working electrode includes an electrode substrate 1 and a sensing layer 2 located on the surface of the electrode substrate;

[0034] The sensing layer 2 includes a sensor layer 21 , which includes an enzyme or a receptor. The enzyme directly or indirectly uses female reproductive hormone as a catalytic substrate, and the receptor is a female reproductive hormone receptor.

[0035] The present invention uses an enzyme or a receptor as a sensing body layer material, the enzyme directly or indirectly uses female reproductive hormone as a catalytic substrate, the receptor is a female reproductive hormone receptor, the enzyme or receptor and female reproductive hormone as specific recognition elements to identify the target hormone, react with the target hormone (such as redox reaction), cause a change in the electrochemical signal, and then realize the detection of female reproductive hormone. In addition, during the process of the enzyme or receptor acting on the female reproductive hormone, it will not be consumed. Therefore, when the working electrode provided by the present invention is used for female reproductive hormone detection, it can be reused and continuous monitoring can be achieved, and the working electrode has strong anti-interference performance and can detect the concentration of female reproductive hormones in a range of 10 to 1000 pM.

[0036] The working electrode in the present invention can realize continuous monitoring of female reproductive hormones. The core lies in that the enzyme will not be consumed in the catalytic reaction (depending on its catalytic cycle characteristics) or the receptor will not be consumed in the reaction, so it can continuously and repeatedly act on new substrate molecules (i.e., new female reproductive hormone molecules).

[0037] Specifically, in the enzyme-catalyzed electrochemical sensing process, the enzyme binds to the substrate and catalyzes the reaction to produce a product. However, the enzyme itself is not consumed. Instead, it returns to its initial state and continues to catalyze new substrate molecules. This entire process can be represented by the following reaction mechanism:

[0038] (1) Substrate recognition and binding:

[0039] Among them, E is enzyme; S is substrate; ES is enzyme-substrate complex.

[0040] (2) Catalytic reaction (conversion of substrate to product): ES → E + P;

[0041] Among them, P is the product; E is the enzyme released after catalysis, which can continue to bind new substrates.

[0042] Theoretically, this enzyme catalytic cycle can be repeated indefinitely, as long as the substrate is continuously supplied and the enzyme is not inactivated (e.g., denatured or lost). Furthermore, in enzyme electrochemical sensors, the enzyme's catalytic action is accompanied by the transfer of electrons, generating an electrical signal. This electron transfer process is also continuous, so the working electrode can operate continuously.

[0043] During receptor-based electrochemical sensing, female reproductive hormone receptors reversibly bind and dissociate from female sex hormones, without depleting the receptors themselves. For example, when estradiol binds to the estradiol receptor, the receptor undergoes a conformational change, transitioning from an inactive state to an active state, where it begins regulating gene transcription. This process is reversible: when estradiol dissociates from the estradiol receptor, the estradiol receptor returns to its inactive state. After a single activation cycle, the estradiol receptor can dissociate from estradiol and remain in a dormant or deactivated state, awaiting reactivation by the next signal. This process ensures that the estradiol receptor can be reused repeatedly in response to different signals.

[0044] For example, when the luteinizing hormone receptor binds to luteinizing hormone, the luteinizing hormone receptor undergoes conformational changes and activates downstream signaling pathways. The luteinizing hormone receptor initiates a series of biochemical reactions, such as the production of cAMP (cyclic adenosine monophosphate) and the activation of PKA (protein kinase A), through interaction with G proteins, thereby regulating related physiological processes within the cell. The deactivation and reactivation of the luteinizing hormone receptor can be repeated. When luteinizing hormone separates from the luteinizing hormone receptor, the luteinizing hormone receptor usually returns to its original inactive state and is ready to bind to new luteinizing hormone molecules. Therefore, the luteinizing hormone receptor is reusable.

[0045] In some embodiments, the thickness of the electrode substrate is 50 to 600 μm, for example, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm or 600 μm.

[0046] In some embodiments, the electrode substrate comprises a metal electrode or a conductive non-metal electrode.

[0047] In some embodiments, the metal electrode includes one of a gold electrode, a platinum electrode, and a platinum-iridium alloy electrode; and the conductive non-metallic electrode includes one of an indium tin oxide (ITO) electrode, a carbon electrode, and a glassy carbon electrode.

[0048] In some embodiments, the female reproductive hormone comprises one of progesterone, follicle-stimulating hormone, luteinizing hormone, and estradiol.

[0049] In some embodiments, the enzyme comprises one of an enzyme that uses progesterone as a direct or indirect catalytic substrate and an enzyme that uses estradiol as a direct or indirect catalytic substrate; the receptor comprises one of a follicle-stimulating hormone receptor (which is a G protein-coupled receptor), a luteinizing hormone receptor, and an estradiol receptor;

[0050] The enzyme that directly or indirectly catalyzes progesterone as a substrate includes one of CYP17A1 (cytochrome P45017A1 enzyme), 3β-HSD (3β-hydroxysteroid dehydrogenase), CYP3A4 (cytochrome P450 3A4 enzyme) and 20β-HSD (20β-hydroxysteroid dehydrogenase); the enzyme that directly or indirectly catalyzes estradiol as a substrate includes one of CYP1A1 (cytochrome P450 1A1 enzyme), CYP1B1 (cytochrome P4501B1 enzyme), HSD17B (17β-hydroxysteroid dehydrogenase), SULTs (sulfotransferases) and UGTs (uridine diphosphate glucuronosyltransferases); the estradiol receptor includes ERα (estrogen receptor α) or ERβ (estrogen receptor β).

[0051] In some embodiments, the thickness of the sensor layer is 10 to 1000 nm, for example, 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm or 1000 nm.

[0052] In some embodiments, as Figure 1 As shown, the working electrode further includes a polymer protective layer 3, which is located on the surface of the sensing layer 2. The polymer protective layer includes at least one of chitosan (whose molecular weight can be 10-2000 kDa), polyurethane (whose molecular weight can be 10-200 kDa), alginate, agarose, collagen, silk fibroin, polytetravinylpyridine-polystyrene block copolymer (whose molecular weight is 100-1000 kDa), polyvinyl alcohol (PVA), and polyethylene glycol (PEG), but is not limited thereto. In some embodiments, the polymer protective layer further includes a crosslinking agent (such as glutaraldehyde).

[0053] In this embodiment, a polymer protective layer with good biocompatibility is provided on the surface of the sensing layer, which can effectively establish a concentration difference between the inside and outside of the membrane layer, thereby effectively expanding the monitoring range of the working electrode, enhancing its service life and stability in the biological environment, reducing possible interference caused by biological fouling, and ensuring the long-term stability of the working electrode in the biological environment.

[0054] In some embodiments, the thickness of the polymer protective layer is 1 to 100 μm, for example, it can be 1 μm, 2 μm, 5 μm, 8 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm or 100 μm, etc.

[0055] In some embodiments, as Figure 1 As shown, the sensing layer 2 further includes a conductive polymer layer 22, which is located between the electrode substrate 1 and the sensing layer 21; the conductive polymer layer includes at least one of poly (3,4-ethylenedioxythiophene) - polystyrene sulfonic acid (PEDOT:PSS), polyaniline, poly (p-phenylene glycol) and polypyrrole.

[0056] In this embodiment, the conductive polymer layer is used as the electroactive material, combined with the polymer protective layer with good biocompatibility, to ensure that the working electrode can operate stably for a long time in a wet liquid environment and effectively expand the monitoring range of the working electrode.

[0057] In some embodiments, the thickness of the polymer conductive layer is 0.1 to 500 nm, for example, 0.1 nm, 0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm or 500 nm.

[0058] In some embodiments, as Figure 1 As shown, the sensing layer 2 further includes a metal nanoparticle layer 23 , which is located between the conductive polymer layer 22 and the sensor layer 21 . The metal nanoparticles in the metal nanoparticle layer are chemically bonded to the enzymes or receptors in the sensor layer.

[0059] While free enzymes are easily lost in solution, metal nanoparticles immobilizing enzymes or receptors on the working electrode surface can maintain their activity for extended periods, extending the working electrode's lifespan and enabling continuous monitoring. Furthermore, the addition of metal nanoparticles enhances electron transfer efficiency, ensuring the sensitivity and stability of the working electrode, making it particularly suitable for long-term monitoring of female reproductive hormones.

[0060] In some embodiments, the metal nanoparticles are gold nanoparticles, and the enzyme or receptor is fixed on the surface of the working electrode by using the interaction between the metal nanoparticles and the -SH groups of the enzyme or receptor to form a chemical bond (for example, the gold nanoparticles form an Au-SH bond with the -SH groups of the enzyme or receptor).

[0061] In some embodiments, the thickness of the metal nanoparticle layer is 0.1 to 100 nm, for example, 0.1 nm, 0.2 nm, 0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm or 100 nm, etc.

[0062] The present invention also provides a method for preparing the working electrode as described above. Figure 2 As shown, the method includes the following steps:

[0063] S1. Providing an electrode substrate;

[0064] S2. Forming a sensing layer on the surface of the electrode substrate, wherein the sensing layer includes a sensor layer, the sensor layer includes an enzyme or a receptor, the enzyme directly or indirectly uses female reproductive hormone as a catalytic substrate, and the receptor is a female reproductive hormone receptor, to obtain the working electrode.

[0065] In some embodiments, a gold electrode, a platinum electrode, a platinum-iridium alloy electrode, an indium tin oxide electrode, a carbon electrode, or a glassy carbon electrode can be directly used as the electrode substrate. Alternatively, the electrode substrate can be prepared using a method combining etching with physical vapor deposition. Using etching combined with physical vapor deposition to prepare the electrode substrate can improve the precision and sensitivity of the working electrode, resulting in a high-precision and high-sensitivity working electrode.

[0066] In this embodiment, etching includes photolithography, laser etching, etc. Physical vapor deposition includes magnetron sputtering, evaporation, etc.

[0067] In some embodiments, the method for preparing the working electrode specifically comprises the following steps:

[0068] S11, providing an electrode substrate;

[0069] S12, forming a conductive polymer layer on the surface of the electrode substrate;

[0070] S13, forming a metal nanoparticle layer on the surface of the conductive polymer layer;

[0071] S14, forming a sensing layer on the surface of the metal nanoparticle layer to obtain the working electrode.

[0072] In some embodiments, the method for preparing the working electrode specifically comprises the following steps:

[0073] S11, providing an electrode substrate;

[0074] S12, forming a conductive polymer layer on the surface of the electrode substrate;

[0075] S13, forming a metal nanoparticle layer on the surface of the conductive polymer layer;

[0076] S14, forming a sensing layer on the surface of the metal nanoparticle layer;

[0077] S15, forming a polymer protective layer on the surface of the sensing layer to obtain the working electrode.

[0078] In step S11, in some specific embodiments, the electrode substrate includes the following steps:

[0079] Providing a substrate, wherein the substrate is a polymer film such as polyethylene, polyurethane, polyethylene terephthalate (PET), polyimide (PI), polycarbonate (PC), polydimethylsiloxane (PDMS), or the substrate is glass or silicon wafer, but is not limited thereto;

[0080] Gold, platinum, platinum-iridium alloy or carbon are deposited on the substrate by etching combined with physical vapor deposition to form an electrode matrix.

[0081] In step S12, in some embodiments, a conductive polymer layer is formed on the surface of the electrode substrate by one of electroplating, drop coating, spin coating, spray coating, doctor blade coating, printing and dip coating.

[0082] In step S13 , in some embodiments, a metal nanoparticle layer is formed on the conductive polymer layer by one of electroplating, drop coating, spray coating, doctor blade coating, printing, and dip coating.

[0083] In step S14, in some embodiments, the enzyme or receptor is prepared into a solution, then coated on the surface of the metal nanoparticle layer, and placed in the dark for a preset time to form a chemical bond between the enzyme or receptor and the metal nanoparticles to form a sensor layer.

[0084] In step S15, in some embodiments, a polymer protective layer is formed on the surface of the sensor layer by one of drop coating, spin coating, spray coating, doctor blade coating, printing, and dip coating. Specifically, a polymer and a crosslinking agent are mixed in a solution, and then drop coating, spin coating, spray coating, doctor blade coating, printing, or dip coating is applied to the surface of the sensor layer. After drying, the polymer protective layer is formed.

[0085] During the drying process, the polymer and the cross-linking agent (such as glutaraldehyde, etc.) react to generate a cross-linked network, and a polymer film with a cross-linked network structure, namely, a polymer protective layer, is formed on the surface of the sensor layer.

[0086] The prepared working electrode was stored at 0-30°C.

[0087] An embodiment of the present invention further provides an electrochemical sensor, wherein the electrochemical sensor includes a counter electrode, a reference electrode, and at least one working electrode as described above in the present invention.

[0088] In some embodiments, the electrochemical sensor includes a working electrode, a counter electrode, and a reference electrode. The material of the sensing layer in the working electrode can be selected from the group consisting of an enzyme with progesterone as a direct or indirect catalytic substrate, an enzyme with estradiol as a direct or indirect catalytic substrate, a follicle-stimulating hormone receptor, a luteinizing hormone receptor, and an estradiol receptor. The electrochemical sensor is a single-channel electrochemical sensor capable of continuously monitoring progesterone, estradiol, follicle-stimulating hormone, or luteinizing hormone.

[0089] In some embodiments, the electrochemical sensor includes two working electrodes, a counter electrode, and a reference electrode, wherein the material of the sensing layer in the two working electrodes can be an enzyme that directly or indirectly catalyzes progesterone or an enzyme that directly or indirectly catalyzes estradiol. The electrochemical sensor is then a two-channel sensor (i.e., a multi-channel electrochemical sensor) that can be used for continuous monitoring of progesterone and estradiol. Of course, the material of the sensing layer of the two working electrodes can also be a follicle-stimulating hormone receptor or a luteinizing hormone receptor, respectively. In this case, the electrochemical sensor can be used for continuous monitoring of follicle-stimulating hormone and luteinizing hormone.

[0090] In some embodiments, the electrochemical sensor includes three working electrodes, a counter electrode, and a reference electrode. The materials of the sensing layers in the three working electrodes can be an enzyme with progesterone as a direct or indirect catalytic substrate, an enzyme with estradiol as a direct or indirect catalytic substrate, and a luteinizing hormone receptor, respectively. This electrochemical sensor is a three-channel sensor (i.e., a multi-channel electrochemical sensor) that can be used for continuous monitoring of progesterone, estradiol, and luteinizing hormone receptors. Of course, the materials of the sensing layers in the three working electrodes can also be selected from other combinations.

[0091] In some embodiments, the electrochemical sensor includes four working electrodes, a counter electrode, and a reference electrode. The materials of the sensing layers in the four working electrodes can be, respectively, an enzyme with progesterone as a direct or indirect catalytic substrate, an enzyme with estradiol as a direct or indirect catalytic substrate, a follicle-stimulating hormone receptor, and a luteinizing hormone receptor. This electrochemical sensor is a four-channel sensor (i.e., a multi-channel electrochemical sensor) that can be used for continuous monitoring of progesterone, estradiol, follicle-stimulating hormone receptor, and luteinizing hormone receptor.

[0092] The core of a biosensor is its biorecognition element, which is highly sensitive to biomarkers such as hormones. Although electrochemical sensors have been widely used to monitor biomolecules such as glucose, uric acid, and amino acids, there are still technical difficulties in monitoring low-concentration and easily interfered biomarkers such as female reproductive hormones. The electrochemical sensor provided by the present invention has high precision, high sensitivity, high specificity and long-term stability, and can continuously monitor female reproductive hormones in real time, and can then accurately determine the time of ovulation, which has important practical significance. In addition, its electrochemical stability and low operating potential enable it to maintain high sensitivity and specificity at low voltage.

[0093] In some embodiments, the counter electrode includes one of a platinum counter electrode, a carbon counter electrode, and a gold counter electrode, but is not limited thereto.

[0094] In some embodiments, the reference electrode includes one of an Ag / AgCl reference electrode, a saturated calomel electrode, and a standard hydrogen electrode, but is not limited thereto.

[0095] The present invention will be further described below with reference to specific examples.

[0096] Example 1

[0097] This embodiment provides an electrochemical sensor (for detecting estradiol), comprising a working electrode, a platinum counter electrode, and an Ag / AgCl reference electrode, wherein the three electrodes are spaced apart.

[0098] The working electrode includes a carbon electrode substrate, a PEDOT:PSS layer located on the surface of the carbon electrode substrate, a gold nanoparticle layer located on the surface of the PEDOT:PSS layer, and a sensor layer located on the surface of the gold nanoparticle layer. The sensor layer is composed of HSD17B, and HSD17B is connected to the gold nanoparticles through Au-SH.

[0099] This embodiment also provides a method for preparing the electrochemical sensor as described above, comprising the following steps:

[0100] (1) Soak the carbon electrode substrate in deionized water, ethanol, and acetone for 10 min, repeat three times, and dry under nitrogen.

[0101] (2) forming a 100 nm thick PEDOT:PSS layer on the carbon electrode substrate;

[0102] 3,4-ethylenedioxythiophene was dissolved in acetonitrile to prepare a 0.3 M solution, and phenyl ethyl sulfonate (its concentration in the solution was 0.03 M) was added. Electroplating was performed at a voltage of 1.2 V for 300 s.

[0103] (3) Take 200 mL of 0.01% HAuCl4·3H2O aqueous solution, heat to boiling under stirring and reflux, immediately add 8 mL of 1% sodium citrate aqueous solution, and vigorously stir for 30 minutes. Slowly cool to room temperature, filter through a 0.22 μm filter membrane to obtain a gold nanoparticle solution (stored at 4°C in the dark); drop the above gold nanoparticle solution on the PEDOT:PSS layer and react in the dark for 8 hours to form a gold nanoparticle layer with a thickness of 50 nm;

[0104] (4) HSD17B was dissolved in PBS (phosphate buffered saline) to obtain an HSD17B enzyme solution with a concentration of 10 mg / L, and then activated at room temperature in a dark place for 4 h. The enzyme solution was dropwise coated on the surface of the gold nanoparticle layer and incubated overnight in the dark to allow the SH in HSD17B to fully react with the Au in the gold nanoparticle layer to form Au-SH, forming a sensor layer with a thickness of 200 nm, thereby obtaining a working electrode.

[0105] (5) The obtained working electrode is spaced apart from a platinum counter electrode and an Ag / AgCl reference electrode to obtain the electrochemical sensor.

[0106] test:

[0107] (1) The electrochemical constant potential method was used to perform a sensing test on a 100 pM estradiol solution. The test results showed that the response current of the electrochemical sensor to a 100 pM estradiol solution was 0.059 mA cm -2 .

[0108] (2) The cyclic voltammetry stability of the working electrode was tested by electrochemical cyclic voltammetry. The test results showed that after 2000 cyclic voltammetry tests (electrochemical cycle voltage was -0.6 to 0.6 V), the electroactivity decay rate of the working electrode was 32%.

[0109] Example 2

[0110] This embodiment provides an electrochemical sensor (for detecting luteinizing hormone), which differs from Embodiment 1 only in that HSD17B in the sensor layer is replaced by a luteinizing hormone receptor.

[0111] This embodiment also provides a method for preparing the electrochemical sensor as described above, which differs from the preparation method in Example 1 only in that in step (4), HSD17B is replaced by a luteinizing hormone receptor.

[0112] test:

[0113] (1) The electrochemical constant potential method was used to perform a sensing test on a luteinizing hormone solution with a concentration of 100mIU / mL. The test results showed that the response current of the electrochemical sensor to the luteinizing hormone solution with a concentration of 100mIU / mL was 0.055mA cm -2 .

[0114] (2) The cyclic voltammetry stability of the working electrode was tested by electrochemical cyclic voltammetry. The test results showed that the electroactivity decay rate of the working electrode was 38% after 2000 cyclic voltammetry tests (the electrochemical cycle voltage range was -0.6 to 0.6 V).

[0115] Example 3

[0116] This embodiment provides an electrochemical sensor (for detecting progesterone), which differs from the embodiment 1 only in that HSD17B in the sensor layer is replaced by CYP3A4.

[0117] This embodiment also provides a method for preparing the electrochemical sensor as described above, which differs from the preparation method in Example 1 only in that in step (4), HSD17B is replaced by CYP3A4.

[0118] test:

[0119] (1) The electrochemical constant potential method was used to perform a sensing test on a progesterone solution with a concentration of 100 pM. The test results showed that the response current of the electrochemical sensor to a progesterone solution with a concentration of 100 pM was 0.068 mA cm -2 .

[0120] (2) The cyclic voltammetry stability of the working electrode was tested by electrochemical cyclic voltammetry. The test results showed that the electroactivity decay rate of the working electrode was 42% after 2000 cyclic voltammetry tests (the electrochemical cycle voltage range was -0.6 to 0.6 V).

[0121] Example 4

[0122] This embodiment provides an electrochemical sensor (for detecting follicle-stimulating hormone), which differs from the first embodiment only in that HSD17B in the sensor layer is replaced with a follicle-stimulating hormone receptor.

[0123] This embodiment also provides a method for preparing the electrochemical sensor as described above, which differs from Example 1 only in that in step (4), HSD17B is replaced by a follicle-stimulating hormone receptor.

[0124] test:

[0125] (1) The electrochemical constant potential method was used to perform a sensing test on a 100mIU / mL follicle-stimulating hormone solution. The test results showed that the response current of the electrochemical sensor to a 100mIU / mL follicle-stimulating hormone solution was 0.71mA cm -2 .

[0126] (2) The cyclic voltammetry stability of the working electrode was tested by electrochemical cyclic voltammetry. The test results showed that the electroactivity decay rate of the working electrode was 36% after 2000 cyclic voltammetry tests (the electrochemical cycle voltage range was -0.6 to 0.6 V).

[0127] Example 5

[0128] This embodiment provides a multi-channel electrochemical sensor (for detecting estradiol, luteinizing hormone, progesterone and follicle-stimulating hormone), including four working electrodes located on a PI membrane, a platinum counter electrode and an Ag / AgCl reference electrode, and the six electrodes are arranged at intervals.

[0129] The four working electrodes are respectively referred to as a first working electrode, a second working electrode, a third working electrode, and a fourth working electrode;

[0130] The first working electrode includes a gold electrode, a PEDOT:PSS layer located on the surface of the gold electrode, a gold nanoparticle layer located on the surface of the PEDOT:PSS layer, and a sensing body layer located on the surface of the gold nanoparticle layer. The sensing body layer is composed of HSD17B, and HSD17B is connected to the gold nanoparticles through Au-SH.

[0131] The second working electrode includes a gold electrode, a PEDOT:PSS layer located on the surface of the gold electrode, a gold nanoparticle layer located on the surface of the PEDOT:PSS layer, and a sensing body layer located on the surface of the gold nanoparticle layer. The sensing body layer is composed of luteinizing hormone receptors, and the luteinizing hormone receptors are connected to the gold nanoparticles through Au-SH.

[0132] The third working electrode comprises a gold electrode, a PEDOT:PSS layer on the surface of the gold electrode, a gold nanoparticle layer on the surface of the PEDOT:PSS layer, and a sensing layer on the surface of the gold nanoparticle layer. The sensing layer is composed of CYP3A4, and CYP3A4 is connected to the gold nanoparticles via Au-SH.

[0133] The fourth working electrode includes a gold electrode, a PEDOT:PSS layer located on the surface of the gold electrode, a gold nanoparticle layer located on the surface of the PEDOT:PSS layer, and a sensing body layer located on the surface of the gold nanoparticle layer. The sensing body layer is composed of follicle-stimulating hormone receptors, and the follicle-stimulating hormone receptors are connected to the gold nanoparticles through Au-SH.

[0134] This embodiment also provides a method for preparing the electrochemical sensor as described above, comprising the following steps:

[0135] (1) Immerse the PI film in deionized water, ethanol, and acetone for 10 min, then ultrasonically clean it three times. Dry it under nitrogen.

[0136] A PI film substrate was covered with photoresist using photolithography technology, and gold was deposited on the PI film using a magnetron sputtering system. The excess was then washed away with a degumming solution to obtain four spaced gold electrodes (the size of each gold electrode was consistent with the carbon electrode substrate in Example 1);

[0137] (2) Using the same method as step (2) in Example 1, a PEDOT:PSS layer with a thickness of 100 nm was formed on the surfaces of the four gold electrodes;

[0138] (3) Using the same method as step (3) in Example 1, gold nanoparticles were deposited on the surface of the four PEDOT:PSS layers, and reacted in the dark for 8 h to form a gold nanoparticle layer with a thickness of 50 nm;

[0139] (4) The HSD17B enzyme solution, the luteinizing hormone receptor solution, the CYP3A4 enzyme solution, and the follicle-stimulating hormone receptor solution (the preparation methods of the four solutions are the same as those in Example 1, Example 2, Example 3, and Example 4, respectively) are placed in a dark place and activated at room temperature for 4 hours. The above solutions are then drop-coated on the surfaces of the four gold nanoparticle layers and left in the dark overnight to allow the SH in the enzyme / receptor to fully react with Au to form Au-SH, forming four sensing layers with a thickness of 200 nm each, thereby obtaining four working electrodes;

[0140] (5) The four working electrodes obtained are spaced apart from each other with a platinum counter electrode and an Ag / AgCl reference electrode to obtain the electrochemical sensor.

[0141] test:

[0142] (1) The electrochemical potentiostatic method was used to perform a sensing test on a 10 pM estradiol solution. The results showed that the response current of the first working electrode to the 10 pM estradiol solution was 0.96 mA cm -2 The results of the electrochemical potentiostatic method for the sensing test of 10mIU / mL luteinizing hormone solution showed that the response current of the second working electrode to the 10mIU / mL luteinizing hormone solution was 0.62mAcm -2 The results of the electrochemical potentiostatic method for the sensing test of 10pM progesterone solution showed that the response current of the third working electrode to the 10pM progesterone solution was 0.68mA cm-2 The electrochemical constant potential method was used to perform a sensing test on a 10mIU / mL follicle-stimulating hormone solution. The results showed that the response current of the fourth working electrode to the 10mIU / mL follicle-stimulating hormone solution was 0.64mA cm -2 .

[0143] (2) The cyclic voltammetry stability of the four working electrodes was tested by electrochemical cyclic voltammetry. The results showed that the electroactivity decay rate of the first working electrode was 18% after 2000 cyclic voltammetry tests (electrochemical cycle window was -0.4 to 0.6 V). Figure 3 After 2000 cyclic voltammetry tests (electrochemical cycle voltage range of -0.6 to 0.6 V), the electroactivity decay rate of the second working electrode was 24.5%; after 2000 cyclic voltammetry tests (electrochemical cycle voltage range of -0.6 to 0.6 V), the electroactivity decay rate of the third working electrode was 19%; and after 2000 cyclic voltammetry tests (electrochemical cycle voltage range of -0.6 to 0.6 V), the electroactivity decay rate of the fourth working electrode was 26%.

[0144] Example 6

[0145] This embodiment provides a multi-channel electrochemical sensor (for detecting estradiol, luteinizing hormone, progesterone and follicle-stimulating hormone). The only difference from Example 5 is that the surfaces of the four working electrode sensor layers are all provided with a polymer protective layer composed of chitosan and glutaraldehyde.

[0146] This embodiment also provides a method for preparing a multi-channel electrochemical sensor, comprising the following steps:

[0147] (1) is the same as step (1) in Example 5.

[0148] (2) The same as step (2) in Example 5.

[0149] (3) is the same as step (3) in Example 5.

[0150] (4) The HSD17B enzyme solution, the luteinizing hormone receptor solution, the CYP3A4 enzyme solution, and the follicle-stimulating hormone receptor solution (the preparation methods of the four solutions are the same as those in Example 1, Example 2, Example 3, and Example 4, respectively) were placed in a dark place and activated at room temperature for 4 h. The above solutions were then drop-coated on the surfaces of the four gold nanoparticle layers and left in the dark overnight to allow the SH in the enzyme / receptor to fully react with Au to form Au-SH, forming four sensor layers with a thickness of 200 nm each.

[0151] (5) 100 mg of chitosan was added to 2 mL of acetic acid aqueous solution (acetic acid content was 1% by mass), glutaraldehyde was added (the addition amount was 1% of the mass of chitosan), and the mixture was thoroughly mixed to obtain a polymer solution;

[0152] The product obtained in step (4) was dip-coated twice in the above polymer solution, with an interval of 15 minutes between the two dip-coatings, and then dried and cured in a 20°C constant temperature box under dark conditions for 10 hours to form a polymer protective layer with a thickness of 50 nm, thereby obtaining four working electrodes;

[0153] (6) The four working electrodes obtained are spaced apart from each other with a platinum counter electrode and an Ag / AgCl reference electrode to obtain the electrochemical sensor.

[0154] test:

[0155] (1) The sensing test of 10 pM estradiol solution was carried out by electrochemical constant potential method. The results showed that the response current of the first working electrode to 10 pM estradiol solution was 0.76 mA cm -2 The results of the electrochemical potentiostatic method showed that the response current of the second working electrode to the 10mIU / mL luteinizing hormone solution was 0.592mAcm -2 The results of the electrochemical constant potential method for the sensing test of 10pM progesterone solution showed that the response current of the third working electrode to the 10pM progesterone solution was 0.608mAcm -2 The electrochemical constant potential method was used to perform a sensing test on a 10mIU / mL follicle-stimulating hormone solution. The results showed that the response current of the fourth working electrode to the 10mIU / mL follicle-stimulating hormone solution was 0.558mA cm -2 .

[0156] (2) The cyclic voltammetry stability of the four working electrodes was tested by electrochemical cyclic voltammetry. The results showed that the electroactivity decay rate of the first working electrode was 11% after 2000 cyclic voltammetry tests (electrochemical cycle window was -0.4 to 0.6 V); the electroactivity decay rate of the second working electrode was 10.5% after 2000 cyclic voltammetry tests (electrochemical cycle voltage range was -0.6 to 0.6 V); the electroactivity decay rate of the third working electrode was 10.3% after 2000 cyclic voltammetry tests (electrochemical cycle voltage range was -0.6 to 0.6 V); and the electroactivity decay rate of the fourth working electrode was 9.9% after 2000 cyclic voltammetry tests (electrochemical cycle voltage range was -0.6 to 0.6 V).

[0157] Example 7

[0158] This embodiment provides an electrochemical sensor, which is composed of the first working electrode in Example 5, a platinum counter electrode, and an Ag / AgCl reference electrode.

[0159] Perform the following test:

[0160] (1) Estradiol was dissolved in phosphate buffer to prepare estradiol solutions with concentrations of 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 pM, respectively. These estradiol solutions were detected in order from low concentration to high concentration using the electrochemical sensor. The results were as follows: Figure 4 As shown, it can be seen that the detection range of the electrochemical sensor for estradiol concentration can reach 1000 pM.

[0161] (2) Estradiol was dissolved in phosphate buffer to prepare estradiol solutions with concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 pM, respectively. These estradiol solutions were detected in order from low concentration to high concentration using the electrochemical sensor. The results were as follows: Figure 5 As shown, it can be seen that the electrochemical sensor can achieve continuous monitoring of estradiol solution from 0 pM to 100 pM.

[0162] (3) Anti-interference performance test: Estradiol was dissolved in phosphate buffer to prepare an estradiol solution with a concentration of 100 pM. The electrochemical sensor was used for detection for 200 s. Then, estriol (E3), ethinylestradiol (Et), progesterone (P4) and hydrocortisone (Hy) were added to the estradiol solution to make the final concentration of 100 pM. The detection was continued for 200 s. Then, estradiol was added to the estradiol solution to make the final concentration of estradiol 600 pM. The detection was continued for 200 s. Then, E3, Et, P4 and Hy were added to the estradiol solution to make the final concentration of estradiol 600 pM. The detection was continued for 200 s. The results are as follows: Figure 6 As shown, it can be seen that the electrochemical sensor has strong detection specificity for estradiol and is resistant to interference.

[0163] (4) Female rats were taken, and the first working electrode, platinum counter electrode and Ag / AgCl reference electrode were implanted subcutaneously in the female rats for continuous monitoring. The in vivo monitoring curve from 0 to 6 hours was as follows: Figure 7 As shown, the results of continuous monitoring in vivo from day 1 to day 28 are as follows Figure 8 As shown, the working electrode or electrochemical sensor provided by the present invention can achieve continuous monitoring in vivo for up to 28 days.

[0164] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A working electrode, characterized in that The working electrode includes an electrode substrate and a sensing layer located on the surface of the electrode substrate; The sensing layer includes a sensor layer, and the sensor layer includes an enzyme or a receptor. The enzyme directly or indirectly uses female reproductive hormone as a catalytic substrate, and the receptor is a female reproductive hormone receptor.

2. The working electrode according to claim 1, characterized in that The working electrode further includes a polymer protective layer, which is located on the surface of the sensing layer and includes at least one of chitosan, polyurethane, alginate, agarose, collagen, silk fibroin, polytetravinylpyridine-polystyrene block copolymer, polyvinyl alcohol and polyethylene glycol.

3. The working electrode according to claim 1 or 2, characterized in that The sensing layer further includes a conductive polymer layer, which is located between the electrode substrate and the sensing layer; the conductive polymer layer includes at least one of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid, polyaniline, poly(p-phenylene glycol) and polypyrrole.

4. The working electrode according to claim 3, characterized in that The sensing layer further comprises a metal nanoparticle layer, which is located between the conductive polymer layer and the sensor layer. The metal nanoparticles in the metal nanoparticle layer are combined with the enzyme or receptor in the sensor layer through chemical bonds.

5. The working electrode according to claim 1, characterized in that The electrode matrix includes a metal electrode or a conductive non-metal electrode.

6. The working electrode according to claim 1, characterized in that The female reproductive hormone includes one of progesterone, follicle-stimulating hormone, luteinizing hormone and estradiol.

7. The working electrode according to claim 1, characterized in that The enzyme includes one of an enzyme that uses progesterone as a direct or indirect catalytic substrate and an enzyme that uses estradiol as a direct or indirect catalytic substrate; the receptor includes one of a follicle-stimulating hormone receptor, a luteinizing hormone receptor and an estradiol receptor; The enzyme that directly or indirectly catalyzes progesterone as a substrate includes one of cytochrome P450 17A1, 3β-hydroxysteroid dehydrogenase, cytochrome P450 3A4, and 20β-hydroxysteroid dehydrogenase; the enzyme that directly or indirectly catalyzes estradiol as a substrate includes one of cytochrome P450 1A1, cytochrome P450 1B1, 17β-hydroxysteroid dehydrogenase, sulfotransferase, and uridine diphosphate glucuronyltransferase; and the estradiol receptor includes estrogen receptor α or estrogen receptor β.

8. A method for preparing a working electrode according to any one of claims 1 to 7, characterized in that: The steps include: providing an electrode substrate; A sensing layer is formed on the surface of the electrode substrate to obtain the working electrode.

9. The preparation method according to claim 8, characterized in that The electrode substrate is prepared by using a method of etching combined with physical vapor deposition; The sensing layer is formed on the surface of the electrode substrate by one of drop coating, spin coating, spray coating, doctor blade coating, printing and dip coating.

10. An electrochemical sensor, characterized in that The electrochemical sensor comprises a counter electrode, a reference electrode and at least one working electrode according to any one of claims 1 to 7.

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