Preparation method and application of nitrogen-sulfur doped carbon dot modified cuprous oxide composite material

Abstract

The invention provides a preparation method and application of a nitrogen and sulfur doped carbon dot modified cuprous oxide composite material. The preparation method comprises the following steps: introducing nitrogen-sulfur doped carbon dots in a cuprous oxide synthesis process to obtain the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material; wherein CuSO4. 5H2O is adopted to synthesize the cuprous oxide. The composite material has the advantages of enhanced electrochemical performance, excellent stability and repeatability and low cost. The electrochemical immunosensor constructed on the basis of the Cu2O and NS-CDs composite material can be used for quantitative analysis of tumor markers (such as AFP).

Description

Technical Field

[0001] This invention relates to the field of nanotechnology, specifically to a method for preparing and applying a nitrogen-sulfur doped carbon dot modified cuprous oxide composite material. Background Technology

[0002] Alpha-fetoprotein (AFP) is the earliest and most widely used blood biomarker for screening hepatocellular carcinoma (HCC). The concentration of AFP in the serum of healthy adults is below 10 μg / L. 80% of HCC patients have elevated AFP levels. The international academic community recommends limiting the reference value for AFP to 20 μg / L. Existing methods such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay have been used to detect AFP. However, ELISA has a cumbersome and time-consuming labeling process and a limited dynamic range. Radioimmunoassay has low sensitivity, long detection time, potential health risks, and requires complex equipment and skilled operators. Therefore, developing efficient, sensitive, and rapid novel AFP detection methods is of significant clinical importance.

[0003] Electrochemical biosensors utilize the interaction between biological receptors and sensor elements to provide accurate qualitative and quantitative information on specific targets, offering advantages such as excellent detection capabilities, high sensitivity, cost-effectiveness, and ease of operation. Antibodies recognize specific antigens and form stable immune complexes. The selection of sensor materials must consider their inherent properties and their ability to immobilize antibodies, which directly affects the sensitivity and detection limit of the immunosensor.

[0004] Noble metal nanoparticles, metal-organic frameworks (MOFs), transition metal sulfides, and semiconductor quantum dots have been used for the quantitative detection of tumor markers. However, the high cost of noble metals, the poor conductivity of MOFs, and the toxicity of transition metal sulfides and semiconductor quantum dots are their main limitations. Therefore, developing an electrochemical sensor material that is low-cost, highly conductive, safe, and highly sensitive is of great significance. Summary of the Invention

[0005] The main objective of this invention is to provide a method for preparing and applying a nitrogen-sulfur doped carbon dot modified cuprous oxide composite material, in order to solve the problems of weak conductivity and high cost of electrochemical immunosensor electrode materials in the prior art.

[0006] To achieve the above objectives, according to a first aspect of the present invention, a method for preparing a nitrogen-sulfur doped carbon dot modified cuprous oxide composite material is provided. The method includes: introducing nitrogen-sulfur doped carbon dots during the synthesis of cuprous oxide to obtain the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material; wherein the above-mentioned cuprous oxide is synthesized using CuSO4·5H2O.

[0007] Furthermore, the above preparation method includes the following steps:

[0008] S1) Glutathione and p-phenylenediamine are mixed in a solvent and subjected to a hydrothermal reaction to obtain nitrogen-sulfur-doped carbon dots;

[0009] S2) CuSO4·5H2O, the above nitrogen-sulfur doped carbon dots, water and sodium hydroxide are mixed and heated to obtain the first solid-liquid mixture;

[0010] S3) The first solid-liquid mixture is mixed with a reducing agent to carry out a reduction reaction, thereby obtaining a second solid-liquid mixture;

[0011] S4) The second solid-liquid mixture was centrifuged, washed and dried sequentially to obtain the above-mentioned nitrogen-sulfur-doped carbon dot modified cuprous oxide composite material.

[0012] The solvents mentioned above are selected from ethanol and water.

[0013] Further, in S1), the molar volume ratio of the glutathione, p-phenylenediamine, ethanol, and water is (0.05~0.10):(0.05~0.2):(5~10):(5~10).

[0014] Preferably, the hydrothermal reaction time is 2-5 hours; the hydrothermal reaction temperature is 120-180°C.

[0015] Preferably, after the above-mentioned hydrothermal reaction and before obtaining the above-mentioned nitrogen-sulfur doped carbon dots, the above-mentioned S1) further includes the steps of first cooling and first drying of the product after the above-mentioned hydrothermal reaction.

[0016] More preferably, the temperature of the first cooling is 20°C to 25°C; and the time of the first cooling is 2 to 5 hours.

[0017] More preferably, the temperature of the first drying is 50~60°C; and the time of the first drying is 24~28h.

[0018] Furthermore, the reducing agent is selected from glucose solution; the mass-volume ratio of CuSO4·5H2O, the nitrogen-sulfur doped carbon dots, water, sodium hydroxide and glucose solution is (300~500):(0.5~1.5):(20~40):(5~10):(5~10);

[0019] The concentration of the sodium hydroxide solution is 1-5 M, and the concentration of the glucose solution is 0.1-0.5 M.

[0020] In the above glucose solution, the amount of glucose is greater than that of Cu in the above CuSO4·5H2O. 2+ The amount of substance;

[0021] Preferably, the temperature of the first solid-liquid mixture is 60°C to 80°C.

[0022] Furthermore, the reduction reaction takes 2 to 5 hours and is carried out at a temperature of 60°C to 80°C.

[0023] Preferably, in S4 above, ethanol and / or water are used for the washing process;

[0024] More preferably, the drying temperature is 65°C to 80°C; and the drying time is 24h to 28h.

[0025] To achieve the above objectives, according to a second aspect of the present invention, a nitrogen-sulfur-doped carbon dot-modified cuprous oxide composite material prepared by the above-described preparation method is provided.

[0026] Preferably, the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material includes a cuprous oxide matrix and the above-mentioned nitrogen-sulfur doped carbon dots uniformly distributed on the surface of the above-mentioned cuprous oxide matrix.

[0027] Preferably, the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material has a dodecahedral structure;

[0028] More preferably, the average diameter of the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material is 1.21µm ± 0.21µm;

[0029] Preferably, the C, N, O, S and Cu elements in the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material are arranged in a manner that is uniformly dispersed on the surface;

[0030] Preferably, the mass ratio of Cu, O, S, C and N elements in the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material is (60~73):(10~25):(2~6):(3~8):(2~5).

[0031] To achieve the above objectives, according to a third aspect of the present invention, a modified electrode is provided, the modified electrode comprising a substrate electrode and a composite material modified on the surface of the substrate electrode, wherein the composite material is selected from a nitrogen-sulfur doped carbon dot modified cuprous oxide composite material prepared by the above preparation method or the above nitrogen-sulfur doped carbon dot modified cuprous oxide composite material.

[0032] To achieve the above objectives, according to a fourth aspect of the present invention, an electrochemical immunosensor is provided, wherein the electrodes of the electrochemical immunosensor include the modified electrodes described above.

[0033] To achieve the above objectives, according to a fifth aspect of the present invention, a method for detecting alpha-fetoprotein is provided, the method comprising: detecting alpha-fetoprotein using the above-described electrochemical immunosensor.

[0034] To achieve the above objectives, according to a sixth aspect of the present invention, an application is provided of the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material prepared by the above preparation method, or the above-described nitrogen-sulfur doped carbon dot modified cuprous oxide composite material, or the above-described modified electrode, or the above-described electrochemical immunosensor, or the above-described method in the detection of tumor markers.

[0035] Preferably, the tumor markers mentioned above include any one of the following: alpha-fetoprotein, carcinoembryonic antigen, or squamous cell carcinoma antigen.

[0036] By applying the technical solution of this invention, nitrogen-sulfur doped carbon dots are introduced during the synthesis of Cu2O using CuSO4·5H2O. The synthesized nitrogen-sulfur doped carbon dot-modified cuprous oxide composite material (Cu2O@NS-CDs) can promote electron transfer on the electrode surface of an electrochemical immunosensor, capture a large number of antigens, and generate electrochemical signals, enabling specific analysis of tumor markers (e.g., alpha-fetoprotein) and distinguishing them from other interfering components (e.g., other antigens and biomolecules). The electrochemical immunosensor with electrodes modified with this composite material can detect alpha-fetoprotein concentrations in the range of 0.001-100 ng / mL, and the electrochemical sensor has an ultra-low detection limit (21.4 fg / mL). Attached Figure Description

[0037] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0038] Figure 1 A schematic diagram illustrating the synthesis and detection of Cu2O@NS-CDs according to an embodiment of the present invention is shown.

[0039] Figure 2 Image (a) shows the XRD patterns of Cu2O, Cu2O@NS-CDs and NS-CDs according to an embodiment of the present invention.

[0040] Figure 2 (b) shows the XRD patterns of Cu2O@NS-CDs with different masses of NS-CDs added according to an embodiment of the present invention.

[0041] Figure 2 (c) shows the FTIR spectra of Cu2O, Cu2O@NS-CDs and NS-CDs according to an embodiment of the present invention.

[0042] Figure 2 (d) shows the FTIR spectra of Cu2O@NS-CDs with different masses of NS-CDs added according to an embodiment of the present invention.

[0043] Figure 3 Image (a) shows a SEM image of Cu2O according to an embodiment of the present invention.

[0044] Figure 3 (b) shows a SEM image of Cu2O@NS-CDs according to an embodiment of the present invention.

[0045] Figure 3 (c) shows a histogram of the particle size distribution of Cu2O according to an embodiment of the present invention.

[0046] Figure 3 (d) shows a histogram of the particle size distribution of Cu2O@NS-CDs according to an embodiment of the present invention.

[0047] Figure 3 (e) shows the EDS spectrum of Cu2O@NS-CDs according to an embodiment of the present invention.

[0048] Figure 3 (f) shows the mass ratio of each element in Cu2O@NS-CDs determined by EDS according to an embodiment of the present invention.

[0049] Figure 4 Image (a) shows the morphology of Cu2O@NS-CDs prepared by introducing 0.5 mg NS-CDs according to an embodiment of the present invention.

[0050] Figure 4 (b) shows an enlarged morphological image of Cu2O@NS-CDs prepared by introducing 0.5 mg NS-CDs according to an embodiment of the present invention.

[0051] Figure 4 (c) shows the morphology of Cu2O@NS-CDs prepared by introducing 0.75 mg NS-CDs according to an embodiment of the present invention.

[0052] Figure 4 Image (d) shows an enlarged morphological view of Cu2O@NS-CDs prepared by introducing 0.75 mg NS-CDs according to an embodiment of the present invention.

[0053] Figure 4 (e) shows the morphology of Cu2O@NS-CDs prepared by introducing 1.0 mg NS-CDs according to an embodiment of the present invention.

[0054] Figure 4 Image (f) shows an enlarged morphological view of Cu2O@NS-CDs prepared by introducing 1.0 mg NS-CDs according to an embodiment of the present invention.

[0055] Figure 5 (a) shows the overall elemental distribution of Cu2O@NS-CDs according to an embodiment of the present invention.

[0056] Figure 5 (b) shows the C element distribution of Cu2O@NS-CDs according to an embodiment of the present invention.

[0057] Figure 5 (c) shows the N element distribution of Cu2O@NS-CDs according to an embodiment of the present invention.

[0058] Figure 5 (d) shows the O element distribution diagram of Cu2O@NS-CDs according to an embodiment of the present invention.

[0059] Figure 5 (e) shows the S element distribution of Cu2O@NS-CDs according to an embodiment of the present invention.

[0060] Figure 5 (f) shows the Cu element distribution of Cu2O@NS-CDs according to an embodiment of the present invention.

[0061] Figure 6 (a) shows the XPS full spectrum of Cu2O@NS-CDs according to an embodiment of the present invention.

[0062] Figure 6 (b) shows a high-resolution XPS spectrum of C 1s according to an embodiment of the present invention.

[0063] Figure 6 (c) shows a high-resolution XPS spectrum of N 1s according to an embodiment of the present invention.

[0064] Figure 6 (d) shows a high-resolution XPS spectrum of O 1s according to an embodiment of the present invention.

[0065] Figure 6 (e) shows a high-resolution XPS spectrum of S 2p according to an embodiment of the present invention.

[0066] Figure 6 (f) shows the XPS full spectrum of Cu 2p according to an embodiment of the present invention.

[0067] Figure 7Image (a) illustrates Bare / GCE, NS-CDs / GCE, Cu2O / GCE, and Cu2O@NS-CDs / GCE according to embodiments of the present invention in the presence of 5 mM [Fe(CN)6]. 3- / 4- The CV in the 0.1 M KCl solution was scanned at a rate of 50 mV / s. -1 .

[0068] Figure 7 (b) illustrates Bare / GCE, NS-CDs / GCE, Cu2O / GCE, and Cu2O@NS-CDs / GCE according to embodiments of the present invention in the presence of 5 mM [Fe(CN)6]. 3- / 4- EIS in a solution of 0.1 M KCl, scan rate 50 mV s -1 .

[0069] Figure 7 (c) shows the CVs of Bare / GCE, Cu2O@NS-CDs / GCE, Cu2O@NS-CDs / Ab / GCE, Cu2O@NS-CDs / Ab / BSA / GCE, and Cu2O@NS-CDs / Ab / BSA / AFP / GCE according to embodiments of the present invention.

[0070] Figure 7 (d) shows the EIS of Bare / GCE, Cu2O@NS-CDs / GCE, Cu2O@NS-CDs / Ab / GCE, Cu2O@NS-CDs / Ab / BSA / GCE, and Cu2O@NS-CDs / Ab / BSA / AFP / GCE according to embodiments of the present invention.

[0071] Figure 7 (e) shows the peak DPV current of Cu2O / GCE, Cu2O / Ab / BSA / GCE, Cu2O@NS-CDs / GCE and Cu2O@NS-CDs / Ab / BSA / AFP / GCE according to embodiments of the present invention.

[0072] Figure 7 (f) shows the response signal of Cu2O@NS-CDs doped with different amounts of NS-CDs (0.5 mg-1.5 mg) according to an embodiment of the present invention for detecting alpha-fetoprotein.

[0073] Figure 8 Image (a) shows Cu2O@NS-CDs / GCE according to an embodiment of the present invention in the presence of 5 mM [Fe(CN)6]. 3- / 4- The CV values ​​in the 0.1 M KCl solution were measured at the following scan rates: 10⁻¹⁰⁰ mV s.-1 .

[0074] Figure 8 (b) shows a linear curve of peak current versus the square root of scan rate for Cu2O@NS-CDs / GCE according to an embodiment of the present invention.

[0075] Figure 8 (c) shows Cu2O@NS-CDs / GCE in PBS (containing 50 ng / mL) at pH 7.4 according to an embodiment of the present invention. -1 In AFP (Automatic Photonic Vibration), the CV scan rate is as follows: 10-100 mV / s -1 .

[0076] Figure 8 (d) shows Cu2O@NS-CDs / GCE in PBS (containing 50 ng / mL) at pH 7.4 according to an embodiment of the present invention. -1 The linear curve of peak current versus the square root of scan rate in AFP.

[0077] Figure 9 (a) shows the electrochemical reaction of 50 ng·mL⁻¹ alpha-fetoprotein on Cu₂O@NS-CDs / Ab / BSA / GCE under different pH conditions of PBS according to embodiments of the present invention.

[0078] Figure 9 (b) shows the electrochemical reaction of 50 ng·mL⁻¹ alpha-fetoprotein on Cu₂O@NS-CDs / Ab / BSA / GCE at different AFP incubation times according to embodiments of the present invention.

[0079] Figure 10 (a) shows the electrochemical detection of 0.001-100 ng / mL according to an embodiment of the present invention. -1 AFP's signal response.

[0080] Figure 10 (b) shows a calibration curve of current versus logarithm of AFP concentration according to an embodiment of the present invention.

[0081] Figure 10 (c) shows an immunosensor according to an embodiment of the present invention for 10 ng mL -1 AFP, mixture (100 ng / mL) -1 Interfering substances and 10 ng mL -1 AFP) and 100 ng mL -1 Response signals of IgG, CEA, BSA, Glu, AA and His.

[0082] Figure 10 (d) shows the same sensor according to an embodiment of the present invention for 10 ng mL -1 Repeatability of AFP measurements (5 times).

[0083] Figure 10 (e) illustrates the detection of 10 ng / mL using five different electrodes according to an embodiment of the present invention. -1 Reproducibility of AFP.

[0084] Figure 10 (f) shows the DPV current response of the immune sensor stability over 17 days according to an embodiment of the present invention.

[0085] Figure 11 The method of this application for detecting alpha-fetoprotein in different clinical serum samples is illustrated according to an embodiment of the present invention. Detailed Implementation

[0086] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0087] Terminology Explanation:

[0088] Cyclic voltammetry (CV) is an electrochemical analytical technique used to study redox reactions in electrochemical systems. By applying a cyclically changing potential to an electrode and measuring the corresponding current response, information about the electrochemical properties of the substance under study can be obtained, including redox potential, reaction kinetics, and diffusion characteristics.

[0089] Glassy carbon electrode (GCE): A commonly used electrode material in electrochemical research, widely applied due to its chemical inertness, good conductivity, and wide potential window. GCE is frequently used as the working electrode in electrochemical sensors, electroanalysis, and other electrochemical experiments.

[0090] Carbon dots (CDs) are a novel class of zero-dimensional carbon nanomaterials with excellent fluorescence properties, low toxicity, good biocompatibility, and chemical inertness. They have broad application prospects in fields such as bioimaging, sensing, photocatalysis, and drug delivery. There are various methods for synthesizing carbon dots, including chemical oxidation, laser ablation, hydrothermal methods, and microwave methods.

[0091] Electrochemical impedance spectroscopy (EIS) is a method for studying electrode processes in electrochemical systems. EIS obtains information by measuring the impedance of a system to an applied alternating current signal. Its applications include the study of batteries, fuel cells, electrochemical sensors, corrosion processes, and electroplating. This technique helps researchers understand electrochemical reaction kinetics, interfacial properties, and the conductivity of materials.

[0092] Antibodies (Abs): Proteins in the immune system that recognize and neutralize foreign substances (such as bacteria and viruses). Antibodies have wide applications in biochemical and molecular biology research, such as in immunoassays (e.g., ELISA) for detecting specific proteins or other molecules.

[0093] Charge transfer resistance (Rct) is an important parameter in electrochemistry, especially in electrochemical impedance spectroscopy (EIS) analysis. It reflects the ease with which charge transfers from the electrode to the electrolyte during an electrochemical reaction. A higher Rct value generally indicates a slower or more impeded charge transfer process, while a lower Rct value indicates a smoother process. This parameter is crucial for studying batteries, fuel cells, corrosion processes, and other systems involving electrochemical reactions.

[0094] Differential pulse voltammetry (DPV) is an electrochemical analysis technique used to study redox reactions in electrochemical systems. DPV measures current changes by applying a pulsed voltage, providing information about analyte concentration and electrochemical behavior. Due to its high sensitivity and resolution, DPV is commonly used for the detection and analysis of trace substances.

[0095] Electrochemical biosensors are devices that combine biorecognition elements and electrochemical signal transducers to detect and measure the concentration or presence of target analytes. Their basic principle is based on the specific interaction between biomolecules (such as enzymes, antibodies, and DNA) and the target analyte, generating a detectable electrochemical signal, such as a change in current, voltage, or conductance. This signal can be further processed and analyzed to provide quantitative or qualitative analytical results. Electrochemical biosensors are widely used in various fields, including medical diagnostics (such as blood glucose monitoring), environmental monitoring (such as water quality testing), food safety, and biotechnology research. They offer advantages such as high sensitivity, high specificity, rapid response, and portability.

[0096] Electrochemical immunosensors are biosensors that combine the high specificity of immunoassay technology with the high sensitivity of electrochemical sensing technology. They are primarily used for the detection and quantification of specific targets (called "analytes," typically proteins, hormones, pathogens, small molecule compounds, etc.) in complex samples (such as blood, urine, and environmental samples). Their working principle is based on the highly specific recognition and binding reaction between antigens and antibodies, converting this biorecognition event into measurable electrochemical signals (such as changes in current, voltage, or impedance).

[0097] Hydrothermal reaction: refers to a chemical reaction carried out in a closed container (such as a high-pressure reactor) at a certain temperature (usually above 100°C) and pressure (spontaneously generated saturated vapor pressure), using water as a solvent or reaction medium.

[0098] Modified electrodes: An important tool in electrochemical research and applications, defined as the purposeful addition of one or more layers of material to the surface of traditional electrodes (such as glassy carbon electrodes, gold electrodes, platinum electrodes, etc.) through physical adsorption, chemical bonding, or biomolecular immobilization to improve the electrode's performance, response characteristics, and selectivity. The modification layer can be inorganic materials, organic materials, polymers, nanomaterials, biomolecules (such as enzymes, antibodies, DNA, etc.), or combinations thereof.

[0099] As mentioned in the background section, existing methods for detecting AFP suffer from drawbacks such as cumbersome and time-consuming labeling steps, limited dynamic range, low sensitivity, and long detection times. Electrochemical biosensors utilize the interaction between biological receptors and sensor elements to provide accurate qualitative and quantitative information on specific targets, offering advantages such as excellent detection capabilities, high sensitivity, and ease of operation. However, existing materials used to fabricate electrodes for electrochemical biosensors have several limitations. For example, noble metal nanoparticles are costly, MOFs have poor conductivity, and transition metal sulfides and semiconductor quantum dots are toxic.

[0100] Cu₂O is an electroactive substance because Cu + / Cu 2+ The reversible oxidation of carbon dots can generate good electrochemical signals. The surface of carbon dots contains abundant oxygen-containing functional groups (such as hydroxyl and carboxyl groups), which can donate electrons and exhibit a certain reducing ability. Nitrogen and sulfur can promote charge-electron transfer at the interface, giving the material good conductivity. In this invention, the inventors originally attempted to modify the interior or surface of Cu₂O with carbon dots, nitrogen, and sulfur to improve the material's conductivity.

[0101] However, during the experiment, it was unexpectedly discovered that when nitrogen-sulfur doped carbon dots were introduced during the synthesis of Cu2O using CuSO4·5H2O to synthesize a nitrogen-sulfur doped carbon doped cuprous oxide composite material, the nitrogen-sulfur doped carbon dots could significantly change the shape of Cu2O, transforming it from a cube to a polyhedron (e.g., a dodecahedron or a sphere), increasing the specific surface area of ​​Cu2O, and further improving the conductivity and electron transport efficiency of the composite material. This composite material can promote electron transfer on the surface of the electrochemical immunosensor electrode, capture a large number of antigens and generate electrochemical signals, enabling specific analysis of tumor markers (e.g., AFP) and distinguishing them from other interfering components (e.g., other antigens and biomolecules). Therefore, the protection scheme of this invention is proposed.

[0102] In a first typical embodiment of the present invention, a method for preparing a nitrogen-sulfur doped carbon dot modified cuprous oxide composite material is provided, the method comprising:

[0103] Nitrogen-sulfur doped carbon dots were introduced during the synthesis of cuprous oxide to obtain the above-mentioned nitrogen-sulfur doped carbon doped cuprous oxide composite material; wherein, CuSO4·5H2O was used to synthesize the above-mentioned cuprous oxide.

[0104] Cu₂O is a typical three-dimensional transition metal oxide and p-type semiconductor material, possessing a large specific surface area and good electrical conductivity. As an electroactive material, Cu… + / Cu 2+ The reversible oxidation of Cu₂O can generate a good electrochemical signal. The various oxidation states of copper provide ideal conditions for electrochemical sensors, and copper-based biosensors exhibit excellent performance in terms of sensitivity, stability, and selectivity.

[0105] Electrolyte crystals (CDs) possess excellent electronic properties, large specific surface area, good water solubility, biocompatibility, chemical stability, ease of synthesis, and low cost, making them a promising candidate for tumor marker detection. The surface and edges of CDs contain numerous hydrophilic functional groups, such as carboxyl, hydroxyl, and amino groups. These functional groups can serve as active centers or provide bonding sites for constructing composite materials, thereby improving the charge transfer rate and stability of the composite material and promoting electrode-electrolyte interactions.

[0106] Double doping (N, S) can induce synergistic coupling effects between heteroatoms, generating more defect sites and surface functional groups, resulting in more active sites, improved intrinsic activity of active sites, higher reaction current, and better electrochemical stability. It also promotes charge-electron transfer at the interface, giving the material good conductivity. Combining CDs with Cu2O can construct composite materials, achieving a synergistic effect and thus improving the conductivity and electrochemical activity of the composite material.

[0107] This application introduces nitrogen-sulfur doped carbon dots during the synthesis of cuprous oxide, resulting in a nitrogen-sulfur doped carbon dot-modified cuprous oxide composite material. The introduction of nitrogen-sulfur dual-doped carbon dots plays a role in regulating the morphology of cuprous oxide, transforming its original cubic structure into a dodecahedron, thus increasing its specific surface area. This demonstrates the regulatory effect of carbon dots on the morphology of the synthesized cuprous oxide. Furthermore, by modifying the electrodes of an electrochemical immunosensor with this composite material, the electrochemical immunosensor achieves sensitive detection of tumor markers (e.g., AFP). In particular, when used to detect AFP in real serum samples, it exhibits the advantages of high specificity (unaffected by interference from other components in the serum) and high sensitivity.

[0108] In a preferred embodiment of the present invention, the preparation method includes the following steps:

[0109] S1) Glutathione and p-phenylenediamine are mixed in a solvent and subjected to a hydrothermal reaction to obtain nitrogen-sulfur doped carbon dots (NS-CDs).

[0110] S2) CuSO4·5H2O, the above nitrogen-sulfur doped carbon dots, water and sodium hydroxide are mixed and heated to obtain the first solid-liquid mixture;

[0111] S3) The first solid-liquid mixture is mixed with a reducing agent to carry out a reduction reaction, thereby obtaining a second solid-liquid mixture;

[0112] S4) The second solid-liquid mixture is centrifuged, washed and dried sequentially to obtain the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material; wherein the solvent is selected from ethanol and water.

[0113] Using glutathione and p-phenylenediamine as carbon sources and dopants, nitrogen-sulfur co-doped carbon dots are formed via a hydrothermal reaction in selected solvents (ethanol and water). Controlling the molar volume ratio, hydrothermal reaction time, and temperature during this process helps to further ensure that the carbon dots have appropriate doping levels and sizes, thereby endowing them with superior electrochemical performance and biocompatibility.

[0114] In a preferred embodiment of the present invention, in S1), the molar volume ratio (mM:mM:mL:mL) of the glutathione, p-phenylenediamine, ethanol, and water is (0.05~0.10):(0.05~0.2):(5~10):(5~10). Using the above-mentioned specific ratio of raw materials to prepare nitrogen-sulfur co-doped carbon dots has beneficial effects such as enhancing the current signal, improving detection sensitivity, and reducing the detection limit.

[0115] In a preferred embodiment of the present invention, the hydrothermal reaction time is 2-5 hours, and the hydrothermal reaction temperature is 120-180°C. The carbon dot material prepared by hydrothermal reaction under these specific conditions exhibits good properties.

[0116] In a preferred embodiment of the present invention, after the hydrothermal reaction and before obtaining the nitrogen-sulfur doped carbon dots, step S1) further includes a first cooling and a first drying step on the product after the hydrothermal reaction. After cooling the product after the hydrothermal reaction to room temperature (e.g., 20-25°C), the dark purple liquid is collected and subjected to a first drying to obtain a powdered product. In a preferred embodiment of the present invention, the temperature of the first cooling is 20°C-25°C; the time of the first cooling is 2-5 hours. In a preferred embodiment of the present invention, the temperature of the first drying is 50-60°C; the time of the first drying is 24-28 hours.

[0117] In a preferred embodiment of the present invention, the reducing agent is selected from a glucose solution; the mass-to-volume ratio (mg:mg:mL:mL:mL) of CuSO4·5H2O, nitrogen-sulfur doped carbon dots, water, sodium hydroxide, and glucose solution is (300~500):(0.5~1.5):(20~40):(5~10):(5~10); the concentration of sodium hydroxide is 1~5 M; the concentration of glucose solution is 0.1~0.5 M; and the amount of glucose in the glucose solution is greater than that in CuSO4·5H2O. 2+ The amount of substance.

[0118] CuSO4·5H2O, nitrogen-sulfur doped carbon dots (NS-CDs), water, and sodium hydroxide were mixed in a specific mass-volume ratio and heated at a specified temperature to form a first solid-liquid mixture. The presence of sodium hydroxide helps control the reaction environment, promoting subsequent morphology regulation of Cu2O and uniform intercalation of NS-CDs. Glucose is a mild reducing agent, and the above specific ratio allows the product to grow slowly, resulting in a better crystal shape.

[0119] It should be noted that in S2 above, when mixing the above components, not all components are mixed at the same time. Instead, CuSO4·5H2O, nitrogen-sulfur doped carbon dots and water are mixed first, and then sodium hydroxide is slowly added. The purpose of this is to fully mix the nitrogen-sulfur doped carbon dots and the regulator, i.e., the nitrogen-sulfur doped carbon dots, first, and then adjust the pH of the solution with sodium hydroxide. Under alkaline conditions, the solution slowly precipitates to obtain the Cu2O product modified with nitrogen-sulfur doped carbon dots.

[0120] Furthermore, in S2) above, before obtaining the first solid-liquid mixture after mixing the above components, the mixed product needs to be heated. In a preferred embodiment of the present invention, the temperature of the first solid-liquid mixture is 60°C to 80°C.

[0121] In a more preferred embodiment of the present invention, the mass-to-volume ratio (mg:mg:mL:mL:mL) of the CuSO4·5H2O, the nitrogen-sulfur doped carbon dots, water, sodium hydroxide, and the glucose solution is 500:(0.5~1.5):40:10:10; for example, 500:0.5:40:10:10, 500:0.6:40:10:10, 500:0.7:40:10:10, 500:0.8 The concentrations of the sodium hydroxide solution are 40:10:10, 500:0.9:40:10:10, 500:1:40:10:10, 500:1.1:40:10:10, 500:1.2:40:10:10, 500:1.3:40:10:10, 500:1.4:40:10:10, and 500:1.5:40:10:10; wherein the concentration of the sodium hydroxide solution is 1M and the concentration of the glucose solution is 0.3M.

[0122] The time and temperature of the reduction reaction may affect the crystallization and growth of the product. In a preferred embodiment of the present invention, the reduction reaction time is 2-5 hours and the reduction reaction temperature is 60°C-80°C.

[0123] To remove residual glucose solution and sodium hydroxide in the nitrogen-sulfur doped carbon dot modified cuprous oxide and prevent them from affecting the performance of the composite material, in a preferred embodiment of the present invention, in S4) above, the precipitate after centrifugation is washed with ethanol and / or water. In a preferred embodiment of the present invention, ethanol is used first, followed by water. The number of washing cycles is determined according to actual needs.

[0124] In a preferred embodiment of the present invention, the drying temperature is 65°C to 80°C, and the drying time is 24h to 28h. Drying under these conditions provides the beneficial effect of thorough drying and preventing particle agglomeration.

[0125] In a second typical embodiment of the present invention, a nitrogen-sulfur-doped carbon dot-modified cuprous oxide composite material prepared by the above-described preparation method is provided.

[0126] The nitrogen-sulfur doped carbon dot-modified cuprous oxide composite material prepared by the above method has the advantages of enhanced electrochemical performance, excellent stability and reproducibility, and low cost. The introduction of NS-CDs significantly improves the electrochemical activity of Cu2O because a synergistic effect is formed between NS-CDs and Cu2O, promoting charge transfer at the electrode / electrolyte interface, thereby enhancing the conductivity and electrocatalytic efficiency of the composite material.

[0127] Modifying the electrodes of electrochemical immunosensors with the aforementioned materials not only enables the detection of tumor markers (e.g., AFP) with high sensitivity, specificity, and stability, but also offers cost-effectiveness and ease of operation, demonstrating broad application prospects in clinical diagnosis and biomedical research.

[0128] In a preferred embodiment of the present invention, the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material includes a cuprous oxide matrix and the above-mentioned nitrogen-sulfur doped carbon dots doped with the above-mentioned cuprous oxide matrix; the above-mentioned nitrogen-sulfur doped carbon dots are uniformly distributed on the surface of the above-mentioned cuprous oxide matrix.

[0129] It should be noted that during the synthesis of the nitrogen-sulfur-doped carbon dot-modified cuprous oxide composite material, the nitrogen-sulfur-doped carbon dots were also being doped in real time. Therefore, the interior (not detected due to limitations in detection conditions) and surface of the cuprous oxide matrix may be modified with nitrogen-sulfur-doped carbon dots. Scanning electron microscopy revealed that the nitrogen-sulfur-doped carbon dots on the surface of the cuprous oxide matrix were uniformly distributed.

[0130] In a preferred embodiment of the present invention, the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material exhibits a dodecahedral structure. This increases the specific surface area of ​​the material, thereby increasing the effective reaction area during catalysis, improving electron transport efficiency, and thus contributing to enhancing the sensitivity of the electrochemical immunosensor.

[0131] In a more preferred embodiment of the present invention, the average diameter of the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material is 1.21µm ± 0.21µm. This composite material has a small size and a large specific surface area, which can further effectively improve the reaction area and electron transport efficiency of the catalytic reaction.

[0132] In a preferred embodiment of the present invention, the C, N, O, S and Cu elements in the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material are arranged in a manner that is uniformly dispersed on the surface.

[0133] In a more preferred embodiment of the present invention, the mass ratio of the above-mentioned Cu element, the above-mentioned O element, the above-mentioned S element, the above-mentioned C element and the above-mentioned N element in the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material is (60~73):(10~25):(2~6):(3~8):(2~5).

[0134] In a third typical embodiment of the present invention, a modified electrode is provided, the modified electrode comprising a substrate electrode and a composite material modified on the surface of the substrate electrode; the composite material is selected from the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material prepared by the above preparation method or the above nitrogen-sulfur doped carbon dot modified cuprous oxide composite material.

[0135] The Cu2O@NS-CDs composite material used to modify the electrode can enhance the catalytic performance and electron transport efficiency of the electrode, which helps to build an electrochemical detection platform with high sensitivity and strong selectivity.

[0136] In a preferred embodiment of the present invention, the electroactive area of ​​the modified electrode is 0.04 cm². 2 ~0.06cm 2 .

[0137] In a fourth typical embodiment of the present invention, an electrochemical immunosensor is provided, which includes the modified electrode described above.

[0138] The aforementioned electrochemical immunosensor, after incubation with alpha-fetoprotein (AFP) antibodies, specifically recognizes and binds to AFP, resulting in a significant decrease in the electrochemical signal. A good linear relationship exists between the peak DPV current and the logarithm (Log C) of AFP concentration, allowing for quantitative analysis of AFP using a linear equation. It exhibits high selectivity for AFP even in complex biological samples (e.g., serum), effectively distinguishing it from other biomolecules such as antigens and other biomolecules, greatly improving the accuracy and reliability of detection.

[0139] Furthermore, this electrochemical immunosensor exhibits extremely high sensitivity for AFP within a concentration range of 0.001–100 ng / mL, with a detection limit as low as 21.4 fg / mL. This performance surpasses that of many sensors reported in existing literature, making it a highly attractive tool for detecting liver cancer biomarkers. This electrochemical immunosensor not only plays a crucial role in the early diagnosis of liver cancer but may also be applied to the detection of other biomarkers (e.g., tumor markers such as alpha-fetoprotein, carcinoembryonic antigen, or squamous cell carcinoma antigen), providing new directions and possibilities for related biomedical research and disease screening.

[0140] In a fifth typical embodiment of the present invention, a method for detecting alpha-fetoprotein is provided, the method comprising: detecting alpha-fetoprotein using the above-described electrochemical immunosensor.

[0141] In a preferred embodiment of the present invention, the method includes the following steps: incubating the electrode of the electrochemical immunosensor and the sample to be tested in a buffer solution for 10 min to 60 min (e.g., 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min); the sample to be tested contains alpha-fetoprotein. In a more preferred embodiment of the present invention, the pH of the buffer solution is 6.4 to 8 (e.g., 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0). Incubating the sample containing alpha-fetoprotein with the electrode under the above conditions results in a larger peak current at the electrode, indicating higher electrode sensitivity.

[0142] It should be noted that the electrodes of the electrochemical immunosensor here are not only modified with nitrogen-sulfur doped carbon dot modified cuprous oxide composite material, but also modified with alpha-fetoprotein antibody and BSA. The alpha-fetoprotein antibody is used to capture alpha-fetoprotein, and the BSA is used to block non-specific active sites.

[0143] The above method for detecting alpha-fetoprotein is not only simple and quick, but also highly sensitive (21.4 fg / mL) and has a wide detection range (0.001-100 ng / mL).

[0144] In a sixth typical embodiment of the present invention, the application of the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material prepared by the above preparation method, or the above-mentioned nitrogen-sulfur doped carbon dot modified cuprous oxide composite material, or the above-mentioned modified electrode, or the above-mentioned electrochemical immunosensor, or the above-mentioned method for detecting alpha-fetoprotein, in the detection of tumor markers is provided.

[0145] Tumor markers are a class of bioactive substances produced by tumor cells themselves or abnormally produced by the body in response to tumors, which can indicate the presence of a tumor or reflect its biological characteristics. They are essentially a core subset of biomarkers and are important tools in clinical oncology for assisting cancer management. Any tumor marker detected based on the principle of antigen-antibody binding is applicable to this application. In a preferred embodiment of the present invention, the tumor marker preferably includes any one of the following: alpha-fetoprotein, carcinoembryonic antigen, or squamous cell carcinoma antigen.

[0146] The nitrogen-sulfur-doped carbon dot-modified cuprous oxide composite material, the electrode of the electrochemical immunosensor, or the electrochemical immunosensor itself, prepared using the above-described method, or the method for detecting AFP, exhibit excellent repeatability, reproducibility, stability, low cost, and ease of operation when detecting tumor markers (e.g., AFP). When used to detect tumor markers (e.g., AFP) in clinical serum, the detection results are comparable to commercial ELISA and ECL methods, demonstrating its potential application in the clinical diagnosis of liver cancer and opening up new directions for research in related fields.

[0147] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0148] Example 1: Preparation of Cu2O@NS-CDs

[0149] NS-CDs were synthesized via a one-step solvothermal method using glutathione and p-phenylenediamine as precursors. 0.0257 g (0.0835 mM) of glutathione and 0.0181 g (0.167 mM) of p-phenylenediamine were dissolved in a mixture of 7.5 mL water and 7.5 mL ethanol. The mixture was stirred for 10 min, transferred to a 50 mL reactor, and heated in an oven at 160 °C for 5 h. After cooling to room temperature, the dark purple product was collected. The dark purple liquid was then collected and dried in an oven at 60 °C to obtain NS-CDs.

[0150] 500 mg CuSO4·5H2O was mixed with 10 mL of water containing 1 mg NS-CDs and dissolved in 40 mL of water under mechanical stirring. 10 mL of 1 M sodium hydroxide solution was slowly added, and the mixture was heated to 60 °C. Then, 10 mL of 0.3 M glucose solution was added, and the mixture was heated at 60 °C for 3.5 h. After cooling to room temperature, the product was centrifuged, washed three times with ethanol and water, and dried at 65 °C for 24 h to obtain Cu2O@NS-CDs (see...). Figure 1 ).

[0151] Example 2: Preparation of Cu2O@NS-CDs using NS-CDs of different masses

[0152] NS-CDs were synthesized via a one-step solvothermal method using glutathione and p-phenylenediamine as precursors. 0.0257 g (0.0835 mM) of glutathione and 0.0181 g (0.167 mM) of p-phenylenediamine were dissolved in a mixture of 7.5 mL water and 7.5 mL ethanol. The mixture was stirred for 10 min, transferred to a 50 mL reactor, and heated in an oven at 160 °C for 5 h. After cooling to room temperature, the dark purple product was collected. The dark purple liquid was then collected and dried in an oven at 60 °C to obtain NS-CDs.

[0153] 500 mg CuSO4·5H2O was mixed with 10 mL of water containing different masses of NS-CDs (0.25 mg–1.75 mg NS-CDs). The mixture was dissolved in 40 mL of water under mechanical stirring. 10 mL of 1 M sodium hydroxide solution was slowly added, and the mixture was heated to 60 °C. Then, 10 mL of 0.3 M glucose solution was added, and the mixture was heated at 60 °C for 3.5 h. After cooling to room temperature, the product was centrifuged, washed three times with ethanol and water, and dried at 65 °C for 24 h to obtain Cu2O@NS-CDs.

[0154] Example 3 Characterization of Cu2O@NS-CDs

[0155] (1) X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR)

[0156] The structures of NS-CDs, Cu2O, and Cu2O@NS-CDs in Example 1 were characterized by XRD, see [link to XRD diagram]. Figure 2 (a) In the figure, the broad diffraction peak centered at 26.38° of NS-CDs is consistent with the standard card (JCPDS No. 41-1487), confirming the presence of partial graphite structure in the amorphous carbon phase and carbon core of NS-CDs. Cu2O and Cu2O@NS-CDs show characteristic peaks at 29.55°, 36.42°, 42.30°, 61.34°, 73.53°, and 77.32°, corresponding to the (110), (111), (200), (220), (311), and (222) crystal planes, respectively. The XRD characteristic peaks of Cu2O and Cu2O@NS-CDs are consistent with the characteristic peaks in the standard card of Cu2O (JCPDS No. 05-0667).

[0157] However, different NS-CD doping amounts show that the diffraction peaks of Cu2O@NS-CDs changed significantly when different masses of NS-CDs were added (i.e., 0.5 mg, 1 mg, and 1.5 mg). This demonstrates the control of product morphology by NS-CDs. It also shows that the intensity of the characteristic peaks at 36.42° and 42.30° changed after compositing with NS-CDs. The intensity of the characteristic peak at 36.42° increased, indicating significant growth on the (111) crystal plane, while the intensity of the characteristic peak at 42.30° decreased, indicating the disappearance of the diffraction peak on the (200) crystal plane. This indicates that the amount of NS-CDs incorporated affects the morphology of the Cu2O@NS-CDs composite material and further influences its performance. See Figure 2 (b) in the middle.

[0158] The functional groups contained in NS-CDs, Cu2O, and Cu2O@NS-CDs in Example 1 were characterized by FTIR spectroscopy, see [link to FTIR spectroscopy]. Figure 2 (c) Cu2O, Cu2O@NS-CDs at 613 cm⁻¹ -1 The characteristic bands at 613 cm⁻¹ are attributed to Cu-O vibrations. Compared to Cu₂O, Cu₂O@NS-CDs show a more pronounced band at 613 cm⁻¹. -1 The greater band intensity at 3430 cm⁻¹ is likely attributed to the reaction of NS-CDs with Cu₂O. NS-CDs exhibit a higher band intensity at 3430 cm⁻¹. -1 and 1650 cm -1 Absorption peaks appear at these locations, corresponding to the vibrations of OH and C=O, respectively. The FT-IR characteristic peaks of Cu₂O@NS-CDs doped with 1 mg and 1.5 mg of NS-CDs are essentially the same. See Figure 2 (d) shows that Cu2O@NS-CDs nanocomposite materials were successfully prepared.

[0159] (2) Scanning electron microscope (SEM)

[0160] The morphology and size of NS-CDs were analyzed using SEM. SEM images show that Cu2O has a cubic morphology (see...). Figure 3 (a) shows a dispersed state and uniform size, with a side length and diagonal length of 1.21µm ± 0.13µm. Figure 3 (c) In this context, NS-CDs are uniformly distributed on Cu2O, the edges of the cubes become smooth, and Cu2O@NS-CDs exhibit a dodecahedral structure (see [link]). Figure 3 (b) of the figure has an average diameter of 1.21µm ± 0.21µm (see [reference]). Figure 3 (d) in the middle.

[0161] After adding different doping amounts (0.5 mg, 0.75 mg, and 1 mg) of NS-CDs during the preparation process, the particle size did not change significantly, but the morphology changed from cubic to dodecahedral (e.g., ...). Figure 4 As shown in (a) to (f) in the figure, this structural change indicates that carbon dots also play a role in regulating the morphology of cuprous oxide during the synthesis process, transforming the original cubic structure of cuprous oxide into a dodecahedron. This demonstrates the regulatory effect of carbon dots on the morphology of synthesized cuprous oxide, which increases the specific surface area of ​​the material and can increase the effective reaction area during the catalytic process.

[0162] The results show that doping with NS-CDs effectively synthesized Cu2O@NS-CDs binary nanocomposites, ensuring the improved catalytic performance of Cu2O@NS-CDs. EDS analysis revealed C, N, O, S, and Cu elemental peaks (see...). Figure 3 (e)). SEM mapping results show that the five elements C, N, O, S and Cu are uniformly dispersed on the surface of Cu2O@NS-CDs (see [reference]). Figure 5 As shown in (a) to (f), it is confirmed that NS-CDs are doped into the Cu2O framework.

[0163] (3) X-ray photoelectron spectroscopy (XPS)

[0164] XPS characterization was performed to analyze the detailed elemental composition and functional groups of Cu2O@NS-CDs in Example 1. Peaks for C 1s (63.66%), N 1s (2.13%), O 1s (22.58%), S 2p (1.37%), and Cu 2p (10.26%) were observed in the XPS full spectrum. Figure 6 (a) in the data is consistent with EDS data. Figure 3 (f)). The results show that Cu2O@NS-CDs were successfully synthesized.

[0165] The C 1s spectrum shows three peaks at 284.8 eV, 286.4 eV, and 288.5 eV, corresponding to C=C / CC, C-OH, and C=O, respectively. Figure 6 (b)). The peak of N 1s at 399.9 eV corresponds to C=N ( Figure 6 (c)). The O 1s spectrum splits into three peaks at 530.6 eV, 532.1 eV, and 533.9 eV, corresponding to Cu-O, C=O / N=O, and C-OH, respectively. Figure 6 (d)). The peak of S 2p at 167.3 eV corresponds to SO4. 2- ( Figure 6 In (e)), the Cu 2p spectrum is divided into two peaks, which are Cu + / Cu0 Cu 2p 3 / 2 (932.8 eV) and Cu + / Cu 0 Cu 2p 1 / 2 (952.6 eV) Figure 6 (f) in the middle.

[0166] Example 4: Electrochemical characterization of Cu2O@NS-CDs and preparation process of the immunosensor

[0167] Preparation method of NS-CDs / GCE: The glassy carbon electrode (GCE) was polished sequentially with alumina powders of particle sizes of 1.5, 0.5, and 0.05 μm, and then thoroughly rinsed with ethanol and ultrapure water. NS-CDs were dispersed in deionized water and ethanol (volume ratio 1:1) and sonicated for 60 min to form a homogeneous suspension of 4 mg / mL. Then, 6 μL of the above homogeneous suspension was placed on the GCE and allowed to air dry at room temperature to obtain NS-CDs / GCE.

[0168] Preparation method of Cu2O / GCE: The glassy carbon electrode (GCE) was polished sequentially with alumina powders of particle sizes of 1.5, 0.5, and 0.05 μm, and then thoroughly rinsed with ethanol and ultrapure water. Cu2O was dispersed in deionized water and ethanol (volume ratio 1:1) and sonicated for 60 min to form a homogeneous suspension of 4 mg / mL. Then, 6 μL of the above homogeneous suspension was placed on the GCE and allowed to air dry at room temperature to obtain Cu2O / GCE.

[0169] Preparation method of Cu2O@NS-CDs / GCE: The glassy carbon electrode (GCE) was polished sequentially with alumina powders of particle sizes of 1.5, 0.5, and 0.05 μm, and then thoroughly rinsed with ethanol and ultrapure water. Cu2O@NS-CDs were dispersed in deionized water and ethanol (volume ratio 1:1) and sonicated for 60 min to form a homogeneous suspension of 4 mg / mL. Then, 6 μL of the above homogeneous suspension was placed on the GCE and allowed to air dry at room temperature to obtain Cu2O@NS-CDs / GCE.

[0170] Preparation of electrochemical immunosensor: Add 6 μL of 10 μg mL... -1After adding antibody (Sina Biological 12177-MM26) solution to Cu2O@NS-CDs / GCE, the Cu2O@NS-CDs / GCE electrode was incubated overnight at 4°C. 1% BSA was added, and the electrode was allowed to air dry to block non-specific active sites. Subsequently, the electrode surface was rinsed with 0.01M PBS buffer (pH 7.4). The sensing performance of the electrochemical immunosensor was then evaluated by modifying the electrode with different concentrations of AFP antigen (purchased from Xibao Biotechnology (Shanghai) Co., Ltd., EKY0032E) after incubation at 37°C for 30 min (see [link to relevant documentation]). Figure 1 ).

[0171] The electrochemical performance of Cu2O, NS-CDs, and Cu2O@NS-CDs in Example 1 was evaluated using CV. Both NS-CDs and Cu2O showed a distinct pair of redox peaks. The electrochemical properties of Cu2O@NS-CDs / GCE were evaluated by comparing CV curves (see [link to CV analysis]). Figure 7 (a)). After recombination, compared with Cu2O / GCE and NS-CDs / GCE, Cu2O@NS-CDs / GCE showed obvious redox peaks and decreased peak current, indicating that the introduction of NS-CDs significantly changed the conductivity.

[0172] EIS is also used to evaluate the interfacial properties of various modified electrodes (see Figure 7 (b) includes Bare / GCE, NS-CDs / GCE, Cu2O / GCE, and Cu2O@NS-CDs / GCE. NS-CDs / GCE exhibits a smaller charge transfer resistance, indicating that NS-CDs have good conductivity. The Rct of Cu2O / GCE is smaller than that of NS-CDs / GCE, indicating that Cu2O has better electron transfer capability. The synergistic effect of the Cu2O@NS-CDs composite material alters the electron transfer on the electrode surface.

[0173] The stepwise fabrication process of the electrochemical immunosensor was characterized using CV and EIS. Initially, Bare / GCE exhibited a pair of characteristic redox peaks, indicating its inherent electrochemical activity (see [link to original text]). Figure 7 (c)). After Cu2O@NS-CDs / GCE modification, the redox peak current showed a decreasing trend, indicating that the electrode surface was effectively modified.

[0174] After incubation with Ab, the current signal of Cu2O@NS-CDs decreased significantly, reflecting the effective deposition of the insulating Ab layer, further verifying the successful binding of Ab on the Cu2O@NS-CDs electrode. Due to the sufficient coverage of non-conductive biomaterials, BSA modification further hindered electron transfer, resulting in a significant decrease in the redox peak current. Finally, with incubation with alpha-fetoprotein, the peak current continuously decreased. The highly specific immune recognition between alpha-fetoprotein and its antibody further hindered electron transfer, leading to the successful fabrication of the final electrochemical immunosensor.

[0175] The construction process of the electrochemical immunosensor was further determined using EIS to identify electrode surface properties (see [link to EIS]). Figure 7 (d) After modification with Cu2O@NS-CDs, the conductivity decreased and the semicircle diameter increased. With the gradual immobilization of Ab, Rct showed an upward trend, indicating that the Ab was modified and combined with Cu2O@NS-CDs, further increasing the impedance and hindering electron transfer on the electrode surface. The further increase in the semicircle diameter of the curve indicates that the BSA modification further weakened the signal and hindered electron transport, demonstrating that BSA successfully blocked non-specific active sites.

[0176] The impedance curve after AFP modification was higher than that after BSA modification, indicating that the antigen was successfully modified. The CV and EIS results were consistent, validating the successful construction of the electrochemical immunosensor. The electrocatalytic activity of Cu2O@NS-CDs / GCE and Cu2O / GCE for AFP was compared using DPV (see [link to DPV]). Figure 7 (e)). Cu2O showed a trend toward AFP sensing performance; however, Cu2O@NS-CDs / GCE showed a significantly increased current difference. The superior detection performance of Cu2O@NS-CDs / GCE can be attributed to the synergistic effect between the composite materials.

[0177] To obtain optimal detection performance, the sensing ability of Cu2O@NS-CDs / GCE with the addition of 0.5 mg, 1 mg, and 1.5 mg of NS-CDs during synthesis was investigated (see...). Figure 7 (f)). Cu2O@NS-CDs with 1 mg of NS-CDs added showed the best detection effect for AFP, therefore the mass of NS-CDs was determined to be 1 mg.

[0178] Example 5: Electroactive area and kinetics of Cu2O@NS-CDs

[0179] At 5mM [Fe(CN)6] 3- / 4- In an aqueous solution of 0.1 M KCl, 10-100 mVs -1The CV curves of Cu2O@NS-CDs / GCE were measured at a scan rate of [missing value]. Figure 8 In (a), the redox peak current increases with increasing scan rate, and the anodic and cathodic peak currents show a good linear relationship with the square root of the scan rate, respectively. Figure 8 (b) in the middle.

[0180] The linear equation for the anode peak current versus the square root of the sweep rate is: I pa =5.8101v 1 / 2 -3.6321. The electroactive area of ​​the modified electrode was calculated using the Randles-Sevcik equation. The calculation formula is as follows: The calculated electroactive area of ​​Cu2O@NS-CDs / GCE is 0.05 cm². 2 This indicates that Cu2O@NS-CDs possess excellent electrochemical activity, stemming from the synergistic effect of Cu2O and NS-CDs, which is beneficial for subsequent electrochemical detection of alpha-fetoprotein.

[0181] With 10-100 mV s -1 The CV curves of AFP on Cu2O@NS-CDs / Ab / BSA / GCE were tested at the scan rate to further understand the reaction kinetics of AFP on Cu2O@NS-CDs / Ab / BSA / GCE. Figure 8 (c)). The redox peak current of AFP on the modified electrode increases regularly with increasing scan rate. There is a significant linear relationship between the current and the square root of the scan rate. Figure 8 (d) indicates that the electrochemical reaction on the electrode surface conforms to a diffusion-controlled process.

[0182] The electrochemical performance of Cu2O@NS-CDs prepared by incorporating different masses of NS-CDs (mass range of 0.5 mg-1.5 mg NS-CDs) was also tested, and the results showed (see...) Figure 7 In (f), Cu2O@NS-CDs with other masses exhibited lower current response and electroactive area than Cu2O, indicating that the performance of Cu2O@NS-CDs is closely related to its morphology and structure during the optimization process. The advantages of the material structure directly affect its electrochemical performance. Even with the same composition, differences in morphology lead to differences in performance, making these materials unsuitable as electrochemical immunoassay probes.

[0183] Example 6: Optimization of experimental conditions for electrochemical detection of AFP

[0184] Methods for detecting AFP using Cu2O@NS-CDs include:

[0185] 1) Disperse Cu2O@NS-CDs in deionized water and ethanol, and sonicate for 60 min to form a homogeneous suspension of 4 mg / mL. Drop 6 μL of this solution onto a GCE and dry at room temperature. Add 6 μL of 10 µg / mL antibody solution and incubate overnight at 4°C. Add 1% BSA and dry at room temperature to block non-specific active sites. Rinse the electrode surface with 0.01 M pH 7.4 phosphate buffered saline (PBS) and dry at room temperature.

[0186] 2) Electrodes modified with different concentrations of AFP antigen were incubated at 37 °C for 30 min. Differential pulse voltammetry (DPV) was used to record the electrocatalytic behavior of the modified electrodes towards AFP within a potential window of 0–0.7 V. The electrolyte was a solution containing 5 mM [Fe(CN)6]. 3- / 4- 0.1 M PBS (pH=7.4) with 0.1 M KCl. A standard curve was plotted between the peak DPV current and the logarithm (logc) of the AFP concentration. Cu2O@NS-CDs were then used in 5 mM [Fe(CN)6] electrolyte solution. 3- / 4- The DPV current values ​​in 0.1 M PBS with 0.1 M KCl and pH=7.4 were used as the blank group.

[0187] 3) Dilute the AFP antigen solution with serum samples to obtain a series of AFP-spiked serum samples with concentrations (0, 0.9, 1, 4, 20, 25, 30, 35 ng / mL). Drop the AFP-spiked serum samples onto Cu2O@NS-CDs / Ab / BSA electrodes and incubate at 37°C for 30 min for DPV testing.

[0188] To obtain optimal detection results, the pH of the buffer solution and the incubation time of the antigen were investigated. DPV was used to study the effects of 50 ng / mL under different pH conditions. -1 The electrochemical reaction of alpha-fetoprotein on Cu2O@NS-CDs / Ab / BSA / GCE showed that, within the pH range of 6.4 to 7.4, the peak current always increased with increasing pH, decreased with further increases to pH 8, and reached a maximum at pH 7.4. Figure 9 (a)). Considering that the normal pH value of human blood is close to 7.4, the pH of the buffer solution was determined to be 7.4.

[0189] To assess the effect of incubation time on the modified electrode, 50 ng mL was added. -1After AFP incubation, Cu2O@NS-CDs / Ab / BSA / AFP / GCE were incubated for 10-60 min to construct electrochemical immunosensors. The peak current always increased with increasing incubation time, but decreased with further increases in incubation time up to 60 min, reaching a maximum value at 30 min. Figure 9 (b)). The modified electrode incubated for 30 min showed the best performance; therefore, the incubation time was determined to be 30 min in subsequent tests.

[0190] Example 7 Sensitivity of Electrochemical Sensor in Detecting AFP in Buffer Solution

[0191] Cu2O@NS-CDs were used as the electrochemical sensing material to analyze the AFP content in serum. A significant decrease in electrochemical signal occurred after Cu2O@NS-CDs / Ab / BSA was incubated with AFP antigen. A good linear relationship was observed between the peak current (DPV) and the logarithm (LogC) of AFP concentration, allowing for quantitative analysis of AFP using this linear equation. Figure 10 (a) in the middle.

[0192] Under optimal conditions (pH=7.4, electrolyte containing 5 mM [Fe(CN)6]), 3- / 4- The AFP sample was dropped onto a Cu2O@NS-CDs / Ab / BSA / GCE electrode and incubated at 37℃ for 30 min with 0.1 M KCl and 0.1 M PBS (pH=7.4). The sensitivity of the electrochemical sensor for quantitative detection of AFP was tested using DPV. As the AFP concentration gradually increased (0.001-100 ng / mL), a series of orderly decreasing current responses were observed. Figure 10 In (a) of the data, the DPV current is linearly related to the logarithm of the AFP concentration. Figure 10 (b)). In the range of 0.001-100 ng / mL, the linear equation is y = -4.2881 + 31.1651 (R0). 2 =0.998), and the detection limit can be calculated to be 21.4 fg / mL based on 3σ / k.

[0193] Example 8: Selectivity, repeatability, reproducibility and stability analysis of AFP detection using an electrochemical sensor.

[0194] To further validate the sensing performance of the Cu2O@NS-CDs electrochemical sensor for AFP, the sensor's selectivity, repeatability, reproducibility, and stability were tested. In clinical trials, selectivity is a crucial indicator for evaluating sensor performance. For specificity analysis, the electrochemical immunosensor was tested for 10 ng / mL... -1 AFP, 100 ng / mL-1 Electrochemical detection of IgG, CEA, BSA, Glu, His, AA, and mixtures of the above substances ( Figure 10 (c) In this context, the electrochemical immunosensor is only effective for 10 ng / mL. -1 AFP and the mixture of the above substances showed a significant current response, while the interfering substances showed no current response even at 10 times the concentration, indicating that the several interfering substances that may exist in the human body have almost no effect on the detection of AFP, and that the immunosensor has high specificity for AFP.

[0195] By using the same electrode to test 10 ng mL -1 AFP performed five measurements to evaluate the repeatability of the electrochemical immunosensor. The results of the five measurements were similar, with a relative standard deviation of 1.67%. Figure 10 (d) indicates that the prepared electrochemical immunosensor has good reproducibility. Five independent electrochemical immunosensors were prepared under the same conditions to evaluate reproducibility. Each of the five independent electrochemical immunosensors detected 10 ng / mL... -1 The AFP measurement results were consistent, with an RSD of 0.32%. Figure 10 (e) of the above), the reproducibility is satisfactory.

[0196] Stability is also important for the practical application of immunosensors. The prepared immunosensors were stored at 4°C when not in use. After 17 days of storage, testing was performed, and the peak current retained 98% of its initial value. Figure 10 (f)). The change in current response may be due to the decrease in biomolecular activity with increasing storage time. The results show that the electrochemical immunosensor has good stability. The above results demonstrate that the proposed sensor has good anti-interference, repeatability, reproducibility and stability, and has good potential for practical applications.

[0197] Example 9: Electrochemical sensor for detecting AFP in serum

[0198] To illustrate the application of electrochemical immunosensors in biomedicine, the recovery rate of AFP in human serum samples was tested using a standard spiking method. The concentrations of the spiked serum samples were 0, 0.9, 1, 4, 20, 25, 30, and 35 ng / mL. -1 The content of alpha-fetoprotein in spiked human serum samples was analyzed by electrochemical methods. Figure 11The electrochemical immunosensor exhibits a significant current response to alpha-fetoprotein (AFP) in serum, indicating that Cu2O@NS-CDs / GCE can be used for the detection of AFP in serum, with recoveries ranging from 99% to 100.5% and relative standard deviations from 0.62% to 5.34%, demonstrating the good practicality of the electrochemical immunosensor. To verify the effective sensing performance of the electrochemical immunosensor, the same serum samples were tested using clinical methods ELISA and ECL. The test results of the proposed method and the clinical methods were almost identical, indicating the potential of this electrochemical immunosensor to detect AFP in real samples and its promising application in the early diagnosis of hepatocellular carcinoma in clinical medicine.

[0199] In summary, this work achieves the advantages of good selectivity, high sensitivity, and rapid and accurate target detection, and has broad application prospects.

[0200] As can be seen from the above description, this invention has the following advantages: It uses glutathione and p-phenylenediamine as precursors to obtain stable nitrogen-sulfur-doped carbon dots; and it synthesizes Cu2O@NS-CDs composite materials by adding NS-CDs during the synthesis of Cu2O using CuSO4·5H2O. This composite material has the advantages of enhanced electrochemical performance, excellent stability and reproducibility, and low cost.

[0201] An electrochemical immunosensor based on Cu2O@NS-CDs composite material can be used for the quantitative analysis of tumor markers (e.g., AFP). NS-CDs have advantages such as good biocompatibility and large specific surface area, are rich in functional groups (especially unreduced -COOH), can form a large number of active sites, and have good antibody (e.g., AFP antibody) anchoring ability. Cu2O has good conductivity and electrocatalytic properties. The composite with NS-CDs achieves synergistic and electronic effects. Cu2O@NS-CDs can promote electron transfer on the electrode surface, thereby capturing a large amount of antigen and generating electrochemical signals.

[0202] The antigen (e.g., AFP) binds specifically to the antibody (e.g., AFP antibody), and non-conductive proteins hinder electron transfer at the electrode surface. The DPV peak current gradually decreases with increasing antigen (e.g., AFP) concentration. A good linear relationship exists between the peak current and the logarithm (logc) of the antigen (e.g., AFP) concentration, allowing for quantitative analysis of the antigen (e.g., AFP) using a linear equation.

[0203] This electrochemical immunosensor exhibits an ultra-low detection limit (21.4 fg / mL) in the range of 0.001–100 ng / mL. Compared to other methods for detecting tumor markers (e.g., AFP), the method described in this application offers advantages such as simple operation, rapid response, and low cost, and has broad application potential.

[0204] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., 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 preparation method of a copper(I) oxide composite material modified by nitrogen-sulfur doped carbon dots, characterized in that, The preparation method comprises: The nitrogen-sulfur doped carbon dots are introduced in the process of synthesizing cuprous oxide to obtain the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material; wherein the cuprous oxide is synthesized by using CuSO4·5H2O.

2. The production method according to claim 1, characterized by, The preparation method comprises the following steps: S1) mixing glutathione and p-phenylenediamine in a solvent and performing a hydrothermal reaction to obtain nitrogen-sulfur doped carbon dots; S2) mixing CuSO4·5H2O, the nitrogen-sulfur doped carbon dots, water and sodium hydroxide and performing heating to obtain a first solid-liquid mixture; S3) mixing the first solid-liquid mixture with a reducing agent to perform a reduction reaction to obtain a second solid-liquid mixture; S4) sequentially performing centrifugation, washing and drying on the second solid-liquid mixture to obtain the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material; The solvent is selected from ethanol and water.

3. The preparation method according to claim 2, characterized in that, In the S1), the molar volume ratio of the glutathione, the p-phenylenediamine, the ethanol and the water is (0.05-0.10):(0.05-0.2):(5-10):(5-10); Preferably, the time of the hydrothermal reaction is 2-5h; the temperature of the hydrothermal reaction is 120-180℃; Preferably, after the hydrothermal reaction, before the nitrogen-sulfur doped carbon dots are obtained, the S1) further comprises the steps of performing first cooling and first drying on the product after the hydrothermal reaction; More preferably, the temperature of the first cooling is 20℃-25℃; the time of the first cooling is 2-5h; More preferably, the temperature of the first drying is 50-60℃; the time of the first drying is 24-28h.

4. The preparation method according to claim 2, characterized in that, The reducing agent is selected from a glucose solution; the mass-volume ratio of CuSO4·5H2O, the nitrogen-sulfur doped carbon dots, water, the sodium hydroxide and the glucose solution is (300-500):(0.5-1.5):(20-40):(5-10):(5-10); The concentration of the sodium hydroxide is 1-5 M; the concentration of the glucose solution is 0.1-0.5 M; The amount of substance of glucose in the glucose solution is greater than the amount of substance of Cu in the CuSO4·5H2O. 2+ The amount of substance of glucose in the glucose solution is greater than the amount of substance of Cu in the CuSO4·5H2O. Preferably, the temperature of the first solid-liquid mixture is 60℃-80℃.

5. The preparation method according to claim 4, characterized in that, The time of the reduction reaction is 2-5h; the temperature of the reduction reaction is 60℃-80℃; Preferably, in the S4), the washing is performed by using ethanol and / or water; More preferably, the temperature of the drying is 65℃-80℃; the time of the drying is 24h-28h.

6. A nitrogen-sulfur doped carbon dot modified cuprous oxide composite material prepared by using the preparation method in any one of claims 1-5; Preferably, the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material comprises a cuprous oxide matrix and the nitrogen-sulfur doped carbon dots doped with the cuprous oxide matrix; the surface of the cuprous oxide matrix is uniformly distributed with the nitrogen-sulfur doped carbon dots; Preferably, the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material has a dodecahedron structure; Preferably, the average diameter of the nitrogen-sulfur doped carbon dot modified cuprous oxide composite material is 1.21µm±0.21µm; Preferably, the C element, the N element, the O element, the S element and the Cu element in the nitrogen-sulfur doped carbon dot modified cuprous oxide composite are arranged in a uniform dispersion manner on the surface; More preferably, the mass ratio of the Cu element, the O element, the S element, the C element and the N element in the nitrogen-sulfur doped carbon dot modified cuprous oxide composite is (60-73):(10-25):(2-6):(3-8):(2-5).

7. A modified electrode, characterized by, The modified electrode comprises a base electrode and a composite material modified on the surface of the base electrode, and the composite material is selected from the nitrogen-sulfur doped carbon dot modified cuprous oxide composite prepared by the preparation method in any one of claims 1-5 or the nitrogen-sulfur doped carbon dot modified cuprous oxide composite in claim 6.

8. An electrochemical immunosensor, characterized by, The electrode of the electrochemical immunosensor comprises the modified electrode in claim 7.

9. A method of detecting alpha-fetoprotein, characterized by, The method comprises: detecting alpha fetoprotein by using the electrochemical immunosensor in claim 8.

10. Use of the nitrogen-sulfur doped carbon dot modified cuprous oxide composite prepared by the preparation method in any one of claims 1-5 or the nitrogen-sulfur doped carbon dot modified cuprous oxide composite in claim 6 or the modified electrode in claim 7 or the electrochemical immunosensor in claim 8 or the method in claim 9 in detection of tumor markers. Preferably, the tumor markers comprise any one of the following: alpha fetoprotein, carcinoembryonic antigen or squamous cell carcinoma antigen.

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