Kit for electrochemical immunodetection of human epididymal protein 4

Abstract

The invention belongs to the technical field of electrochemical detection, and particularly relates to a kit for electrochemical immunodetection of human epididymal protein 4. The kit for electrochemical immunodetection of the human epididymal protein 4 comprises a modified electrode, a solution for capturing an antibody Ab2 bioconjugate and an H2O2 solution, the modified electrode comprises a substrate electrode and a capture antibody Ab1 bioconjugate arranged on the surface of the substrate electrode. The electrochemical immunodetection kit provided by the invention is used for detecting HE4, has the advantages of high sensitivity, quick response, easiness in miniaturization and low cost, and effectively solves the problems of insufficient sensitivity, low specificity, poor stability and the like in the prior art; and a novel and efficient detection method is provided for early diagnosis of ovarian cancer.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection technology, specifically relating to a kit for electrochemical immunoassay of human epididymal protein 4. Background Technology

[0002] Human epididymal protein 4 (HE4) has become an important clinical biomarker for epithelial ovarian cancer, one of the deadliest gynecological malignancies worldwide. Compared to carbohydrate antigen 125 (CA125), HE4 exhibits higher sensitivity and specificity in the early stages of ovarian cancer and has been approved by the U.S. Food and Drug Administration (FDA) for use in combination with CA125 for risk assessment and disease recurrence monitoring. Increasing clinical evidence suggests that HE4 levels are closely related to tumor stage, treatment response, and overall survival, and its reliable quantitative detection provides a promising approach for early diagnosis and prognostic assessment of ovarian cancer patients.

[0003] Despite the clinical significance of HE4, currently used detection methods—such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, and radioimmunoassay—still have many limitations. While ELISA is widely used, it has a long testing cycle, is complex to operate, and exhibits significant batch-to-batch variability. Chemiluminescent immunoassay, although highly automated, relies heavily on sophisticated instruments and is costly, limiting its application in resource-constrained environments. Radioimmunoassay, due to the use of radioactive reagents, raises safety and waste disposal concerns. Furthermore, these methods often fall short in detecting extremely low concentrations of HE4 in the serum of patients with early-stage ovarian cancer and are not suitable for rapid, point-of-care testing.

[0004] In summary, there is currently a lack of a sensitive, rapid, and cost-effective detection platform to achieve accurate quantification of HE4 in complex clinical samples. Summary of the Invention

[0005] The purpose of this invention is to provide a kit for the electrochemical immunoassay of human epididymal protein 4 (HE4). The electrochemical immunoassay kit provided by this invention is for the detection of HE4 and has the advantages of high sensitivity, rapid response, easy miniaturization and low cost. It effectively solves the problems of insufficient sensitivity, low specificity and poor stability of the prior art, and provides a novel and efficient detection method for the early diagnosis of ovarian cancer.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a kit for electrochemical immunoassay of human epididymal protein 4, comprising a modified electrode, a solution of a capture antibody Ab2 bioconjugate, and an H2O2 solution; the modified electrode comprises a base electrode and a capture antibody Ab1 bioconjugate disposed on the surface of the base electrode, the capture antibody Ab1 bioconjugate being a bioconjugate formed by capture antibody Ab1 and Au / PANI nanoparticles, the Au / PANI nanoparticles comprising polyaniline and gold nanoparticles loaded on the surface of the polyaniline; the capture antibody Ab2 bioconjugate is a bioconjugate formed by capture antibody Ab2 and PCN-222-Fe@PtCu nanoparticles, the PCN-222-Fe@PtCu nanoparticles comprising a PCN-222-Fe metal-organic framework and PtCu nanodendritic particles loaded on the PCN-222-Fe metal-organic framework; The method for preparing the modified electrode includes the following steps: The base electrode was modified with a solution of the capture antibody Ab1 bioconjugate to obtain the modified electrode. The concentration of the capture antibody Ab1 bioconjugate in the solution was 1.5~2.5 mg / mL; the concentration of the capture antibody Ab2 bioconjugate in the solution was 2~5 mg / mL; and the concentration of H2O2 in the H2O2 solution was 7.5~12.5 mmol / L.

[0007] Preferably, the modified electrode is replaced with a base electrode and a solution of the capture antibody Ab1 bioconjugate; the concentration of the capture antibody Ab1 bioconjugate in the solution is 1.5~2.5 mg / mL.

[0008] Preferably, the concentration of the capture antibody Ab1 bioconjugate in the solution is 2 mg / mL.

[0009] Preferably, the Au / PANI nanoparticles have a particle size of 200~300nm.

[0010] Preferably, the method for preparing the capture antibody Ab1 bioconjugate includes the following steps: A solution of the capture antibody Ab1 and a solution of Au / PANI nanoparticles were mixed and incubated to obtain the bioconjugate of the capture antibody Ab1. The concentration of the capture antibody Ab1 in the capture antibody Ab1 solution was 0.05~0.1 ng / mL, the concentration of Au / PANI nanoparticles in the Au / PANI nanoparticle solution was 1~2 mg / mL, and the volume ratio of the Au / PANI nanoparticle solution to the capture antibody Ab1 solution was 100:1. The incubation time was 2~3 h.

[0011] Preferably, the method for preparing the capture antibody Ab2 bioconjugate includes the following steps: A solution of PCN-222-Fe@PtCu nanoparticles and a solution of the capture antibody Ab2 were mixed and incubated to obtain the bioconjugate of the capture antibody Ab2.

[0012] Preferably, the concentration of PCN-222-Fe@PtCu nanoparticles in the solution is 1~5 mg / mL, the concentration of capture antibody Ab2 in the solution is 1~10 μg / mL, and the volume ratio of the PCN-222-Fe@PtCu nanoparticle solution to the capture antibody Ab2 solution is 1:1; the incubation time is 8~12 h, and the incubation temperature is 1~4℃.

[0013] Preferably, the PCN-222-Fe@PtCu nanoparticles have a particle size of 200~300 nm; the preparation method of the PCN-222-Fe@PtCu nanoparticles includes the following steps: K2PtCl4, CuCl2, cationic surfactant and water are mixed to obtain a mixture; the mixture is then reacted with an aqueous ascorbic acid solution to obtain the PtCu nanodendritic particles. PCN-222-Fe nanoparticles were obtained by mixing tetracarboxyphenylporphyrin iron, zirconium oxychloride octahydrate (ZrOCl2·8H2O), benzoic acid, and an organic solvent. The solutions of PtCu nanodendritic particles and PCN-222-Fe nanoparticles were mixed to obtain PCN-222-Fe@PtCu nanoparticles. The concentration of PtCu nanodendritic particles in the PtCu nanodendritic particle solution was 0.7~0.75 mg / mL, and the concentration of PCN-222-Fe nanoparticles in the PCN-222-Fe nanoparticle solution was 1~3 mg / mL. The volume ratio of the PtCu nanodendritic particle solution to the PCN-222-Fe nanoparticle solution was 3:7.

[0014] Preferably, the concentration of H2O2 in the H2O2 solution is 10 mmol / L.

[0015] Preferably, the substrate electrode is a glassy carbon electrode, and the glassy carbon electrode is a polished glassy carbon electrode.

[0016] This invention provides a kit for electrochemical immunoassay of human epididymal protein 4, comprising a modified electrode, a solution of a capture antibody Ab2 bioconjugate, and an H2O2 solution; the modified electrode includes a base electrode and a capture antibody Ab1 bioconjugate disposed on the surface of the base electrode, wherein the capture antibody Ab1 bioconjugate is a bioconjugate formed by the capture antibody Ab1 and Au / PANI nanoparticles, and the Au / PANI nanoparticles include polyaniline and gold nanoparticles loaded on the surface of the polyaniline; the capture antibody Ab2 bioconjugate is a bioconjugate formed by the capture antibody Ab2 and PCN-222-Fe@PtCu nanoparticles, wherein PCN-222-Fe@PtCu nanoparticles are... The tCu nanoparticles comprise a PCN-222-Fe metal-organic framework and PtCu nanodendritic particles loaded on the PCN-222-Fe metal-organic framework. The preparation method of the modified electrode includes the following steps: modifying the substrate electrode with a solution of a capture antibody Ab1 bioconjugate to obtain the modified electrode. The concentration of the capture antibody Ab1 bioconjugate in the solution is 1.5–2.5 mg / mL; the concentration of the capture antibody Ab2 bioconjugate in the solution is 2–5 mg / mL; and the concentration of H2O2 in the H2O2 solution is 7.5–12.5 mmol / L. This invention, through reasonable electrode modification and signal amplification strategies, enables ultra-trace detection of disease biomarkers, with a detection limit reaching the fg / mL level. Furthermore, the kit provided by this invention requires only a very small amount of sample, can adapt to complex biological matrices such as serum, and is easily integrated with portable devices, demonstrating significant potential for point-of-care testing (POCT). This invention addresses electrode dilution and signal amplification: Au / PANI nanoparticles exhibit excellent conductivity and a high specific surface area. Their abundant gold nanoparticles connect to antibodies via Au-NH2 bonds, thereby improving binding efficiency and signal stability. Simultaneously, the bimetallic nanomaterial PtCu nanodendritic particles (NDs) possess peroxidase-like activity. The large inner surface of PCN-222-Fe provides abundant anchoring sites for PtCu NPs, and its regular nanopores confine the PtCu NPs within the pores, preventing migration and aggregation during the reaction, thus achieving strong electrochemical signal amplification without the need for natural enzymes. In summary, this invention uses the capture antibody Ab1 bioconjugate to immobilize the capture antibody and utilizes the capture antibody Ab2 bioconjugate to achieve signal amplification. The kit provided by this invention not only improves detection sensitivity and specificity but also shows promising application prospects, potentially becoming an effective platform for the early diagnosis of ovarian cancer.The results of the examples show that the kit provided by this invention achieves a limit of detection of 35 fg / mL for the ovarian cancer biomarker HE4, with a broad linear range (0.0005~10 ng / mL). The kit specifically responds to HE4 only in the presence of multiple interfering proteins, indicating its outstanding specificity. Repeatability tests show a relative standard deviation of less than 5%, demonstrating the stability and reliability of the kit. After storage at 4 °C for 28 days, it retains more than 90% of its initial signal intensity, indicating excellent long-term stability. In summary, this invention, through the synergistic design of gold / polystyrene nanospheres and PCN-222-Fe@PtCu nanozymes, effectively solves the problems of insufficient sensitivity, low specificity, and poor stability in existing technologies, providing a novel and efficient detection method for the early diagnosis of ovarian cancer. Attached Figure Description

[0017] Figure 1 Morphology and elemental distribution characterization of Au / PANI nanoparticles, PtCu nanodendritic particles, PCN-222-Fe nanoparticles and PCN-222-Fe@PtCu nanoparticles prepared for the examples; Figure 2 A comparison chart showing the optimization results of experimental conditions for electrochemical immunosensors; Figure 3 Analytical performance graph of the electrochemical immunosensor prepared for the example; Figure 4 The performance evaluation diagram of the electrochemical immunosensor prepared for the example is shown. Detailed Implementation

[0018] This invention provides a kit for electrochemical immunoassay of human epididymal protein 4, comprising a modified electrode, a solution of a capture antibody Ab2 bioconjugate, and an H2O2 solution; the modified electrode comprises a base electrode and a capture antibody Ab1 bioconjugate disposed on the surface of the base electrode, the capture antibody Ab1 bioconjugate being a bioconjugate formed by capture antibody Ab1 and Au / PANI nanoparticles, the Au / PANI nanoparticles comprising polyaniline and gold nanoparticles loaded on the surface of the polyaniline; the capture antibody Ab2 bioconjugate is a bioconjugate formed by capture antibody Ab2 and PCN-222-Fe@PtCu nanoparticles, the PCN-222-Fe@PtCu nanoparticles comprising a PCN-222-Fe metal-organic framework and PtCu nanodendritic particles loaded on the PCN-222-Fe metal-organic framework; The method for preparing the modified electrode includes the following steps: The base electrode was modified with a solution of the capture antibody Ab1 bioconjugate to obtain the modified electrode. The concentration of the capture antibody Ab1 bioconjugate in the solution was 1.5~2.5 mg / mL; the concentration of the capture antibody Ab2 bioconjugate in the solution was 2~5 mg / mL; and the concentration of H2O2 in the H2O2 solution was 7.5~12.5 mmol / L.

[0019] In this invention, unless otherwise specified, all raw materials / components used in preparation are commercially available products well-known to those skilled in the art. Unless otherwise specified, all percentages in this invention refer to mass percentages. Unless otherwise specified, all solutions in this invention are aqueous solutions of water or PBS solutions; for example, an Au / PANI solution is an aqueous solution of Au / PANI. Room temperature in this invention generally refers to a temperature between 15°C and 25°C, typically defined as 25°C. In this invention, the capture antibody Ab1 is a mouse anti-human antibody (Ab1). The capture antibody Ab2 is a mouse anti-human antibody (Ab2). Capture antibodies Ab1 and Ab2 were purchased from Sangon Biotech (Shanghai) Co., Ltd. The PBS solution in this invention has a molar concentration of 0.01 mol / L and a pH of 7.4.

[0020] This invention allows the modified electrode to be replaced by a base electrode and a solution of a bioconjugate of the capture antibody Ab1. Specifically, this invention provides an electrochemical immunoassay kit for detecting human epididymal protein 4, comprising a base electrode, a solution of a bioconjugate of the capture antibody Ab1, a solution of a bioconjugate of Ab2, and an H2O2 solution; the bioconjugate of the capture antibody Ab1 is a bioconjugate formed by the capture antibody Ab1 and an Au / PANI solution; the bioconjugate of the capture antibody Ab2 is a bioconjugate formed by the capture antibody Ab2 and PCN-222-Fe@PtCu nanoparticles.

[0021] The electrochemical immunoassay kit for detecting human epididymal protein 4 provided by this invention may include a substrate electrode. The substrate electrode is preferably a glassy carbon electrode (GCE). The glassy carbon electrode is preferably a polished glassy carbon electrode. The polishing treatment preferably includes polishing the glassy carbon electrode with a polishing agent. The polishing agent is preferably Al2O3 polishing powder, and the particle size of the Al2O3 polishing powder is preferably 0.03~0.05 μm. After polishing, the invention further includes washing the polishing product with water to obtain the polished glassy carbon electrode. The water washing is preferably done with deionized water.

[0022] The electrochemical immunoassay kit for detecting human epididymal protein 4 provided by this invention may include a solution of a capture antibody Ab1 bioconjugate (Au / PANI@Ab1). In this invention, the capture antibody Ab1 bioconjugate solution is a PBS solution of the capture antibody Ab1 bioconjugate. The concentration of the capture antibody Ab1 bioconjugate in the solution is preferably 1.5~2.5 mg / mL, and in the examples it can be 1.5, 2, or 2.5 mg / mL.

[0023] In this invention, the method for preparing the capture antibody Ab1 bioconjugate includes the following steps: A solution of the capture antibody Ab1 and a solution of Au / PANI nanoparticles are mixed and incubated to obtain the bioconjugate of the capture antibody Ab1. In this invention, the concentration of the capture antibody Ab1 in the solution is 0.05~0.1 ng / mL, and in the examples, it can be 0.1 ng / mL. In this invention, the particle size of the Au / PANI nanoparticles is 200~300 nm. The concentration of Au / PANI nanoparticles in the solution is preferably 1~2 mg / mL, and in the examples, it can be 2 mg / mL. The preparation method of the Au / PANI nanoparticles preferably includes: sequentially mixing and aging aniline, HAuCl4 solution, and sodium dodecyl sulfate solution to obtain the Au / PANI nanoparticles. In this invention, the molar concentration of HAuCl4 in the HAuCl4 solution is preferably 2 mM. The molar concentration of the sodium dodecyl sulfate solution is preferably 40 mM. The volume ratio of aniline to the HAuCl4 solution is preferably 1:1. The volume ratio of aniline to sodium dodecyl sulfate solution is preferably 1:1. Mixing is carried out under stirring. The mixing temperature is preferably room temperature. The mixing time is preferably 20-30 minutes. Aging is natural aging, and the natural aging time is preferably 7-8 hours. After aging, the resulting reaction solution undergoes solid-liquid separation. The solid product from the solid-liquid separation is washed sequentially with water and anhydrous ethanol; finally, it is freeze-dried to obtain Au / PANI nanoparticles.

[0024] In this invention, the volume ratio of the Au / PANI nanoparticle solution to the capture antibody Ab1 solution is preferably 100:1. The incubation temperature is preferably room temperature, the incubation is preferably carried out under stirring conditions, and the incubation time is preferably 2-3 hours. The capture antibody Ab1 bioconjugate is obtained.

[0025] The electrochemical immunoassay kit for detecting human epididymal protein 4 provided by this invention may include a modified electrode. The modified electrode includes a base electrode and a capture antibody Ab1 bioconjugate disposed on the surface of the base electrode.

[0026] In this invention, the preparation method of the modified electrode includes the following steps: modifying the base electrode with a solution of a capture antibody Ab1 bioconjugate to obtain the modified electrode. In this invention, the concentration of the capture antibody Ab1 bioconjugate in the solution is 1.5~2.5 mg / mL, preferably 2 mg / mL. The modification preferably includes: coating the surface of the base electrode with the capture antibody Ab1 bioconjugate solution, followed by drying and blocking treatments. The coating is preferably drop-coating. The drying is preferably natural drying at room temperature. The blocking treatment is preferably performed using a BSA solution, wherein the mass content of BSA in the BSA solution is preferably 1 wt%. This invention preferably uses a BSA solution to block non-specific sites.

[0027] The electrochemical immunoassay kit for detecting human epididymal protein 4 provided by this invention includes a solution of a capture antibody Ab2 bioconjugate (PCN-222-Fe@PtCu@Ab2). In this invention, the capture antibody Ab2 bioconjugate solution is a PBS solution of the capture antibody Ab2 bioconjugate. The concentration of the capture antibody Ab2 bioconjugate in the solution is 2-5 mg / mL, and in the examples it can be 2 mg / mL, 3 mg / mL, 4 mg / mL or 5 mg / mL.

[0028] In this invention, the method for preparing the capture antibody Ab2 bioconjugate preferably includes the following steps: A solution of PCN-222-Fe@PtCu nanoparticles and a solution of the capture antibody Ab2 were mixed and incubated to obtain the bioconjugate of the capture antibody Ab2.

[0029] In this invention, the particle size of the PCN-222-Fe@PtCu nanoparticles is preferably 200~300nm, and in the embodiments it can be 250nm.

[0030] In this invention, the preparation method of the PCN-222-Fe@PtCu nanoparticles preferably includes the following steps: K2PtCl4, CuCl2, cationic surfactant and water were mixed to obtain a mixture. The mixture was reacted with an aqueous ascorbic acid solution to obtain the PtCu nanodendritic particles; PCN-222-Fe nanoparticles were obtained by mixing tetracarboxyphenylporphyrin iron (Fe-TCPP), ZrOCl2·8H2O, benzoic acid (BA), and an organic solvent. The solution of PtCu nanodendritic particles and the solution of PCN-222-Fe nanoparticles are mixed to obtain PCN-222-Fe@PtCu nanoparticles.

[0031] This invention involves mixing K₂PtCl₄, CuCl₂, a cationic surfactant, and water to obtain a mixture. In this invention, the cationic surfactant can be hexadecyltrimethylammonium chloride (CTAC). The water is preferably deionized water. The preferred mass ratio of K₂PtCl₄, CuCl₂, and the cationic surfactant is (38~38.5):(12~12.5):(630~650), and in the examples, it can be 38.19:12.37:640. The preferred volume ratio of K₂PtCl₄ to water is (38~38.5) mg:40 mL, and in the examples, it can be 38.19 mg:40 mL. The mixing is preferably carried out under ultrasonic conditions, and the ultrasonic mixing time is preferably 10~15 min.

[0032] After obtaining the mixture, the present invention reacts the mixture with an ascorbic acid aqueous solution to obtain the PtCu nanodendritic particles. In the present invention, the ascorbic acid concentration in the ascorbic acid aqueous solution is preferably 17-18 mg / mL, and in the examples, it can be 17.6 mg / mL. The mass ratio of K2PtCl4 to the volume ratio of the ascorbic acid aqueous solution is preferably (38-38.5) mg:5 mL, and in the examples, it can be 38.19 mg:5 mL. The reaction temperature is preferably 80-90℃. The reaction is carried out under stirring. The reaction time is preferably 4-5 min. After the reaction is completed, the temperature is lowered to room temperature, and then solid-liquid separation, water washing, and drying are performed sequentially to obtain the PtCu nanodendritic particles. The solid-liquid separation is preferably centrifugal separation. Deionized water is used for water washing, and the number of water washings is preferably 3 times. The drying is preferably vacuum drying, and the vacuum drying temperature is preferably 40-45℃.

[0033] This invention involves mixing tetracarboxyphenylporphyrin iron (Fe-TCPP), ZrOCl2·8H2O, and benzoic acid (BA) with an organic solvent to obtain PCN-222-Fe nanoparticles. In this invention, the preferred mass ratio of Fe-TCPP, ZrOCl2·8H2O, and BA is 1:3:33. The organic solvent can be N,N-dimethylformamide (DMF). The preferred mixing temperature is 85-90°C; the preferred mixing time is 5-6 h. The reaction occurs during the mixing process. After mixing, this invention preferably involves sequentially performing solid-liquid separation and washing of the obtained reaction solution to obtain the PCN-222-Fe nanoparticles. The solid-liquid separation is preferably centrifugation, with a preferred centrifugation speed of 10000-12000 rpm and a preferred centrifugation time of 15-20 min. The preferred washing reagent is DMF.

[0034] After obtaining the PtCu nanodendritic particles and PCN-222-Fe nanoparticles, the present invention mixes the solutions of the PtCu nanodendritic particles and PCN-222-Fe nanoparticles to obtain the PCN-222-Fe@PtCu nanoparticles. In the present invention, the concentration of PtCu nanodendritic particles in the PtCu nanodendritic particle solution is 0.7~0.75 mg / mL, the concentration of PCN-222-Fe nanoparticles in the PCN-222-Fe nanoparticle solution is 1~3 mg / mL, and the volume ratio of the PtCu nanodendritic particle solution to the PCN-222-Fe nanoparticle solution is 3:7.

[0035] In this invention, the concentration of PCN-222-Fe@PtCu nanoparticles in the solution is 1-5 mg / mL, and in the examples, it can be 1, 2, 3, 4, or 5 mg / mL. The solution of the capturing antibody Ab2 is a PBS solution of the capturing antibody Ab2. The concentration of the capturing antibody Ab2 in the solution is preferably 1-10 μg / mL. The volume ratio of the PCN-222-Fe@PtCu nanoparticle solution to the capturing antibody Ab2 solution is preferably 1:1. The incubation temperature is preferably 2-4°C. The incubation time is preferably 8-12 h. After the incubation, the present invention preferably separates the solid and liquid of the reaction solution obtained from the incubation, and disperses the obtained precipitate in PBS solution to obtain a solution of the bioconjugate of the capturing antibody Ab2. The solid-liquid separation is preferably centrifugation, and the centrifugation speed is preferably 6000-7000 r / min. The centrifugation time is preferably 3-5 min. After centrifugation, the supernatant was discarded to remove unbound Ab2, yielding a precipitate. The PBS solution had a molar concentration of 0.01 mol / L and a pH of 7.4.

[0036] The electrochemical immunoassay kit provided by this invention includes an H2O2 solution. The H2O2 solution is preferably a PBS solution of H2O2. The PBS solution has a molar concentration of 0.01 mol / L and a pH of 7.4. The concentration of H2O2 in the H2O2 solution is preferably 10 mmol / L.

[0037] The preferred method of using the electrochemical immunoassay kit for detecting human epididymal protein 4 provided by the present invention includes: The substrate electrode is modified with a solution of the bioconjugate of the antibody Ab1 to obtain the modified electrode; the modified electrode is incubated with HE4 solution (first incubation) to obtain the loaded electrode; the loaded electrode is incubated with a solution of the bioconjugate of the antibody Ab2 to obtain the electrochemical immunosensor; the electrochemical immunosensor is tested in H2O2 solution using differential pulse voltammetry. The first incubation temperature is preferably room temperature, and the time is preferably 10-80 min, which can be 10, 20, 40, 60, or 80 min in the examples. The second incubation temperature is preferably room temperature, and the time is preferably 10-80 min, which can be 10, 20, 40, 60, or 80 min in the examples. The conditions for the differential pulse voltammetry preferably include: a potential range of 0 to -0.6 V, a pulse amplitude of 0.07 V, a pulse width of 0.05 s, and a sampling interval of 0.0167 s.

[0038] The electrochemical immunoassay kit for detecting human epididymal protein 4 provided by this invention is a sensitive, rapid, and cost-effective novel detection platform to achieve accurate quantification of HE4 in complex clinical samples.

[0039] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1 Preparation of PtCu nanodendritic particles 38.19 mg K₂PtCl₄, 12.37 mg CuCl₂, and 640 mg CTAC were added to 40 mL of deionized water to form a flocculent mixture, which was then sonicated for 10 min. During sonication, 5 mL of ascorbic acid aqueous solution (17.6 mg / mL) was added. -1 The mixture was vigorously stirred in an oil bath at 90 °C for 5 min and then cooled to room temperature. During the reaction, the solution color changed from white to transparent brown within 5 min, and then to opaque black. The product was separated by centrifugation, washed three times with deionized water, and dried under vacuum at 40 °C overnight to obtain PtCu nanodendritic particles.

[0041] Preparation of PCN-222-Fe@PtCu nanoparticles 100 mg Fe-TCPP, 300 mg ZrOCl2·8H2O, and 3.3 g benzoic acid (BA) were dissolved in 100 mL N,N-dimethylformamide (DMF) using a 250 mL flask. The solution was transferred to an oil bath and heated at 90 °C with gentle stirring for 5 h. After centrifugation at 10,000 rpm for 20 min and washing three times with DMF, PCN-222-Fe nanoparticles were obtained.

[0042] 3 mL of the synthesized PtCu nanodendritic particles (0.75 mg / mL) solution was added to 7 mL of PCN-222-Fe nanoparticle solution (concentration 3 mg / mL), and stirred for 12 hours to obtain PCN-222-Fe@PtCu nanoparticles. The product was separated by centrifugation, washed three times with deionized water, and dried under vacuum at 40°C overnight.

[0043] Synthesis of PCN-222-Fe@PtCu@Ab2 (or PCN-222-Fe@PtCu-Ab2) 0.5 mL of the synthesized homogeneous aqueous solution of PCN-222-Fe@PtCu nanoparticles (concentration of PCN-222-Fe@PtCu nanoparticles: 3 mg / mL) was mixed with 0.5 mL of Ab2 solution (10 μg / mL), and incubated overnight (8–12 h) at 4 °C with stirring to achieve effective cross-linking of the antibody and nanomaterials, forming stable Pt-NH2 bonds. The mixture was centrifuged at 6000 r / min for 3 min, and the supernatant was discarded to remove unbound Ab2. The precipitate was dispersed in 1 mL of PBS (0.01 M, pH 7.4) and stored at 4 °C for subsequent electrochemical detection.

[0044] Preparation of Au / PANI@Ab1 bioconjugate Au / PANI nanomaterials were synthesized using a one-step method. The preparation steps were as follows: Aniline (10 mL), HAuCl4 aqueous solution (2 mM, 10 mL), and sodium dodecyl sulfate aqueous solution (SDs, 40 mM, 10 mL) were vigorously mixed for 30 minutes by shaking. The reaction mixture was then allowed to age naturally at room temperature for 8 hours. The product was separated and purified, washed three times with deionized water and anhydrous ethanol, and then freeze-dried for 12 hours to obtain Au / PANI nanoparticles. 1.5 mL of a solution containing 2 mg / mL Au / PANI nanoparticles was stirred until homogeneous, and then 15 μL of an Ab1 solution (0.1 ng·mL⁻¹) was added. -1The mixture was stirred and incubated for another 2 hours to promote effective coupling of Ab1 to the Au / PANI surface, yielding Au / PANI@Ab1. After the reaction was complete, Au / PANI@Ab1 was stored at 4°C for subsequent experiments.

[0045] Construction of a sandwich-type electrochemical immunosensor First, the glassy carbon electrode (GCE) was thoroughly polished with 0.05 μm Al2O3 polishing powder, and then rinsed with deionized water; subsequently, Au / PANI@Ab1 (2 mg·mL) was added dropwise. -1 GCE was modified with PBS solution and allowed to air dry at room temperature. After washing, 1 wt% BSA (10 μL) was added to block non-specific sites. The electrode was then incubated with different concentrations of HE4 solution (10 μL) for 1 h to ensure specific binding of HE4 to Au / PANI@Ab1; subsequently, the modified electrode was incubated with Ab2 bioconjugate PBS solution (PCN-222-Fe@PtCu@Ab2, 10 μL, 3 mg·mL⁻¹). -1 Incubate at room temperature for 60 min. Differential pulse voltammetry (DPV) was performed in 5 mL PBS containing 10 mM H2O2; the potential range was 0 to -0.6 V, the pulse amplitude was 0.07 V, the pulse width was 0.05 s, and the sampling interval was 0.0167 s.

[0046] Example 2: Morphology and structural characterization of nanomaterials First, the morphology and structure of Au / PANI nanoparticles, PtCu nanodendritic particles, and PCN-222-Fe@PtCu nanoparticles were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), and high-angle annular dark-field scanning transmission electron microscopy (HAADF-TEM). Figure 1 As shown in Figure A, SEM results revealed that the Au / PANI nanoparticles exhibit a regular spherical structure with good dispersibility, and the particle size is mainly concentrated between 200 and 300 nm. The spherical structure of these particles is rich in gold nanoparticles on its surface, providing numerous active sites for efficient antibody immobilization and also exhibiting good electrical conductivity, laying the foundation for the subsequent specific recognition of the HE4 antigen.

[0047] Further TEM and HRTEM images showed that the PtCu nanodendritic particles exhibited a typical dendritic morphology with an average particle size of approximately 20 nm. Figure 1 B and Figure 1 (See illustration b). This branching structure significantly increases the specific surface area and the number of exposed active sites, which is beneficial for catalytic reactions. HAADF-STEM images further illustrate the clear branching morphology of the PtCu nanodendritic particles. Figure 1 The presence of C in the figure demonstrates the successful synthesis of the PtCu nanocomposite.

[0048] Furthermore, HAADF-TEM combined energy dispersive spectroscopy (EDS) elemental distribution analysis showed that Pt and Cu elements were uniformly distributed in the overall structure of the synthesized PtCu nanodendritic particles. Figure 1 (D, E, F). The uniform alloying structure means that Pt provides high catalytic activity, while Cu further enhances catalytic performance and reduces costs through synergistic effects. This uniform elemental distribution is expected to enhance its peroxidase-like activity, thereby effectively amplifying the electrochemical response signal to H2O2.

[0049] Moreover, such as Figure 1 As shown in G, the obtained PCN-222-Fe nanoparticles (PCN-222-Fe NP) exhibit a spindle-shaped shape, with a length of 200 ± 20 nm. TEM results show that PCN-222-Fe is loaded with a large number of PtCu nanodendritic particles (…). Figure 1 (H in the sample). Subsequently, XPS analysis was used to analyze the elemental composition of the PCN-222-Fe@PtCu nanocomposite. Figure 1 The XPS spectrum of the nanocomposite material shown in Figure I contains peaks for C 1s, Cu 2p, Fe 2p, N 1s, Pt 4f, O 1s, Zr 3d, and Cl 2p. This further confirms the successful synthesis of PCN-222-Fe@PtCu nanoparticles.

[0050] In summary, Figure 1 The morphology and elemental composition of Au / PANI nanoparticles and PtCu nanodendritic particles, as well as the morphological distribution of PCN-222-Fe particles and PCN-222-Fe@PtCu nanoparticles, were characterized. Figure 1 A in the image is a TEM image, showing that the Au / PANI nanoparticles have a regular spherical structure with a particle size of approximately 200~300 nm. Figure 1 B in the image represents TEM and HRTEM images, showing that the PtCu nanodendritic particles exhibit a dendritic structure with an average particle size of approximately 20 nm. Figure 1 Illustration b in the image is a high-resolution magnified view of a specific area. Figure 1 C in the image is a HAADF-TEM image, which further visually demonstrates the clear branching structure of PtCu nanodendritic particles; Figure 1 D, E, and F in the figure represent the elemental mapping results of HAADF-TEM combined with EDS, indicating that Pt and Cu elements are uniformly distributed in the nano-dendritic structure, which verifies the successful synthesis of the PtCu alloy structure. Figure 1The G, H, and I molecules in the composite material were further verified by TEM and XPS, confirming the successful synthesis of the PCN-222-Fe@PtCu nanocomposite. Au / PANI nanoparticles, with their excellent conductivity and high specific surface area, can serve as an ideal antibody immobilization substrate, while PCN-222-Fe@PtCu nanoparticles, due to their structural and compositional advantages, can act as a highly efficient signal amplification probe. This rational combination lays a solid foundation for constructing a sandwich-type electrochemical immunosensor with ultra-high sensitivity.

[0051] Example 3: Optimization of Experimental Variables for Biosensing Strategies To achieve the best analytical performance of the proposed electrochemical immunosensor, several experimental parameters were systematically optimized. Figure 2 To optimize experimental conditions for electrochemical immunosensors. Figure 2 In the figure, A represents the effect of the concentration of Au / PANI@Ab1 (0.5~2.5 mg / mL) on the current response. The results show that the signal reaches its maximum value at 2.0 mg / mL. Figure 2 In the figure, B represents the effect of the concentration (1~5 mg / mL) of the PCN-222-Fe@PtCu-Ab2 nanoprobe on the current response, with the optimal response obtained at 3.0 mg / mL. Figure 2 C in the figure represents the effect of the incubation time (10~80 min) of captured HE4 and PCN-222-Fe@PtCu-Ab2 on the current response, with the signal reaching a plateau at 60 min. Figure 2 In the figure, D represents the effect of H2O2 concentration (2.5~12.5 mM) on the catalytic current response, and the results show that the signal is strongest at 10 mM. Error bars represent the standard deviation of three independent measurements.

[0052] First, the effect of Au / PANI@Ab1 concentration on the current response was investigated. For example... Figure 2 As shown in Figure A, the signal intensity steadily increased from 0.5 mg / mL to 2.0 mg / mL, reaching a maximum at 2.0 mg / mL. However, with further increases in concentration, the current response decreased, possibly due to electron transport being hindered by an excessively thick film. Therefore, 2.0 mg / mL was determined to be the optimal concentration for Au / PANI@Ab1. Subsequently, the concentration of the PCN-222-Fe@PtCu-Ab2 solution was optimized, as this parameter directly affects catalytic efficiency and non-specific adsorption. Figure 2As shown in Figure B, the current response of the PCN-222-Fe@PtCu-Ab2 nanoprobe significantly increased within the concentration range of 1–3 mg / mL, reaching a peak at 3.0 mg / mL. With further increases in concentration, the signal decreased, possibly due to steric hindrance and particle aggregation. Therefore, 3.0 mg / mL was selected as the optimal PCN-222-Fe@PtCu-Ab2 probe concentration. Further investigation was conducted on the incubation time after the addition of PCN-222-Fe@PtCu-Ab2. Figure 2 As shown in Figure C, the current response rapidly increased with increasing incubation time from 10 min to 60 min, reaching a plateau at 60 min; further extending the incubation time did not bring a significant increase. Therefore, 60 min is considered a suitable incubation time. Finally, the effect of H2O2 concentration on the detection signal was evaluated. As the catalytic substrate of PCN-222-Fe@PtCu nanozyme, the concentration of H2O2 is crucial to the current response. Figure 2 As shown in Figure D, the current gradually increases with increasing H2O2 concentration, reaching a maximum at 10 mM. However, when the concentration further increases to 12.5 mM, the signal decreases, possibly due to enhanced side reactions and increased background current. Therefore, 10 mM H2O2 was determined to be the optimal substrate concentration for subsequent experiments.

[0053] Example 4: Analytical performance of the immunosensor used to detect HE4 Under optimized experimental conditions, an immunosensor was used to detect different concentrations of HE4 protein. The analytical performance of the designed electrochemical immunosensor for HE4 was systematically evaluated. Figure 3 The analytical performance of the proposed electrochemical immunosensor is demonstrated. Figure 3 In this context, A represents the DPV response of the immunosensor at different HE4 concentrations (a→g: 0.0005, 0.001, 0.01, 0.1, 0.5, 1 and 10 ng / mL). Figure 3 In the figure, B represents the calibration curve of peak current versus the logarithm of HE4 concentration. Error bars represent the standard deviation of three independent measurements. For example... Figure 3 As shown in Figure A, the differential pulse voltammetry (DPV) response gradually weakens with increasing HE4 concentration (a→g: 0.0005, 0.001, 0.01, 0.1, 0.5, 1, and 10 ng / mL), exhibiting a typical concentration-dependent signal change. Within the range of 0.0005–10 ng / mL, a good linear correlation is observed between the peak current and the logarithm of the HE4 concentration, with the regression equation: I = -74.84 - 19.61 × Ig C HE4 , (R 2 = 0.987) (see Figure 3(B) The limit of detection (LOD), calculated at 3σ / slope, was 35 fg / mL, indicating that the immunosensor possesses extremely high sensitivity. These results demonstrate that the platform constructed in this invention can achieve reliable and highly sensitive detection of HE4, providing potential application value for the early diagnosis of ovarian cancer.

[0054] Example 5: Specificity, repeatability, and stability analysis First, the specificity of the constructed immune sensor was evaluated. Under the same conditions, its response to HE4 was compared with that of several potential tumor markers that interfere with tumors, including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), and carbohydrate antigen 199 (CA199). Figure 4 This is for the performance evaluation of the proposed electrochemical immunosensor. Figure 4 In the figure, A represents specificity analysis: comparing the detection results of different tumor markers (AFP, CEA, CA125, CA199) with HE4, only HE4 showed a significant signal response. Figure 4 B in the figure represents the repeatability test: five independently prepared electrodes were used for detection, and the results showed that the response was highly consistent with an RSD of <5%. Figure 4 The "C" in the figure represents the stability study: after storage at 4 °C for 1–28 days, the immunosensor retained more than 90% of its initial response after 28 days of storage. The error bars represent the standard deviation of three independent measurements.

[0055] like Figure 4 As shown in A, the current changes of these interfering proteins and the blank control are negligible, while a significant signal response is generated in the presence of HE4, demonstrating that the immunosensor has high specificity.

[0056] The repeatability of the detection method was further evaluated. Five independently prepared immunosensors were used to detect the same concentration of HE4. Figure 4 As shown in B, the current response of each measurement showed only slight differences, with a relative standard deviation (RSD) of less than 5%, indicating that the immunosensor has excellent preparation consistency and detection repeatability.

[0057] Simultaneously, the storage stability of the immunosensor was investigated. Its current response to HE4 was measured after storage at 4 °C for 1, 7, 14, 21, and 28 days, respectively. Figure 4 As shown in C, even after 28 days of storage, the current signal can still maintain more than 90% of the initial value, indicating that the immunosensor has good long-term stability.

[0058] In summary, the proposed immunosensor demonstrates satisfactory performance in terms of specificity, repeatability, and stability, and can be used for reliable detection of HE4 in complex samples, showing potential for practical application.

[0059] As demonstrated by the above embodiments, the electrochemical immunoassay kit based on Au / PANI nanospheres and PCN-222-Fe@PtCu nanocomposite constructed in this invention exhibits significant performance advantages over existing technologies. By modifying the glassy carbon electrode surface with Au / PANI nanoparticles, this invention not only enhances the conductivity of the electrode interface but also achieves efficient and stable antibody coupling through Au-NH2 bonds, thereby improving the capture efficiency of target molecules and the stability of the detection signal. Simultaneously, the PCN-222-Fe@PtCu nanoparticles exhibit excellent peroxidase-like activity, achieving significant signal amplification in the presence of H2O2, primarily attributed to the synergistic effect between PCN-222-Fe and PtCu. The synergistic effect of the above interface modification and catalytic amplification strategy enables the sensor to achieve a limit of detection of 35 fg / mL for the ovarian cancer biomarker HE4, with a broad linear range (0.0005~10 ng / mL).

[0060] Furthermore, the electrochemical immunoassay kit provided by this invention exhibits a specific response to HE4 only in the presence of multiple interfering proteins, demonstrating its outstanding specificity. Repeatability test results show that the relative standard deviation is less than 5%, indicating that its preparation process is stable and reliable. After being stored at 4 °C for 28 days, it can still maintain more than 90% of the initial signal intensity, indicating its excellent long-term stability.

[0061] In summary, the electrochemical immunoassay kit provided by this invention, through the synergistic design of Au / PANI nanoparticles and PCN-222-Fe@PtCu nanocomposite, effectively solves the problems of insufficient sensitivity, low specificity, and poor stability in existing technologies, providing a novel and efficient detection method for the early diagnosis of ovarian cancer.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A kit for electrochemical immunoassay of human epididymal protein 4, characterized by, The solution comprises a modified electrode, a capture antibody Ab2 bioconjugate solution and an H2O2 solution; the modified electrode comprises a base electrode and a capture antibody Ab1 bioconjugate arranged on the surface of the base electrode, wherein the capture antibody Ab1 bioconjugate is a bioconjugate of a capture antibody Ab1 and Au / PANI nanoparticles, and the Au / PANI nanoparticles comprise polyaniline and gold nanoparticles loaded on the surface of the polyaniline; the capture antibody Ab2 bioconjugate is a bioconjugate of a capture antibody Ab2 and PCN-222-Fe@PtCu nanoparticles, and the PCN-222-Fe@PtCu nanoparticles comprise PCN-222-Fe metal organic frameworks and PtCu nanocluster particles loaded on the PCN-222-Fe metal organic frameworks; The preparation method of the modified electrode comprises the following steps: The base electrode is modified by using a capture antibody Ab1 bioconjugate solution to obtain the modified electrode, wherein the concentration of the capture antibody Ab1 bioconjugate in the capture antibody Ab1 bioconjugate solution is 1.5-2.5 mg / mL; the concentration of the capture antibody Ab2 bioconjugate in the capture antibody Ab2 bioconjugate solution is 2-5 mg / mL; and the concentration of H2O2 in the H2O2 solution is 7.5-12.5 mmol / L.

2. The kit for electrochemical immunoassay of human epididymal protein 4 according to claim 1, characterized in that, The base electrode and the capture antibody Ab1 bioconjugate solution are used to replace the modified electrode, and the concentration of the capture antibody Ab1 bioconjugate in the capture antibody Ab1 bioconjugate solution is 1.5-2.5 mg / mL.

3. The kit for electrochemical immunoassay of human epididymal protein 4 according to claim 1, characterized in that, The concentration of the capture antibody Ab1 bioconjugate in the capture antibody Ab1 bioconjugate solution is 2 mg / mL.

4. The kit for electrochemical immunoassay of human epididymal protein 4 according to claim 1, characterized in that, The particle size of the Au / PANI nanoparticles is 200-300 nm.

5. The kit for electrochemical immunoassay of human epididymal protein 4 according to claim 1 or 4, characterized in that, The preparation method of the capture antibody Ab1 bioconjugate comprises the following steps: The capture antibody Ab1 solution and the Au / PANI nanoparticle solution are mixed and incubated to obtain the capture antibody Ab1 bioconjugate, wherein the concentration of the capture antibody Ab1 in the capture antibody Ab1 solution is 0.05-0.1 ng / mL, the concentration of the Au / PANI nanoparticles in the Au / PANI nanoparticle solution is 1-2 mg / mL, the volume ratio of the Au / PANI nanoparticle solution to the capture antibody Ab1 solution is 100:1, and the incubation time is 2-3 h.

6. The kit for electrochemical immunoassay of human epididymal protein 4 according to claim 1, characterized in that, The preparation method of the capture antibody Ab2 bioconjugate comprises the following steps: The PCN-222-Fe@PtCu nanoparticle solution and the capture antibody Ab2 solution are mixed and incubated to obtain the capture antibody Ab2 bioconjugate.

7. The kit for electrochemical immunoassay of human epididymal protein 4 according to claim 6, characterized in that, The concentration of the PCN-222-Fe@PtCu nanoparticles in the solution of the PCN-222-Fe@PtCu nanoparticles is 1-5 mg / mL, the concentration of the capture antibody Ab2 in the solution of the capture antibody Ab2 is 1-10 μg / mL, and the volume ratio of the solution of the PCN-222-Fe@PtCu nanoparticles to the solution of the capture antibody Ab2 is 1:1; the incubation time is 8-12 h, and the incubation temperature is 1-4℃.

8. The kit for electrochemical immunoassay of human epididymal protein 4 according to claim 1, characterized in that, The particle size of the PCN-222-Fe@PtCu nanoparticles is 200-300 nm; and the preparation method of the PCN-222-Fe@PtCu nanoparticles comprises the following steps: K2PtCl4, CuCl2, a cationic surfactant and water are mixed to obtain a mixture; the mixture and an ascorbic acid aqueous solution are mixed to react, so as to obtain the PtCu nanobrush particles; Tetracarboxyphenyl porphyrin iron, zirconium oxychloride octahydrate and benzoic acid and an organic solvent are mixed to obtain PCN-222-Fe nanoparticles; The solution of the PtCu nanobrush particles and the solution of the PCN-222-Fe nanoparticles are mixed to obtain the PCN-222-Fe@PtCu nanoparticles; the concentration of the PtCu nanobrush particles in the solution of the PtCu nanobrush particles is 0.7-0.75 mg / mL, the concentration of the PCN-222-Fe nanoparticles in the solution of the PCN-222-Fe nanoparticles is 1-3 mg / mL, and the volume ratio of the solution of the PtCu nanobrush particles to the solution of the PCN-222-Fe nanoparticles is 3:

7.

9. The kit for electrochemical immunoassay of human epididymal protein 4 according to claim 1 or 2, characterized in that, The concentration of H2O2 in the H2O2 solution is 10 mmol / L.

10. The kit for electrochemical immunoassay of human epididymal protein 4 according to claim 1 or 2, characterized in that, The base electrode is a glassy carbon electrode, and the glassy carbon electrode is a glassy carbon electrode after polishing treatment.

Images