CeO2-Pd NPs composite material, immunosensor, preparation method and application

Abstract

The invention relates to the technical field of immunosensors, in particular to a CeO2-Pd NPs composite material, an immunosensor, a preparation method and application. The preparation method of the CeO2-Pd NPs composite material comprises the following steps: dissolving cerium salt and a polyvinylpyrrolidone solution in an organic solvent, adding an inorganic solvent, and carrying out a second heating reaction to obtain cerium dioxide; dissolving potassium salt, L-ascorbic acid and polyvinylpyrrolidone in an inorganic solvent, carrying out a third heating reaction, adding a palladium salt solution, and continuing the third heating reaction to obtain Pd NPs, namely palladium nanoparticles; the preparation method comprises the following steps: dispersing cerium dioxide in an inorganic solvent, and adding Pd NPs to obtain a CeO2-Pd NPs composite material; the invention also provides the CeO2-Pd NPs composite material prepared by the method, an immunosensor and a preparation method. The problem that an existing sensor is low in sensitivity when used for detecting DBP is solved.

Description

Technical Field

[0001] This invention relates to the field of immune sensor technology, specifically to a CeO2-Pd NPs composite material, an immune sensor, its preparation method, and its application. Background Technology

[0002] Dibutyl phthalate (DBP) is an organic compound belonging to the phthalate class. Due to its low volatility and good stability, it is commonly used as a plasticizer to increase the flexibility and low-temperature resistance of plastics, and has wide applications in industrial production and daily life. However, DBP has a certain degree of toxicity. Leakage or excessive addition during production and use can cause extremely serious adverse effects on the environment and human health. Therefore, timely and accurate detection of DBP content is crucial. However, existing sensors for DBP content detection suffer from poor sensitivity.

[0003] Electrochemiluminescence (ECL)-based sensors have advantages such as wide detection range, low background signal, and fast response speed, and have been extensively and deeply studied in related fields. Based on this, developing a material that can improve the sensitivity of the sensor and fabricating an ECL immunosensor for the detection of DBP is of great practical significance. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a CeO2-Pd NPs composite material, an immunosensor, a preparation method and application, to solve the problem of low sensitivity of existing sensors for detecting DBP.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a CeO2-Pd NPs composite material includes the following steps: Cerium salt and polyvinylpyrrolidone solution are dissolved in a second organic solvent, and then an inorganic solvent is added to carry out a second heating reaction to obtain cerium dioxide (CeO2). Potassium salt, L-ascorbic acid (AA), and polyvinylpyrrolidone (PVP) were dissolved in an inorganic solvent and subjected to a third heating reaction. Then, a palladium salt solution was added, and the third heating reaction was continued to obtain Pd NPs, i.e., palladium (Pd) nanoparticles. Cerium dioxide was dispersed in an inorganic solvent, Pd NPs were added, and the mixture was stirred overnight to obtain a CeO2-Pd NPs composite material.

[0006] Based on the aforementioned technical methods, a synergistic interface between the CeO2 support and Pd nanoparticles was formed through a stepwise construction and physical composite strategy. CeO2, as a high specific surface area oxide support, not only effectively disperses and anchors Pd nanoparticles through the steric hindrance effect of polyvinylpyrrolidone, preventing their aggregation and exposing more active sites, but also its own CeO2... 3 + / Ce 4+ Redox pairs can participate in electron transport cycles; Pd NPs provide high-density catalytic active centers. The metal-oxide heterostructure formed by their combination significantly enhances the interfacial charge transfer efficiency and the electron enrichment effect of the catalytically active region. This results in a significant amplification of the DBP detection signal (typically an ECL signal) when used as a sensor, thereby greatly improving the sensing sensitivity. This effectively solves the problem of low sensitivity in existing sensors for DBP detection.

[0007] Preferably, the temperature of the second heating reaction is 160~180 ℃, and the time of the second heating reaction is 8~10 h.

[0008] Preferably, the temperature of the second heating reaction is 160 °C, and the time of the second heating reaction is 8 h.

[0009] Preferably, the temperature of the third heating reaction is 80~90 ℃, the time for the third heating reaction is 10~15 min, and the time for continuing the third heating reaction is 3~4 h.

[0010] Preferably, the temperature of the third heating reaction is 80 °C, the time for the third heating reaction is 10 min, and the time for continuing the third heating reaction is 3 h.

[0011] Preferably, the cerium salt is selected from cerium nitrate or cerium nitrate hexahydrate.

[0012] Preferably, the potassium salt is selected from potassium bromide.

[0013] Preferably, the palladium salt is selected from sodium tetrachloropalladium(II)ate.

[0014] The palladium salt solution is an aqueous solution of palladium salt.

[0015] Preferably, the second organic solvent is selected from ethylene glycol.

[0016] Preferably, the inorganic solvent is selected from water.

[0017] Preferably, the preparation method of CeO2-Pd NPs composite material includes the following steps: Cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and polyvinylpyrrolidone (PVP) (Mw = 58000) solution were dissolved in ethylene glycol (EG), then deionized water was added, and the mixture was transferred to a high-pressure reactor for a second heating reaction. The pale purple precipitate was collected, washed with deionized water and anhydrous ethanol in sequence, dried overnight, and ground to obtain cerium dioxide, i.e., CeO2. Potassium bromide (KBr), L-ascorbic acid (AA), and polyvinylpyrrolidone (PVP) (Mw = 58000) were dissolved in water and subjected to a third heating reaction with magnetic stirring. Then, Na2PdCl4 aqueous solution was added, and the mixture was stirred magnetically to continue the third heating reaction. Finally, the solid product was collected by centrifugation and washed with water to obtain Pd nanoparticles, namely Pd NPs. Cerium dioxide was dispersed in water, Pd nanoparticles were added, and the mixture was stirred overnight to obtain a CeO2-Pd NPs composite material.

[0018] Preferably, the mass ratio of the cerium nitrate hexahydrate to the PVP (Mw = 58000) solution is 5:2.

[0019] Preferably, the ratio of the aqueous solution of KBr, AA, PVP (Mw = 58000) and Na2PdCl4 is 100mg:20mg:35mg:1mL.

[0020] Preferably, the concentration of Na2PdCl4 in the Na2PdCl4 aqueous solution is 19 mg / mL. -1 .

[0021] The present invention also provides a CeO2-Pd NPs composite material prepared by the preparation method described herein.

[0022] The present invention also provides an application of CeO2-Pd NPs composite material prepared by the preparation method described herein in a sensor.

[0023] Preferably, in the sensor, the CeO2-Pd NPs composite material serves as a co-reaction promoter.

[0024] The present invention also provides an immune sensor, comprising a luminescent body that binds to an antigen, and a CeO2-Pd NPs composite material prepared by the preparation method described in the present invention.

[0025] Preferably, it includes the following steps: Isoluminol and 2-aminoterephthalic acid were dissolved in a first organic solvent, and then glutaraldehyde (GA) was added. The mixture was stirred in the dark to obtain solution A. Polyether F127 was dissolved in a mixed solvent, and then copper salt was added to obtain solution B; Solution A and solution B were mixed and subjected to a first heating reaction to obtain a dual-ligand Cu-MOF (copper-based metal-organic framework) composite material; The dual-ligand Cu-MOF composite material was dissolved in a buffer solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and antigen solution were added. After incubation, bovine serum albumin was added to obtain a luminescent body that binds to the antigen. An aqueous solution of the CeO2-Pd NPs composite material was dropped onto the electrode surface, followed by the sequential addition of Ab (DBP antibody), bovine serum albumin (BSA), dibutyl phthalate (DBP), and a luminescent solution that binds to the antigen. After incubation, the immunosensor was obtained.

[0026] By using a copper-based metal-organic framework (MOF) to crosslink isoluminol and 2-aminoterephthalic acid (2-aminoterephthalic acid) with glutaraldehyde, followed by coordination with copper ions, a dual-ligand Cu-MOF composite material was obtained. This Cu-MOF composite material, used as the luminescent agent in an ECL sensor, exhibits excellent ECL performance under H₂O₂ co-reactant conditions, effectively addressing the poor ECL signal stability issue of isoluminol (ABEI). It shows significant application potential in bioanalysis.

[0027] Pluronic F127, also known as polyether F127, is a triblock copolymer of polyethylene oxide (PEO), polypropylene oxide (PPO), and polyethylene oxide (PEO). It is a nonionic surfactant with good biocompatibility and water solubility. The average molecular weight of Pluronic F127 is approximately 12,600 g / mol, with PEO comprising about 70% and PPO about 30%.

[0028] The ternary ECL signal amplification system constructed in this invention, consisting of a luminescent agent, a co-reactant, and a co-reaction promoter, effectively improves detection sensitivity. Specifically, the introduction of CeO2-Pd NPs as a co-reaction promoter significantly promotes the activation of superoxide anion radicals (O2). •− The generation of H2O2, a co-reactant, enables dual amplification of the ECL signal. Furthermore, since excessively high concentrations of the co-reactant H2O2 may destroy antigen-antibody activity, the introduction of a co-reaction promoter effectively avoids the requirement for high concentrations of co-reactants in traditional ECL systems, thus achieving highly sensitive detection of the target analyte.

[0029] Preferably, the temperature of the first heating reaction is 120~130 ℃, and the time of the first heating reaction is 12 h.

[0030] Preferably, the temperature of the first heating reaction is 120 °C.

[0031] Preferably, the mass ratio of isoluminol, 2-aminoterephthalic acid, polyether F127 and copper salt is 4:3:4:8.

[0032] Preferably, the copper salt is selected from one or both of copper nitrate and copper nitrate trihydrate.

[0033] Preferably, the first organic solvent is selected from N,N-dimethylformamide (DMF).

[0034] Preferably, the mixed solvent is selected from a mixture of water and anhydrous ethanol.

[0035] Preferably, the method for preparing the immune sensor includes the following steps: The glassy carbon electrode (GCE) was polished with alumina powder, and then an aqueous solution of CeO2-Pd NPs composite material was dropped onto the electrode surface. Subsequently, Ab (DBP antibody) solution, bovine serum albumin (BSA) solution, dibutyl phthalate (DBP) and antigen-binding luminescent solution were dropped onto the electrode surface in sequence to modify the electrode surface. The electrode was then incubated at 4°C for 2 hours. After each modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances or biomolecules, thus obtaining the immunosensor.

[0036] Preferably, the Ab (DBP antibody) solution is a PBS solution containing Ab (DBP antibody).

[0037] Preferably, the bovine serum albumin (BSA) solution is a PBS solution containing bovine serum albumin (BSA).

[0038] Preferably, the antigen-binding luminescent solution is a PBS solution containing an antigen-binding luminescent agent.

[0039] Preferably, the preparation method of the dual-ligand Cu-MOF composite material includes the following steps: Isoluminol and 2-aminoterephthalic acid were dissolved in N,N-dimethylformamide (DMF), and then glutaraldehyde (GA) was added. The mixture was stirred in the dark to obtain solution A. Polyether F127 was dissolved in a mixed solution of water and ethanol (volume ratio of water to ethanol 1:1), and then Cu(NO3)2·3H2O was added to obtain solution B; Solution A and solution B were mixed and sonicated, then transferred to a high-pressure reactor for the first heating reaction. The resulting product was then centrifuged to obtain a precipitate. The precipitate was washed repeatedly with water, DMF, and ethanol until the supernatant was clear. The precipitate was then dried and ground to obtain Cu-ABEI, i.e., the dual-ligand Cu-MOF composite material.

[0040] The preparation method of the dual-ligand Cu-MOF composite material of the present invention involves crosslinking isoluminol and 2-aminoterephthalic acid with glutaraldehyde, followed by coordination with copper ions to obtain the dual-ligand Cu-MOF composite material. The dual-ligand Cu-MOF composite material, as a luminescent material, has a stable and excellent ECL signal and has great application potential in bioanalysis.

[0041] The present invention also provides a dual-ligand Cu-MOF composite material prepared by the method of the present invention.

[0042] The dual-ligand Cu-MOF composite material of the present invention is obtained by self-assembling isoluminol and 2-aminoterephthalic acid using a copper-centered MOF material as a metal-organic framework. The dual-ligand Cu-MOF composite material is used as a luminescent material and exhibits excellent ECL performance under the condition of H2O2 as a co-reactant.

[0043] The present invention also provides an application of the dual-ligand Cu-MOF composite material prepared by the method of the present invention as a light emitter of an ECL sensor.

[0044] The present invention also provides a photoluminescent material that binds to an antigen, which is made using the antigen and a dual-ligand Cu-MOF composite material prepared by the preparation method described in the present invention.

[0045] The present invention also provides a method for preparing a photoluminescent body that binds to an antigen, comprising the following steps: The dual-ligand Cu-MOF composite material was dissolved in a buffer solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and an antigen solution were added. After incubation, bovine serum albumin (BSA) was added to obtain the antigen-binding luminescent body Cu-ABEI-antigen.

[0046] Preferably, the antigen is selected from DBP antigen.

[0047] Preferably, the method for preparing a luminescent body conjugated with an antigen includes the following steps: The Cu-ABEI was dissolved in PBS buffer solution and ultrasonically dispersed. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and DBP antigen solution were added. The mixture was incubated overnight at 4 °C with shaking. Bovine serum albumin (BSA) was then added to block non-specific binding sites. Unbound antigen was removed by centrifugation to obtain the product. The obtained product was redispersed in 1 mL of PBS solution (pH 7.4) to obtain the Cu-ABEI-antigen solution containing the bound antigen for later use.

[0048] Preferably, the ratio of the Cu-ABEI, EDC, NHS and DBP antigen solution is 1.5 mg: 0.4 mmol: 0.1 mmol: 250 µL.

[0049] Preferably, the solvent for the DBP antigen solution is PBS solution (pH 7.4), and the concentration of DBP antigen in the DBP antigen solution is 4.4 µg / mL. -1 .

[0050] The present invention also provides an immune sensor comprising a CeO2-Pd NPs composite material prepared by the preparation method described in the present invention and a Cu-ABEI-antigen solution that binds to an antigen.

[0051] A competitive immunosensor for detecting DBP was constructed using CeO2-Pd NPs as a substrate and Cu-ABEI as a luminescent agent. CeO2-Pd NPs provide abundant sites for H2O2 catalysis and antibody immobilization, effectively enhancing the ECL response and thus improving the sensor's sensitivity. The ECL immunosensor constructed based on this strategy achieves sensitive detection of the environmental pollutant DBP, overcoming the problem of low sensitivity in existing sensors for DBP detection.

[0052] The present invention also provides an application of the immunosensor prepared by the method described herein as an ECL immunosensor.

[0053] Preferably, the immunosensor is used as an ECL immunosensor to detect dibutyl phthalate (DBP).

[0054] Preferably, the detection limit of the immunosensor used as an ECL immunosensor for detecting dibutyl phthalate (DBP) is 0.263 pg / mL. -1 .

[0055] Preferably, the immunosensor, as an ECL immunosensor, has a detection limit as low as 0.263 pg / mL for detecting dibutyl phthalate (DBP) in polluted water. -1 .

[0056] Preferably, the polluted water includes polluted tap water and polluted lake water.

[0057] The beneficial effects of this invention are: The method for preparing CeO2-Pd NPs composite material of the present invention, through a stepwise construction and physical composite strategy, forms a synergistic interface between CeO2 support and Pd nanoparticles, and effectively disperses and anchors Pd NPs through the steric hindrance effect of polyvinylpyrrolidone, preventing their aggregation and exposing more active sites, while simultaneously protecting CeO2 support. 3+ / Ce 4+ Redox pairs can participate in electron transport cycles; Pd NPs provide high-density catalytic active centers. The metal-oxide heterostructure formed by the combination of the two significantly enhances the interfacial charge transfer efficiency and the electron enrichment effect of the catalytic active region, which makes it a significant amplification of the DBP detection signal (usually the ECL signal) when used as a sensor, thereby greatly improving the sensing sensitivity and showing great application potential in bioanalysis.

[0058] The CeO2-Pd NPs composite material of the present invention, by using CeO2 with a high specific surface area as an oxide support and combining it with Pd NPs with high-density catalytic active centers, and introducing the CeO2-Pd NPs composite material into a sensor as a co-reaction promoter, significantly promotes the catalytic activity of superoxide anion radicals (SOPs). The generation of H2O2, a co-reactant, enables dual amplification of the ECL signal. Furthermore, since excessively high concentrations of the co-reactant H2O2 may destroy antigen-antibody activity, the introduction of a co-reaction promoter effectively avoids the requirement for high concentrations of co-reactants in traditional ECL systems, thus achieving highly sensitive detection of the target analyte and successfully constructing a highly efficient ECL immunosensor.

[0059] The immunosensor of the present invention constructs a competitive ECL immunosensor using CeO2-Pd NPs as a substrate and Cu-ABEI as a luminescent material. CeO2-Pd NPs provide abundant sites for H2O2 catalysis and antibody immobilization, effectively enhancing the ECL response and thus improving the sensor's sensitivity. This enables sensitive detection of the environmental pollutant DBP and has significant potential for widespread application in the field of immunosensor technology. Attached Figure Description

[0060] Figure 1 This is a flowchart of the preparation method for an immune sensor; Figure 2 The XPS spectra of the dual-ligand Cu-MOF composite material prepared in Example 1 are shown below. 2A is the full-area XPS spectrum, 2B is the fine XPS spectrum of Cu 2p orbitals, 2C is the fine XPS spectrum of O 1s orbitals, 2D is the fine XPS spectrum of N 1s orbitals, 2E is the fine XPS spectrum of C 1s orbitals, and 2F is the Auger electron spectrum of Cu. Figure 3 The images shown are SEM, TEM, and EDS images of the dual-ligand Cu-MOF composite material prepared in Example 1. In this image, 3A is the SEM image, 3B is the TEM image and a magnified view of a part, and 3C is the EDS mapping spectrum. Figure 4 Characterization of CeO2-Pd NPs composite material and CeO2 prepared in Example 3, wherein 4A is the XRD pattern of CeO2 and CeO2-Pd NPs, 4B is the SEM image of CeO2, 4C is the TEM image and a magnified view of Pd NPs, 4D is the SEM image of CeO2-Pd NPs, 4E is the EDS mapping spectrum of CeO2-Pd NPs, and 4F is the UV-Vis absorption spectrum of CeO2, Pd NPs, and CeO2-Pd NPs; Figure 5 These are characterization diagrams of the biosensor, where 5A is the AC impedance spectrum corresponding to electrodes with different modified layers, and 5B is the cyclic voltammetry curve corresponding to electrodes with different modified layers. Figure 6 The figures show the ECL response results under different conditions. In the figures, 6A represents the ECL response results corresponding to different H2O2 concentrations, 6B represents the ECL response results corresponding to different pH values, 6C represents the ECL response results corresponding to different Cu-ABEI concentrations, and 6D represents the ECL response results corresponding to different CeO2-Pd NPs concentrations. Figure 7 Figure 7A shows the ECL response of the electrodes modified with Cu-ABEI, CeO2 / Cu-ABEI, and CeO2-Pd NPs / Cu-ABEI in PBS solution. Figure 7B shows the response of the electrodes modified with Cu-ABEI, CeO2 / Cu-ABEI, and CeO2-Pd NPs / Cu-ABEI in PBS solution containing 0.1 mmol / L. -1 ECL response of H2O2 in PBS solution, 7C is a comparison of the catalytic performance of CeO2 and CeO2-Pd NPs, and 7D is a schematic diagram of the ECL mechanism; Figure 8 The graph shows the performance evaluation results of the sensor, where 8A represents different concentrations of DBP (a~g: 1 pg mL). -1 ~ 1µgmL-1 The graph shows the ECL response, DBP detection calibration curve (8B), stability of the ECL immunosensor under fixed potential scanning (8C), selectivity of the ECL immunosensor for different analytes (8D), a: blank, b: DMP, c: DINP, d: BBP, e: DBP, f: DMP + DBP, g: DINP + DBP, h: BBP + DBP, reproducibility of the ECL immunosensor (8E), and the six competitive immunosensors Cu-ABEI-antigen / DBP / BSA / Ab / CeO2-Pd NPs / GCE prepared using the method described in Example 4 (1-6 on the x-axis). The graph also shows the storage stability of the ECL immunosensor (n = 5). Detailed Implementation

[0061] The following description, with reference to preferred embodiments, illustrates the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are merely illustrative of the present invention and not intended to limit the scope of protection of the present invention.

[0062] The reagents used in the following examples included: dibutyl phthalate (DBP), DBP antigen, and DBP antibody, purchased from Shenzhen Antibio Biotechnology Co., Ltd. Isoluminol (ABEI), 2-aminoterephthalic acid, copper nitrate trihydrate (Cu(NO3)2·3H2O), glutaraldehyde (GA), cerium nitrate hexahydrate (Ce(NO3)3·6H2O), potassium bromide (KBr), and polyether (F127) were purchased from Shanghai Maclean Industrial Co., Ltd. Polyvinylpyrrolidone (PVP, Mw = 58000), ethylene glycol (EG), L-ascorbic acid (AA), sodium chloropalladium (Na2PdCl4), anhydrous ethanol, and N,N-dimethylformamide (DMF) were provided by Sinopharm Chemical Reagents. All reagents used were of analytical grade purity, and ultrapure water (18.25 MΩ cm⁻¹) was used in all experiments. -1 ).

[0063] The experimental instruments and equipment used in the following examples are shown in Table 1.

[0064] Table 1 lists the experimental instruments and equipment. Example 1 A method for preparing a dual-ligand Cu-MOF composite material includes the following steps: S1. Dissolve 0.1 g isoluminol and 0.075 g 2-aminoterephthalic acid in 10 mL N,N-dimethylformamide (DMF), then add 500 µL of 1% glutaraldehyde and stir in the dark for 48 h to obtain solution A. S2. Dissolve 100 mg of polyether F127 in 20 mL of a mixed solution of water and ethanol (water to ethanol volume ratio 1:1), sonicate until completely dissolved, and then add 0.2 g of Cu(NO3)2·3H2O to obtain solution B; S3. Mix solution A obtained in S1 and solution B obtained in S2 and sonicate for 20 min. Transfer the mixture to a high-pressure reactor and react at 120℃ for 12 h. Centrifuge the product to obtain a precipitate. Wash the precipitate repeatedly with water, DMF and ethanol until the supernatant is clear. Then dry the precipitate under vacuum at 60℃ for 12 h. Grind the product to obtain Cu-ABEI, i.e., the dual-ligand Cu-MOF composite material.

[0065] Example 2 A method for preparing a Cu-ABEI-antigen that binds to an antigen includes the following steps: 1.5 mg of Cu-ABEI obtained in Example 1 was dissolved in 750 µL of PBS solution (pH 7.4), ultrasonically dispersed, and then 0.4 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 0.1 mmol of N-hydroxysuccinimide (NHS), and 250 µL of a 4.4 µg / mL solution were added. -1 The DBP antigen solution was incubated overnight at 4°C with shaking. 100 μL of 0.1% bovine serum albumin (BSA) was added to the incubated solution to block non-specific binding sites. Unbound antigen was then removed by centrifugation to obtain the product. The product was redispersed in 1 mL of PBS solution (pH 7.4) to obtain the Cu-ABEI-antigen solution containing DBP antigen.

[0066] Example 3 A method for preparing a CeO2-Pd NPs composite material includes the following steps: S1. Dissolve 500 mg of cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and 200 mg of polyvinylpyrrolidone (PVP) (Mw = 58000) in 15 mL of ethylene glycol (EG), then add 1 mL of deionized water, stir continuously for 30 min to obtain a clear solution, transfer the clear solution to a 50 mL high-pressure reactor, heat at 160 °C for 8 h, collect the light purple precipitate, wash the precipitate three times with deionized water and anhydrous ethanol, dry the product at 70 °C overnight, grind to obtain CeO2, and store for later use; S2. Dissolve 300 mg potassium bromide (KBr), 60 mg L-ascorbic acid (AA), and 105 mg polyvinylpyrrolidone (PVP) (Mw = 58000) in 8 mL of water, then heat at 80°C with magnetic stirring for 10 min. Subsequently, add 3 mL of a solution containing 19 mg / mL potassium bromide. -1 The Na2PdCl4 aqueous solution was heated at 80℃ for 3 h. Finally, the solid product was collected by centrifugation and washed three times with water to obtain Pd nanoparticles. The Pd nanoparticles were dispersed in 5 mL of water to obtain an aqueous solution of Pd nanoparticles (Pd NPs) for later use. S3. Take 10 mg of CeO2 prepared in S1 and ultrasonically disperse it in 10 mL of water. Then add 2 mL of Pd nanoparticle aqueous solution prepared in S2 and stir overnight to obtain CeO2-Pd NPs composite material.

[0067] Example 4 like Figure 1 As shown, a method for preparing an immune sensor includes the following steps: The glassy carbon electrode (GCE) was polished with alumina powder, and then 10 µL of the 1 mg·mL solution prepared in Example 3 was dropped onto the electrode surface. -1 An aqueous solution of CeO2-Pd NPs composite material was used to modify the electrode surface, followed by the sequential addition of 10 µL of a 1.5 µg·mL⁻¹ solution to the electrode surface. -1The electrode surface was modified sequentially with PBS solution (pH 7.4) of Ab (DBP antibody), 3 µL of PBS solution (pH 7.4) of 1% bovine serum albumin (BSA), 10 µL of dibutyl phthalate (DBP), and 10 µL of PBS solution (pH 7.4) of Cu-ABEI-antigen, the luminescent material bound to DBP antigen prepared in Example 2. The modified layers were then incubated at 4 °C for 2 h. After each modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances or biomolecules, resulting in the competitive immunosensor Cu-ABEI-antigen / DBP / BSA / Ab / CeO2-Pd NPs / GCE.

[0068] Detection and Analysis 1) Characterization of Cu-ABEI The Cu-ABEI prepared in Example 1 was analyzed using X-ray photoelectron spectroscopy. The results are as follows: Figure 2 As shown.

[0069] from Figure 2 As shown in A, Cu-ABEI is composed of four elements: Cu, O, N, and C. The fine spectrum of the Cu 2p orbital was measured, and the results are as follows... Figure 2 As shown in Figure B, the two peaks at 933.61 eV and 953.30 eV correspond to Cu 2p, respectively. 3 / 2 and Cu 2p 1 / 2 Furthermore, small oscillating satellite peaks appeared at approximately 945 eV and 965 eV. To further determine the valence state of Cu, Auger electron spectroscopy was performed, and the results obtained by least squares fitting are as follows: Figure 2 As shown in Figure F, a strong peak is observed at 916.50 eV after fitting, indicating that Cu exists in Cu-ABEI in the form of Cu. + The fine spectrum of the O 1s orbital was studied, and the results are as follows: Figure 2 As shown in Figure C, the fitting peak at 531.50 eV indicates that O in the MOF material is most likely in the form of C=O. Furthermore, the fine spectrum of the N 1s orbital is shown in Figure C. Figure 2 As shown in Figure D, the fitted peak at 399.70 eV indicates that N exists in the form of CNH. The fine XPS spectrum of the C 1s orbital is shown below. Figure 2 As shown in Figure E, the sample contains carbon, primarily in the unsaturated carbon (C=C) chemical state. Therefore, the C element spectrum exhibits an asymmetric structure, with the C=C peak dominating (284.0 eV). Thus, during charge correction, the peak position of the C=C chemical state is used as the reference, and it is calibrated to 284.0 eV. The peaks of other elements are also shifted accordingly. This achieves XPS data calibration.

[0070] The Cu-ABEI prepared in Example 1 was analyzed by scanning electron microscopy (SEM), transmitted electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDS). The results are as follows: Figure 3 As shown.

[0071] from Figure 3 As shown in A, Cu-ABEI exhibits a flower-like morphology with a wrinkled, lamellar surface. Further TEM analysis revealed its internal structure... Figure 3 As can be seen from B, Cu-ABEI forms uniformly sized solid flower-shaped spheres, with a size of approximately 2–3 µm. Furthermore, Figure 3 The EDS mapping spectrum of C showed the presence and distribution of four elements: Cu, O, N, and C, further indicating the successful synthesis of the material.

[0072] 2) Characterization of CeO2-Pd NPs X-ray diffraction (XRD) was used to perform XRD tests on the CeO2 composite materials prepared in S1 and S3 of Example 3, respectively. Scanning electron microscopy (SEM) was used to perform SEM tests on the CeO2 composite materials prepared in S1 and S3 of Example 3, respectively. Transmission electron microscopy (TEM) was used to perform TEM tests on the Pd nanoparticles (Pd NPs) prepared in S2 of Example 3, respectively. Energy dispersive X-ray spectroscopy (EDS) was used to perform EDS tests on the CeO2-Pd NPs composite materials prepared in S3 of Example 3, respectively. UV-Vis absorption spectrophotometry was used to perform UV-Vis absorption spectrophotometry tests on the CeO2 composite materials prepared in S1, S2, and S3 of Example 3, respectively. The results are as follows: Figure 4 As shown.

[0073] from Figure 4 As shown in Figure A, the diffraction peaks of the synthesized CeO2 at 28.5°, 33.1°, 47.5°, and 56.3° correspond to the (111), (200), (220), and (311) crystal planes in the standard card (PDF#43-1002), respectively. The XRD pattern of the CeO2-Pd NPs composite material, while exhibiting characteristic CeO2 peaks, also shows a distinct diffraction peak at 40.2° belonging to the (111) crystal plane of Pd NPs (PDF#87-0638), thus proving the successful synthesis of the material. Figure 4B shows that CeO2 consists of uniformly sized, slightly rough-surfaced nanospheres, with a size of approximately 200 nm. The synthesized Pd NPs were characterized by TEM, such as... Figure 4 As shown in Figure C, the particles exhibit a uniform square-round morphology with a particle size ranging from approximately 15 to 20 nm. They are found to have distinct lattice fringes, with a calculated lattice spacing of 0.227 nm, corresponding to the (111) crystal plane of Pd NPs. Furthermore, SEM images of the CeO2-Pd NPs composite material... Figure 4 As can be seen in D, the surface of the CeO2 nanospheres has some bright spots, which is presumably due to the excellent electrical conductivity and small size of the noble metal Pd NPs. Figure 4 D). From Figure 4 The EDS mapping spectrum shown in Figure E reveals the presence and distribution of Ce, O, and Pd, further confirming the successful synthesis of the materials. Finally, qualitative analysis of the three materials—CeO2, Pd NPs, and CeO2-Pd NPs—was conducted using UV-Vis spectroscopy results. Figure 4 As shown in F. From Figure 4 As shown in F, CeO2 has a wide characteristic peak in the wavelength range of 280~385 nm, Pd NPs have a characteristic peak at 420 nm, and CeO2-Pd NPs have an obvious characteristic peak in an even wider wavelength range of 280~450 nm, indicating the successful preparation of the composite material.

[0074] 3) Electrochemical characterization of biosensors with different numbers of modified layers 3.1) Polish the glassy carbon electrode (GCE) with alumina powder, and then drop 10 µL of the 2 mg·mL solution prepared in S3 of Example 3 onto the electrode surface. -1 An aqueous solution of CeO2-Pd NPs composite material was used to modify the electrode surface as a single layer. After natural drying, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances, resulting in the CeO2-Pd NPs / GCE electrode (b).

[0075] 3.2) Polish the glassy carbon electrode (GCE) with alumina powder, and then drop 10 µL of the 2 mg·mL solution prepared in S3 of Example 3 onto the electrode surface. -1 An aqueous solution of CeO2-Pd NPs composite material was prepared, and then 10 µL of a 1.5 µg·mL⁻¹ was dropped onto the electrode surface. -1The electrode surface was modified with two layers of Ab (DBP antibody) aqueous solution and then incubated at 4 °C for 2 h. After each layer modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances or biomolecules, resulting in the Ab / CeO2-Pd NPs / GCE electrode (c).

[0076] 3.3) Polish the glassy carbon electrode (GCE) with alumina powder, and then drop 10 µL of the 2 mg·mL solution prepared in S3 of Example 3 onto the electrode surface. -1 An aqueous solution of CeO2-Pd NPs composite material was prepared, and then 10 µL of a 1.5 µg·mL⁻¹ material was added dropwise to the electrode surface. -1 The electrode surface was modified with a three-layer mixture of 3 µL of 1% bovine serum albumin (BSA) aqueous solution and 3 µL of 1% bovine serum albumin (DBP antibody) aqueous solution. The mixture was then incubated at 4 °C for 2 h. After each layer modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances or biomolecules, resulting in the BSA / Ab / CeO2-Pd NPs / GCE electrode (d).

[0077] 3.4) Polish the glassy carbon electrode (GCE) with alumina powder, and then drop 10 µL of the 2 mg·mL solution prepared in S3 of Example 3 onto the electrode surface. -1 An aqueous solution of CeO2-Pd NPs composite material was prepared, and then 10 µL of a 1.5 µg·mL⁻¹ solution was added dropwise to the electrode surface. -1 The electrode surface was modified with four layers: an aqueous solution of Ab (DBP antibody), 3 µL of a 1% bovine serum albumin (BSA) aqueous solution, and 10 µL of dibutyl phthalate (DBP). The electrode was then incubated at 4 °C for 2 h. After each layer modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances or biomolecules, resulting in the DBP / BSA / Ab / CeO2-Pd NPs / GCE electrode (e).

[0078] Ag / AgCl and Pt were used as the reference and counter electrodes, respectively. The unmodified glassy carbon electrode (GCE) (a), the CeO2-Pd NPs / GCE electrodes prepared with the different modification layers (b), Ab / CeO2-Pd NPs / GCE electrodes (c), BSA / Ab / CeO2-Pd NPs / GCE electrodes (d), DBP / BSA / Ab / CeO2-Pd NPs / GCE electrodes (e), or the electrochemically competitive ECL immunosensor Cu-ABEI-antigen / DBP / BSA / Ab / CeO2-Pd NPs / GCE (f) constructed in Example 4 were used as working electrodes for electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The parameters set during the tests were as follows: photomultiplier tube high voltage of 600 V, voltage scan range of 0 ~ 0.7 V, and scan rate of 0.1 V s. -1 The test solution was 10 mL of PBS with different concentrations of H2O2 (pH = 8). The results are as follows: Figure 5 As shown.

[0079] Figure 5 A and Figure 5 In section B, the black curve corresponds to the measurement results of the unmodified glassy carbon electrode (a) as the working electrode; the red curve corresponds to the measurement results of the monolayer modified CeO2-Pd NPs / GCE electrode (b) as the working electrode; the blue curve corresponds to the measurement results of the bilayer modified Ab / CeO2-Pd NPs / GCE electrode (c) as the working electrode; the green curve corresponds to the measurement results of the trilayer modified BSA / Ab / CeO2-Pd NPs / GCE electrode (d) as the working electrode; the purple curve corresponds to the measurement results of the quadruple modified DBP / BSA / Ab / CeO2-Pd NPs / GCE electrode (e) as the working electrode; and the yellow curve corresponds to the measurement results of the five-layer modified sensor Cu-ABEI-antigen / DBP / BSA / Ab / CeO2-Pd NPs / GCE (f), i.e., the measurement results of the ECL immunosensor constructed in Example 4 as the working electrode.

[0080] The electrochemical behavior of the proposed biosensor was characterized using EIS and CV tests to verify the layer-by-layer modification process of the glassy carbon electrode. Figure 5 As shown in A, after layer-by-layer modification with CeO2-Pd NPs, Ab, BSA, DBP, and Cu-ABEI-antigen, the diameter of the semicircle gradually increases, which means that the conductivity decreases and the impedance increases, indicating that the aforementioned substances hinder electron transport. From Figure 5The CV test results of B show a decreasing trend of oxidation peak, which also proves the successful modification of the above substances, thus proving the successful preparation of the electrochemical competitive ECL immunosensor.

[0081] 4) Condition optimization test Effect of Cu-ABEI concentration The ECL test employed a standard three-electrode system, using Ag / AgCl and Pt as the reference and counter electrodes, respectively. Cu-ABEI prepared in Example 1 was added to deionized water to prepare mixed solutions with concentrations of 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. These mixed solutions were then modified onto glassy carbon electrodes (GCE) and used as the working electrodes to test the corresponding ECL response signals. The parameters set during the test were as follows: photomultiplier tube high voltage of 600 V, voltage scan range of 0 ~ 0.7 V, and scan rate of 0.1 V s. -1 The test substrate was 10 mL with a concentration of 0.2 mmol / L. -1 The results of the PBS solution containing H2O2 (pH = 8) are as follows: Figure 6 As shown in C.

[0082] The effect of pH The ECL test employed a standard three-electrode system, using Ag / AgCl and Pt as the reference and counter electrodes, respectively. The Cu-ABEI prepared in Example 1 was dissolved in deionized water to prepare a mixed solution with a Cu-ABEI concentration of 1.5 mg / mL. This mixed solution was then added dropwise onto a glassy carbon electrode (GCE) as the working electrode, and the corresponding ECL response signal was measured. The parameters set during the test were as follows: photomultiplier tube high voltage of 600 V, voltage scan range of 0 ~ 0.7 V, and scan rate of 0.1 V s. -1 The test substrate was 10 mL with a concentration of 0.2 mmol / L. -1 The ECL responses of PBS solutions with different pH values ​​for H2O2, such as Figure 6 As shown in B.

[0083] The effect of H2O2 The ECL test employed a standard three-electrode system, using Ag / AgCl and Pt as the reference and counter electrodes, respectively. The Cu-ABEI prepared in Example 1 was added to deionized water to prepare a mixed solution with a Cu-ABEI concentration of 1.5 mg / mL. This mixed solution was then added dropwise onto a glassy carbon electrode (GCE) as the working electrode, and the corresponding ECL response signal was measured. The parameters set during the test were as follows: photomultiplier tube high voltage of 600 V, voltage scan range of 0 ~ 0.7 V, and scan rate of 0.1 V s. -1 The test substrate was 10 mL of PBS with different concentrations of H2O2 (pH = 8) to measure the ECL response, as shown below. Figure 6 As shown in Figure A.

[0084] Effect of CeO2-Pd NPs concentration The ECL test employed a standard three-electrode system, using Ag / AgCl and Pt as the reference and counter electrodes, respectively. First, CeO2-Pd NPs prepared in S3 of Example 3 were added to deionized water to prepare mixed solutions with CeO2-Pd NPs concentrations of 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. These mixed solutions were then added dropwise to modify a glassy carbon electrode (GCE) to form a modification layer. Next, Cu-ABEI prepared in Example 1 was added to deionized water to prepare a mixed solution with a Cu-ABEI concentration of 1.5 mg / mL. This mixed solution was also added dropwise to modify a glassy carbon electrode (GCE) to form two modification layers, which were then used as the working electrode to test the corresponding ECL response signal. The parameters set during the test were as follows: photomultiplier tube high voltage of 600 V, voltage scan range of 0 ~ 0.7 V, and scan rate of 0.1 V s. -1 The test substrate was 10 mL with a concentration of 0.2 mmol / L. -1 The ECL response of PBS solution containing H2O2 (pH = 8) is as follows: Figure 6 As shown in D.

[0085] from Figure 6 As shown in A, within a certain range, the ECL response reaches its maximum value with changes in H2O2 concentration; therefore, the optimal concentration is 0.2 mmol / L. -1 .from Figure 6 As shown in B, the ECL signal gradually increases with pH as it increases from 6.0 to 8.5, indicating that an alkaline environment is conducive to luminescence. However, a strongly alkaline environment may lead to antigen-antibody inactivation; therefore, 8.0 was chosen as the optimal pH. Figure 6 From C, we know that at a Cu-ABEI concentration of 1.5 mg / mL... -1At that time, the ECL intensity reaches its maximum value. From Figure 6 As shown in D, the optimal concentration of CeO2-Pd NPs is 1 mg / mL. -1 .

[0086] 5) ECL mechanism research To explore possible luminescence mechanisms, a glassy carbon electrode (GCE) was polished with alumina powder. Then, the electrode surface was successively supplemented with 1 mg / mL Cu-ABEI aqueous solution prepared in Example 1, 1 mg / mL CeO2 aqueous solution prepared in S1 of Example 3, 1 mg / mL Cu-ABEI aqueous solution prepared in Example 1, 1 mg / mL CeO2-Pd NPs composite material prepared in S3 of Example 3, and 1 mg / mL Cu-ABEI aqueous solution prepared in Example 1. This resulted in three modified electrodes: a Cu-ABEI modified electrode, a CeO2 / Cu-ABEI modified electrode, and a CeO2-Pd NPs / Cu-ABEI modified electrode, respectively. The electrodes were then subjected to tests in pure PBS solution and a solution containing 0.1 mmol / L... -1 In a PBS solution of H2O2, using electrodes modified with the different materials mentioned above as working electrodes, and employing Ag / AgCl and Pt as reference and counter electrodes respectively, a standard three-electrode system for ECL testing was constructed to test the ECL response of the electrodes modified with the different materials. The results are as follows: Figure 7 As shown.

[0087] from Figure 7 As can be seen from A, under test conditions without H2O2, the ECL signal of the Cu-ABEI modified electrode is almost negligible, while under test conditions containing 0.1 mmol L... -1 In the PBS buffer solution containing H2O2, the ECL signal of Cu-ABEI can reach around 6000, indicating that H2O2 is an indispensable co-reactant for Cu-ABEI luminescence. Meanwhile, the signals of the CeO2 / Cu-ABEI and CeO2-Pd NPs / Cu-ABEI modified electrodes increased to 1.1k and 1.6k respectively in the presence of H2O2, achieving dual signal amplification. Furthermore, in the test buffer solution without H2O2, the signals of the CeO2 / Cu-ABEI and CeO2-Pd NPs / Cu-ABEI modified electrodes were similar to, or even negligible compared to, Cu-ABEI, indicating that the enhanced ECL signals of CeO2 and CeO2-Pd NPs are due to the catalytic effect of H2O2, rather than that of Cu-ABEI. Meanwhile, from... Figure 7As shown in D, the emission peak positions of the ECL spectrum of CeO2-Pd NPs / Cu-ABEI are similar to those of Cu-ABEI, but the ECL response is enhanced, which also indicates that the addition of CeO2-Pd NPs does not directly change the ECL properties of Cu-ABEI. To analyze the dual amplification mechanism of the ECL signal, the ECL peak positions are investigated. Figure 7 As shown in Figure B, compared with pure Cu-ABEI, the ECL peak position shifted significantly positively and the ECL response increased significantly after further modification of the electrode with CeO2, indicating that the introduction of CeO2 produced effective catalysis. However, the introduction of CeO2-Pd NPs did not produce a significant shift in the peak position. Therefore, further analysis of the cyclic voltammetry curves of the materials yielded the following results: Figure 7 As shown in Figure C, the peak current of CeO2-Pd NPs is significantly larger than that of CeO2, indicating that the introduction of noble metal Pd NPs accelerates electron transfer and further realizes the dual amplification of the ECL signal.

[0088] Based on the above research, the luminescence mechanism can be understood as follows: Figure 7 As shown in D and routes 1, 2 and ECL emission below, with increasing potential, Cu-ABEI is first oxidized to generate reducing free radicals ( (Formula I), then reacts with O2 to produce less (Formula II). Furthermore, under the catalysis of CeO2-Pd NPs, H2O2 electrolysis generates more active intermediates. and OH • (Equation III). For the ECL launch process, and / OH • The reaction produces Cu-ABEI*, which in turn generates strong ECL emission (Formulas IV and V).

[0089] Route 1: Route 2: ECL launch: 6) Sensor performance evaluation The ECL immunosensor constructed in Example 4 was used to quantitatively detect DBP, and a working curve was plotted. The corresponding competitive sensing mechanism is as follows: Figure 1As shown, after sequentially modifying the electrode surface with DBP standard samples and Cu-ABEI (Cu-ABEI-antigen) that binds to DBP antigen, both can specifically bind to DBP antibodies. Therefore, DBP and Cu-ABEI-antigen compete for antibody binding, and the ECL response intensity depends on the signal probe Cu-ABEI-antigen. Furthermore, the small molecule characteristics of the DBP standard enhance its binding ability to antibodies; therefore, from... Figure 8 As shown in A, with the increase of DBP concentration, the binding rate of Cu-ABEI-antigen decreases, and the ECL signal decreases. Figure 8 A, a, b, c, d, e, f, and g correspond to DBP concentrations of 0.001 pg / mL, respectively. -1 0.01 pg mL -1 0.1 pg mL -1 1 pg mL -1 10 pg mL -1 100 pg mL -1 and 1000 pg mL -1 The plotted working curve is as follows: Figure 8 As shown in B, at 1 pg mL -1 ~ 1µg mL -1 Within the specified concentration range, the logarithms of ECL signal and DBP concentration exhibit a good linear relationship, with the corresponding linear equation being I = -1982lg c + 9194, R 2 = 0.998, compared with other detection methods (Table 2), this sensor has a wider detection range and a detection limit as low as 0.263 pg mL. -1 .

[0090] Table 2 shows the detection range results obtained by different detection methods. To verify the stability of the sensor, a DBP concentration of 1 µg / mL was used. -1 At that time, the results were obtained by continuously scanning 14 cycles on one electrode as follows: Figure 8 As shown in Figure C, the peak ECL intensity varies slightly across different periods, with a calculated RSD of 1.74%, indicating good stability of the sensor. To illustrate the sensor's selectivity, 1 ng / mL... -1 DBP was used as a control sample, while dimethyl phthalate (DMP), diisononyl phthalate (DINP), and butyl benzyl phthalate (BBP) at the same concentrations were used as interfering agents for comparison. The selectivity of the ECL immunosensor was investigated by testing the ECL response to DBP and other interfering agents. Figure 8 As shown in Figure D, the ECL responses of interfering substances such as DMP, DINP, and BBP are close to the ECL signal of the blank sample, while the ECL response of the mixed sample is almost identical to that of DBP (1 ng mL). -1 DBP + 10 ng mL -1 The presence of interfering substances indicates that the sensor has good selectivity. Furthermore, under the same conditions, the ECL signals of six competitive immunosensors (Cu-ABEI-antigen / DBP / BSA / Ab / CeO2-Pd NPs / GCE) prepared using the method in Example 4, respectively, were detected as working electrodes to study the reproducibility of the biosensors. The results showed only minor differences (…). Figure 8 E). Finally, the storage stability of the ECL immune sensor was tested, and the results are as follows: Figure 8 As shown in Figure F, after storage at 4°C for 15 days, the ECL signal decreased by only 16.3% of its initial value, demonstrating good storage stability. In summary, the biosensor constructed in this invention exhibits excellent ECL performance and can be used for trace detection of the target DBP.

[0091] 7) Sample testing To verify the practical application feasibility of the ECL immunosensor of the present invention, and to verify the practical application feasibility of the proposed aptamer sensor, the ECL immunosensor prepared in Example 4 was used to quantitatively detect tap water and lake water using the standard addition method. Before detection, the water samples were filtered and centrifuged to remove suspended impurities. The results are shown in Table 3.

[0092] Table 3. Analytical results of DBP in water samples As shown in Table 3, the spiked recoveries ranged from 96.2% to 103.4%, and the RSDs ranged from 1.0% to 2.8%, all within acceptable ranges. Therefore, this demonstrates that the ECL immunosensor developed in this invention is a feasible method for DBP detection and has promising application prospects.

[0093] In summary, the CeO2-Pd NPs composite material of the present invention, by using CeO2 with a high specific surface area as an oxide support and combining it with Pd NPs with high-density catalytic active centers, and introducing the CeO2-Pd NPs composite material into the sensor as a co-reaction promoter, provides abundant sites for H2O2 catalysis and antibody immobilization, significantly promoting the catalysis of superoxide anion radicals (… The generation of [a specific substance] enables dual amplification of the ECL signal, which is beneficial for the subsequent construction of the immunosensor. Furthermore, since excessively high concentrations of the co-reactant H2O2 may destroy antigen-antibody activity, the introduction of a co-reaction promoter effectively avoids the requirement for high concentrations of co-reactants in traditional ECL systems, thus achieving highly sensitive detection of the target analyte. In addition, CeO2-Pd NPs are excellent bioimmobilizers with Pd-NH2 coordination bonds, which can be used to connect antibodies, thereby successfully constructing a highly efficient ECL immunosensor.

[0094] The immunosensor of this invention, constructed using CeO2-Pd NPs as a substrate and Cu-ABEI as a luminescent agent, is a competitive immunosensor for the sensitive detection of the environmental pollutant DBP, with a detection limit as low as 0.263 pg / mL. -1 Furthermore, the method of this invention broadens the design and application of CeO2-Pd NPs and the detection means of DBP, has important practical value, and provides ideas for the detection of other pollutants.

[0095] The above embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A method for preparing a CeO2-Pd NPs composite material, characterized in that, Includes the following steps: Cerium salt and polyvinylpyrrolidone solution are dissolved in a second organic solvent, and then an inorganic solvent is added to carry out a second heating reaction to obtain cerium dioxide; Potassium salt, L-ascorbic acid, and polyvinylpyrrolidone were dissolved in an inorganic solvent and subjected to a third heating reaction. Then, palladium salt solution was added, and the third heating reaction was continued to obtain Pd NPs. Cerium dioxide was dispersed in an inorganic solvent, and Pd NPs were added to obtain a CeO2-Pd NPs composite material.

2. The method for preparing the CeO2-Pd NPs composite material according to claim 1, characterized in that, The temperature of the second heating reaction is 160~180 ℃, and the time of the second heating reaction is 8~10 h; And / or, the temperature of the third heating reaction is 80~90 ℃, the time for the third heating reaction is 10~15 min, and the time for continuing the third heating reaction is 3~4 h; And / or, the cerium salt is selected from cerium nitrate or cerium nitrate hexahydrate; And / or, the potassium salt is selected from potassium bromide; And / or, the palladium salt is selected from sodium tetrachloropalladium(II)ate; And / or, the second organic solvent is selected from ethylene glycol; And / or, the inorganic solvent is selected from water.

3. A CeO2-Pd NPs composite material prepared by the preparation method described in claim 1 or claim 2.

4. The application of a CeO2-Pd NPs composite material prepared by the preparation method described in claim 1 or claim 2 in a sensor.

5. The application according to claim 4, characterized in that, In the sensor, the CeO2-Pd NPs composite material serves as a co-reaction promoter.

6. An immune sensor, characterized in that, It includes a luminescent body that binds to an antigen, and a CeO2-Pd NPs composite material prepared by the preparation method as described in claim 1 or claim 2.

7. The method for preparing the immune sensor according to claim 6, characterized in that, Includes the following steps: Isoluminol and 2-aminoterephthalic acid were dissolved in a first organic solvent, and then glutaraldehyde was added. The mixture was stirred in the dark to obtain solution A. Polyether F127 was dissolved in a mixed solvent, and then copper salt was added to obtain solution B; Solution A and solution B were mixed and subjected to a first heating reaction to obtain a dual-ligand Cu-MOF composite material. The dual-ligand Cu-MOF composite material was dissolved in a buffer solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and antigen solution were added. After incubation, bovine serum albumin was added to obtain a luminescent body that binds to the antigen. An aqueous solution of the CeO2-Pd NPs composite material was dropped onto the electrode surface, followed by the sequential addition of DBP antibody, bovine serum albumin, dibutyl phthalate, and a luminescent solution that binds to the antigen. After incubation, the immunosensor was obtained.

8. The method for preparing the immune sensor according to claim 7, characterized in that, The temperature of the first heating reaction is 120~130 ℃, and the time of the first heating reaction is 12 h; And / or, the mass ratio of isoluminol, 2-aminoterephthalic acid, polyether F127 and copper salt is 4:3:4:8; And / or, the copper salt is selected from one or both of copper nitrate and copper nitrate trihydrate; And / or, the first organic solvent is selected from N,N-dimethylformamide; And / or, the mixed solvent is selected from a mixture of water and anhydrous ethanol.

9. An immunosensor prepared by the method described in claim 7 or claim 8, used as an ECL immunosensor.

10. An application as described in claim 9, characterized in that, The immunosensor is used as an ECL immunosensor to detect dibutyl phthalate. And / or, the detection limit of the immunosensor used as an ECL immunosensor for detecting dibutyl phthalate is 0.263 pg mL. -1 .

Images