A dual-ligand Cu-MOF composite material, an immunosensor, a preparation method and application thereof

Abstract

The present application relates to the technical field of immunosensor, in particular to a kind of double ligand Cu-MOF composite material, immunosensor and preparation method and application.The preparation method of double ligand Cu-MOF composite material includes the following steps: isorumi and 2-amino terephthalic acid are dissolved in first organic solvent, then glutaraldehyde is added, stirring is carried out in dark condition, and solution A is obtained;Polyether F127 is dissolved in mixed solvent, then copper salt is added, and solution B is obtained;Solution A and solution B are mixed, first heating reaction is carried out, and Cu-ABEI is obtained.The present application also provides double ligand Cu-MOF composite material, antigen-binding luminophore and immunosensor and preparation method obtained by the method of the present application.The present application solves the problem of poor ECL signal stability of isorumi, and also solves the problem of low sensitivity of existing sensor for detecting DBP.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immunosensors, in particular to a dual-ligand Cu-MOF composite material, an immunosensor, and a preparation method and application thereof. BACKGROUND

[0002] Dibutyl phthalate (DBP) is an organic compound belonging to phthalate esters. Due to its low volatility and good stability, it is often used as a plasticizer to increase the flexibility and low-temperature resistance of plastics, and has a wide range of applications in industrial production and daily life. However, DBP has certain toxicity, and in the production and use process, once leakage or excessive addition occurs, it will cause extremely serious adverse effects on the environment and human health. Therefore, it is crucial to detect the content of DBP in a timely and accurate manner. However, the existing sensors for detecting the content of DBP have the problem of poor sensitivity.

[0003] The sensor based on electrochemiluminescence (ECL) has the advantages of wide detection range, low background signal, and fast response speed, and has been widely and deeply researched in related fields. Therefore, it is of great practical significance to develop an ECL immunosensor for detecting DBP.

[0004] Copper-based metal organic framework (Cu-MOF) is a kind of porous material composed of metal copper ions and organic ligands, which has the characteristics of large surface area, strong framework flexibility, adjustable pore, and can be "tailored" according to actual needs. With these characteristics, different ligand copper-based MOFs have been developed for sensing. For example, a novel electrochemical biosensor was prepared using a Cu-MOF catalytic material modified with gold nanoparticles for detecting microRNA. However, there is currently no related report on the preparation of an ECL immunosensor for detecting DBP.

[0005] As a derivative of luminol, isoluminol (ABEI) has good luminescence performance, but its ECL signal stability is poor, which to a large extent limits its wide application in the ECL field. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a dual-ligand Cu-MOF composite material, an immunosensor, and a preparation method and application thereof, to solve the problem of poor ECL signal stability of isoluminol (ABEI), and also to solve the problem of low sensitivity of the existing sensors for detecting DBP.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] A preparation method of a dual-ligand Cu-MOF composite material, comprising the following steps:

[0009] dissolving isorumi no and 2-amino terephthalic acid in a first organic solvent, then adding glutaraldehyde (GA), stirring in the dark to obtain solution A;

[0010] dissolving polyether F127 in a mixed solvent, then adding a copper salt to obtain solution B;

[0011] mixing solution A and solution B to perform a first heating reaction to obtain a double-ligand Cu-MOF composite material, namely Cu-ABEI;

[0012] The mass ratio of isorumi no, 2-amino terephthalic acid, polyether F127 and copper salt is 4:3:4:8; the first organic solvent is selected from N,N-dimethylformamide (DMF); and the mixed solvent is selected from a mixture of water and anhydrous ethanol.

[0013] According to the above technical means, by taking the MOF material with copper as the central ion as the metal organic framework, isorumi no and 2-amino terephthalic acid are crosslinked by glutaraldehyde, and then coordinated with copper ions to obtain a double-ligand Cu-MOF composite material. The double-ligand Cu-MOF composite material is used as the luminophore of the ECL sensor, which has excellent ECL performance under the condition that H2O2 is the coreactant, effectively solves the problem of poor ECL signal stability of isorumi no (ABEI), and has great application potential in biological analysis.

[0014] Among them, the full name of polyether F127 is Pluronic F127, which is a polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) triblock copolymer. Polyether F127 is a non-ionic surfactant with good biocompatibility and water solubility. The average molecular weight of polyether F127 is about 12600 g / mol, wherein PEO accounts for about 70 % of the molecular weight, and PPO accounts for about 30 % of the molecular weight.

[0015] Preferably, the temperature of the first heating reaction is 120-130 ℃, and the time of the first heating reaction is 12 h.

[0016] Preferably, the temperature of the first heating reaction is 120 ℃.

[0017] Preferably, the copper salt is selected from one or both of copper nitrate and copper nitrate trihydrate.

[0018] Preferably, the preparation method of the double-ligand Cu-MOF composite material comprises the following steps:

[0019] Isoluminol and 2-amino terephthalic acid are dissolved in N,N-dimethylformamide (DMF), then glutaraldehyde (GA) is added, and the solution is stirred in the dark to obtain solution A;

[0020] Polyether F127 is dissolved in a mixed solution of water and ethanol (volume ratio of water to ethanol 1:1), then Cu(NO3)2·3H2O is added to obtain solution B;

[0021] Solution A and solution B are mixed and ultrasonically treated, then transferred to a high-pressure reaction kettle for first heating reaction, and then the obtained product is centrifuged to obtain a precipitate, which is washed with water, DMF and ethanol alternately for multiple times until the supernatant is clear, and then the precipitate is dried and ground to obtain Cu-ABEI, i.e., a dual-ligand Cu-MOF composite material.

[0022] The application further provides a dual-ligand Cu-MOF composite material prepared by the method.

[0023] The application further provides application of the dual-ligand Cu-MOF composite material prepared by the method as a luminophore of an ECL sensor.

[0024] The application further provides a luminophore binding to an antigen, which is prepared by using an antigen and the dual-ligand Cu-MOF composite material prepared by the method.

[0025] The application further provides a preparation method of the luminophore binding to the antigen, which comprises the following steps:

[0026] The dual-ligand Cu-MOF composite material is dissolved in a buffer solution, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and an antigen solution are added, and then bovine serum albumin (BSA) is added after incubation to obtain the luminophore Cu-ABEI-antigen binding to the antigen.

[0027] Preferably, the antigen is selected from DBP antigens.

[0028] Preferably, the preparation method of the luminophore binding to the antigen comprises the following steps:

[0029] The Cu-ABEI is dissolved in a PBS buffer solution, ultrasonic dispersion is performed, then 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and a DBP antigen solution are added, and overnight incubation is performed under the condition that the temperature is 4 DEG C, then bovine serum albumin (BSA) is added to block non-specific binding sites, and then centrifugation is performed to remove unbound antigen, thereby obtaining a product; the product is redispersed in 1 mL of a PBS solution (pH 7.4) to obtain a luminescent body Cu-ABEI-antigen solution to which the antigen is bound, which is ready for use.

[0030] 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.

[0031] Preferably, the solvent of the DBP antigen solution is a PBS solution (pH 7.4), and the concentration of the DBP antigen in the DBP antigen solution is 4.4 µg / mL. -1 .

[0032] The application further provides an immunosensor comprising the CeO2-Pd NPs composite material and the luminescent body to which the antigen is bound prepared by the preparation method.

[0033] By taking the CeO2-Pd NPs as a substrate and the Cu-ABEI as a luminescent body, a competitive immunosensor is constructed for detecting DBP. The CeO2-Pd NPs provide abundant sites for catalysis of H2O2 and fixation of antibodies, effectively enhance the ECL response, and thus improve the sensitivity of the sensor. The ECL immunosensor constructed based on the above strategy realizes sensitive detection of the environmental pollutant DBP, and solves the problem of low sensitivity of the existing sensor for detecting DBP.

[0034] Preferably, the preparation method of the CeO2-Pd NPs composite material comprises the following steps:

[0035] The cerium salt and the polyvinylpyrrolidone solution are dissolved in a second organic solvent, then an inorganic solvent is added, and a second heating reaction is performed to obtain ceria;

[0036] The potassium salt, L-ascorbic acid and polyvinylpyrrolidone are dissolved in an inorganic solvent, a third heating reaction is performed, then a palladium salt solution is added, and a third heating reaction is continued to obtain Pd NPs;

[0037] The ceria is dispersed in an inorganic solvent, and the Pd NPs are added to obtain the CeO2-Pd NPs composite material.

[0038] Preferably, the potassium salt is selected from potassium bromide.

[0039] Preferably, the palladium salt is selected from sodium tetrachloropalladate(II).

[0040] The palladium salt solution is a palladium salt aqueous solution.

[0041] Through the step-by-step construction and physical compounding strategy, a synergistic interface between the CeO2 carrier and the Pd nanoparticles is formed. 3+ / Ce 4+ The redox pair can participate in the electron transfer cycle; the Pd NPs provide a high-density catalytically active center, and the metal-oxide heterostructure formed after the compounding of the two significantly enhances the interface charge transfer efficiency and the electron enrichment effect of the catalytically active region, so that the sensor used for DBP detection signal (usually ECL signal) is significantly amplified, thereby greatly improving the sensing sensitivity.

[0042] Preferably, the preparation method of the CeO2-Pd NPs composite material comprises the following steps:

[0043] The cerium salt and a polyvinylpyrrolidone (PVP) solution are dissolved in a second organic solvent, then water is added, and a second heating reaction is performed to obtain cerium dioxide;

[0044] Potassium bromide, L-ascorbic acid (AA) and polyvinylpyrrolidone (PVP) are dissolved in water, a third heating reaction is performed, then a Na2PdCl4 aqueous solution is added, and a third heating reaction is continued to obtain Pd nanoparticles, namely Pd NPs;

[0045] The cerium dioxide is dispersed in water, the Pd nanoparticles are added, and stirring is performed overnight to obtain the CeO2-Pd NPs composite material.

[0046] The preparation method of the CeO2-Pd NPs composite material of the present application forms a synergistic interface between the CeO2 carrier and the Pd nanoparticles through a step-by-step construction and physical compounding strategy, and effectively disperses and anchors the Pd NPs through the steric hindrance effect of polyvinylpyrrolidone to prevent agglomeration to expose more active sites, while the Ce 3+ / Ce 4+The redox pair can participate in the electron transfer cycle; Pd NPs provide high-density catalytically active centers, and strong adsorption and rapid catalytic conversion of DBP molecules. The metal-oxide heterostructure formed after the combination of the two significantly enhances the interface charge transfer efficiency and the electron enrichment effect of the catalytically active region, so that the signal (ECL signal) for DBP detection as a sensor is significantly amplified, thereby greatly improving the sensing sensitivity and having great application potential in biological analysis.

[0047] Preferably, the temperature of the second heating reaction is 160-180 ℃, and the time of the second heating reaction is 8-10 h.

[0048] Preferably, the temperature of the second heating reaction is 160 ℃, and the time of the second heating reaction is 8 h.

[0049] Preferably, the temperature of the third heating reaction is 80-90 ℃, the time of the third heating reaction is 10-15 min, and the time of the continued third heating reaction is 3-4 h.

[0050] Preferably, the temperature of the third heating reaction is 80 ℃, the time of the third heating reaction is 10 min, and the time of the continued third heating reaction is 3 h.

[0051] Preferably, the cerium salt is selected from cerium nitrate or cerium nitrate hexahydrate.

[0052] Preferably, the second organic solvent is selected from ethylene glycol.

[0053] Preferably, the method for preparing the CeO2-Pd NPs composite material comprises the following steps:

[0054] The cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and polyvinylpyrrolidone (PVP) (Mw = 58000) solution are dissolved in ethylene glycol (EG), then deionized water is added, and the mixture is transferred to a high-pressure reaction kettle for a second heating reaction. The light purple precipitate is collected, washed with deionized water and anhydrous ethanol in sequence, dried overnight, ground, and then CeO2 is obtained.

[0055] Potassium bromide (KBr), L-ascorbic acid (AA), and polyvinylpyrrolidone (PVP) (Mw = 58000) are dissolved in water, and a third heating reaction is performed under magnetic stirring. Then, an aqueous Na2PdCl4 solution is added, and the mixture is magnetically stirred to continue the third heating reaction. Finally, the solid product is collected by centrifugation and washed with water to obtain Pd nanoparticles, i.e., Pd NPs.

[0056] CeO2 was dispersed in water, Pd nanoparticles were added, and stirring was performed overnight to obtain a CeO2-Pd NPs composite material.

[0057] Preferably, the mass ratio of the cerium nitrate hexahydrate to the PVP (Mw = 58000) solution is 5:2.

[0058] Preferably, the ratio of the KBr, AA, PVP (Mw = 58000) and Na2PdCl4 aqueous solution is 100 mg:20 mg:35 mg:1 mL.

[0059] Preferably, the concentration of Na2PdCl4 in the Na2PdCl4 aqueous solution is 19 mg mL -1 .

[0060] The application further provides a CeO2-Pd NPs composite material prepared by the preparation method.

[0061] The CeO2-Pd NPs composite material of the application, by compounding CeO2 with a high specific surface area as an oxide support and Pd NPs with a high density of catalytically active centers, and introducing the CeO2-Pd NPs composite material into a sensor as a co-reaction promoter, significantly promotes the generation of superoxide anion radicals (O2 ), and realizes double amplification of the ECL signal. Since a too high concentration of the co-reaction agent H2O2 may destroy the antigen-antibody activity, by introducing the co-reaction promoter, the requirement for a high concentration of the co-reaction agent in the traditional ECL system can be effectively avoided, that is, high-sensitivity detection of the target substance can be realized.

[0062] The application further provides an application of the CeO2-Pd NPs composite material prepared by the preparation method in a sensor.

[0063] Preferably, in the sensor, the CeO2-Pd NPs composite material serves as a co-reaction promoter.

[0064] The application further provides a preparation method of an immunosensor, comprising the following steps:

[0065] A CeO2-Pd NPs composite material aqueous solution is added dropwise on the surface of an electrode, and then Ab (DBP antibody), bovine serum albumin (BSA), dibutyl phthalate (DBP) and a luminophore solution combined with an antigen are sequentially added dropwise on the surface of the electrode, and incubation is performed to obtain the immunosensor.

[0066] The prepared light emitter, co-reaction agent and co-reaction promoter ternary ECL signal amplification system effectively improves the detection sensitivity. The CeO2-Pd NPs are introduced as the co-reaction promoter to significantly promote the generation of superoxide anion radicals (O2-·), and the ECL signal is doubled.

[0067] Preferably, the preparation method of the immunosensor comprises the following steps:

[0068] The glassy carbon electrode (GCE) is polished with aluminum oxide powder, and then the CeO2-Pd NPs composite aqueous solution is added dropwise on the surface of the electrode, followed by sequentially adding Ab (DBP antibody) solution, bovine serum albumin (BSA) solution, dibutyl phthalate (DBP) and light emitter solution combined with antigen on the surface of the electrode to modify the surface of the electrode, respectively, and then incubated at 4℃ for 2h.

[0069] Preferably, the Ab (DBP antibody) solution is a PBS solution containing Ab (DBP antibody).

[0070] Preferably, the bovine serum albumin (BSA) solution is a PBS solution containing bovine serum albumin (BSA).

[0071] Preferably, the light emitter solution combined with antigen is a PBS solution containing light emitter combined with antigen.

[0072] The application further provides an application of the immunosensor prepared by the preparation method of the application as an ECL immunosensor.

[0073] The application further provides an application of the immunosensor prepared by the preparation method of the application as an ECL immunosensor for detecting dibutyl phthalate (DBP).

[0074] Preferably, the detection limit of the immunosensor as an ECL immunosensor for detecting dibutyl phthalate (DBP) in contaminated water is 0.263 pg mL -1 .

[0075] Preferably, the immunosensor is used as an ECL immunosensor for detecting dibutyl phthalate (DBP) in contaminated water, with a detection limit as low as 0.263 pg mL -1 .

[0076] Preferably, the contaminated water includes contaminated tap water and contaminated lake water.

[0077] Advantages of the present application:

[0078] The preparation method of the dual-ligand Cu-MOF composite material of the present application is as follows: isorhodamine and 2-amino terephthalic acid are crosslinked by glutaraldehyde, and then coordinated with copper ions to obtain the dual-ligand Cu-MOF composite material, which is used as a luminophore and has stable and excellent ECL signals, and has great application potential in biological analysis.

[0079] The dual-ligand Cu-MOF composite material of the present application is obtained by self-assembly of isorhodamine and 2-amino terephthalic acid with a copper-centered ion MOF material as a metal organic framework, and the dual-ligand Cu-MOF composite material is used as a luminophore, which exhibits excellent ECL performance under the condition of H2O2 as a co-reactant.

[0080] The immunosensor of the present application is constructed by using CeO2-Pd NPs as a substrate and Cu-ABEI as a luminophore, and a competitive ECL immunosensor is constructed, and CeO2-Pd NPs provide abundant sites for catalysis of H2O2 and immobilization of antibodies, effectively enhance the ECL response, and thus improve the sensitivity of the sensor, and realize sensitive detection of environmental pollutants DBP, and has popularization and application value in the technical field of immunosensors. BRIEF DESCRIPTION OF DRAWINGS

[0081] Figure 1 A flowchart of the preparation method of the immunosensor;

[0082] Figure 2 XPS spectra of the dual-ligand Cu-MOF composite material prepared in Example 1, wherein 2A is a full-area XPS energy spectrum, 2B is an XPS fine spectrum of a Cu 2p orbital, 2C is an XPS fine spectrum of an O 1s orbital, 2D is an XPS fine spectrum of an N 1s orbital, 2E is an XPS fine spectrum of a C 1s orbital, and 2F is an Auger electron spectrum of Cu;

[0083] Figure 3SEM, TEM and EDS mapping of the bi-ligand Cu-MOF composite prepared in Example 1, wherein 3A is the SEM image, 3B is the TEM image, 3C is the EDS mapping of all elements, and 3D is the EDS mapping of single element;

[0084] Figure 4 Characterization of the CeO2-Pd NPs composite and CeO2 prepared in Example 3, wherein 4A is the XRD pattern of CeO2, CeO2-Pd NPs, 4B is the SEM image of CeO2, 4C1 is the TEM image of Pd NPs, 4C2 is the local magnification of the TEM image of Pd NPs, 4D is the SEM image of CeO2-Pd NPs, 4E is the EDS mapping of CeO2-Pd NPs, and 4F is the UV-Vis absorption spectrum of CeO2, Pd NPs and CeO2-Pd NPs;

[0085] Figure 5 Characterization of the biosensor, wherein 5A is the EIS spectrum of the electrodes with different modification layers, and 5B is the cyclic voltammogram of the electrodes with different modification layers;

[0086] Figure 6 ECL response results under different conditions, wherein 6A is the ECL response results corresponding to different concentrations of H2O2, 6B is the ECL response results corresponding to different pH values, 6C is the ECL response results corresponding to different concentrations of Cu-ABEI, and 6D is the ECL response results corresponding to different concentrations of CeO2-Pd NPs;

[0087] Figure 7 Results of the luminescence mechanism research, wherein 7A is the ECL response of the electrodes modified with Cu-ABEI, CeO2 / Cu-ABEI and CeO2-Pd NPs / Cu-ABEI in PBS solution, 7B is the ECL 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 H2O2, 7C is the comparison of the catalytic performance of CeO2 and CeO2-Pd NPs, and 7D is the ECL mechanism diagram;

[0088] Figure 8 Performance evaluation results of the sensor, wherein 8A is the ECL response of the sensor to different concentrations of DBP (a~g: 1 pg mL -1 ~ 1µgmL -18A: ECL response of the ECL immunosensor, 8B: DBP detection calibration curve, 8C: stability of the ECL immunosensor under fixed potential scanning, 8D: selectivity of the ECL immunosensor to different detection objects, a: blank, b: DMP, c: DINP, d: BBP, e: DBP, f: DMP + DBP, g: DINP + DBP, h: BBP + DBP in the abscissa, 8E: reproducibility of the ECL immunosensor, 1-6 in the abscissa represent 6 competitive immunosensors Cu-ABEI-antigen / DBP / BSA / Ab / CeO2-Pd NPs / GCE prepared by the preparation method in Example 4, 8F: storage stability of the ECL immunosensor (n = 5). DETAILED DESCRIPTION

[0089] The advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of the preferred embodiments. The present application can also be implemented or applied by different specific embodiments, and the details in the specification can be modified or changed based on different views and applications without departing from the spirit of the present application. It should be understood that the preferred embodiments are only for illustrating the present application, but not for limiting the protection scope of the present application.

[0090] The reagents used in the following examples include: dibutyl phthalate (DBP), DBP antigen, DBP antibody purchased from Shenzhen Anti Biological Technology Co., Ltd. Heteroluminol (ABEI), 2-amino terephthalic acid, copper nitrate trihydrate (Cu(NO3)2·3H2O), glutaraldehyde (GA), cerium nitrate hexahydrate (Ce(NO3)3·6H2O), potassium bromide (KBr), polyether (F127). Polyvinylpyrrolidone (PVP, Mw = 58000), ethylene glycol (EG), L-ascorbic acid (AA), sodium chloropalladate (Na2PdCl4), anhydrous ethanol, N, N-dimethylformamide (DMF) are provided by National Pharmaceutical Chemical Reagent. The purity of the reagents used is analytical grade, and ultrapure water (18.25 MΩ cm -1 ).

[0091] The experimental instruments and equipment used in the following examples are shown in Table 1.

[0092] Table 1 is the experimental instruments and equipment

[0093]

[0094] Example 1

[0095] A preparation method of a dual-ligand Cu-MOF composite material, comprising the following steps:

[0096] S1, 0.1 g of isoluminol and 0.075 g of 2-amino terephthalic acid were dissolved in 10 mL of N,N-dimethylformamide (DMF), then 500 μL of 1% glutaraldehyde was added, and stirred in the dark for 48 h to obtain solution A;

[0097] S2, 100 mg of polyether F127 was dissolved in 20 mL of a mixed solution of water and ethanol (volume ratio of water to ethanol 1:1), and ultrasonically dissolved, then 0.2 g of Cu(NO3)2·3H2O was added to obtain solution B;

[0098] S3, solution A obtained in S1 and solution B obtained in S2 were mixed and ultrasonically treated for 20 min, and then transferred to a high-pressure reaction kettle and reacted at a temperature of 120°C for 12 h. The obtained product was centrifuged to obtain a precipitate, which was washed with water, DMF and ethanol alternately for several times until the supernatant was clear, and then the precipitate was dried under vacuum at a temperature of 60°C for 12 h. The obtained product was ground to obtain Cu-ABEI, i.e. a dual-ligand Cu-MOF composite material.

[0099] Example 2

[0100] A preparation method of a luminescent body Cu-ABEI-antigen binding antigen, comprising the following steps:

[0101] 1.5 mg of Cu-ABEI obtained in Example 1 was dissolved in 750 μL of PBS solution (pH 7.4) and ultrasonically dispersed, then 0.4 mmol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 0.1 mmol of N-hydroxysuccinimide (NHS) and 250 μL of DBP antigen (antigen) solution with a concentration of 4.4 μg mL -1 were added, and then incubated overnight at a temperature of 4°C with shaking; 100 μL of 0.1% bovine serum albumin (BSA) was added to the solution after incubation to block the non-specific binding sites, and then centrifuged to remove the unbound antigen to obtain a product; the obtained product was redispersed in 1 mL of PBS solution (pH 7.4) to obtain a luminescent body Cu-ABEI-antigen solution binding DBP antigen.

[0102] Example 3

[0103] A preparation method of a CeO2-Pd NPs composite material, comprising the following steps:

[0104] S1, 500 mg of cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and 200 mg of polyvinylpyrrolidone (PVP) (Mw = 58000) were dissolved in 15 mL of ethylene glycol (EG), then 1 mL of deionized water was added, and after continuous stirring for 30 min, a clear solution was obtained. The clear solution was transferred to a 50 mL high-pressure reaction kettle, and heated at a temperature of 160 °C for 8 h. The light purple precipitate product was collected, washed with deionized water and anhydrous ethanol three times in turn, and then the product was dried at a temperature of 70 °C overnight. After grinding, CeO2 was obtained and stored for later use;

[0105] S2, 300 mg of potassium bromide (KBr), 60 mg of L-ascorbic acid (AA) and 105 mg of polyvinylpyrrolidone (PVP) (Mw = 58000) were dissolved in 8 mL of water, then heated at a temperature of 80 °C for 10 min under magnetic stirring, then 3 mL of Na2PdCl4 aqueous solution with a concentration of 19 mg mL -1 was added and heated at a temperature of 80 °C for 3 h. Finally, the solid product was collected by centrifugation and washed with water three times to obtain Pd nanoparticles. The Pd nanoparticles were dispersed in 5 mL of water to obtain a Pd nanoparticle (Pd NPs) aqueous solution, which was stored for later use;

[0106] S3, 10 mg of CeO2 prepared in S1 was ultrasonically dispersed in 10 mL of water, then 2 mL of Pd nanoparticle aqueous solution prepared in S2 was added, and stirred overnight to obtain a CeO2-Pd NPs composite material.

[0107] Example 4

[0108] As Figure 1 shown, a preparation method of an immunosensor includes the following steps:

[0109] A glassy carbon electrode (GCE) was polished with aluminum oxide powder, then 10 µL of CeO2-Pd NPs composite aqueous solution with a concentration of 1 mg·mL -1 prepared in Example 3 was added dropwise on the surface of the electrode to modify the surface of the electrode, and then 10 µL of Pd NPs aqueous solution with a concentration of 1.5 µg·mL -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.

[0110] Detection and Analysis

[0111] 1) Characterization of Cu-ABEI

[0112] The Cu-ABEI prepared in Example 1 was analyzed using X-ray photoelectron spectroscopy. The results are as follows: Figure 2 As shown.

[0113] 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 2As shown in E, the sample contains carbon element, and mainly exists in the form of unsaturated carbon (C=C), so the C element spectrum presents an asymmetric structure, and the C=C peak is dominant (284.0 eV). Therefore, during the charging correction, the peak position of the C=C chemical state is taken as the reference, and it is calibrated to 284.0 eV, and the peaks of other elements are also moved by the corresponding numerical value. The XPS data calibration is realized.

[0114] The Cu-ABEI prepared in Example 1 was subjected to SEM test by using a scanning electron microscope, TEM test by using a transmission electron microscope, and EDS test by using an energy dispersive X-ray spectrometer, and the results are shown in Figure 3 .

[0115] As shown in Figure 3 A, the Cu-ABEI presents a flower ball-like morphology, and the surface is a wrinkled sheet structure. Further, the internal structure thereof was studied by TEM, and it can be seen from Figure 3 B that the Cu-ABEI is a solid flower ball with uniform size, and the size is about 2-3 µm. In addition, Figure 3 C and Figure 3 D, the existence and distribution of Cu, O, N and C four elements are presented, which further indicates the successful synthesis of the material.

[0116] 2) Characterization of CeO2-Pd NPs

[0117] The CeO2 prepared in S1 and the CeO2-Pd NPs composite material prepared in S3 of Example 3 were subjected to XRD test by using an X-ray diffractometer, respectively, and the CeO2 prepared in S1 and the CeO2-Pd NPs composite material prepared in S3 of Example 3 were subjected to SEM test by using a scanning electron microscope, respectively, the Pd nanoparticles (Pd NPs) prepared in S2 of Example 3 were subjected to TEM test by using a transmission electron microscope, the CeO2-Pd NPs composite material prepared in S3 of Example 3 was subjected to EDS test by using an energy dispersive X-ray spectrometer, and the CeO2 prepared in S1, the Pd nanoparticles (Pd NPs) prepared in S2 and the CeO2-Pd NPs composite material prepared in S3 of Example 3 were subjected to ultraviolet-visible absorption spectrum test by using an ultraviolet-visible spectrophotometer, respectively, and the results are shown in Figure 4 .

[0118] As shown in Figure 4As can be seen in 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 spectrum of the composite CeO2-Pd NPs has obvious diffraction peaks at 40.2°, which belong to the (111) crystal plane of Pd NPs (PDF #87-0638) in addition to the characteristic peaks of CeO2, thus proving the successful synthesis of the material. From Figure 4 B, it can be seen that the CeO2 is a nanosphere with uniform size and a slightly rough surface, with a size of about 200 nm. The synthesized Pd NPs were characterized by TEM, as shown in Figure 4 C1 and 4C2, which exhibit a uniform square-round morphology with a particle size in the range of about 15-20 nm. It was found that they have obvious lattice fringes, and the calculated lattice fringe spacing is 0.227 nm, which corresponds to the (111) crystal plane of Pd NPs (PDF #87-0638) Figure 4 C2). In addition, from the SEM Figure 4 D, it can be seen that the surface of the CeO2 nanosphere is covered with some bright spots, which are presumably due to the excellent electrical conductivity and small size of the noble metal Pd NPs Figure 4 D). From the TEM Figure 4 E, it can be seen that the EDS mapping spectrum shows the presence and distribution of Ce, O and Pd, further proving the successful synthesis of the material. Finally, qualitative analysis of the three materials CeO2, Pd NPs and CeO2-Pd NPs was performed by analyzing the results of the UV-visible spectrum, as shown in Figure 4 F. From Figure 4 F, it can be seen that CeO2 has a relatively wide characteristic peak in the wavelength range of 280-385 nm, Pd NPs has a characteristic peak at 420 nm, and CeO2-Pd NPs has a clear characteristic peak in a wider wavelength range of 280-450 nm, indicating the successful preparation of the composite material.

[0119] 3) Electrochemical characterization of biosensors with different modification layer numbers

[0120] 3.1) The glassy carbon electrode (GCE) was polished with aluminum oxide powder, and then 10 μL of the CeO2-Pd NPs composite aqueous solution prepared in S3 of Example 3 with a concentration of 2 mg·mL -1 was added to the electrode surface for single-layer modification, and then naturally dried. After modification, the electrode surface was rinsed with PBS solution (pH 7.4) to remove unbound substances, obtaining a CeO2-Pd NPs / GCE electrode (b).

[0121] 3.2) The glassy carbon electrode (GCE) was polished with alumina powder, then 10 μL of the CeO2-Pd NPs composite aqueous solution with a concentration of 2 mg·mL-1 prepared in S3 of Example 3 was added dropwise on the electrode surface, followed by 10 μL of the Ab (DBP antibody) aqueous solution with a concentration of 1.5 μg·mL-1 added dropwise on the electrode surface in sequence, so as to modify the electrode surface with two layers, and then incubated at a temperature of 4 ℃ for 2 h, wherein the electrode surface was rinsed with the PBS solution (pH 7.4) after each layer of modification to remove the unbound substances or biological molecules, so as to obtain the Ab / CeO2-Pd NPs / GCE electrode (c). -1 -1

[0122] 3.3) The glassy carbon electrode (GCE) was polished with alumina powder, then 10 μL of the CeO2-Pd NPs composite aqueous solution with a concentration of 2 mg·mL-1 prepared in S3 of Example 3 was added dropwise on the electrode surface, followed by 10 μL of the Ab (DBP antibody) aqueous solution with a concentration of 1.5 μg·mL-1 and 3 μL of the bovine serum albumin (BSA) aqueous solution with a mass fraction of 1 % added dropwise on the electrode surface in sequence, so as to modify the electrode surface with three layers, and then incubated at a temperature of 4 ℃ for 2 h, wherein the electrode surface was rinsed with the PBS solution (pH 7.4) after each layer of modification to remove the unbound substances or biological molecules, so as to obtain the BSA / Ab / CeO2-Pd NPs / GCE electrode (d). -1 -1

[0123] 3.4) The glassy carbon electrode (GCE) was polished with alumina powder, then 10 μL of the CeO2-Pd NPs composite aqueous solution with a concentration of 2 mg·mL-1 prepared in S3 of Example 3 was added dropwise on the electrode surface, followed by 10 μL of the Ab (DBP antibody) aqueous solution with a concentration of 1.5 μg·mL-1, 3 μL of the bovine serum albumin (BSA) aqueous solution with a mass fraction of 1 % and 10 μL of the dibutyl phthalate (DBP) added dropwise on the electrode surface in sequence, so as to modify the electrode surface with four layers, and then incubated at a temperature of 4 ℃ for 2 h, wherein the electrode surface was rinsed with the PBS solution (pH 7.4) after each layer of modification to remove the unbound substances or biological molecules, so as to obtain the DBP / BSA / Ab / CeO2-Pd NPs / GCE electrode (e). -1 -1

[0124] ​​​​​​Ag / AgCl and Pt were used as reference electrode and counter electrode respectively, and unmodified glassy carbon electrode (GCE) (a), CeO2-Pd NPs / GCE electrode (b) prepared by different modification layers mentioned above, Ab / CeO2-Pd NPs / GCE electrode (c), BSA / Ab / CeO2-Pd NPs / GCE electrode (d), DBP / BSA / Ab / CeO2-Pd NPs / GCE electrode (e) or the electrochemical competitive ECL immunosensor Cu-ABEI-antigen / DBP / BSA / Ab / CeO2-Pd NPs / GCE (f) constructed in Example 4 were used as working electrode respectively to conduct EIS and CV tests. The parameters set during the test were as follows: the high voltage of photomultiplier was 600 V, the voltage scanning range was 0 ~ 0.7 V, and the scanning rate was 0.1 V s -1 . The test base solution was 10 mL PBS with different concentrations of H2O2 (pH = 8). The results are shown in Figure 5 .

[0125] Figure 5 In A and Figure 5 B, the black curve corresponds to the determination result of unmodified glassy carbon electrode (a) as working electrode; the red curve corresponds to the determination result of single-layer modified CeO2-Pd NPs / GCE electrode (b) as working electrode; the blue curve corresponds to the determination result of double-layer modified Ab / CeO2-Pd NPs / GCE electrode (c) as working electrode; the green curve corresponds to the determination result of three-layer modified BSA / Ab / CeO2-Pd NPs / GCE electrode (d) as working electrode; the purple curve corresponds to the determination result of four-layer modified DBP / BSA / Ab / CeO2-Pd NPs / GCE electrode (e) as working electrode; and the yellow curve corresponds to the determination result of five-layer modified sensor Cu-ABEI-antigen / DBP / BSA / Ab / CeO2-Pd NPs / GCE (f) as working electrode, i.e. the determination result of ECL immunosensor as working electrode constructed in Example 4.

[0126] The electrochemical behavior of the proposed biosensor was characterized by EIS and CV tests to verify the layer-by-layer modification process of the glassy carbon electrode. From Figure 5 A, it can be seen that after layer-by-layer modification of 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, and the above-mentioned substances hinder the transmission of electrons. From Figure 5The CV test results of B show that the oxidation peak decreases, which proves the successful modification of the above-mentioned substance, i.e., the successful preparation of the electrochemical competitive ECL immunosensor.

[0127] 4) Condition optimization test

[0128] Influence of Cu-ABEI concentration

[0129] The ECL test adopts a standard three-electrode system, Ag / AgCl and Pt are used as the reference electrode and the counter electrode respectively, the Cu-ABEI prepared in Embodiment 1 is added into deionized water to prepare a mixed solution with a Cu-ABEI concentration of 1.5 mg / mL, and the mixed solution is dropwise modified on a glassy carbon electrode (GCE) to serve as a working electrode, and the corresponding ECL response signal is tested. The parameters set during the test are as follows: the high voltage of the photomultiplier is 600 V, the voltage scanning range is 0 ~ 0.7 V, and the scanning rate is 0.1 V s -1 . The test base solution is 10 mL of a PBS solution with a H2O2 concentration of 0.2 mmol L -1 H2O2 (pH = 8), and the results are shown in Figure 6 C.

[0130] Influence of pH

[0131] The ECL test adopts a standard three-electrode system, Ag / AgCl and Pt are used as the reference electrode and the counter electrode respectively, the Cu-ABEI prepared in Embodiment 1 is added into deionized water to prepare a mixed solution with a Cu-ABEI concentration of 1.5 mg / mL, and the mixed solution is dropwise modified on a glassy carbon electrode (GCE) to serve as a working electrode, and the corresponding ECL response signal is tested. The parameters set during the test are as follows: the high voltage of the photomultiplier is 600 V, the voltage scanning range is 0 ~ 0.7 V, and the scanning rate is 0.1 V s -1 . The test base solution is 10 mL of a PBS solution with a H2O2 concentration of 0.2 mmol L -1 H2O2 (pH = 8), and the results are shown in Figure 6 B.

[0132] Influence of H2O2

[0133] The ECL test adopts a standard three-electrode system, Ag / AgCl and Pt are used as the reference electrode and the counter electrode respectively, the Cu-ABEI prepared in Example 1 is added to deionized water to prepare a mixed solution with a Cu-ABEI concentration of 1.5 mg / mL, and the mixed solution is added dropwise to modify the glassy carbon electrode (GCE), then the glassy carbon electrode (GCE) is used as the working electrode to test the corresponding ECL response signal. The parameters set during the test are as follows: the high voltage of the photomultiplier tube is 600 V, the voltage scanning range is 0~0.7 V, and the scanning rate is 0.1 V s -1 . The ECL response corresponding to the PBS solution with different concentrations of H2O2 (pH = 8) is shown in Figure 6 A.

[0134] Effect of CeO2-Pd NPs concentration

[0135] The ECL test adopts a standard three-electrode system, Ag / AgCl and Pt are used as the reference electrode and the counter electrode respectively, the CeO2-Pd NPs prepared in S3 of Example 3 are 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 respectively, and the mixed solutions are added dropwise to modify the glassy carbon electrode (GCE) to form a modified layer, then the Cu-ABEI prepared in Example 1 is added to deionized water to prepare a mixed solution with a Cu-ABEI concentration of 1.5 mg / mL, and the mixed solution is added dropwise to modify the glassy carbon electrode (GCE) to form two modified layers, which are used as the working electrode to test the corresponding ECL response signal. The parameters set during the test are as follows: the high voltage of the photomultiplier tube is 600 V, the voltage scanning range is 0~0.7 V, and the scanning rate is 0.1 V s -1 . The ECL response corresponding to the PBS solution with a concentration of 0.2 mmol L -1 H2O2 (pH = 8) is shown in Figure 6 D.

[0136] As can be seen from Figure 6 A, within a certain range, the ECL response has a maximum value with the change of H2O2 concentration, so the optimal concentration is 0.2 mmol L -1 . As can be seen from Figure 6 B, during the process of increasing the pH from 6.0 to 8.5, the ECL signal gradually increases with the increase of the pH, which indicates that the alkaline environment is conducive to the luminescence, but the strong alkali environment may cause the inactivation of the antigen and antibody, so 8.0 is selected as the optimal pH. As can be seen from Figure 6 C, when the Cu-ABEI concentration is 1.5 mg mL-1 ECL intensity reached the maximum value. From Figure 6 It can be seen from Fig. 8D that the optimal concentration of CeO2-Pd NPs is 1 mg mL -1 .

[0137] 5)ECL mechanism research

[0138] To explore the possible mechanism of ECL, the glassy carbon electrode (GCE) was polished with alumina powder, and then the electrode surface was added with Cu-ABEI aqueous solution with a concentration of 1 mg / mL prepared in Example 1, CeO2 aqueous solution with a concentration of 1 mg / mL prepared in S1 of Example 3 and Cu-ABEI aqueous solution with a concentration of 1 mg / mL prepared in Example 1, successively, and CeO2-Pd NPs composite material aqueous solution with a concentration of 1 mg / mL prepared in S3 of Example 3 and Cu-ABEI aqueous solution with a concentration of 1 mg / mL prepared in Example 1, successively, to obtain three kinds of electrodes modified in different ways, which are Cu-ABEI modified electrode, CeO2 / Cu-ABEI modified electrode and CeO2-Pd NPs / Cu-ABEI modified electrode, respectively. Then, the above electrodes modified with different materials were used as working electrodes, Ag / AgCl and Pt were used as reference electrode and counter electrode, respectively, to construct a standard three-electrode system for ECL test, and the ECL response of the above electrodes modified with different materials was tested. The results are shown in -1 Fig. 8. Figure 7

[0139] From Figure 7 A, it can be seen that the ECL signal of Cu-ABEI modified electrode can be almost negligible under the test condition without H2O2, while the ECL signal of Cu-ABEI can reach about 6000 in PBS buffer solution containing 0.1 mmol L -1 H2O2, which indicates that H2O2 is an indispensable co-reactant for Cu-ABEI ECL. The signals of CeO2 / Cu-ABEI and CeO2-Pd NPs / Cu-ABEI modified electrodes are increased to 1.1k and 1.6k, respectively, in the presence of H2O2, realizing the double amplification of the signal. In addition, the signals of CeO2 / Cu-ABEI and CeO2-Pd NPs / Cu-ABEI modified electrodes are similar to that of Cu-ABEI, which can be almost negligible, in the test buffer solution without H2O2, which indicates that the ECL signal enhancement of CeO2 and CeO2-Pd NPs is due to the catalytic effect on H2O2, rather than Cu-ABEI. At the same time, from Figure 7 ​As 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.

[0140] 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).

[0141] Route 1:

[0142]

[0143] Route 2:

[0144]

[0145] ECL launch:

[0146]

[0147] 6) Sensor performance evaluation

[0148] 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 .

[0149] Table 2 shows the detection range results obtained by different detection methods.

[0150]

[0151] 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... -1DBP 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.

[0152] 7) Sample testing

[0153] 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.

[0154] Table 3. Analytical results of DBP in water samples

[0155]

[0156] 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.

[0157] In summary, the present application is obtained by cross-linking isorumi no and 2-amino terephthalic acid by glutaraldehyde and then coordinating with copper ions to obtain a dual-ligand Cu-MOF (Cu-ABEI). The material has excellent ECL performance and great application potential in biological analysis. The introduction of CeO2-Pd NPs as a co-reaction promoter provides abundant sites for the catalysis of H2O2 and the immobilization of antibodies, realizes the dual amplification of the ECL signal and is conducive to the subsequent construction of an immunosensor. Then, a competitive immunosensor is constructed for the sensitive detection of environmental pollutants DBP using CeO2-Pd NPs as a substrate and Cu-ABEI as a luminophore, and the detection limit is as low as 0.263 pg mL -1 In addition, the method of the present application widens the design application of Cu-MOF and the detection means of DBP, has important practical value and provides a train of thought for the detection of other pollutants.

[0158] The above examples are only preferred embodiments for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art on the basis of the present application is within the protection scope of the present application.

Claims

1. An application of an immunosensor for detecting dibutyl phthalate, characterized in that, Including CeO2-Pd NPs composite materials, and luminescent organisms that bind antigens; The immunosensor is used as a competitive ECL immunosensor to detect dibutyl phthalate. The antigen-binding luminescent body is made of the antigen and a dual-ligand Cu-MOF composite material. The preparation method of the dual-ligand Cu-MOF composite material 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 mass ratio of isoluminol, 2-aminoterephthalic acid, polyether F127 and copper salt is 4:3:4:8; The first organic solvent is selected from N,N-dimethylformamide; The mixed solvent is selected from a mixture of water and anhydrous ethanol; The preparation method of the CeO2-Pd NPs composite material includes the following steps: Potassium bromide, L-ascorbic acid and polyvinylpyrrolidone were dissolved in water and subjected to a third heating reaction. Then, Na2PdCl4 aqueous solution was added and the third heating reaction was continued to obtain Pd nanoparticles. Cerium dioxide was dispersed in water, Pd nanoparticles were added, and the mixture was stirred overnight to obtain a CeO2-Pd NPs composite material.

2. The application of the immunosensor according to claim 1 for detecting dibutyl phthalate, characterized in that, The temperature of the first heating reaction is 120~130 ℃, and the heating reaction time is 12 h; And / or, the copper salt is selected from one or both of copper nitrate and copper nitrate trihydrate.

3. The application of the immunosensor according to claim 1 for detecting dibutyl phthalate, wherein the method for preparing the antigen-binding luminescent material comprises the following steps: 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.

4. The application of the immunosensor according to claim 3 for detecting dibutyl phthalate, characterized in that, The antigen is selected from DBP antigen.

5. The application of the immunosensor according to claim 1 for detecting dibutyl phthalate, characterized in that, 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.

6. The application of the immunosensor according to claim 1 for detecting dibutyl phthalate, characterized in that, The method for preparing the immune sensor includes the following steps: An aqueous solution of 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.

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