Preparation method of chemiluminescence enzyme-linked immunosorbent assay sensor for progesterone detection

Abstract

The invention relates to a preparation method of a chemiluminescence enzyme-linked immunosorbent assay sensor for progesterone detection. According to the sensor, magnetic Fe3O4 nanoparticles serve as a core, a surface functionalization modification acrylamide molecular layer is adopted, a polymer serves as an imprinting shell layer, the imprinting shell layer for eluting imprinting molecules has recognition sites for progesterone molecules, chemiluminescence immunoassay is conducted after the imprinting shell layer is combined with artificial enzyme, detection on the progesterone molecules is achieved, and the detection limit is 5.52 * 10 <-11 > g / L. The preparation method comprises the following steps: firstly, modifying the surfaces of magnetic Fe3O4 nanoparticles, then preparing a progesterone-imprinted magnetic nano molecularly imprinted polymer by taking progesterone as an imprinted molecule, acrylic acid as a functional monomer, N, N-dimethyl bisacrylamide as a cross-linking agent and azodiisobutyronitrile as an initiator, and finally, preparing the progesterone-imprinted magnetic nano molecularly imprinted polymer. The artificial antibody after elution of the target molecule has a specific recognition site for progesterone, the progesterone artificial antibody is labeled by an artificial enzyme, and chemiluminescence immunoassay is carried out in a luminol-H2O2 system to realize trace detection of progesterone.

Description

Technical Field

[0001] The invention relates to the field of material science, and in particular to a method for preparing a chemiluminescent enzyme-linked immunosorbent sensor for detecting progesterone. Background Art

[0002] Steroid hormones, also known as steroid hormones, include androgens, progestins, corticosteroids and estrogens, which are derived from cholesterol in vertebrates. Endogenous steroid hormones participate in a variety of human physiological activities. Their metabolic abnormalities are highly correlated with a variety of diseases and are very important for fertility, growth, metabolism, and immune function. Among them, sex hormones (progesterone, androgen, estrogen) are related to the development of many diseases, such as polycystic ovary syndrome, breast cancer, prostate cancer, Alzheimer's disease, depression, etc. However, their concentration in the human body is low and they are greatly interfered by the matrix, making it difficult to accurately detect. As one of the steroid hormones, progesterone plays an irreplaceable role in maintaining human physical and mental health. Its concentration in the body is low. Literature reports that the content in plasma and serum ranges from picograms to nanograms per milliliter, and there are large differences in concentration. In addition to the differences caused by different analytical methods, the progesterone concentration is also affected by factors such as gender, age, and environment, so it is necessary to establish a highly sensitive progesterone hormone quantitative detection technology.

[0003] There are many methods for detecting steroid hormones, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS), which is a highly sensitive and specific detection method that can detect multiple steroid hormones at the same time. For example, a study has developed a method that can simultaneously detect 13 steroid hormones in serum, including pregnenolone, progesterone, etc. LC-MS / MS technology has become the "gold standard" method for steroid hormone detection due to its high throughput and accuracy; a detection method based on double derivatization treatment to analyze steroid hormones, this method improves the retention and separation of steroid hormones on reversed-phase liquid chromatography through derivatization reactions, and at the same time improves the mass spectrometry detection sensitivity of low-abundance steroid hormones; gas chromatography-mass spectrometry analysis (GC-MS) This method uses mass spectrometry for detection after gas chromatography separation, which is suitable for the analysis of steroid hormones; liquid chromatography-mass spectrometry analysis (LC-MS) is similar to LC-MS / MS. This method is also based on the combination of liquid chromatography and mass spectrometry for the detection of steroid hormones. Zhou Fang et al. published an invention patent (CN112763602A) "A rapid detection method for steroid hormones in serum". The invention relates to the field of hormone detection technology and discloses a rapid detection method for steroid hormones in serum, including the steps of hormone calibration preparation, sample preparation, ultra-high performance liquid chromatography separation, tandem quadrupole mass spectrometry detection, single-point quantitative analysis, etc. The detection method innovatively uses isotope-labeled [13C, 2H 3]-Methoxyamine (hydrochloride) derived hormone calibrator, unisotope-labeled methoxyamine derived serum sample, the two derivative products are mixed in a certain volume ratio, separated by ultra-high performance liquid chromatography, detected by triple quadrupole tandem mass spectrometry, and quantified by single-point method, which can quickly and accurately detect 13 steroid hormones in serum samples.

[0004] At present, immunoassay is the most promising rapid detection technology. The sensitivity of chemiluminescent immunoassay is very high, which enables it to detect extremely low concentrations of substances, which is of great significance for the early diagnosis of diseases. Xu Zhuangjian et al. disclosed the invention patent (CN201610495810.8) "A method and process for rapid detection of urinary steroid hormones by immunochemiluminescence". The invention uses β-glucuronidase to enzymatically hydrolyze steroid hormones in urine. This is because steroid hormones often exist in the form of glucuronidation in the body. Enzymatic hydrolysis can release free steroid hormones, which is convenient for subsequent detection. Then, based on the detection method of immune response, specific antibodies bind to the antigen of steroid hormones to form an antigen-antibody complex. In this patent, after the antibody binds to the steroid hormone, the presence of the complex is detected by chemiluminescence, thereby realizing the quantitative analysis of steroid hormones. Wang Xianjun et al. disclosed the invention patent (CN201510227904.2) "A quantitative detection method for luteinizing hormone (LH)". The invention uses a sandwich method to determine the LH level in serum, using anti-LH antibodies as coating antibodies, adding LH calibrators or serum to be tested, and then adding another anti-LH antibody marker for reaction. After sufficient washing, a luminescent substrate is added to determine its luminescence intensity (RLU). The LH content in the sample can be calculated based on the standard curve, and the RLU value of the sample increases with the increase in LH concentration. Chemiluminescent immunoassay can accurately quantify the LH level in the sample due to its high sensitivity and specificity, which is crucial for diagnosing and monitoring LH-related disease states. The advantage of this detection method is its high sensitivity and specificity, which can provide accurate LH quantitative results, which is helpful for clinical diagnosis and treatment monitoring.

[0005] Although the above-mentioned methods such as gas chromatography, liquid chromatography and chemiluminescence immunoassay have high sensitivity, they use large instruments, are expensive and have poor selectivity. Therefore, it is necessary to seek a convenient and highly selective method for identifying target analytes. Molecular imprinting polymers have unique structural heterogeneity, so they have high selectivity for target molecules. At present, some teams have conducted extensive research on it. Hu Xiaozhong et al. have published an invention patent (CN101177501A) "A method for preparing a progesterone molecular imprinting polymer", proposing a method for preparing a progesterone molecular imprinting molecule to achieve the enrichment and purification of progesterone drug residues in cosmetics. The progesterone molecular imprinting polymer is a progesterone molecular imprinting polymer that polymerizes the template molecule progesterone, the functional monomer tetravinylpyridine, the cross-linking agent N, N-methylenebisacrylamide and the initiator azobisisobutyronitrile into a progesterone molecular imprinting polymer. After elution and drying, it is used as an adsorbent to adsorb the progesterone drug residues in the test component of the cosmetic sample, thereby establishing a rapid, sensitive and convenient method for detecting progesterone in cosmetics.

[0006] In summary, compared with natural antibodies, molecular imprinting polymers as biomimetic antibodies not only have better thermal stability and chemical stability, but can also be used under a wider range of conditions, including extreme pH values ​​and temperatures, while antibodies may lose their activity under these conditions, and will not cause immune responses, making them potentially advantageous in in vivo applications. Surface molecular imprinting technology can directly establish imprinting sites on the surface of the carrier that are identical or similar in structure to the target to be separated. The prepared molecular imprinting polymers have recognition sites that are complementary to the function and shape of the template and have high affinity and selectivity for the target molecule. Molecular imprinting polymers are polymers that mimic the interaction mechanism between receptors and antibodies and have specific recognition of template molecules. The preparation method of molecular imprinting polymers is to dissolve the template (target) molecule, functional monomers and cross-linking agents in an appropriate solvent, and form highly cross-linked polymers by free radical polymerization under certain experimental and reaction conditions. After polymerization, the template is removed. This step is usually eluted using a simple weakly acidic organic solvent or a Soxhlet extraction device. After elution, nano-sized template cavities are left. The shape, size and functional groups of the cavities are complementary to the binding sites of the template molecules. Molecularly imprinted polymers have many advantages: high selectivity, reusability, high chemical and physical stability (resistant to high temperature, high pressure, acid and alkali), low production cost and easy preparation.

[0007] Natural enzymes play an extremely important role in the field of immunoassays. However, some inherent defects of natural enzymes, such as high cost and poor stability, limit their application. In order to overcome these limitations, researchers began to explore enzyme mimics based on nanomaterials, which have been proven to be viable alternatives to natural enzymes. These nanomaterials include covalent organic frameworks (COFs), carbon nanomaterials, and precious metal nanoparticles. They not only show broad application potential in the fields of environmental protection, energy resources, and biosensors, but also show significant advantages in cost-effectiveness, ease of preparation, and stability. The key advantage of these nanomaterial-based enzyme mimics is that they can simulate the catalytic specificity and efficiency of natural enzymes. Cheng Jianhua et al. published an invention patent (CN113106079A) "An iron-based metal organic framework material for fixing peroxidase and its preparation method and application". The material prepared by this invention is similar to H 2 O 2 It has excellent affinity with TMB and can efficiently catalyze H 2 O 2 TMB is oxidized to a blue color product and then detected by H 2 O 2 The paper shows that a method for preparing peroxidase fixed on an iron-based metal organic framework material and its application in the removal of bisphenol A has the advantages of low cost, good stability and simple preparation. Luo Shuai et al. from Southwest University published an academic paper (Water dispersible cobalt single-atom catalysts with efficient chemiluminescence enhancement for sensitive bioassay) using hybrid MOFs Fe 2 O 3 / MIL-100(Fe) was used as the carrier and the cobalt capsule was prepared by solvothermal method. Compared with the original MOFs MIL-100(Fe), the carrier has a higher loading capacity, and the loading content of cobalt is as high as 4.69%. 2 O 3 The injection of cobalt atoms into MIL-100(Fe) greatly increased the specific surface area of ​​charge carriers by 68 times. -1 The CSACs can catalyze H 2 O 2 Produces a large amount of active oxygen, greatly enhancing the luminol-H 2 O 2CSACs also exhibit satisfactory dispersibility in aqueous media. Benefiting from these attractive properties, CSACs can be used as sensitive signal probes in chemiluminescent immunoassays to detect carbendazim in Chinese herbal medicines. Therefore, enzyme mimics based on nanomaterials are becoming an important alternative in the field of chemiluminescent immunoassays due to their low cost, easy preparation, and excellent stability, as well as their catalytic specificity and efficiency.

[0008] Chemiluminescent immunoassay is a detection method that combines chemiluminescence and immune response, with the advantages of high sensitivity, strong specificity, and simple operation. This technology is widely used in clinical diagnosis, environmental monitoring, food safety, and drug analysis. It consists of two parts: the immune response system and the chemiluminescent analysis system. In the presence of antigens and antibodies, the binding region of the antibody binds to the epitope of the antigen to form an immune complex. The chemiluminescent analysis system uses chemiluminescent substances to form an excited intermediate under the catalysis of the catalyst and the oxidation of the oxidant. When this intermediate returns to a stable ground state, it emits photons. The intensity of the luminescent signal is linearly related to the concentration of the analyte under certain conditions, so that the content of the analyte can be determined.

[0009] The present invention mainly prepares magnetic nanoparticle artificial antibodies with specific recognition ability for progesterone, and performs trace detection of progesterone by chemiluminescence immunoassay, which overcomes the problem of poor selectivity of chemiluminescence and provides recognition signals for molecular imprinting recognition of progesterone. At the same time, there is no report on using progesterone as a target molecule, imprinting it to prepare magnetic nanoparticles, and detecting progesterone by chemiluminescence immunoassay. The present invention uses magnetic nanoparticles as cores and polymers as imprinted shells, eluting progesterone imprinted molecules located in the shells, forming a cavity structure with complementary structure, size and functional groups to the imprinted molecules inside the shells, and the imprinted shells of the eluted imprinted molecules have specific recognition sites for progesterone molecules, and selective recognition and detection of target analyte progesterone molecules are achieved by chemiluminescence immunoassay technology. Summary of the invention

[0010] Purpose of the invention: In order to make up for the deficiencies of the prior art, the present invention invents a method for preparing a chemiluminescent enzyme-linked immunosorbent assay for progesterone detection. The method has the advantages of simple preparation steps, high sensitivity, high selectivity, strong specificity, multiple binding sites, reusability and low cost.

[0011] The present invention is realized by the following technical scheme: a method for preparing a chemiluminescent enzyme-linked immunosorbent sensor for progesterone detection, characterized in that: the sensor is a magnetic Fe 3 O 4The nanoparticles are cores, the surface is functionally modified with an acrylamide molecular layer, the polymer is an imprinted shell layer, the progesterone imprinted molecules located in the shell layer are eluted, and a cavity structure having a structure, size and functional group complementary to the imprinted molecules is formed inside the shell layer. The imprinted shell layer of the eluted imprinted molecules has a specific recognition site for the progesterone molecules, and the selective recognition and detection of the target analyte progesterone molecules is achieved by combining with an artificial enzyme and performing chemiluminescent immunoassay. The preparation process of the chemiluminescent enzyme-linked immunosorbent sensor includes the following three steps: The first step is Fe 3 O 4 Preparation of artificial antibodies based on magnetic nanoparticles: Fe 3 O 4 Magnetic nanoparticles coated with SiO 2 Shell, 3-aminopropyltriethoxysilane ethyl oxygen hydrolyzes, and reacts with Fe through silanization reaction. 3 O 4 @SiO 2 The free silanol groups on the surface of the Fe react to form -Si-O-Si-, thereby grafting amino groups to obtain Fe 3 O 4 @NH 2 , the synthesized Fe 3 O 4 @NH 2 Place it in a 100mL three-necked flask, dry it thoroughly, add 20-40mL of toluene solution and 50-150mg of anhydrous potassium carbonate, disperse it evenly by ultrasonication, deoxygenate it with nitrogen for 5 minutes, put it in an ice bath, stir it for 10 minutes, then add 1-3mL of acryloyl chloride, continue mechanical stirring, react for 4-6 hours, rinse it with ultrapure water for 3 times, and dry it in a vacuum drying oven for 20-24 hours to obtain Fe 3 O 4 @AA nanoparticles, take progesterone and acrylic acid in a molar ratio of 1:6 to 10:6 and dissolve them in a three-necked flask of acetonitrile solution, stir mechanically at 20 °C for 3 to 5 h to mix, then add 0.2 to 0.8 g of the Fe 3 O 4 @AA magnetic nanoparticles were ultrasonically dispersed, and then 1.80 ~ 1.90 mL N, N-dimethylbisacrylamide and 50 ~ 65 mg azobisisobutyronitrile were added. After nitrogen was introduced for 10 min, the polymerization reaction was stirred at 55 ~ 65 °C for 22 ~ 26 h to obtain the polymerization product Fe 3 O 4 @AA@MIPs nanoparticles were extracted by Soxhlet extraction using a mixed solution of methanol and acetic acid with a volume ratio of 7:3 as the elution solvent to elute Fe 3 O 4@AA@MIPs nanoparticles contain progesterone, and the Fe 3 O 4 @AA@MIPs were placed in a vacuum oven and dried for 24 h to obtain magnetic Fe 3 O 4 @AA@MIPs artificial antibodies based on magnetic nanoparticles; The second step is the preparation of artificial enzyme-labeled progesterone antibody: 3.2 ~ 3.8 mg Co-MOFs Fe 2 O 3 / MIL-100(Fe) was dispersed in 0.5 ~ 1.5 mL of dilution buffer containing 20 mg / mL 4-(maleimido)phenyl isocyanate and activated at 20 °C for 3 s. 2 O 3 / MIL-100(Fe) for 1 h, the solution was removed by centrifugation, and the activated Co-MOFs Fe 2 O 3 / MIL-100(Fe) was redispersed in 0.5 ~ 1.5 mL phosphate buffer solution containing 20 ~ 80 μg progesterone artificial antibody. After incubation for 10 ~ 14 h, a 0.05 ~ 0.15 M glycine solution was added to the above buffer solution to quench the excess activated groups to terminate the reaction. After filtration, the artificial enzyme-labeled progesterone antibody was obtained and redispersed in 1.0 mL PBS buffer solution; 1.3 The third step is the chemiluminescent enzyme-linked immunosorbent sensor: the progesterone-BSA conjugate was diluted to 2.0 μg / mL with coating buffer and coated into the wells of a 96-well microplate at 37°C for 2 to 4 h. After the microplate was washed, 150 μL of superblock blocking buffer was used to block the coated microplate wells for 1 to 2 h. The blocked microplate wells were washed again, and 40 to 60 μL of progesterone sample solution and an equal volume of artificial enzyme-labeled progesterone antibody suspension were injected. The solution was incubated for 1 h. After washing to remove excess reactants, 100 μL of 1.0×10 -6 M luminol solution and 50 μL of 0.1MH 2 O 2 The solution is injected into each microplate well to collect the chemiluminescent immunoassay intensity for quantifying progesterone, thereby achieving progesterone detection.

[0012] Compared with the prior art, the advantages of the present invention are: In the present invention, we use the magnetic nanoparticles imprinted biomimetic antibodies and artificial enzymes prepared based on molecular imprinting technology to achieve trace detection of progesterone by using chemiluminescence. Magnetic nanoparticles imprinted biomimetic antibodies are particularly suitable as a detection tool for progesterone. First, magnetic nanoparticles are prepared, and then the surface of the magnetic nanoparticles is modified to prepare magnetic Fe 3 O 4 Artificial antibodies, in magnetic Fe 3 O 4 The progesterone imprinted molecules located in the shell are eluted in the artificial antibody, and the interior of the shell forms a cavity structure complementary to the imprinted molecule structure, size and functional group, and the microspheres of the eluted imprinted molecules have specific recognition sites for the target analyte molecules. Secondly, the molecular imprinted biomimetic antibody is combined with the chemiluminescent artificial enzyme-linked immunosorbent assay, and the specificity of molecular imprinting is combined with the sensitivity of chemiluminescence. This magnetic molecular imprinted biomimetic antibody with recognition effect on progesterone molecules is based on nanotechnology and molecular imprinting technology, and shows high selectivity and high sensitivity detection of progesterone target molecules. Therefore, a chemiluminescent enzyme-linked immunosorbent sensor for progesterone detection prepared by the present invention has the advantages of simple preparation steps, high selectivity, strong sensitivity, strong specificity, multiple binding sites, reusability, low cost, etc.

[0013] Compared with the prior art, Gao Daming et al. published an invention patent (CN202110969739.3) "A method for preparing a nano-titanium dioxide artificial antibody shell for the recognition and degradation of chlorpyrifos", which discloses a method for preparing a nano-titanium dioxide artificial antibody shell for the recognition and degradation of chlorpyrifos. The method uses chemiimmunoluminescence to detect trace amounts of artificial enzyme-labeled antigen molecules with a detection limit of 1.98×10 -10 mg / L, used in immunoassay technology, it has the advantages of high sensitivity, high specificity, simplicity, speed, ease of operation and high stability.

[0014] The present invention is firstly Fe 3 O 4 Preparation of artificial antibodies based on magnetic nanoparticles: Fe 3 O 4 Magnetic nanoparticles coated with SiO 2 Shell, 3-aminopropyltriethoxysilane ethyl oxygen hydrolyzes, and reacts with Fe through silanization reaction. 3 O 4 @SiO 2 The free silanol groups on the surface of the Fe react to form -Si-O-Si-, thereby grafting amino groups to obtain Fe 3 O 4 @NH 2 , the synthesized Fe 3 O4 @NH 2 Place it in a 100 mL three-necked flask, dry it thoroughly, add 20 ~ 40 mL of toluene solution and 50 ~ 150 mg of anhydrous potassium carbonate, disperse it evenly by ultrasonication, deoxygenate it with nitrogen for 5 min, put it in an ice bath, stir it for 10 min, then add 1 ~ 3 mL of acryloyl chloride, continue mechanical stirring, react for 4 ~ 6 h, rinse it with ultrapure water 3 times, and dry it in a vacuum drying oven for 20 ~ 24 h to obtain Fe 3 O 4 @AA magnetic nanoparticles, take progesterone and acrylic acid in a molar ratio of 1:6 to 10:6 and dissolve them in a three-necked flask of acetonitrile solution, stir mechanically at 20 °C for 3 to 5 h to mix, then add 0.2 to 0.8 g of the Fe 3 O 4 @AA magnetic nanoparticles were ultrasonically dispersed, and then 1.80 ~ 1.90 mL N, N-dimethylbisacrylamide and 50 ~ 65 mg azobisisobutyronitrile were added. After nitrogen was introduced for 10 min, the polymerization reaction was stirred at 55 ~ 65 °C for 22 ~ 26 h to obtain the polymerization product Fe 3 O 4 @AA@MIPs magnetic nanoparticles were extracted by Soxhlet extraction using a mixed solution of methanol and acetic acid with a volume ratio of 7:3 as the elution solvent to elute Fe 3 O 4 @AA@MIPs magnetic nanoparticles contain progesterone, and the Fe 3 O 4 @AA@MIPs were placed in a vacuum oven and dried for 24 h to obtain Fe 3 O 4 @AA@MIPs artificial antibodies based on magnetic nanoparticles; The second step is the preparation of artificial enzyme-labeled progesterone antibody: 3.2 ~ 3.8 mg Co-MOFs Fe 2 O 3 / MIL-100(Fe) was dispersed in 0.5 ~ 1.5 mL of dilution buffer containing 20 mg / mL 4-(maleimido)phenyl isocyanate and activated at 20 °C for 3 s. 2 O 3 / MIL-100(Fe) for 1 h, the solution was removed by centrifugation, and the activated Co-MOFs Fe 2 O 3 / MIL-100(Fe) was redispersed in 0.5 ~ 1.5 mL phosphate buffer solution containing 20 ~ 80 μg progesterone artificial antibody. After incubation for 10 ~ 14 h, a 0.05 ~ 0.15 M glycine solution was added to the above buffer solution to quench the excess activated groups to terminate the reaction. After filtration, the artificial enzyme-labeled progesterone antibody was obtained and redispersed in 1.0 mL PBS buffer solution; The third step is the chemiluminescent enzyme-linked immunosorbent sensor: the progesterone-BSA conjugate was diluted to 2.0 μg / mL with coating buffer and coated into the wells of a 96-well microplate at 37°C for 2 to 4 h. After the microplate was washed, 150 μL of superblock blocking buffer was used to block the coated microplate wells for 1 to 2 h. The blocked microplate wells were washed again, and 40 to 60 μL of progesterone sample solution and an equal volume of artificial enzyme-labeled progesterone antibody suspension were injected. The solution was incubated for 1 h. After washing to remove excess reactants, 100 μL of 1.0×10 -6 M luminol solution and 50 μL of 0.1MH 2 O 2 The solution is injected into each microplate well to collect the chemiluminescent immunoassay intensity for quantifying progesterone, thereby achieving progesterone detection.

[0015] In summary, the chemiluminescent enzyme-linked immunosorbent assay sensor obtained in the present invention can be used to detect progesterone: First, the artificial antibody prepared above with progesterone as the imprinting molecule has a corresponding cavity structure with the progesterone after binding with the artificial enzyme after elution, and can recognize progesterone with high selectivity.

[0016] Second, the prepared magnetic nanomaterial artificial antibody has strong magnetism under the action of an external magnetic field, which facilitates separation and purification and reduces losses during the separation and purification process.

[0017] Third: Compared with the traditional molecular imprinting technology, the present invention combines chemiluminescent immunoassay. By adding newly prepared artificial enzymes, this enzyme mimic based on nanomaterials shows the advantages of low cost, easy preparation and excellent stability. In addition, if its catalytic specificity and efficiency can be comparable to those of natural enzymes, we use it to replace natural enzymes to participate in chemiluminescent immunoreaction and build a better chemiluminescent immunoassay platform. Fourthly, the enzyme-labeled antigen enters the recognition site of the prepared artificial antibody, and the chemiluminescence instrument is used to detect the change in luminescence intensity before and after the enzyme-labeled antigen binds to the antibody to achieve the detection of progesterone. This method has high sensitivity and good selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The magnetic Fe prepared by the present invention3 O 4 Technology roadmap for progesterone detection using nanoparticle-mimetic antibodies.

[0019] Figure 2 The Fe prepared by the present invention 3 O 4 Magnetic response diagram of nanoparticles (A) and hysteresis loop spectrum (B).

[0020] Figure 3 The Fe prepared by the present invention 3 O 4 (A)Fe 3 O 4 @SiO 2 (B) Fe 3 O 4 SEM images of @AA@MIPs (C) and Fe 3 O 4 TEM image of @AA@MIPs (D).

[0021] Figure 4 The Fe prepared by the present invention 3 O 4 , Fe 3 O 4 @SiO 2 , Fe 3 O 4 @SiO 2 @NH 2 , Fe 3 O 4 @AA@MIPs、Fe 3 O 4 @AA@NIPs、Fe 3 O 4 Infrared spectrum of the target molecules after elution in @AA@MIPs.

[0022] Figure 5 The Fe prepared by the present invention 3 O 4 @AA@MIPs thermodynamic adsorption (A) and kinetic adsorption (B) of progesterone and its structural analogue estradiol.

[0023] Figure 6 The artificial enzyme Co-MOFs Fe prepared by the present invention 2 O 3 SEM image (A) and TEM image (B) of / MIL-100(Fe).

[0024] Figure 7 The artificial enzyme MOFs Fe prepared by the present invention 2 O 3 / EDS full spectrum of MIL-100(Fe).

[0025] Figure 8 It is the ultraviolet spectrum of the progesterone-BSA conjugate prepared by the present invention.

[0026] Fig. 9 The graph is a graph showing the variation of the chemiluminescence intensity of the magnetic nanoparticle artificial antibody prepared by the present invention with the progesterone concentration in the same time period.

[0027] Fig.10 is the parameter equation of the progesterone to chemiluminescence ratio in the present invention.

[0028] Figure 1 This is the technical roadmap for the detection of progesterone by synthetic magnetic nanoparticle biomimetic antibodies prepared in this embodiment. First, Fe 3 O 4 Nanoparticles; then the Stöber method was used to 3 O 4 The surface of the nanoparticles is coated with a silica layer; then APTES is used to graft amino groups onto the silica layer and then polymerized with acryloyl chloride to obtain Fe 3 O 4 @SiO 2 @AA, and then the template molecule progesterone, the functional monomer acrylic acid, the crosslinking agent N, N-dimethylbisacrylamide and the initiator AIBN were synthesized by the imprinting method. 3 O 4 @AA@MIPs. Finally, the prepared artificial enzyme catalyst Co-MOFs Fe 2 O 3 / MIL-100(Fe) labeled magnetic artificial antibody and exposed to luminol-H 2 O 2 The system was used for chemiluminescent immunoassay of progesterone.

[0029] Figure 2 The Fe prepared in this example 3 O 4The response diagram of nanoparticles under an external magnetic field (A) shows that the magnetic nanoparticles originally suspended in the solvent can be quickly separated, and the solvent becomes clear with almost no residue, indicating that the magnetic nanoparticles not only have the ability to respond quickly to an external magnetic field, but also show the high purity and high dispersibility of the nanoparticles in the preparation process, and there is no non-magnetic impurities. After the external magnetic field disappears, the magnetic ferroferric oxide nanoparticles can be resuspended in the solvent with slow shaking without agglomeration or non-dispersion, indicating that the magnetism of the prepared magnetic ferroferric oxide nanoparticles disappears after the external magnetic field is removed. This may be due to its coercive force being close to zero, and its rapid separation characteristics indicate that its saturation magnetization is high. (B) is the prepared Fe 3 O 4 The HM spectrum of the nanospheres responding to an external magnetic field shows that the initial magnetization curve coincides with other curves and the saturation magnetization intensity is 77.6 emu / g, which shows that the prepared material has excellent responsiveness to the magnetic field. During the magnetization and demagnetization of the material under the action of an external magnetic field, the hysteresis loop is almost a straight line, indicating that the hysteresis loop shows almost no residual magnetization and coercive force, which means that when the external magnetic field is removed, the ferroferric oxide nanoparticles can quickly return to the unmagnetized state and will not retain any magnetism. This property is crucial for the application of magnetic materials.

[0030] Figure 3 (A) is the Fe prepared in this example 3 O 4 Nanoparticles are spherical nanoparticles with uniform particle size distribution of about 200nm and rough surface. They not only provide a magnetic core for molecular imprinting polymers, but also increase their specific surface area and improve the adsorption capacity. 3 O 4 @SiO 2 (B) with Fe 3 O 4 The surface is smoother and the particle size has increased by about 10nm-20nm, which indicates that a thin layer of silicon is modified on the surface of ferroferric oxide. 3 O 4 SEM and Figure (D) of @AA@MIPs are Fe 3 O 4 TEM of @AA@MIPs shows that compared with the unimprinted nanoparticles, the imprinted nanoparticles have an obvious thin layer on the outside, and the core-shell structure can be seen more clearly. 3 O 4 The surface of @AA@MIPs becomes rough, the overall particle size increases to 240nm-270nm, and the surface is uniformly covered with an imprinting layer of about 30nm-40nm.

[0031] Figure 4 For this example, Fe 3 O 4 , Fe 3 O 4 @SiO 2 , Fe 3 O 4 @SiO 2 @NH 2 , Fe 3 O 4 @AA@MIPs、Fe 3 O 4 @AA@NIPs、Fe 3 O 4 Fourier transform infrared spectra of progesterone eluted from @AA@MIP. (a) 533 cm -1 The absorption peaks on the left and right are the characteristic absorption peaks of the stretching vibration of the Fe-O metal tetrahedron, 1066 cm -1 The absorption peaks on the left and right are the double frequency peaks of Fe-O bonds, 947 cm -1 The absorption peaks on the left and right are CH out-of-plane bending vibration peaks, indicating that the magnetic Fe 3 O 4 The successful synthesis of nanoparticles; (b) 1055 cm -1 , 947 cm -1 and 817 cm -1 The peak at 1639 cm in (c) is the stretching vibration peak of Si-O-, indicating the formation of a silicon dioxide layer. -1 The NH bending vibration peak is at 3420 cm -1 The absorption peak at 2959 cm-1 is the NH stretching vibration peak. Both groups of absorption peaks are amino vibration peaks, indicating that the amino group is successfully modified. -1 The characteristic absorption peak at 1455 cm -1 The left and right sides are CH bending vibration peaks, indicating the carboxyl vibration peak of acrylic acid, reflecting the successful polymerization of acrylic acid on the surface of magnetic beads; 1725 cm -1 There are strong absorption peaks on the left and right, which should be the C=O stretching vibration peak and the absorption peak of unsaturated aldehyde, as well as 877 cm -1 , 799 cm -1 Out-of-plane bending vibration of the extracyclic CH bonds indicates successful imprinting of progesterone; (e) 1729 cm -1 The absorption peak at 40° disappears, which is due to the successful removal of the template.

[0032] Figure 5The isothermal adsorption curves of various materials prepared in this example for progesterone and progesterone structural analog estradiol at room temperature (Figure A). It can be seen from the figure that the maximum adsorption of progesterone molecules by magnetic imprinting materials is about 126 μmol / L, which is 1.34 times the maximum adsorption of progesterone molecules by ordinary imprinting materials (about 94 μmol / L) and 3.15 times the maximum adsorption of progesterone molecules by non-imprinting materials (about 40 μmol / L). Figure B shows the isothermal adsorption of progesterone and progesterone structural analog estradiol E by the prepared materials. 2 From the kinetic adsorption curve, we can see that Fe 3 O 4 The time for @AA@MIPs to reach equilibrium adsorption of progesterone was about 100 min, and the adsorption amount was about 122 μmol / L, which was about 1.3 times the maximum adsorption amount of progesterone by common imprinted materials (about 93 μmol / L) and 2.9 times the maximum adsorption amount of progesterone by non-imprinted materials (42 μmol / L), which was also close to the maximum adsorption amount obtained by thermodynamics. Before the equilibrium adsorption amount was saturated, the rate at which magnetic imprinted materials adsorbed molecules from the solution phase was faster than that of common imprinted materials and non-imprinted materials. The time required for the magnetic imprinted material to adsorb the maximum equilibrium adsorption amount in the solution phase was about 100 min, while the time for the common imprinted material and non-imprinted material to adsorb the maximum equilibrium adsorption amount was about 120 min and 200 min respectively. This indicates that the magnetic imprinted material has more recognition sites than ordinary imprinted materials, and that the imprinted material has a higher selective recognition of progesterone because of its ability of selective recognition and specific adsorption, as well as the specific interaction between the imprinted sites and the target molecules, achieving efficient adsorption performance, which also reflects the successful preparation of progesterone-imprinted magnetic molecular imprinted polymers.

[0033] Figure 6 The artificial enzyme Co-MOFs Fe synthesized in this example 2 O 3 / MIL-100(Fe) form, Figure 6 (A) SEM of the synthesized catalyst at 100K and Figure 6 (B) TEM shows that Co-doped MOFs Fe 2 O 3 / MIL 100(Fe) was found to be an agglomerate with a flower-like structure, presenting a compact spherical structure and a size of approximately 80 nm.

[0034] Figure 7 The artificial enzyme Co-MOFs Fe synthesized in this example 2 O 3 / EDS full spectrum of MIL-100(Fe). The cobalt content in the catalyst measured by EDS is 5.6%, revealing the successful doping of Co.

[0035] Figure 8 This is the UV spectrum of the progesterone-BSA conjugate synthesized in this example. The maximum characteristic absorption peak wavelengths of progesterone and BSA are 242 nm and 279 nm, respectively, and the maximum characteristic absorption peak wavelength of the progesterone-BSA conjugate is 250 nm, which is shifted compared with the maximum characteristic absorption peaks of progesterone and BSA, indicating that progesterone is successfully coupled to the carrier protein BSA.

[0036] Fig. 9 This is a graph showing the change in chemiluminescence intensity with progesterone concentration during the same time period in this example. 2 O 2 and optimal pH, the chemiluminescence analyzer was used to detect once every 2 minutes during the period of 0-40min, and the graph of the chemiluminescence intensity corresponding to different concentrations of progesterone versus time was shown. Under the action of different concentrations of progesterone, the chemiluminescence intensity of the reaction continued to decrease until it was almost 0, and the downward trend changed from rapid to gentle. This was due to the catalysis of the artificial enzyme and the continuous consumption of the reaction raw materials. By comparing the chemiluminescence intensity corresponding to the curves of different progesterone concentrations at 0min, it can be observed that there is a gradual downward trend, which is the result of competition between progesterone and the artificial enzyme for binding to progesterone.

[0037] Fig.10 is the parameter equation of the progesterone to chemiluminescence ratio in the present invention. First, take Fig.10 The maximum luminescence point in the sample was fitted with the standard equation using the luminescence intensity as the ordinate and the progesterone concentration as the abscissa. The detection limit could reach 5.52×10 -11 g / L, here we choose 3 times the standard deviation (3SD). DETAILED DESCRIPTION

[0038] A method for preparing a chemiluminescent enzyme-linked immunosorbent sensor for progesterone detection, characterized in that: the sensor is a magnetic Fe 3 O 4 The nanoparticles are cores, the surface is functionally modified with an acrylamide molecular layer, the polymer is an imprinted shell layer, the progesterone imprinted molecules located in the shell layer are eluted, and a cavity structure having a structure, size and functional group complementary to the imprinted molecules is formed inside the shell layer. The imprinted shell layer of the eluted imprinted molecules has a specific recognition site for the progesterone molecules, and the selective recognition and detection of the target analyte progesterone molecules is achieved by combining with an artificial enzyme and performing chemiluminescent immunoassay. The preparation process of the chemiluminescent enzyme-linked immunosorbent sensor includes the following three steps: The first step is Fe 3 O 4 Preparation of artificial antibodies based on magnetic nanoparticles: Fe 3 O4 Magnetic nanoparticles coated with SiO 2 Shell, 3-aminopropyltriethoxysilane ethyl oxygen hydrolyzes, and reacts with Fe through silanization reaction. 3 O 4 @SiO 2 The free silanol groups on the surface of the Fe react to form -Si-O-Si-, thereby grafting amino groups to obtain Fe 3 O 4 @NH 2 , the synthesized Fe 3 O 4 @NH 2 Place it in a 100mL three-necked flask, dry it thoroughly, add 20-40mL of toluene solution and 50-150mg of anhydrous potassium carbonate, disperse it evenly by ultrasonication, deoxygenate it with nitrogen for 5 minutes, put it in an ice bath, stir it for 10 minutes, then add 1-3mL of acryloyl chloride, continue mechanical stirring, react for 4-6 hours, rinse it with ultrapure water for 3 times, and dry it in a vacuum drying oven for 20-24 hours to obtain Fe 3 O 4 @AA nanoparticles, take progesterone and acrylic acid in a molar ratio of 1:6 to 10:6 and dissolve them in a three-necked flask of acetonitrile solution, stir mechanically at 20 °C for 3 to 5 h to mix, then add 0.2 to 0.8 g of the Fe 3 O 4 @AA nanoparticles were dispersed by ultrasonication, and then 1.80 ~ 1.90 mL N, N-dimethylbisacrylamide and 50 ~ 65 mg azobisisobutyronitrile were added. After nitrogen was introduced for 10 min, the polymerization reaction was stirred at 55 ~ 65 °C for 22 ~ 26 h to obtain the polymerization product Fe 3 O 4 @AA@MIPs nanoparticles were extracted by Soxhlet extraction using a mixed solution of methanol and acetic acid with a volume ratio of 7:3 as the elution solvent to elute Fe 3 O 4 @AA@MIPs magnetic nanoparticles contain progesterone, and the Fe 3 O 4 @AA@MIPs were placed in a vacuum oven and dried for 24 h to obtain Fe 3 O 4 @AA@MIPs artificial antibodies based on magnetic nanoparticles; The second step is the preparation of artificial enzyme-labeled progesterone antibody: 3.2 ~ 3.8 mg Co-MOFs Fe 2 O 3 / MIL-100(Fe) was dispersed in 0.5 ~ 1.5 mL of dilution buffer containing 20 mg / mL 4-(maleimido)phenyl isocyanate and activated at 20 °C for 3 s. 2 O 3 / MIL-100(Fe) for 1 h, the solution was removed by centrifugation, and the activated Co-MOFs Fe 2 O 3 / MIL-100(Fe) was redispersed in 0.5 ~ 1.5 mL phosphate buffer solution containing 20 ~ 80 μg progesterone artificial antibody. After incubation for 10 ~ 14 h, a 0.05 ~ 0.15 M glycine solution was added to the above buffer solution to quench the excess activated groups to terminate the reaction. After filtration, the artificial enzyme-labeled progesterone antibody was obtained and redispersed in 1.0 mL PBS buffer solution; The third step is the chemiluminescent enzyme-linked immunosorbent sensor: the progesterone-BSA conjugate was diluted to 2.0 μg / mL with coating buffer and coated into the wells of a 96-well microplate at 37°C for 2 to 4 h. After the microplate was washed, 150 μL of superblock blocking buffer was used to block the coated microplate wells for 1 to 2 h. The blocked microplate wells were washed again, and 40 to 60 μL of progesterone sample solution and an equal volume of artificial enzyme-labeled progesterone antibody suspension were injected. The solution was incubated for 1 h. After washing to remove excess reactants, 100 μL of 1.0×10 -6 M luminol solution and 50 μL of 0.1MH 2 O 2 The solution is injected into each microplate well to collect the chemiluminescent immunoassay intensity for quantifying progesterone, thereby achieving progesterone detection. Specific embodiments

[0039] Use Fe 3 O 4 The magnetic nanoparticles were used as cores and the surface was functionalized with an acrylamide molecular layer to prepare Fe-based nanoparticles that can recognize progesterone molecules. 3 O 4 Magnetic nanoparticle artificial antibodies are labeled with artificial enzymes, and progesterone is detected by chemiluminescent enzyme-linked immunosorbent assay.

[0040] The first step is Fe 3 O 4 Preparation of artificial antibodies based on magnetic nanoparticles: Fe 3 O 4 Magnetic nanoparticles coated with SiO 2 Shell, 3-aminopropyltriethoxysilane ethyl oxygen hydrolyzes, and reacts with Fe through silanization reaction.3 O 4 @SiO 2 The free silanol groups on the surface of the Fe react to form -Si-O-Si-, thereby grafting amino groups to obtain Fe 3 O 4 @NH 2 , the synthesized Fe 3 O 4 @NH 2 Place it in a 100 mL three-necked flask, dry it thoroughly, add 30 mL of toluene solution and 100 mg of anhydrous potassium carbonate, disperse it evenly with ultrasound, deoxygenate it with nitrogen for 5 min, put it in an ice bath, stir it for 10 min, then add 2 mL of acryloyl chloride, continue mechanical stirring, react for 5 h, rinse it with ultrapure water 3 times, and dry it in a vacuum drying oven for 22 h to obtain Fe 3 O 4 @AA nanoparticles, take progesterone and acrylic acid in a molar ratio of 1:6 and dissolve them in a three-necked flask of acetonitrile solution, stir mechanically for 4 h at 20 °C to mix, then add 0.5 g of the Fe 3 O 4 @AA nanoparticles were dispersed by ultrasonication, and then 1.89 mL N, N-dimethylbisacrylamide and 62 mg azobisisobutyronitrile were added. After nitrogen was introduced for 10 min, the polymerization reaction was stirred at 60 °C for 24 h to obtain the polymerization product Fe 3 O 4 @AA@MIPs magnetic nanoparticles were extracted by Soxhlet extraction using a mixed solution of methanol and acetic acid with a volume ratio of 7:3 as the elution solvent to elute Fe 3 O 4 @AA@MIPs magnetic nanoparticles contain progesterone, and the Fe 3 O 4 @AA@MIPs were placed in a vacuum oven and dried for 24 h to obtain Fe 3 O 4 @AA@MIPs artificial antibodies based on magnetic nanoparticles; The second step is the preparation of artificial enzyme-labeled progesterone antibody: 36 mg Co-MOFs Fe 2 O 3 / MIL-100(Fe) was dispersed in 1 mL of dilution buffer containing 20 mg / mL 4-(maleimido)phenyl isocyanate and activated at 20 °C for 3 s to 4 s of Co-MOFs Fe. 2 O 3 / MIL-100(Fe) for 1 h, the solution was removed by centrifugation, and the activated Co-MOFs Fe 2 O 3 / MIL-100(Fe) was redispersed in 1 mL phosphate buffer solution containing 50 μg of progesterone artificial antibody. After incubation for 12 h, a 0.1 M glycine solution was added to the above buffer solution to quench the excess activated groups to terminate the reaction. After filtration, the artificial enzyme-labeled progesterone antibody was obtained and redispersed in 1.0 mL PBS buffer solution. The third step is the chemiluminescent enzyme-linked immunosorbent sensor: the progesterone-BSA conjugate was diluted to 2.0 μg / mL with coating buffer and coated into the wells of a 96-well microplate at 37°C for 3 h. After the microplate was washed, 150 μL of super block blocking buffer was used to block the coated microplate wells for 1.5 h. The blocked microplate wells were washed again, and 50 μL of progesterone sample solution and an equal volume of artificial enzyme-labeled progesterone antibody suspension were injected and incubated for 1 h. After washing to remove excess reactants, 100 μL of a 1.0×10 -6 M luminol solution and 50 μL of 0.1MH 2 O 2 The solution is injected into each microplate well to collect the chemiluminescent immunoassay intensity for quantifying progesterone, thereby achieving progesterone detection.

Claims

1. A method for preparing a chemiluminescent enzyme-linked immunosorbent assay sensor for progesterone detection, characterized in that: The sensor is a core of magnetic Fe3O4 nanoparticles, a surface functionalized acrylamide molecular layer, a polymer as an imprinted shell layer, and the progesterone imprinted molecules located in the shell layer are eluted. The inner part of the shell layer forms a hole structure complementary to the structure, size and functional group of the imprinted molecules. The imprinted shell layer of the eluted imprinted molecules has a specific recognition site for the progesterone molecules. The selective recognition and detection of the progesterone molecules is achieved by combining with an artificial enzyme and performing chemiluminescent immunoassay. The preparation process of the chemiluminescent enzyme-linked immunosorbent sensor includes the following three steps: 1.1 The first step is the preparation of artificial antibodies of Fe3O4 magnetic nanoparticles: the surface of Fe3O4 magnetic nanoparticles is coated with SiO2 shell layer, and the ethyl oxygen of 3-aminopropyltriethoxysilane is hydrolyzed, and then reacts with the free silanol group on the surface of Fe3O4@SiO2 through silanization reaction to generate -Si-O-Si-, thereby grafting amino groups to obtain Fe3O4@NH2, and the synthesized Fe3O4@NH2 is placed in a 100mL three-necked flask, and after being fully dried, 20~40mL of toluene solution and 50~150mg of anhydrous potassium carbonate are added, ultrasonically dispersed uniformly, nitrogen deoxygenated for 5min, ice bath, stirred for 10min, and then 1~3mL of acryloyl chloride is added, mechanical stirring is continued, the reaction is carried out for 4~6h, and then rinsed with ultrapure water for 3 times, and dried in a vacuum drying oven for 20~24h to obtain Fe3O4@AA magnetic nanoparticles, and the molar ratio of progesterone to acrylic acid is 1:6~ The mixture was dissolved in an amount of 10:6 in a three-necked flask of acetonitrile solution, and mechanically stirred at 20 °C for 3 to 5 h for mixing. Subsequently, 0.2 to 0.8 g of the Fe3O4@AA magnetic nanoparticles prepared above were added, and ultrasonic dispersion was performed. Then, 1.80 to 1.90 mL of N, N-dimethylbisacrylamide and 50 to 65 mg of azobisisobutyronitrile were added. After nitrogen was introduced for 10 min, the polymerization reaction was stirred at 55 to 65 °C for 22 to 26 h to obtain the polymerization product Fe3O4@AA@MIPs nanoparticles. The progesterone in the Fe3O4@AA@MIPs magnetic nanoparticles was eluted using a Soxhlet extractor with a mixed solution of methanol and acetic acid in a volume ratio of 7:3 as an elution solvent. The Fe3O4@AA@MIPs after eluting progesterone was placed in a vacuum oven and dried for 24 h to obtain artificial antibodies of Fe3O4@AA@MIPs magnetic nanoparticles that recognize progesterone. 1.2 The second step is the preparation of artificial enzyme-labeled progesterone antibody: 3.2 ~ 3.8 mg Co-MOFs Fe2O3 / MIL-100(Fe) was dispersed in 0.5 ~ 1.5 mL of dilution buffer containing 20 mg / mL 4-(maleimido)phenyl isocyanate, and the hydroxyl groups on Co-MOFs Fe2O3 / MIL-100(Fe) were activated at 20°C for 1 h. The above solution was removed by centrifugation, and the activated Co-MOFs Fe2O3 / MIL-100(Fe) was redispersed in 0.5 ~ 1.5 mL of phosphate buffer solution containing 20 ~ 80 μg of progesterone artificial antibody. After incubation for 10 ~ 14 h, a 0.05 ~ 0.15 M glycine solution was added to the above buffer solution to quench the excess activated groups to terminate the reaction. After filtration, the artificial enzyme-labeled progesterone antibody was obtained and redispersed in 1.0 mL of PBS buffer solution; 1.3 The third step is the chemiluminescent enzyme-linked immunosorbent sensor: the progesterone-BSA conjugate was diluted to 2.0 μg / mL with coating buffer and coated into the wells of a 96-well microplate at 37°C for 2 to 4 h. After the microplate was washed, 150 μL of superblock blocking buffer was used to block the coated microplate wells for 1 to 2 h. The blocked microplate wells were washed again, and 40 to 60 μL of progesterone sample solution and an equal volume of artificial enzyme-labeled progesterone antibody suspension were injected. The solution was incubated for 1 h. After washing to remove excess reactants, 100 μL of 1.0×10 -6 M luminol solution and 50 μL of 0.1 M H2O2 solution were injected into each microplate well to collect the chemiluminescent immunoassay intensity for quantification of progesterone, thereby realizing the detection of progesterone.

2. The method for preparing a chemiluminescent enzyme-linked immunosorbent assay sensor for progesterone detection according to claim 1, characterized in that: The magnetic separation in the preparation of the sensor is the aggregation of the magnetic nanoparticles themselves under the action of an external magnetic field.

3. The method for preparing a chemiluminescent enzyme-linked immunosorbent assay sensor for progesterone detection according to claim 1, characterized in that: In the preparation of the sensor, the progesterone molecules in the polymer imprinted shell are antigen molecules, template molecules, target molecules, and target analytes.

4. The method for preparing a chemiluminescent enzyme-linked immunosorbent assay sensor for progesterone detection according to claim 1, characterized in that: In the preparation of the sensor, Fe3O4@AA@MIPs, AA is acrylamide molecule coupled and modified on the surface of magnetic ferroferric oxide and has a functional monomer effect, and MIPs is a molecular imprinting polymer coated on the surface of magnetic ferroferric oxide, that is, an artificial antibody.

5. The method for preparing a chemiluminescent enzyme-linked immunosorbent assay sensor for progesterone detection according to claim 1, characterized in that: In the preparation of the sensor, the artificial enzyme-labeled progesterone antibody is an enzyme-labeled antigen molecule.

6. The method for preparing a chemiluminescent enzyme-linked immunosorbent assay sensor for progesterone detection according to claim 1, characterized in that: The thickness of the imprinted shell layer in the preparation of the sensor can be controlled by changing the amount of the cross-linking agent and the functional monomer.

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