A nanozyme and its preparation method and its application in detecting estrogen

Abstract

The present invention relates to a nanoenzyme and a preparation method thereof and an application thereof in detecting estrogen, belonging to the field of detection technology. The present invention uses vitamin B6 as a ligand to synthesize a MOF and a Cu and Co ion node combination to form a framework structure compound Co / Cu@MOF, wherein Co / Cu@MOF has laccase-like activity and photocatalytic performance, and the introduction of Co ions enhances Cu + / Cu 2+ Electron transfer ability. Under near-infrared irradiation, the hot electrons generated by Co / Cu@MOF can greatly reduce the activation energy of the reaction and accelerate the reaction. In the presence of 4-aminoantipyrine and near-infrared light, Co / Cu@MOF can catalyze the oxidation of estradiol, estriol and diethylstilbestrol, producing a typical color change from colorless to wine red, thereby establishing a new method for rapid detection of estrogen with a highly sensitive colorimetric probe, which can be used for the detection of estrogen in urine and blood, and has the characteristics of simple operation, strong specificity, high sensitivity and rapidity.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and specifically relates to a nanozyme and a preparation method thereof and an application thereof in detecting estrogen. Background Art

[0002] Steroid hormones, also known as steroid hormones, have a clear role in body development, immune regulation and fertility control. Estrogen is an important type of sex hormone in women, among which estradiol is the most biologically active type of estrogen in women. The content of estrogen is closely related to many diseases, and also has a significant impact on the human endocrine system, cardiovascular system, metabolic system, etc. In addition, as women age, especially during menopause, estrogen levels drop sharply, which can cause mood disorders and even osteoporosis. Therefore, timely quantitative analysis of estrogen in serum is of great significance for studying the mechanism of disease development, disease development and changes, timely supplementation of estrogen, and clinical drug development.

[0003] Laccases are an important class of multi-copper oxidases that catalyze the single-electron oxidation of many important biological and environmental organic compounds. 2+ Complexes such as Cu 2+ -Imidazole, Cu 2+ -Porphyrin, Cu 2+ -metal organic frameworks (MOFs) have been developed to mimic laccases and are called laccase-like nanozymes. Although great progress has been made in the research of laccase-like nanozymes, most of them are based on Cu 2+ The nanozymes are constructed by covalent coordination of metal ions and are mainly concentrated on the active center Cu. However, there are few studies on laccase-like nanozymes with dual metal ions. Improving the activity of nanozymes by physical stimulation is also an important research direction. Summary of the invention

[0004] In view of the problems raised in the background technology, the present invention provides a nanozyme and a preparation method thereof and its application in detecting estrogen. 6 MOF was synthesized as a ligand and combined with Cu and Co ion nodes to form a framework structure compound Co / Cu@MOF. Co / Cu@MOF has laccase-like activity and photocatalytic properties. The introduction of Co ions enhances the activity of Cu + / Cu 2+Electron transfer ability. Under near-infrared light irradiation, the hot electrons generated by Co / Cu@MOF greatly reduce the activation energy of the reaction and accelerate the reaction. In the presence of 4-aminoantipyrine (4-AP) and near-infrared light, Co / Cu@MOF can catalyze the oxidation of estradiol, estriol and diethylstilbestrol, producing a typical color change from colorless to wine red. A new method for rapid detection of estrogen with a highly sensitive colorimetric probe was established. This method is used for the detection of estrogen in urine and blood, with a detection limit of 6-38ng / L; and the detection method has the characteristics of simple operation, strong specificity, high sensitivity and rapidity.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] The present invention provides a method for preparing a nanozyme, comprising the following steps:

[0007] (1) Vitamin B 6 Add to methanol, stir and dissolve to obtain solution A;

[0008] (2) Put Zn(NO 3 ) 2 6H 2 O, CuCl 2 and CoCl 2 Add to methanol, stir and dissolve to obtain solution B;

[0009] (3) Add solution A to solution B and stir to obtain a mixed solution;

[0010] (4) Add the mixed solution into a high-pressure reactor and heat to 100-140°C for 2-4 hours;

[0011] (5) Cool the reaction solution to room temperature and filter out large particles of impurities using a microfiltration membrane to obtain a clear solution;

[0012] (6) Centrifuging the clear liquid to obtain a supernatant;

[0013] (7) The supernatant was vacuum dried to obtain Co / Cu@MOF nanozyme.

[0014] Furthermore, vitamin B6 and Zn(NO 3 ) 2 6H 2 The mass ratio of O is 1:1; CuCl 2 With CoCl 2 The mass ratio of is 1:1; a 0.22 μm filter membrane is used in step (5); and the drying temperature in step (7) is 50-70°C.

[0015] Furthermore, in step (1), 0.060-0.10 g of vitamin B6 Add 5-10 mL of methanol and stir to dissolve to obtain solution A; in step (2), add 0.060-0.10 g Zn(NO 3 ) 2 6H 2 O, 0.03-0.05 mg CuCl 2 and 0.03-0.05mgCoCl 2 Add 5-10 mL of methanol and stir to dissolve to obtain solution B; in step (3), solution A and solution B are mixed for 5-10 minutes.

[0016] The present invention also provides the Co / Cu@MOF nanozyme obtained by the preparation method of the present invention.

[0017] The present invention also provides the use of the Co / Cu@MOF nanozyme prepared by the present invention in the determination of estrogen levels.

[0018] Furthermore, the estrogen is estradiol, estriol and diethylstilbestrol.

[0019] The present invention also provides a method for detecting estrogen levels using the nanozyme obtained by the present invention, which specifically comprises the following steps:

[0020] S1, prepare sample measurement solution.

[0021] S2, add the sample test solution into the ELISA plate, then add Co / Cu@MOF nanozyme and 4-aminopyrine, add PBS buffer solution to the fixed volume and mix well.

[0022] S3, irradiated with 808 nm near-infrared light, and measured absorbance at a wavelength of 500-510 nm.

[0023] S4, substituting the measured absorbance into the standard curve equation to obtain the estrogen concentration in the sample.

[0024] The standard curve equations include standard curve equations of estradiol E2, estriol E3 and diethylstilbestrol DES.

[0025] Furthermore, the preparation method of the sample measuring solution is:

[0026] When the sample to be tested is serum: add ethyl acetate to the sample to be tested, vortex and then centrifuge to obtain a supernatant and a residue; add ethyl acetate to the residue for extraction, and then centrifuge to obtain a supernatant; combine the two extraction supernatants, blow dry with nitrogen, and add pH 7.4 PBS buffer to obtain a sample test solution.

[0027] When the sample to be tested is urine: centrifuge the urine, take the supernatant, and store it at 4°C in the dark; before testing, add 0.1% perchloric acid to the supernatant, mix well, centrifuge at 8000 r / min for 5 min, take the supernatant, and prepare the sample test solution.

[0028] Furthermore, in step S2, the concentration of the Co / Cu@MOF nanozyme is 1 mg / mL, the addition amount is 5-10 μL, the concentration of 4-aminopyrine is 1 mg / mL, and the volume is 5-10 μL; the PBS buffer solution is pH 7.4 PBS buffer; in step S3, the power of the near-infrared lamp is 1.25-1.50 W / cm 2 , the irradiation time is 10min.

[0029] Furthermore, the standard curve equations of estradiol E2, estriol E3 and diethylstilbestrol DES are obtained by the following steps:

[0030] (1) Add 10 μL of E2 standard solution with a concentration of 0.012-36 μg / L, E3 standard solution with a concentration of 0.02-25 μg / L, or DES standard solution with a concentration of 0.045-15 μg / L to the ELISA plate;

[0031] (2) Add 5 μL of 1 mg / mL Co / Cu@MOF and 5 μL of 1 mg / mL 4-aminopyrine, then add 0.1 mmol / L pH 7.4 PBS buffer solution to 50 μL and mix well.

[0032] (3) Irradiate under 808 nm near-infrared light for 10 min, measure the absorbance at a wavelength of 505 nm, and plot the standard curve equation with E2, E3 or DES concentration as the horizontal axis and absorbance as the vertical axis.

[0033] Beneficial effects of the present invention:

[0034] The Co / Cu@MOF nanozyme synthesized in the present invention has laccase-like activity and photocatalytic performance. The introduction of Co ions enhances the photocatalytic activity of Cu + / Cu 2+ Electron transfer ability. Under near-infrared irradiation, the hot electrons generated by the excitation of Co / Cu@MOF can greatly reduce the activation energy of the reaction and accelerate the reaction. In the presence of 4-aminoantipyrine (4-AP) and near-infrared light, Co / Cu@MOF can catalyze the oxidation of estrogens including estradiol, estriol and diethylstilbestrol, producing a typical color change from colorless to wine red. This can establish a new method for the rapid detection of estrogen using a highly sensitive colorimetric probe.

[0035] The estrogen detection method established by the present invention has good specificity and high detection sensitivity. The established method can not only determine the total amount of estradiol, estriol and diethylstilbestrol, but also can determine them separately when the three exist independently. The established method can be used for the detection of estrogen in urine and blood, with a detection limit of 6-38 ng / L, reaching or even exceeding the detection sensitivity of liquid chromatography-mass spectrometry.

[0036] The estrogen determination method established by the present invention has the characteristics of fewer processing steps, shorter time, lower processing cost, simple operation, and no need for large-scale instruments and equipment, and has great advantages in actual detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 TEM image of Co / Cu@MOF prepared in Example 1 of the present invention;

[0038] Figure 2 This is the XRD pattern of Co / Cu@MOF prepared in Example 1 of the present invention;

[0039] Figure 3 This is a graph of the laccase-like activity of Co / Cu@MOF in Example 1 of the present invention;

[0040] Figure 4 In the figure, Figure a is the Michaelis-Menten equation of Co / Cu@MOF+4-AP oxidizing 2,4-DP under infrared light 808 nm irradiation for 10 min, and Figure b is the Michaelis-Menten equation of Co / Cu@MOF+4-AP oxidizing 2,4-DP under natural light irradiation;

[0041] Figure 5 The UV absorption spectra and linear equations of estradiol (a and b), estriol (c and d) and diethylstilbestrol (e and f) of Co / Cu@MOF+4-AP were detected after irradiation with infrared light at 808 nm for 10 min.

[0042] Figure 6 This is the effect of coexisting substances on estrogen detection. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0044] The method for preparing the nanozyme comprises the following steps:

[0045] (1) 0.060-0.10 g of vitamin B 6 Add 5-10 mL of methanol, stir to dissolve, and obtain solution A.

[0046] (2) Add 0.060-0.10 g Zn(NO 3 ) 2 6H 2 O, 0.03-0.05 mg CuCl 2 and 0.03-0.05 mg CoCl 2 Add 5-10 mL of methanol, stir to dissolve, and obtain solution B.

[0047] (3) Quickly add solution A into solution B and stir for 5-10 min to obtain a mixed solution.

[0048] (4) Add the mixed solution into a polytetrafluoroethylene high-pressure reactor and place it in a microwave digester heated to 100-140°C at 60 W for 2-4 h.

[0049] (5) Cool the reaction solution to room temperature and filter out large particles of impurities using a 0.22 μm filter membrane to obtain a clear solution.

[0050] (6) Centrifuge the supernatant at 8000-10000 r / min for 10-15 min to obtain the supernatant.

[0051] (7) The supernatant was dried under vacuum at 60 °C for 12-24 h to obtain Co / Cu@MOF nanozyme.

[0052] The obtained Co / Cu@MOF nanoenzyme is used in the method for detecting estrogen levels, comprising the following steps:

[0053] S1, prepare sample measurement solution.

[0054] S2, add the sample test solution to the ELISA plate, then add 5-10 μL of 1 mg / mL Co / Cu@MOF nanozyme and 5-10 μL of 1 mg / mL 4-aminopyrine, add pH 7.4 PBS buffer solution to 50 μL and mix well.

[0055] S3, at 808 nm, 1.25-1.50 W / cm 2 After irradiation with near-infrared light for 10 min, the absorbance was measured at a wavelength of 500-510 nm.

[0056] S4, substituting the measured absorbance into the standard curve equation to obtain the estrogen concentration in the sample.

[0057] In order to more clearly illustrate the present invention, it is described in detail through the following examples.

[0058] Example 1

[0059] Preparation of Co / Cu@MOF nanozymes

[0060] 0.060 g vitamin B 6 Add 5 mL of methanol and stir to dissolve into solution A; 0.060 g Zn(NO 3 ) 2 6H 2 O, 0.03 mg CuCl 2 and 0.03 mg CoCl 2 Add 5 mL of methanol and stir to dissolve to obtain solution B; quickly add solution A to solution B and stir for 5 minutes to obtain a mixed solution; transfer the mixed solution to a polytetrafluoroethylene high-pressure reactor, place it in a microwave digester at 60 W and heat it to 120°C for 3 hours. After the reaction is completed, naturally cool it to room temperature, use a 0.22 μm filter membrane to remove large particles of impurities, and then centrifuge at 8000 r / min for 15 minutes. The supernatant is vacuum dried at 60°C for 12 hours. There is no special requirement for the drying vacuum degree. The drying temperature is mainly controlled to obtain Co / Cu@MOF nanozyme.

[0061] Physical property analysis of the obtained Co / Cu@MOF nanozyme:

[0062] (1) Morphological characteristics

[0063] Figure 1 TEM image of Co / Cu@MOF nanozyme. Figure 1 It can be seen that: Co / Cu@MOF nanozymes present specific spherical and lamellar structures;

[0064] Figure 2 This is the X-ray diffraction (XRD) pattern of Co / Cu@MOF nanozyme. The characteristic peaks of Co / Cu@MOF are at 26.3° and 59.2°, corresponding to the diffraction peaks of (101) and (211).

[0065] (2) Evaluation of Co / Cu@MOF nanozyme activity

[0066] The laccase-like activity was evaluated using 2,4-chlorophenol (2,4-DP) and 4-aminopyrine (4-AP) as catalytic reaction substrates. 100 μL of 5 mmol / L 2,4-DP, 100 μL of 10 mmol / L 4-AP, and 5 μL of 1 mg / mL Co / Cu@MOF were added to a 5 mL stoppered colorimetric tube. The pH 7.4 PBS buffer solution was made up to 3 mL and shaken. The 2,4-DP and 4-AP were heated at 1.40 W / cm 2, 808 nm near-infrared light or natural light for 10 min, and measure the absorbance at 505 nm. The results are as follows Figure 3 .pass Figure 3 It can be seen that compared with no irradiation under 808 nm near-infrared light, irradiation under near-infrared light can significantly enhance the laccase activity of Co / Cu@MOF, and Co / Cu@MOF exhibits stronger laccase activity.

[0067] (3) Determination of Michaelis-Menten catalytic kinetic parameters

[0068] The results of the test are as follows Figure 4 (ab) and Table 1. Michaelis constants of Co / Cu@MOF for substrate 2,4-DP K m The reaction rate constant is 0.98×10 -2 mmol / L min, 2,4-DP after infrared light irradiation K m is 0.126 mmol / L, and the reaction rate constant is 1.72×10 -2 mmol / L min, indicating that irradiation increased the affinity of Co / Cu@MOF nanozyme with the substrate and the reaction rate.

[0069] Table 1 Michaelis-Menten catalytic kinetic parameters

[0070]

[0071] Preparation of working curves for estradiol (E2), estriol (E3) and diethylstilbestrol (DES)

[0072] Add 10 μL of 0.012-36 μg / L E2 standard solution or 0.02-25 μg / L E3 standard solution or 0.045-15 μg / L DES standard solution to the ELISA plate, add 5 μL of 1 mg / mL Co / Cu@MOF, 5 μL of 1 mg / mL 4-aminopyrine (4-AP), and 0.1 mmol / L pH 7.4 PBS buffer solution to 50 μL, mix well, irradiate under 808 nm near-infrared light for 10 min, measure the absorbance A at a wavelength of 505 nm, and draw a standard curve with E2, E3 or DES concentration as the horizontal axis and absorbance A as the vertical axis to obtain the regression equation, see Figure 5 ; The regression equation, correlation coefficient, relative standard deviation and linear range are shown in Table 2.

[0073] Table 2 Linear equation, correlation coefficient, relative standard deviation, linear range

[0074]

[0075] Determination of estrogen in serum using Co / Cu@MOF nanozyme

[0076] The estrogen levels in the serum of 12 healthy non-pregnant adult women aged 20-44 were measured as follows:

[0077] (1) Sample pretreatment: Take 0.5 mL of serum sample, add 1 mL of ethyl acetate, vortex for 1 min, centrifuge at 13000 r / min for 3 min, remove the supernatant, add 1 mL of ethyl acetate to the remaining residue for a second extraction, transfer the supernatant after centrifugation, combine the supernatants of the two extractions, dry them with nitrogen at 40°C, add 1 mL of pH 7.4 PBS buffer to prepare the sample test solution.

[0078] (2) Determination of estrogen in serum: Add 10 μL of the sample solution in step (1) to the ELISA plate, add 5 μL of 1 mg / mL Co / Cu@MOF, add 5 μL of 1 mg / mL 4-aminopyrine (4-AP), add 0.1 mmol / L pH7.4 PBS buffer solution to 50 μL, mix well, irradiate under 808 nm near-infrared light for 10 min, measure the absorbance A at a wavelength of 505 nm, substitute into the E2 regression equation, and the average value of the estrogen content in serum is determined to be between 0.090±0.050-0.475±0.045 ng / mL.

[0079] Example 2

[0080] (1) Preparation of Co / Cu@MOF nanozymes

[0081] 0.10g vitamin B 6 Add 8 mL of methanol and stir to dissolve into solution A. 0.10 g Zn(NO 3 ) 2 6H 2 O, 0.05 mg CuCl 2 and 0.05 mg CoCl 2 Add 10 mL of methanol and stir to dissolve to obtain solution B. Quickly add solution A to solution B and stir for 10 min. Then transfer the stirred mixed solution to a polytetrafluoroethylene high-pressure reactor and place it in a microwave digester at 60 W and heat to 120 °C for 4 h. After the reaction is completed, naturally cool to room temperature, use a 0.22 μm filter membrane to remove large particles, and then centrifuge at 10000 r / min for 10 min. The supernatant is vacuum dried at 60 °C for 12-24 h to obtain Co / Cu@MOF nanozyme.

[0082] (2) Preparation of E2 working curve: same as in Example 1.

[0083] (3) Determination of estrogen in urine samples: 50 mL of mid-morning urine was collected from 10 women for the determination of estrogen levels. All urine samples were centrifuged at 3000 rpm for 10 min, and the supernatant was collected and stored at 4°C in the dark to prepare the sample test solution.

[0084] (4) Sample pretreatment method: Add 0.1% perchloric acid to the collected urine sample, mix well, centrifuge at 8000 r / min for 5 min, take the supernatant, and prepare the sample test solution.

[0085] (5) Determination of estrogen in urine samples: As in Example 1, the average value of the estrogen content in urine samples was determined to be between 47.23±1.10-125±5.30 ng / mL.

[0086] Recovery and precision investigation

[0087] Select any two test samples, and add three different concentrations of E2 standard solutions respectively; each concentration is measured three times in parallel, and the spiked recovery rate is calculated, and the relative standard deviation RSD is calculated. The results are shown in Table 3; the spiked recovery rate of E2 is measured to be between 98.6% and 112.1%, and the RSD is between 2.43% and 4.08%, which shows that the method of the present invention has high accuracy and precision.

[0088] Table 3 Sample spike recovery and RSD (n = 3)

[0089]

[0090] Investigation of the specificity of the method for detecting estrogen levels using Co / Cu@MOF nanozymes

[0091] E2, E3 and DES and other coexisting substances (ascorbic acid, vitamin E, citric acid, Mg 2+ , Ca 2+ 、Zn 2+ , Cu 2+ 、SO 4 2+ 、 P4, testosterone, DBP, BSA, CPL, BPA) were mixed to detect the effect of coexisting ions on estrogen in the serum detection system. The results are shown in the attached Figure 6 .

[0092] 15 times ascorbic acid, 20 times vitamin E and 60 times citric acid concentration do not interfere with the determination of estrogen. Large amounts of inorganic ions, such as Mg 2+ , Ca 2+ , Zn 2+ , Cu2+ and SO 4 2- The influence on the determination is negligible. 10-fold concentration of progesterone (P4), testosterone, dibutyl phthalate (DBP), bovine serum albumin (BSA), chloramphenicol (CPL), and bisphenol A (BPA) interfere with the detection system. The results show that the detection system has strong anti-interference ability.

[0093] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a nanozyme, characterized in that: The following steps are involved: (1) Add vitamin B6 to methanol and stir to dissolve to obtain solution A; (2) Add Zn(NO3)2·6H2O, CuCl2 and CoCl2 into methanol and stir to dissolve to obtain solution B; (3) Add solution A to solution B and stir to obtain a mixed solution; (4) Add the mixed solution into a high-pressure reactor and heat to 100-140°C for 2-4 hours; (5) Cool the reaction solution to room temperature and filter out large particles of impurities using a microfiltration membrane to obtain a clear solution; (6) Centrifuging the clear liquid to obtain a supernatant; (7) The supernatant was vacuum dried to obtain Co / Cu@MOF nanozyme.

2. The preparation method according to claim 1, characterized in that: The mass ratio of vitamin B6 to Zn(NO3)2·6H2O is 1:1; the mass ratio of CuCl2 to CoCl2 is 1:1; a 0.22 μm filter membrane is used in step (5); and the drying temperature in step (7) is 50-70°C.

3. The preparation method according to claim 1, characterized in that: In step (1), 0.060-0.10 g of vitamin B6 is added to 5-10 mL of methanol and stirred to dissolve to obtain solution A. In step (2), 0.060-0.10 g of Zn(NO3)2·6H2O, 0.03-0.05 mg of CuCl2 and 0.03-0.05 mg of CoCl2 are added to 5-10 mL of methanol and stirred to dissolve to obtain solution B. In step (3), solution A and solution B are mixed for 5-10 minutes.

4. Co / Cu@MOF nanozyme obtained using the preparation method as described in any one of claims 1 to 3.

5. Use of the Co / Cu@MOF nanozyme as claimed in claim 4 in the determination of estrogen levels.

6. The use according to claim 5, wherein the estrogen is estradiol, estriol and diethylstilbestrol.

7. The method for detecting estrogen levels using the nanozyme according to claim 4, characterized in that: The steps include: S1, prepare sample measurement solution; S2, adding the sample test solution to the ELISA plate, then adding Co / Cu@MOF nanozyme and 4-aminopyrine, adding PBS buffer solution to the fixed volume and mixing; S3, irradiated with 808 nm near-infrared light and measured absorbance at a wavelength of 500-510 nm; S4, substituting the measured absorbance into the standard curve equation to obtain the estrogen concentration in the sample; The standard curve equations include standard curve equations of estradiol E2, estriol E3 and diethylstilbestrol DES.

8. The method according to claim 7, characterized in that The method for preparing the sample measuring solution is as follows: When the sample to be tested is serum, add ethyl acetate to the sample to be tested, vortex and centrifuge to obtain a supernatant and a residue; add ethyl acetate to the residue for extraction, extract and centrifuge to obtain a supernatant; combine the two extraction supernatants, blow dry with nitrogen, add pH7.4 PBS buffer, and obtain a sample test solution; When the sample to be tested is urine, the urine is centrifuged, the supernatant is taken, and stored at 4°C in the dark; before the test, 0.1% perchloric acid is added to the supernatant, mixed evenly, centrifuged at 8000 r / min for 5 min, the supernatant is taken, and the sample test solution is prepared.

9. The method according to claim 7, characterized in that In step S2, the concentration of the Co / Cu@MOF nanozyme is 1 mg / mL, the addition amount is 5-10 μL, the concentration of 4-aminopyrine is 1 mg / mL, and the volume is 5-10 μL; the PBS buffer solution is pH 7.4 PBS buffer; in step S3, the power of the near-infrared lamp is 1.25-1.50 W / cm 2 , the irradiation time is 10min.

10. The method according to claim 7, characterized in that The standard curve equation is obtained by the following steps: (1) Add 10 μL of E2 standard solution with a concentration of 0.012-36 μg / L, E3 standard solution with a concentration of 0.02-25 μg / L, or DES standard solution with a concentration of 0.045-15 μg / L to the ELISA plate; (2) Add 5 μL of 1 mg / mL Co / Cu@MOF and 5 μL of 1 mg / mL 4-aminopyrine, then add 0.1 mmol / L pH 7.4 PBS buffer solution to 50 μL and mix well. (3) Irradiate under 808 nm near-infrared light for 10 min, measure the absorbance at a wavelength of 505 nm, and draw a standard curve with E2, E3 or DES concentration as the horizontal axis and absorbance as the vertical axis to obtain the standard curve equation.

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