A colorimetric-fluorescence dual-modal cortisol detection kit and method

Abstract

The application provides a colorimetric-fluorescent dual-mode cortisol detection kit and method; it comprises a detection tube, a magnetic bead modified antibody solution, a Cu-MOF modified cortisol solution and a tetramethylbenzidine TMB solution. In the detection process, the magnetic bead modified antibody solution, the Cu-MOF modified cortisol solution and the sample to be detected are mixed and reacted, then the magnetic beads are separated and reacted with the TMB solution; the absorbance of the reaction product at 650nm wavelength and the fluorescence intensity at 520nm wavelength are detected; and the cortisol concentration in the sample to be detected is obtained according to the ratio of the two. The application utilizes the characteristics that Cu-MOF has fluorescence characteristics and mimics peroxidase activity at the same time, so that the absorbance and fluorescence intensity are obtained at the same time in the same cortisol detection system, and the application utilizes the ratio of the absorbance at 650nm wavelength and the fluorescence intensity at 520nm wavelength as an index for judging the cortisol concentration.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanobiological sensing and biological detection, and relates to a cortisol detection and analysis technology, in particular to a colorimetric-fluorescent dual-mode cortisol detection kit and method. BACKGROUND

[0002] Cortisol is a kind of autonomic secretion of human body, is a kind of typical stress marker, plays an important role in regulating blood pressure, blood sugar and carbohydrate metabolism and other physiological processes. After secretion, cortisol enters the circulatory system and can be detected in various body fluids. Under normal circumstances, the body can well control the secretion of cortisol and regulate the content of cortisol in the blood, however, when the human body bears greater physiological and psychological stress, it can cause long-term high cortisol level, and further cause related personnel's blood sugar to rise, appetite to increase, body weight to rise and extreme fatigue and the like, which seriously affects people's physical and mental health. Therefore, through the detection of cortisol, the physical and mental stress situation can be effectively analyzed, and effective basis is provided for timely psychological health intervention and intervention.

[0003] At present, the cortisol detection methods at home and abroad mainly include chromatographic separation method, mass spectrometric quantitative analysis method and enzyme-linked immunoassay technology and the like. Although these technologies can effectively monitor cortisol within the normal physiological change range, they need precise laboratory equipment, are expensive in price, high in detection cost and lack necessary specificity, and therefore need complex sample pretreatment steps such as solid phase extraction, and are difficult to apply to real-time monitoring. With the continuous development of biological sensing technology, there is an urgent need for a new type of high-efficiency and accurate cortisol detection means. SUMMARY

[0004] The purpose of the present application is to overcome the deficiencies of the prior art, and to provide a dual-mode sensing kit design method for cortisol detection in saliva based on Cu-MOF.

[0005] In a first aspect, the present application provides a colorimetric-fluorescent dual-mode cortisol detection kit, which comprises a detection tube, a magnetic bead modified antibody solution, a Cu-MOF modified cortisol solution and a tetramethylbenzidine TMB solution. In the detection process, the magnetic bead modified antibody solution, the Cu-MOF modified cortisol solution and the measured sample are mixed and reacted, and then the magnetic beads are separated and reacted with the TMB solution; the absorbance A 650nm 650nm wavelength and the fluorescence intensity F 520nm 520nm wavelength of the detection reaction product are detected; and the cortisol concentration in the measured sample is obtained according to the relationship formula or relationship curve between the ratio A 650nm / F 520nm and the cortisol concentration.

[0006] Preferably, the concentration of the magnetic bead-modified antibody solution is 0.3 μM to 1 μM; the concentration of the Cu-MOF-modified cortisol solution is 0.2 μM to 1 μM.

[0007] Preferably, the sample to be tested is saliva.

[0008] Preferably, the Cu-MOF modified cortisol solution is obtained by mixing a cortisol standard solution with a CuCl2 aqueous solution, adding 4,4-bipyridine, reacting, centrifuging, and dissolving the precipitate after rinsing in pure water.

[0009] Preferably, in the Cu-MOF-modified cortisol solution, the concentration of CuCl2 is 4mM to 10mM, the concentration of 4,4-bipyridine is 16mM to 40mM, and the concentration of the cortisol solution is 30µg / mL to 70µg / mL; the volume ratio of the cortisol solution, CuCl2, and 4,4-bipyridine is 1:20:20.

[0010] Preferably, the magnetic bead-modified antibody solution is obtained by mixing N-hydroxysuccinimide-modified magnetic beads with a cortisol antibody solution; separating the magnetic beads and adding a 5% BSA blocking reagent, mixing and blocking the reaction; and separating the magnetic beads, washing them, and placing them in a PBS solution.

[0011] Preferably, in the magnetic bead-modified antibody solution, the magnetic bead content is 2 mg / mL to 5 mg / mL, and the cortisol antibody content is 3 mg / mL to 7.5 mg / mL; the volume ratio of magnetic beads to cortisol antibodies is 5:1.

[0012] In a second aspect, the present invention provides a colorimetric-fluorescence dual-modality cortisol detection method, characterized in that it comprises the following steps:

[0013] Step 1: Mix the magnetic bead-modified antibody solution, the Cu-MOF-modified cortisol solution and the sample to be tested for reaction.

[0014] Step 2: Separate the magnetic beads from the reaction product obtained in step 1 and add TMB solution to react; detect the absorbance of the obtained product at a wavelength of 650 nm. 650nm and the fluorescence intensity F at a wavelength of 520 nm 520nm .

[0015] Step 3: Calculate the absorbance A at 650nm wavelength 650nm The fluorescence intensity F at 520 nm 520nm The ratio A 650nm / F 520nm ; According to the ratio A 650nm / F 520nm The cortisol concentration in the tested sample is obtained by using a relationship equation or a relationship curve with the cortisol concentration.

[0016] Preferably, the reaction condition of step one is 20-40 min at room temperature.

[0017] Preferably, the reaction time of step two is 5-15 min.

[0018] Preferably, the ratio of the magnetic bead modified antibody solution, the Cu-MOF modified cortisol solution and the sample to be detected is 1:1:1.

[0019] The present application has the following advantages.

[0020] 1. The present application utilizes Cu-MOF which has both fluorescence characteristics and peroxidase-like activity to simultaneously obtain absorbance and fluorescence intensity in the same cortisol detection system, and the present application uses the ratio of absorbance A 650nm at 650 nm wavelength to fluorescence intensity F 520nm at 520 nm wavelength as an index to judge the concentration of cortisol, which significantly improves the accuracy of cortisol detection, realizes a low detection limit of 0.0020 μg / L and a large detection range of 0.07-1349 μg / L.

[0021] 2. The present application realizes specific detection through the competitive binding of Cu-MOF modified cortisol and cortisol sample to magnetic bead antibody, improves the specificity of detection, and reduces the interference of other substances. With the help of magnetic beads, the Cu-MOF modified antibody not involved in the reaction can be effectively separated, the operation process is simplified, and the detection time is reduced. In addition, the Cu-MOF used in the present application as catalase can not only catalyze the coloring of TMB, but also amplify the signal and protect the antibody from long-term high temperature. The addition of TMB induces the absorbance and fluorescence characteristics of Cu-MOF to realize the accurate dual-mode quantitative detection of cortisol.

[0022] 3. The detection method of the present application has a short detection time, which can be as low as 30 minutes, and the whole detection process is relatively simple and efficient, without the need for sample pretreatment. The operation is simple, does not require complex instruments and cumbersome operation steps, and is easy to popularize and apply. The accuracy is high, and through the design of dual-mode detection and specific binding, the accuracy and reliability of the detection results are improved. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the transmission electron microscope (TEM) characterization diagram of the Cu-MOF modified cortisol of the present application;

[0024] Figure 2 is the absorption spectrum diagram of the catalytic activity of TMB induced Cu-MOF modified cortisol;

[0025] Figure 3 This is a diagram showing the verification results of the binding of the magnetic bead antibody prepared by the present invention to cortisol;

[0026] Figure 4 It is a schematic diagram of the method of the present invention;

[0027] Figure 5 This is a graph showing the absorbance results of the present invention for detecting different concentrations of cortisol;

[0028] Figure 6 This is a standard curve diagram of absorbance for detecting different concentrations of cortisol according to the present invention;

[0029] Figure 7 is a standard curve diagram of absorbance / fluorescence intensity for detecting different concentrations of cortisol according to the present invention;

[0030] Figure 8 This is the absorption spectrum of the commercial kit for detecting cortisol built in Nanjing, China;

[0031] Figure 9 This is the standard curve of the commercial kit for detecting cortisol in Nanjing, China;

[0032] Figure 10 This is the absorption spectrum of cortisol detected by the commercial kit of Abcam Company;

[0033] Figure 11 This is the standard curve of cortisol detection using the commercial kit of Abcam Company. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] Example 1

[0036] A Cu-MOF-based dual-modal sensing kit for detecting cortisol in saliva comprises the following substances: a magnetic bead-modified antibody solution, a Cu-MOF-modified cortisol solution, and a tetramethylbenzidine (TMB) solution.

[0037] Specifically, the Cu-MOF modified cortisol solution is obtained by mixing a cortisol standard solution with a CuCl2 aqueous solution, adding 4,4-bipyridine, reacting, centrifuging, and rinsing, and dissolving the precipitate in 500 μL of pure water.

[0038] Specifically, the magnetic bead-modified antibody solution is obtained by mixing N-hydroxysuccinimide-modified magnetic beads with a cortisol antibody solution; separating the magnetic beads and adding a 5% BSA blocking reagent, mixing, and blocking the reaction; and washing the separated magnetic beads and placing them in a PBS solution.

[0039] In addition, in order to obtain the standard curve, and verify whether the prepared magnetic bead modified antibody solution and Cu-MOF modified cortisol solution are effective, a corresponding configuration of a variety of different concentrations of cortisol sample diluent, PBS buffer and PBST solution is also needed.

[0040] Specifically, the PBST solution is a mixed solution of 0.1 M PBS and 0.01% Tween-20.

[0041] The Cu-MOF used in this embodiment has a large specific surface area, large porosity and high stability as a metal organic framework. It can be compounded with a variety of nanomaterials, and can be used as a signal tracer, a catalyst and a carrier in immunoassay. The cortisol detection and analysis method based on the Cu-MOF bimodal sensor ingeniously utilizes the biomimetic peroxidase activity and fluorescence characteristics of Cu-MOF, as well as the characteristics of magnetic beads for simple and rapid separation of liquids, has the advantages of precision and sensitivity, on-site rapid detection, etc., is convenient for establishing an on-site rapid detection and analysis platform for disease markers in body fluids, and is expected to become a new tool in the field of disease marker detection and analysis and be popularized.

[0042] Example 2:

[0043] A preparation method of a bimodal sensing kit for cortisol detection in saliva based on Cu-MOF is as follows:

[0044] a. Preparation of Cu-MOF modified cortisol: 100 μL of 50 μg / mL cortisol standard solution and 2 mL of 6.7 mM CuCl2 aqueous solution were placed in a clean beaker, mixed thoroughly at room temperature for 10 minutes, and then 2 mL of 26.8 mM 4,4-pyridine was added to the mixture for 20 minutes. The reaction solution was centrifuged at 13,000 rpm for 5 minutes. The supernatant was discarded, and the precipitate was rinsed with ethanol twice and ultrapure water once. The centrifuged precipitate was dissolved in 500 μL of pure water to obtain a Cu-MOF modified cortisol solution. As shown in Figure 1 TEM characterization can find that the Cu-MOF modified cortisol is successfully prepared.

[0045] 5 μL of the prepared Cu-MOF modified cortisol solution was taken and added to 150 μL of TMB color developing liquid. The solution was placed in a 37°C oven for 15 minutes. The reacted solution was placed in an enzyme marker, the spectral range was set to 525 nm-755 nm, the spectral region to be scanned was selected, and the absorption spectrum was scanned. As shown in Figure 2 It can be found that there is a characteristic absorption peak at 650 nm, indicating that the Cu-MOF modified cortisol has biomimetic peroxidase activity and can catalyze TMB color development.

[0046] b. Preparation of magnetic bead modified antibody: Take 1 mL of 3.2 mg / mL N-hydroxysuccinimide modified magnetic beads in a centrifuge tube, add 200 μL of 5.7 mg / mL cortisol antibody solution, and vortex mix the centrifuge tube on a vortex shaker for 30 seconds to mix the magnetic beads and cortisol antibody solution uniformly. Place the centrifuge tube on a rotary instrument for 2 hours, separate the magnetic beads using a magnetic stand, and aspirate and discard the supernatant. Add 1 mL of 5% BSA blocking reagent to the magnetic beads, vortex mix the centrifuge tube on a vortex shaker for 30 seconds, and then place the centrifuge tube on a rotary instrument for 30 minutes to reduce non-specific adsorption. After the completion of the blocking reaction, separate the magnetic beads using a magnetic stand, and discard the supernatant. Wash the magnetic beads with PBS buffer again for 3 times to completely remove the unbound blocking reagent and other impurities. Finally, store the magnetic bead modified antibody in 500 μL of PBS solution.

[0047] Verify the successful preparation of the magnetic bead antibody by the traditional ELISA method, and the specific steps are as follows: (1) Coating cortisol solid phase antigen: Dilute the cortisol-BSA conjugate with carbonate buffer (pH=9.6), and add the diluted cortisol-BSA conjugate to the 96-well plate at a coating amount of 100 μL per well, and stand at 4°C overnight; (2) BSA blocking: Take out the 96-well plate coated overnight, wash the multi-well plate with PBST washing solution to remove excess free cortisol-BSA conjugate, repeat 4 times, then block the empty binding sites with 2% BSA solution, 200 μL per well, 37°C for 3 hours; (3) Add sample and magnetic bead antibody: After blocking, wash the plate with PBST washing solution 4 times to remove excess BSA, then add 50 μL of cortisol sample and 50 μL of magnetic bead antibody solution to the multi-well plate, incubate at 37°C for 1 hour, at this time the solid phase antigen in the multi-well plate competes with the cortisol in the sample to bind the cortisol magnetic bead antibody; (4) Add HRP-IgG: After washing the plate with PBST washing solution 4 times, add HRP-IgG, incubate at 37°C for 30 minutes; (5) Color development reaction: After incubation, wash the plate with PBST washing solution 4 times to remove excess unbound substances from the multi-well plate, then add 100 μL of TMB color developing liquid to each well, react at 37°C for 15 minutes, and finally add 100 μL of 2M HCl solution to each well to stop the color development reaction; (6) Detect color development results: Detect the absorbance of the above color developing liquid at 450 nm wavelength with an enzyme marker. As shown in Figure 3 , a characteristic absorption peak at 450 nm can be found, indicating that the prepared magnetic bead antibody can bind to cortisol, proving the successful preparation of the magnetic bead antibody.

[0048] Example 3

[0049] As shown in Figure 4 , a colorimetric-fluorescent dual-mode cortisol detection method includes the following steps:

[0050] Step one, take 50 μL of magnetic bead modified antibody solution with a concentration of 0.5 μM and 50 μL of Cu-MOF modified cortisol solution with a concentration of 0.3 μM into the detection tube, corresponding to 50 μL of the measured sample, react at room temperature for 30 minutes, so that cortisol and magnetic bead antibody are fully combined.

[0051] Step two, place the reacted solution on a magnetic stand for 1 minute, discard the supernatant and add 150 μL of TMB solution, react for 5 minutes; place the reacted 96-well polystyrene microwell plate into an enzyme label instrument, read the absorbance A at 650 nm and the fluorescence intensity F at 520 nm 650nm 520nm .

[0052] According to the competitive binding principle, the more cortisol sample content, the less Cu-MOF modified cortisol combined with magnetic bead antibody, the different fluorescence intensity and absorbance caused by the biomimetic peroxidase activity and fluorescence characteristics of Cu-MOF, thereby constructing a colorimetric-fluorescence dual-mode sensing kit to detect cortisol concentration in saliva.

[0053] Step three, calculate the ratio A 650nm / F 520nm of the absorbance A at 650 nm and the fluorescence intensity F at 520 nm 650nm 520nm ; according to the obtained ratio A 650nm / F 520nm , compare and find the cortisol concentration of the measured sample on the standard curve of cortisol concentration, or calculate the cortisol concentration of the measured sample through the functional expression of the standard curve.

[0054] The standard curve is obtained by experimental fitting, and the specific process is as follows: prepare cortisol sample diluent: dilute cortisol sample solution with PBST solution to the following different concentrations: 0.072 μg / L, 0.144 μg / L, 2.158 μg / L, 10.792 μg / L, 31.585 μg / L, 53.962 μg / L, 269.811 μg / L, 1349.055 μg / L. Take multiple groups of 50 μL of magnetic bead modified antibody solution with a concentration of 0.5 μM and 50 μL of Cu-MOF modified cortisol solution with a concentration of 0.3 μM, respectively, and add them to different holes of the 96-well polystyrene microwell plate, corresponding to 50 μL of cortisol sample diluent with different concentrations, react for 30 minutes; place the reacted solution on a magnetic stand for 1 minute, extract the supernatant into the blank holes of the 96-well plate, and finally add 100 μL of TMB solution, react for 5 minutes; place the reacted 96-well polystyrene microwell plate into an enzyme label instrument, read the absorbance A at 650 nm and the fluorescence intensity F at 520 nm 650nm ​​and the fluorescence intensity F at 520 nm 520nm , calculate the ratio A of the two 650nm / F 520nm ; According to the concentration of different cortisol sample dilutions and their corresponding ratio A 650nm / F 520nm , establish a standard curve of this ratio and cortisol concentration. The functional expression of the standard curve obtained in this embodiment is: 650nm / F 520nm =-0.007log(x)+0.0203, where x represents the cortisol concentration.

[0055] Comparative Example 1

[0056] A method for detecting cortisol. The difference between this comparative example and Example 3 is that the absorbance A is not used. 650nm The fluorescence intensity F at 520 nm 520nm The ratio A 650nm / F 520nm As an indicator for establishing a standard curve and detecting cortisol; instead, the absorbance at 650 nm was used. 650nm As an indicator for establishing a standard curve and detecting cortisol.

[0057] In order to verify the advantages of this embodiment in cortisol detection, a comparison between Example 3 and Comparative Example 1 is described as follows:

[0058] The absorbance A used in Comparative Example 1 650nm The relationship between cortisol concentration and Figure 5 As shown in Figure 2, the absorbance value gradually decreases with the increase of cortisol concentration. Figure 6 As shown, according to the least squares curve fitting algorithm, the cortisol concentration and A 650nm It can be seen that Comparative Example 1 shows good linearity in the concentration range of 2.16-53.96 μg / L. The linear relationship is A 650nm =-0.774log(x)+2.863, where x represents the cortisol concentration, the linearity is 0.99, and the detection limit is 0.9904μg / L.

[0059] The ratio of the absorbance at 650 nm and the fluorescence intensity at 520 nm used in Example 3 is A 650nm / F 520nm The relationship between cortisol concentration and Figure 7 As shown, according to the least squares curve fitting algorithm, the cortisol concentration and A 650nm / F 520nm The sensor shows good linearity in the concentration range of 0.07-1349μg / L. The linear relationship is A 650nm / F520nm = -0.007log(x) + 0.0203, where x represents the concentration of cortisol, the linearity is 0.99, and the detection limit is 0.0020 μg / L. The detection range covers the cortisol concentration range in saliva of normal people to high stress patients.

[0060] It can be seen that, compared with detecting the concentration of cortisol by absorbance alone, the colorimetric-fluorescence dual-mode cortisol detection method proposed in Example 3 expands the detection range by nearly 4 orders of magnitude and reduces the detection limit by 495 times. The experimental results prove that Example 3 can accurately detect a wide range of cortisol concentrations in the sample to be tested.

[0061] Example 4

[0062] Verification of a colorimetric-fluorescence dual-mode cortisol detection method:

[0063] The concentration of cortisol in the sample solution to be tested is obtained by Example 3, and the detected concentration is compared with the result detected by the standard detection method (liquid chromatography-tandem mass spectrometry (LC-MS / MS)). The comparison results are shown in Table 1, the recovery rate of Example 3 detection is in the range of 97.56%-102.63%, and the relative standard deviation (RSD) is less than 2.9%. Recovery rate = (measured value / true value) x 100%, relative standard deviation (RSD) = standard deviation / sample mean x 100%. The experimental results prove that the colorimetric-fluorescence dual-mode cortisol detection method provided by Example 3 can accurately detect the concentration of cortisol in the sample to be tested with unknown concentration.

[0064] Table 1. Recovery rate and relative standard deviation of the method for detecting cortisol

[0065] Serial number Standard method (µg / L) The present method (µg / L) Recovery rate (%) RSD (%) 1 3.42 3.51 102.63 2.9 2 12.28 11.98 97.56 2.3 3 20.61 20.97 101.75 1.4

[0066] Example 5

[0067] Performance comparison of a colorimetric-fluorescence dual-mode cortisol detection method with existing cortisol kits:

[0068] In order to further verify the performance of the cortisol detection method of the present application, the colorimetric-fluorescence dual-mode cortisol detection method provided by Example 3 is compared with domestic and foreign commercial cortisol kits in performance. The kits used for comparison all use enzyme-linked immunoassay technology. Figure 8 and Figure 9The absorption spectrum and standard curve chart of the domestic Nanjing Jiancheng commercial kit for detecting cortisol are respectively. The concentration of the measured substance cortisol is 40 µg / L, 80 µg / L, 160 µg / L, 320 µg / L and 640 µg / L. With the increase of the concentration of the measured substance cortisol, the absorbance gradually decreases. The numerical value of the absorbance is linearly related to the concentration of cortisol. The linear equation is y = 3.11-1.03x, the linearity is 0.989, the linear range is 40-640 µg / L, and the detection limit is 28.9 µg / L. Figure 10 and Figure 11 The absorption spectrum and standard curve chart of the foreign Abeam company commercial kit for detecting cortisol are respectively. The concentration of the measured substance cortisol is 1 µg / L, 5 µg / L, 10 µg / L, 20 µg / L and 100 µg / L. With the increase of the concentration of the measured substance cortisol, the absorbance gradually decreases. The numerical value of the absorbance is linearly related to the concentration of cortisol. The linear equation is y = 2.19-0.92x, the linearity is 0.993, the linear range is 1-100 µg / L, and the detection limit is 0.5 µg / L.

[0069] From the detection limit result: the detection limit of the Nanjing Jiancheng commercial kit is 28.9 µg / L, the detection limit of the Abeam company commercial kit is 0.5 µg / L, and the detection limit of the method is 0.0020 µg / L, which is lower than the above-mentioned commercial kits by two orders of magnitude.

[0070] From the detection range: the detection range of the Nanjing Jiancheng commercial kit is 40-640 µg / L, the detection range of the Abeam company commercial kit is 1-100 µg / L, and the detection range of the method is 0.07-1349 µg / L, which is three orders of magnitude larger than the detection range of the commercial kit.

[0071] From the operation steps: the above-mentioned traditional kits all involve a pretreatment step of 96-well plates, including coating cortisol solid-phase antigen on the 96-well plate (4℃, 12 hours) - washing the plate - BSA blocking (37℃, 3 hours) - washing the plate and a series of processes. The storage and shelf life of the pretreated 96-well plate have strict requirements. The kit designed in the application does not need to pretreat the 96-well plate, and can directly use the unmodified 96-well plate to carry out the experiment, which relaxes the requirements for the storage conditions and is simple and practical.

[0072] The experimental results prove that the method has a low detection limit and a wide detection range, can accurately detect the concentration of cortisol in the sample and is simple and convenient to operate.

[0073] It should be noted that the present disclosure and specific embodiments are intended to demonstrate the practical application of the technical solutions provided by the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the scope of protection of the claims shall fall within the scope of protection of the present invention.

Claims

1. A colorimetric-fluorescence dual-modality cortisol detection kit; characterized in that: The method comprises a detection tube, a magnetic bead-modified antibody solution, a Cu-MOF-modified cortisol solution, and a tetramethylbenzidine (TMB) solution. During the detection process, the magnetic bead-modified antibody solution, the Cu-MOF-modified cortisol solution, and the sample to be tested are mixed and reacted, and then the magnetic beads are separated and reacted with the TMB solution. The absorbance A of the reaction product at a wavelength of 650 nm is detected. 650nm and the fluorescence intensity F at a wavelength of 520 nm 520nm ; According to the ratio A 650nm / F 520nm The cortisol concentration in the tested sample is obtained by using a relationship equation or a relationship curve with the cortisol concentration; The Cu-MOF modified cortisol solution is obtained by mixing a cortisol standard solution with a CuCl2 aqueous solution, adding 4,4'-bipyridine, reacting, centrifuging, and rinsing the resulting precipitate and dissolving it in pure water; The magnetic bead-modified antibody solution is prepared by mixing N-hydroxysuccinimide-modified magnetic beads with a cortisol antibody solution; separating the magnetic beads and adding a 5% BSA blocking reagent, mixing and blocking the reaction; and separating the magnetic beads, washing them, and placing them in a PBS solution.

2. A colorimetric-fluorescence dual-modality cortisol detection kit according to claim 1, characterized in that: The concentration of the magnetic bead-modified antibody solution is 0.3 μM to 1 μM; the concentration of the Cu-MOF-modified cortisol solution is 0.2 μM to 1 μM.

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