Colorimetric-fluorescent bimodal cortisol detection kit and method

Abstract

The invention provides a colorimetric-fluorescent dual-mode cortisol detection kit and a method. The kit comprises a detection tube, a magnetic bead modified antibody solution, a Cu-MOF modified cortisol solution and a tetramethyl benzidine TMB solution. In the detection process, after a magnetic bead modified antibody solution, a Cu-MOF modified cortisol solution and a detected sample are mixed and react, magnetic beads are separated out and react with a TMB solution; detecting the absorbance of the reaction product at the wavelength of 650 nm and the fluorescence intensity of the reaction product at the wavelength of 520 nm; and obtaining the cortisol concentration in the detected sample according to the ratio of the two. According to the method, the characteristic that Cu-MOF has the fluorescence characteristic and simulates peroxidase activity is utilized, two indexes including absorbance and fluorescence intensity are obtained in the same cortisol detection system at the same time, and the ratio of the absorbance with the wavelength of 650 nm to the fluorescence intensity with the wavelength of 520 nm is used as an index for judging the cortisol concentration; the accuracy of cortisol detection is obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biosensing and bio-detection, and relates to a cortisol detection and analysis technology, and specifically to a colorimetric-fluorescence dual-modal cortisol detection kit and method. Background Art

[0002] Cortisol is a steroid hormone secreted by the human body. It is a typical stress marker and plays an important role in regulating physiological processes such as blood pressure, blood sugar and carbohydrate metabolism. After secretion, cortisol enters the circulatory system and can be detected in a variety of 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 is under greater physical and psychological stress, it can lead to long-term high levels of cortisol, which in turn leads to increased blood sugar, increased appetite, weight gain and extreme fatigue in the relevant personnel, seriously affecting people's physical and mental health. Therefore, through the detection of cortisol, we can effectively analyze the physical and mental stress situation and provide an effective basis for timely mental health intervention and intervention.

[0003] At present, the main methods for cortisol detection at home and abroad include chromatographic separation, mass spectrometry quantitative analysis and enzyme-linked immunosorbent assay. Although these technologies can effectively monitor cortisol within the normal physiological range, they require sophisticated laboratory equipment, are expensive, have high detection costs, and lack the necessary specificity. Therefore, complex sample pretreatment steps such as solid phase extraction are required, making it difficult to apply to real-time monitoring. With the continuous development of biosensor technology, a new, efficient and accurate method for cortisol detection is urgently needed. Summary of the invention

[0004] The purpose of the present invention is to provide a Cu-MOF-based dual-modal sensing kit design method for detecting cortisol in saliva in view of the deficiencies of the prior art.

[0005] In the first aspect, the present invention provides a colorimetric-fluorescence dual-modality cortisol detection kit; the kit 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 at a wavelength of 650 nm of the reaction product 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 can be obtained by using a relationship equation or a relationship curve with 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, and dissolving the precipitate after reaction, centrifugation, and 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 the magnetic beads to the cortisol antibody is 5:1.

[0012] In a second aspect, the present invention provides a colorimetric-fluorescence dual-modal cortisol detection method, characterized in that it comprises the following steps: Step 1: Mix the magnetic bead-modified antibody solution, the Cu-MOF-modified cortisol solution and the sample to be tested for reaction.

[0013] Step 2: Separate the magnetic beads from the reaction product obtained in step 1, and add TMB solution to react; detect the absorbance A of the obtained product at a wavelength of 650 nm. 650nm and the fluorescence intensity F at a wavelength of 520 nm 520nm .

[0014] Step 3: Calculate the absorbance A at 650nm 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 can be obtained by using a relationship equation or a relationship curve with the cortisol concentration.

[0015] Preferably, the reaction conditions of step 1 are: reaction at room temperature for 20 min to 40 min.

[0016] Preferably, the reaction time of step 2 is 5 min to 15 min.

[0017] Preferably, the ratio of the magnetic bead-modified antibody solution, the Cu-MOF-modified cortisol solution, and the sample to be tested is 1:1:1.

[0018] The present invention has the following beneficial effects.

[0019] 1. The present invention utilizes Cu-MOF, which has both fluorescence properties and the characteristics of mimicking peroxidase activity, to simultaneously obtain two indicators, absorbance and fluorescence intensity, in the same cortisol detection system. In addition, the present invention utilizes the absorbance A at a wavelength of 650 nm. 650nm The fluorescence intensity F at 520 nm 520nm The ratio of 20 μg / L to 1349 μg / L was used as an indicator for judging the cortisol concentration, which significantly improved the accuracy of cortisol detection and achieved a low detection limit of 0.0020 μg / L and a large detection range of 0.07-1349 μg / L.

[0020] 2. The present invention realizes specific detection through competitive binding of Cu-MOF-modified cortisol and cortisol samples to magnetic bead antibodies, thereby improving the specificity of detection and reducing interference from other substances. With the help of magnetic beads, Cu-MOF-modified antibodies that do not participate in the reaction can also be effectively separated, simplifying the operation process and reducing the detection time. In addition, the Cu-MOF used in the present invention, as a catalase, can not only catalyze the coloring of TMB, but also amplify the signal and protect the antibody from long-term high temperature. Accurate dual-modal quantitative detection of cortisol is achieved by adding TMB to induce the absorption and fluorescence properties of Cu-MOF.

[0021] 3. The detection method of the present invention has a short detection time, which can be as low as 30 minutes. The entire detection process is relatively simple and efficient, and no sample pretreatment is required. The operation is simple and convenient, and no complex instruments and cumbersome operation steps are required, which is easy to promote and apply. The method has high precision, and the accuracy and reliability of the detection results are improved through designs such as dual-modal detection and specific binding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron microscope (TEM) characterization image of Cu-MOF modified cortisol of the present invention; Figure 2 is an absorption spectrum diagram of the catalytic activity of Cu-MOF modified cortisol induced by TMB of the present invention; Figure 3 This is a verification result diagram of the binding of the magnetic bead antibody prepared by the present invention to cortisol; Figure 4 It is the principle diagram of the method of the present invention; Figure 5 This is a graph showing the absorbance results of the present invention for detecting different concentrations of cortisol; Figure 6 is a standard curve diagram of absorbance for detecting different concentrations of cortisol in the present invention; Figure 7 is a standard curve diagram of absorbance / fluorescence intensity for detecting different concentrations of cortisol in the present invention; Figure 8 This is the absorption spectrum of the commercial kit for detecting cortisol built in Nanjing, China; Fig. 9 This is the standard curve of the commercial kit for detecting cortisol in Nanjing, China; Fig.10 This is the absorption spectrum of cortisol detected by the commercial kit of Abcam Company; Fig.11 This is the standard curve for detecting cortisol using the commercial kit of Abcam Company. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1 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.

[0025] Specifically, the Cu-MOF modified cortisol solution is obtained by mixing a cortisol standard solution with a CuCl2 aqueous solution, adding 4,4-bipyridine, and dissolving the precipitate after reaction, centrifugation, and rinsing in 500 µL pure water.

[0026] 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.

[0027] In addition, in order to obtain a standard curve and verify whether the prepared magnetic bead-modified antibody solution and Cu-MOF-modified cortisol solution are effective, it is also necessary to configure a variety of cortisol sample dilutions, PBS buffer solutions, and PBST solutions of different concentrations.

[0028] Specifically, the PBST solution is a mixed solution of 0.1 M PBS and 0.01% Tween-20.

[0029] The Cu-MOF used in the present embodiment has a large specific surface area, macroporosity 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, catalyst and carrier in an immunoassay. The cortisol detection and analysis method based on the Cu-MOF bimodal sensor, ingeniously utilizes the bionic peroxidase activity and fluorescence characteristics of Cu-MOF, and the characteristics of the simple and rapid separation of liquids by magnetic beads, with the advantages of accurate sensitivity, rapid on-site detection, it is easy to establish a rapid on-site 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 promote it.

[0030] Embodiment 2: A method for preparing a dual-modal sensing kit for detecting cortisol in saliva based on Cu-MOF is as follows: a. Preparation of Cu-MOF modified cortisol: Use a one-step self-assembly method to place 100µL of 50µg / mL cortisol standard solution and 2mL of 6.7mM CuCl2 aqueous solution into a clean beaker. After fully mixing at room temperature for 10 minutes, add 2mL of 26.8mM 4,4-bipyridine to the above mixture for 20 minutes. Centrifuge the reaction solution at 13000rpm for 5 minutes. Discard the supernatant, rinse the precipitate with ethanol twice and ultrapure water once, take the centrifuged precipitate and dissolve it in 500ul pure water to obtain a Cu-MOF modified cortisol solution. Figure 1 As shown, TEM characterization revealed that Cu-MOF-modified cortisol was successfully prepared.

[0031] Take 5 μL of the Cu-MOF-modified cortisol solution prepared above and add 150 μL of TMB colorimetric solution. Place the solution in a 37°C oven for 15 minutes. Place the reacted solution in an ELISA reader, set the scanning range to 525 nm-755 nm, select the area to be scanned, and perform absorption spectrum scanning. Figure 2 As shown, a characteristic absorption peak can be found at 650 nm, indicating that Cu-MOF-modified cortisol has bionic peroxidase activity and can catalyze TMB color development.

[0032] b. Preparation of magnetic beads modified with antibodies: 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, place the centrifuge tube on a vortex oscillator and vortex for 30 seconds to evenly mix the magnetic beads and cortisol antibody solution. Place the centrifuge tube on a rotating disk for 2 hours, separate the magnetic beads using a magnetic rack, and aspirate and discard the supernatant. Add 1 mL of 5% BSA blocking reagent to the magnetic beads, place the centrifuge tube on a vortex oscillator and vortex for 30 seconds, and then place the centrifuge tube on a rotating disk for 30 minutes to reduce nonspecific adsorption. After the blocking reaction is completed, separate the magnetic beads using a magnetic rack and discard the supernatant. Wash the magnetic beads again with PBS buffer for 3 times to completely remove unbound blocking reagent and other impurities. Finally, store the magnetic bead-modified antibodies in 500 µL PBS solution.

[0033] The successful preparation of magnetic bead antibodies was verified by the traditional ELISA method. The specific steps are as follows: (1) Coating cortisol solid phase antigen: dilute the cortisol-BSA conjugate with carbonate buffer (pH = 9.6), add the diluted cortisol-BSA conjugate to a 96-well plate, and coat each well with 100 μL. Place it at 4°C overnight; (2) BSA blocking: remove the 96-well plate after overnight coating, wash the multi-well plate with PBST washing solution to remove excess free cortisol-BSA conjugate, repeat 4 times, and then block the remaining binding sites with 2% BSA solution, 200 μL per well. μL, block at 37℃ for 3 hours; (3) Add sample and magnetic bead antibody: After blocking, wash the plate 4 times with PBST washing solution to remove excess BSA, then add 50μL of cortisol sample and 50μL of magnetic bead antibody solution to the multi-well plate, incubate at 37℃ for 1 hour, at this time, the solid phase antigen in the multi-well plate competes with the cortisol in the sample for binding to the cortisol magnetic bead antibody; (4) Add HRP-IgG: First wash the plate 4 times with PBST washing solution, then add HRP-IgG, incubate at 37℃ for 30 minutes; (5) Color reaction: After incubation, wash the plate 4 times with PBST washing solution to remove excess substances not bound to the multi-well plate, then add 100 μL of TMB colorimetric solution to each well, react at 37℃ for 15 minutes, and finally add 100μL of 2M HCl solution to each well to terminate the color reaction; (6) Detect color development results: Use an enzyme reader to detect the absorbance of the above colorimetric solution at a wavelength of 450 nm. Figure 3 As shown, a characteristic absorption peak can be found at 450 nm, indicating that the prepared magnetic bead antibody can bind to cortisol, proving the successful preparation of the magnetic bead antibody.

[0034] Example 3 like Figure 4 As shown, a colorimetric-fluorescence dual-modality cortisol detection method comprises the following steps: Step 1: Take 50 μL of 0.5 μM magnetic bead-modified antibody solution and 50 μL of 0.3 μM Cu-MOF-modified cortisol solution and add them into the detection tube, add 50 μL of the sample to be tested, and react at room temperature for 30 minutes to allow cortisol to fully combine with the magnetic bead antibody.

[0035] Step 2: Place the reacted solution on a magnetic stand for 1 minute, discard the supernatant and add 150 μL TMB solution, react for 5 minutes; place the reacted 96-well polystyrene microplate into an ELISA reader and read the absorbance at 650 nm. 650nm and the fluorescence intensity F at 520 nm 520nm .

[0036] According to the principle of competitive binding, the higher the cortisol content in the sample, the less Cu-MOF-modified cortisol will bind to the magnetic bead antibody. The bionic peroxidase activity and fluorescence properties of Cu-MOF lead to different fluorescence intensity and absorbance, thus constructing a colorimetric-fluorescence dual-modal sensing kit to detect the concentration of cortisol in saliva.

[0037] Step 3: Calculate the absorbance A at 650nm 650nm The fluorescence intensity F at 520 nm 520nm The ratio A 650nm / F 520nm ; According to the obtained ratio A 650nm / F 520nm , compare and find the cortisol concentration of the sample to be tested on the standard curve of cortisol concentration, or calculate the cortisol concentration of the sample to be tested through the function expression of the standard curve.

[0038] The standard curve was obtained by experimental fitting, and the specific process was as follows: Preparation of cortisol sample dilution solution: dilution of the cortisol sample solution with PBST solution into the following different concentrations: 0.072µg / L, 0.144µg / L, 2.158µg / L, 10.792µg / L, 31.585µg / L, 53.962µg / L, 269.811µg / L, 1349.055µg / L. Take multiple groups of 50 μL of 0.5 μM magnetic bead-modified antibody solution and 50 μL of 0.3 μM Cu-MOF-modified cortisol solution and add them to different wells of a 96-well polystyrene microplate, and add 50 μL of cortisol sample dilutions of different concentrations to react for 30 minutes; place the reacted solution on a magnetic stand for 1 minute, extract the supernatant into a blank well of the 96-well plate, and finally add 100 μL of TMB solution and react for 5 minutes; put the reacted 96-well polystyrene microplate into an ELISA reader and read the absorbance A at 650 nm. 650nm and the fluorescence intensity F at 520 nm 520nm , calculate the ratio A of the two650nm / 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.

[0039] Comparative Example 1 A method for detecting cortisol. The difference between this comparative example and Example 3 is that 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.

[0040] 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: 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.

[0041] 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 linear relationship between the sensor and the concentration range of 0.07-1349 μg / L is as follows: 650nm / F 520nm=-0.007log(x)+0.0203, where x represents the cortisol concentration, the linearity is 0.99, and the detection limit is 0.0020μg / L. The detection range covers the range of cortisol concentration in saliva from normal people to high-stress patients.

[0042] It can be seen that compared with the detection of cortisol concentration by absorbance alone, the colorimetric-fluorescence dual-modal 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.

[0043] Example 4 Validation of a colorimetric-fluorescence dual-modal cortisol detection method: The cortisol concentration of the sample solution to be tested was obtained by Example 3, and the detected concentration was compared with the results of the standard detection method (liquid chromatography-tandem mass spectrometry (LC-MS / MS)). The comparison results are shown in Table 1. The recovery rate detected in Example 3 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) × 100%, relative standard deviation (RSD) = standard deviation / sample mean × 100%. The experimental results show that the colorimetric-fluorescence dual-modal cortisol detection method provided in Example 3 can accurately detect the cortisol concentration in the sample to be tested with unknown concentration.

[0044] Table 1. Recovery rate and relative standard deviation of cortisol detected by the method of the present invention 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 Example 5 Performance comparison of a colorimetric-fluorescence dual-modal cortisol detection method and existing cortisol kits: In order to further verify the performance of the cortisol detection method of the present invention, the colorimetric-fluorescence dual-modality cortisol detection method provided in Example 3 was compared with domestic and foreign commercial cortisol kits. The kits used for comparison all adopted enzyme-linked immunosorbent assay technology. Figure 8 and Fig. 9 They are the absorption spectrum and standard curve of the commercial kit for detecting cortisol in Nanjing, China. The concentration of cortisol to be tested is 40µg / L, 80µg / L, 160µg / L, 320µg / L, and 640µg / L. As the concentration of cortisol to be tested increases, the absorbance gradually decreases. And the absorbance value 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. Fig.10 and Fig.11This is the absorption spectrum and standard curve of cortisol detected by the commercial kit of Abcam Company. The concentration of cortisol to be tested is 1µg / L, 5µg / L, 10µg / L, 20µg / L, and 100µg / L. As the concentration of cortisol to be tested increases, the absorbance gradually decreases. And the absorbance value 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.

[0045] From the detection limit results: the detection limit of Nanjing Jiancheng's commercial kit is 28.9 µg / L, the detection limit of Abcam's commercial kit is 0.5 µg / L, and the detection limit of the method of the present invention is 0.0020μg / L, which is 2 orders of magnitude lower than the above commercial kits.

[0046] In terms of detection range: the detection range of Nanjing Jiancheng's commercial test kit is 40-640µg / L, the detection range of Abcam's commercial test kit is 1-100µg / L, and the detection range of the method of the present invention is 0.07-1349μg / L, which is 3 orders of magnitude larger than the detection range of commercial test kits.

[0047] In terms of the operation steps: the above-mentioned traditional kits all involve the pretreatment step of 96-well plates, including a series of processes such as coating the cortisol solid phase antigen on the 96-well plate (4°C, 12 hours) - washing the plate - BSA blocking (37°C, 3 hours) - washing the plate. The storage and use period of the pretreated 96-well plate are strictly required. The kit designed by the present invention does not need to pretreat the 96-well plate, and the unmodified 96-well plate can be used directly to carry out the experiment, which relaxes the requirements for storage conditions and is simple and practical.

[0048] The experimental results show that the method of the present invention 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.

[0049] It should be stated that the content and specific implementation methods of the present invention are intended to demonstrate the practical application of the technical solution provided by the present invention and should not be interpreted 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 protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A colorimetric-fluorescence dual-modality cortisol detection kit; characterized in that: The method comprises a detection tube, an antibody solution modified with magnetic beads, a cortisol solution modified with Cu-MOF and a tetramethylbenzidine TMB solution; during the detection process, the antibody solution modified with magnetic beads, the cortisol solution modified with Cu-MOF 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 can be obtained by using a relationship equation or a relationship curve with the cortisol concentration.

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.

3. A colorimetric-fluorescence dual-modality cortisol detection kit according to claim 1, characterized in that: The Cu-MOF modified cortisol solution is obtained by mixing a cortisol standard solution with a CuCl2 aqueous solution, adding 4,4-bipyridine, and dissolving the precipitate after reaction, centrifugation, and rinsing in pure water.

4. A colorimetric-fluorescence dual-modality cortisol detection kit according to claim 3, characterized in that: 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.

5. A colorimetric-fluorescence dual-modality cortisol detection kit according to claim 1, characterized in that: 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.

6. A colorimetric-fluorescence dual-modality cortisol detection kit according to claim 5, characterized in that: In the magnetic bead-modified antibody solution, the magnetic bead content is 2 mg / mL to 5 mg / mL, the cortisol antibody content is 3 mg / mL to 7.5 mg / mL; and the volume ratio of the magnetic beads to the cortisol antibody is 5:

1.

7. A colorimetric-fluorescence dual-modality cortisol detection method; characterized in that: The following steps are involved: Step 1: Mix the magnetic bead-modified antibody solution, the Cu-MOF-modified cortisol solution and the sample to be tested for reaction; Step 2: Separate the magnetic beads from the reaction product obtained in step 1, and add TMB solution to react; detect the absorbance A of the obtained product at a wavelength of 650 nm. 650nm and the fluorescence intensity F at a wavelength of 520 nm 520nm ; Step 3: Calculate the absorbance A at 650nm 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 can be obtained by using a relationship equation or a relationship curve with the cortisol concentration.

8. A colorimetric-fluorescence dual-modality cortisol detection method according to claim 7, characterized in that: The reaction conditions of step 1 are: reaction at room temperature for 20 min to 40 min.

9. A colorimetric-fluorescence dual-modality cortisol detection method according to claim 7, characterized in that: The reaction time of step 2 is 5 min to 15 min.

10. A colorimetric-fluorescence dual-modality cortisol detection method according to claim 7, characterized in that: The ratio of the magnetic bead-modified antibody solution, the Cu-MOF-modified cortisol solution, and the sample to be tested is 1:1:1.

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