A method for detecting adrenaline based on a high laccase activity and high fluorescence performance Cu-MB colorimetric-fluorescent dual-mode sensor

Abstract

The application discloses a method for detecting adrenaline by using a colorimetric-fluorescent dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance, and comprises the following steps: constructing a dual-function copper-based nanoscale enzyme Cu-MB; constructing a colorimetric-fluorescent dual-mode adrenaline sensor; mixing and incubating EP with different concentrations and Cu-MB to react, measuring the ultraviolet absorption curve of the sample at a wavelength of 350-650 nm, taking the concentration of EP as the abscissa and the absorbance value at 485 nm as the ordinate, and establishing an ultraviolet standard curve of EP; measuring the fluorescence emission curve of the sample at a wavelength of 380-600 nm under an excitation wavelength of 350 nm, taking the concentration of EP as the abscissa and the fluorescence intensity value at 435 nm as the ordinate, and establishing a fluorescence standard curve of EP; and measuring the maximum ultraviolet absorbance value and the fluorescence intensity value of the sample to be detected, and substituting the values into the ultraviolet standard curve or the fluorescence standard curve of EP respectively to obtain the concentration of EP in the sample to be detected.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical detection, and relates to a method for detecting adrenaline based on a high-laccase-activity and high-fluorescence-performance dual-function copper-based nanosensor (Cu-MB), in particular to a method for detecting adrenaline based on a high-laccase-activity and high-fluorescence-performance Cu-MB colorimetric-fluorescence dual-mode sensor, or a method for visualizing detecting adrenaline by means of a high-laccase-activity and high-fluorescence-performance Cu-MB hydrogel and a smart phone. BACKGROUND

[0002] Laccase is the most commonly used green catalyst, which can promote the oxidation of various phenolic compounds and effectively remove pollutants. However, due to the inherent defects of laccase, including denaturation by the surrounding environment and high cost of purification process, its practical application is greatly hindered. At present, nanosensors that can simulate enzyme activity have attracted great interest from researchers due to their high stability, low toxicity and low cost. However, it has been observed that most of the developed nanosensors exhibit activities similar to peroxidase (POD) and oxidase (OXD). Therefore, developing nanosensors with laccase-like activity can increase the diversity of nanosensors and expand their application prospects in analytical chemistry. In recent years, researchers have designed nanosensors by simulating the active sites of natural enzymes, making them have similar or even stronger catalytic activity than natural enzymes. The active center of laccase is composed of copper ions combined with specific amino acid residues. This discovery helps to explore new copper-based nanosensors with similar chemical structures to enzymes.

[0003] At present, most of the laccase-like activities observed in nanosensors are limited to the construction of analytical sensors using a single colorimetric signal. Single-mode sensors are easily affected by external influences and operator errors, leading to false results. Notably, dual-mode sensors have the ability to self-validate and self-calibrate by collecting different signals, resulting in more accurate detection results. Developing dual-function nanosensors can achieve multi-modal analysis of target objects, and such enzymes combine enzyme-like catalytic activity and fluorescence properties, thereby improving the accuracy of detection. Therefore, dual-function nanosensors are currently an important direction for the development and research of nanosensor biosensors.

[0004] In the central nervous system (CNS), epinephrine (EP) is a catecholamine neurotransmitter secreted by the adrenal medulla, which has the function of activating the sympathetic nervous system, raising blood pressure, enhancing heart contractility and promoting muscle activity. The change of the level of EP can not only be used for the treatment of various diseases such as arrhythmia, bronchial asthma and allergic reaction, but also can be used for the prediction of diseases such as central nervous system, angina pectoris and brain injury. Therefore, in order to facilitate disease diagnosis and drug analysis, it is necessary to quantify EP. However, the current method for measuring the level of EP has high cost, long time consumption, and is limited by the use of precise instruments, which is difficult to operate and cannot be detected in real time. Therefore, it is crucial to use a convenient, portable and visual method to detect trace EP. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the existing detection method, such as low sensitivity, long detection time, high cost and complicated steps, and to synthesize a new nanoenzyme Cu-MB. A colorimetric and fluorescent sensing system is established by using Cu-MB, and this new dual-mode analysis system is used for the detection of EP.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] A method for detecting epinephrine based on high laccase activity and high fluorescence performance Cu-MB colorimetric-fluorescent dual-mode sensor, comprising the following steps:

[0008] Step (a), constructing a dual-functional copper-based nanoscale enzyme Cu-MB: copper nitrate and 2-amino terephthalic acid are dissolved in N,N-dimethylformamide (DMF) to obtain solution A; 2-methyl imidazole is dissolved in a mixed solution of ethanol and DMF to obtain solution B; solution A is slowly added to solution B, and after the addition is completed, the reaction is heated, and after the reaction is completed, it is naturally cooled, centrifuged, and the precipitate is washed with ethanol and water to obtain a dual-functional copper-based nanoscale enzyme Cu-MB;

[0009] Step (b), constructing a colorimetric-fluorescent dual-mode epinephrine (EP) sensor: mixing different concentrations of epinephrine (EP) and Cu-MB to obtain an incubation system, and carrying out incubation reaction, after the reaction is completed, the ultraviolet absorption curve of the sample is measured at a wavelength of 350-650 nm, and the concentration of EP is taken as the abscissa, and the absorbance value at 485 nm is taken as the ordinate, to establish the ultraviolet standard curve of EP; the fluorescence emission curve of the sample at a wavelength of 380-600 nm is measured at an excitation wavelength of 350 nm, and the concentration of EP is taken as the abscissa, and the absorbance value at 485 nm and the fluorescence intensity value at 435 nm are taken as the ordinate, to establish the fluorescence standard curve of EP;

[0010] Step (c), sample detection: the maximum ultraviolet absorbance value and the fluorescence intensity value of the unknown EP concentration of the sample to be detected are measured according to step (b), the maximum ultraviolet absorbance value is substituted into the ultraviolet standard curve of EP in step (b) to obtain the EP concentration in the sample to be detected, or the fluorescence intensity value is substituted into the fluorescence standard curve of EP in step (b) to obtain the EP concentration in the sample to be detected.

[0011] In step (a), the molar ratio of 2-amino terephthalic acid, 2-methyl imidazole and copper nitrate is (0.8-0.2):(0.2-0.8):0.4, preferably (0.8-0.5):(0.2-0.5):0.4, and more preferably 0.5:0.5:0.4.

[0012] The volume ratio of ethanol and DMF is (1-2):(2-1), preferably 1:2.

[0013] The temperature of the reaction is 130-160°C, preferably 140°C.

[0014] The heating time is 3-6h, preferably 5h.

[0015] The centrifugal speed is 10000-14000rpm, preferably 12000rpm; the centrifugal time is 4-8min, preferably 6min.

[0016] In step (b), preferably, ultrapure water is used to prepare Cu-MB dispersion and EP solutions with different concentrations respectively, 20-30μL of EP solution with different concentrations, 30-40μL of Cu-MB dispersion and Tris-HCl buffer solution are mixed to obtain an incubation system with a final volume of 250-350μL, and an incubation reaction is carried out, after the reaction is completed, the ultraviolet absorption curve of the sample is measured at 350-650nm, the ultraviolet standard curve of EP is established with the concentration of EP as the abscissa and the absorbance value at 485nm as the ordinate; the fluorescence emission curve of the sample is measured at an excitation wavelength of 350nm and a wavelength of 380-600nm, the ultraviolet standard curve of EP is established with the absorbance value at 485nm and the concentration of EP as the abscissa, and the fluorescence standard curve of EP is established with the fluorescence intensity value at 435nm as the ordinate and the concentration of EP as the abscissa.

[0017] The concentration of the Cu-MB dispersion is 1mg / mL.

[0018] In the incubation system, the final concentration of Cu-MB is 33-166 μg / mL, preferably 100-166 μg / mL, more preferably 100-140 μg / mL, and most preferably 100 μg / mL; in the incubation system, the final concentration of EP is 5-50 μg / mL, specifically, the final concentration of EP is 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL.

[0019] The Tris-HCl buffer solution is a Tris-HCl buffer solution with a pH of 3 to 8, preferably a Tris-HCl buffer solution with a pH of 7 to 8, and more preferably a Tris-HCl buffer solution with a pH of 7.4 and 50 mM.

[0020] The incubation reaction temperature is 20-60° C., preferably 35-45° C., more preferably 37° C.; the incubation reaction time is 3-25 minutes, preferably 10-25 minutes, more preferably 10 minutes.

[0021] Specifically, 20 μL of EP solutions of different concentrations, 30 μL of Cu-MB dispersion, and 250 μL of Tris-HCl buffer solution were mixed to obtain an incubation system, wherein the final volume of the incubation system was 300 μL.

[0022] In step (c), preferably, 20 to 30 μL of the sample to be tested, 30 to 40 μL of Cu-MB dispersion and Tris-HCl buffer solution are mixed to obtain an incubation system, and the final volume of the incubation system is 250 to 350 μL; the incubation reaction is carried out, and after the reaction is completed, the absorbance value at 485 nm and the fluorescence intensity value at 435 nm are respectively measured, and the absorbance value at 485 nm is substituted into the ultraviolet standard curve of EP in step (b) to obtain the EP concentration in the sample to be tested, and the fluorescence intensity value at 435 nm is substituted into the fluorescence standard curve of EP in step (b) to obtain the EP concentration in the sample to be tested.

[0023] More preferably, 20 μL of the sample to be tested, 30 μL of the Cu-MB dispersion and 250 μL of Tris-HCl buffer solution are mixed to obtain an incubation system.

[0024] In the incubation system, the final concentration of Cu-MB is 33-166 μg / mL, preferably 100-166 μg / mL, more preferably 100-140 μg / mL, and most preferably 100 μg / mL.

[0025] The Tris-HCl buffer solution is a Tris-HCl buffer solution with pH 3-8, preferably a Tris-HCl buffer solution with pH 7-8, and more preferably a Tris-HCl buffer solution with pH 7.4 and 50 mM.

[0026] The temperature of the incubation reaction is 20-60°C, preferably 35-45°C, and more preferably 37°C; and the time of the incubation reaction is 3-25 minutes, preferably 10-25 minutes, and more preferably 10 minutes.

[0027] As a further preferred embodiment of the method for detecting adrenaline by using the colorimetric-fluorescent dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance according to the application, the method further comprises:

[0028] Step (d), preparation of hydrogel: an agarose solution is prepared by using a Tris-HCl buffer solution, and the agarose solution and the Cu-MB dispersion liquid are mixed in a volume ratio of 8:1 to obtain a mixed solution, the mixed solution is dropped into a mold, and the hydrogel is obtained after cooling to room temperature;

[0029] Step (e), visual detection based on hydrogel: different concentrations of EP solution are respectively dropped on the hydrogel, and the reaction is carried out at room temperature; different colors are obtained corresponding to different concentrations of EP, and a colorimetric chart under natural light and under 365 nm ultraviolet light is respectively constructed; color recognition application programs are used to record color pictures under natural light and under 365 nm ultraviolet light, and the colors are converted into RGB values; a colorimetric standard curve is established by taking the concentration of EP as the abscissa and the ratio of red channel to blue channel R / B under natural light as the ordinate; and a fluorescence standard curve is established by taking the concentration of EP as the abscissa and the ratio of green channel to blue channel G / B under 365 nm ultraviolet light as the ordinate.

[0030] Step (f), an unknown EP concentration sample to be tested is treated according to step (e), and the sample to be tested is qualitatively determined based on the colorimetric chart; the RGB values of the unknown EP concentration sample to be tested under natural light and under 365 nm ultraviolet light are obtained according to step (e), the ratio of red channel to blue channel R / B under natural light and the ratio of green channel to blue channel G / B under 365 nm ultraviolet light are obtained, the R / B under natural light is substituted into the colorimetric standard curve of step (e) to obtain the EP concentration in the sample to be tested, or the G / B under 365 nm ultraviolet light is substituted into the fluorescence standard curve of step (e) to obtain the EP concentration in the sample to be tested.

[0031] In step (d), preferably, the preparation method of the agarose solution is as follows: agarose powder is added into a Tris-HCl buffer solution, and the agarose is dissolved by microwave heating.

[0032] The mold is a cover of a centrifuge tube.

[0033] The final concentration of the agarose is 5 to 20 mg / mL, preferably 15 mg / mL; in the mixed solution, the final concentration of Cu-MB is 0.05 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL.

[0034] The Tris-HCl buffer solution is a Tris-HCl buffer solution with a pH of 3 to 8, preferably a Tris-HCl buffer solution with a pH of 7.4 and 50 mM.

[0035] Preferably, the agarose solution at a temperature of 40° C. and the Cu-MB Cu-MB dispersion are mixed to obtain a mixed solution.

[0036] In step (e), the amount of the EP solution used is 25 to 35 μL, preferably 30 μL.

[0037] The concentration of the EP solution is 5 to 50 μg / mL. Specifically, the concentration of the EP solution is 5, 10, 20, 30, 40, or 50 μg / mL.

[0038] Different colors correspond to different EP concentrations: under natural light, the color of the hydrogel gradually changes to orange with increasing EP concentration; under 365nm ultraviolet light, the fluorescence gradually weakens with increasing EP concentration.

[0039] The test sample is a normal human plasma sample. Sample pretreatment: The plasma sample is mixed with 10% trichloroacetic acid (TCA) at a volume ratio of 1:1. The mixture is cooled in an ice-water bath for 10 minutes to precipitate plasma proteins and eliminate interference. The mixture is then centrifuged at 12,000 rpm for 15 minutes. The supernatant is collected and the pH is adjusted to 7.4 by adding 1 mM NaOH solution.

[0040] The detection mechanism of the method of the present invention is ( Figure 1 ): A fluorescent laccase mimetic Cu-MB was synthesized by a hydrothermal method. Compared with Cu-MB, it not only has higher catalytic activity but also has excellent fluorescence properties. In view of this property, we developed a colorimetric-fluorescence dual-mode sensor for detecting EP. Cu-MB nanozyme can promote the generation of orange-yellow oxidation products, which can be used for colorimetric detection. Due to the internal filter effect (IFE), the oxidation products of EP will quench the fluorescence of Cu-MB, thereby realizing fluorescence mode detection. In addition, a hydrogel platform and a smartphone were used to realize on-site detection of EP.

[0041] Compared with the prior art, the present invention has the following significant advantages:

[0042] 1. In this study, Cu-MB nanozymes with high laccase-like activity and high fluorescence properties were synthesized.

[0043] 2. Single channels are often easily affected by many factors such as operating conditions and biological environment. This study constructed a fluorescence-colorimetric dual-mode sensor, which has higher accuracy and can use the different signals collected to reduce the incidence of false positive and negative results.

[0044] 3. Hydrogel-based sensors were developed for visual detection of adrenaline.

[0045] 4. Cu-MB nanozyme has good activity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a schematic diagram of the principle of detecting EP based on the Cu-MB colorimetric / fluorescence dual-mode sensor of the present invention.

[0047] Figure 2 The UV absorption curves of the detection system constructed in Example 1 when different concentrations of EP were added.

[0048] Figure 3 The UV standard curve of different concentrations of EP was added to the detection system constructed in Example 1.

[0049] Figure 4 The fluorescence curves of the detection system constructed in Example 1 when different concentrations of EP were added.

[0050] Figure 5 The fluorescence standard curve of the detection system constructed in Example 1 with different concentrations of EP added was obtained.

[0051] Figure 6 is the effect of different conditions on the colorimetric sensing system; among them, a is the effect of different pH on the ultraviolet signal response, b is the effect of different incubation temperatures on the ultraviolet signal response, c is the effect of different incubation times on the ultraviolet signal response, and d is the effect of different Cu-MB concentrations on the ultraviolet signal response.

[0052] Figure 7 is the effect of different conditions on the fluorescence sensing system; among them, a is the effect of different pH on the fluorescence signal response, b is the effect of different incubation temperatures on the fluorescence signal response, c is the effect of different incubation times on the fluorescence signal response, and d is the effect of different Cu-MB concentrations on the fluorescence signal response.

[0053] Figure 8 Results of laccase activity testing of Cu-MB prepared with different ratios of 2-aminoterephthalic acid and 2-methylimidazole.

[0054] Figure 9Comparison diagram of different enzyme activities; among them, a is the double reciprocal curve of Cu-MB, b is the double reciprocal curve of Cu-(BDC-NH2), c is the double reciprocal curve of Cu-MI, d is the Michaelis-Menten curve of Cu-MB, e is the Michaelis-Menten curve of Cu-(BDC-NH2), and f is the Michaelis-Menten curve of Cu-MI.

[0055] Figure 10 This is a visual detection diagram of EP by the hydrogel detection system constructed in Example 6; wherein, a is a schematic diagram of the visual detection principle, b is an image of the color change of the hydrogel under natural light after adding different concentrations of EP, and c is an image of the fluorescence color change of the hydrogel under ultraviolet light after adding different concentrations of EP. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is described in more detail with reference to the accompanying drawings and specific embodiments. Although the following are preferred specific embodiments of the present invention, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein.

[0057] The inventors studied the properties of Cu-MB using colorimetric and fluorescence methods. In addition, the Michaelis-Menton constants (K m ) were compared, indicating that Cu-MB had stronger laccase activity.

[0058] Example 1

[0059] A method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance Cu-MB, comprising the following steps:

[0060] Step (a), constructing a bifunctional copper-based nanozyme Cu-MB: dissolving copper nitrate and 2-aminoterephthalic acid in N,N-dimethylformamide (DMF) to obtain solution A; dissolving 2-methylimidazole in a mixed solution of ethanol and DMF to obtain solution B; slowly adding solution A to solution B while constantly stirring, the final concentration of 2-aminoterephthalic acid in the reaction system is 1.25 mM, the molar ratio of 2-aminoterephthalic acid, 2-methylimidazole and copper nitrate is 0.5:0.5:0.4, and the volume ratio of ethanol and DMF is 1:2; the reaction system is transferred to an autoclave, heated in an oven at 140°C for 5 hours, the reaction solution is naturally cooled, and centrifuged at 12000 rpm for 6 minutes to obtain a precipitate, which is washed 4 times with ethanol and water. The bifunctional copper-based nanozyme Cu-MB is dispersed in ultrapure water to obtain a Cu-MB dispersion with a concentration of 1 mg / mL;

[0061] Step (b), constructing a colorimetric-fluorescence dual-mode epinephrine (EP) sensor: ultrapure water was used to prepare EP solutions of different concentrations, 20 μL of EP solutions of different concentrations, 30 μL of Cu-MB dispersion of step (a) and 250 μL of Tris-HCl buffer solution (50 mM, pH 7.4) were mixed to obtain an incubation system with a final volume of 300 μL, wherein the final concentration of Cu-MB in the incubation system was 100 μg / mL, and the final concentration of EP was 0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL and 50 μg / mL; the reaction was incubated at 37°C for 10 minutes. After the reaction was completed, the ultraviolet absorption curve of the sample was measured at a wavelength of 350-650 nm ( Figure 2 ), with the absorbance at 485 nm and the concentration of EP as the horizontal axis, the UV standard curve of EP was established: y = 0.00494x + 0.10022, R 2 =0.999( Figure 3 ); the fluorescence emission curve of the sample at a wavelength of 380 to 600 nm was measured at an excitation wavelength of 350 nm ( Figure 4 ), with the fluorescence intensity at 435 nm as the ordinate and the concentration of EP as the abscissa, a fluorescence standard curve of EP was established: y = 129.6748x + 209.526, R 2 =0.999( Figure 5 );

[0062] Step (c), sample detection: 20 μL of the sample to be tested, 30 μL of Cu-MB dispersion and 250 μL of Tris-HCl buffer solution (50 mM, pH 7.4) were mixed, the final concentration of Cu-MB in the incubation system was 100 μg / mL, and the reaction was carried out at 37°C for 10 minutes. After the reaction, the absorbance value at 485 nm and the fluorescence intensity value at 435 nm were respectively measured. The absorbance value at 485 nm was substituted into the ultraviolet standard curve of EP in step (b) to obtain the EP concentration in the sample to be tested. The fluorescence intensity value at 435 nm was substituted into the fluorescence standard curve of EP in step (b) to obtain the EP concentration in the sample to be tested;

[0063] Among them, sample pretreatment: the plasma sample was mixed with 10% trichloroacetic acid (TCA) in a volume ratio of 1:1, cooled in an ice water bath for 10 minutes to precipitate the protein in the plasma and eliminate its interference, centrifuged at 12000 rpm for 15 minutes, and the supernatant was collected and NaOH solution (1mM) was added to adjust the pH to 7.4 to obtain the sample to be tested.

[0064] Example 2

[0065] Based on the colorimetric sensor constructed in step (a) and step (b) of Example 1, the effects of different pH, temperature, time and concentration on the ultraviolet signal response were investigated.

[0066] (1) Effect of different pH on UV signal response

[0067] 20 μL of EP solution (prepared with ultrapure water), 40 μL of Cu-MB dispersion (dispersed in ultrapure water, initial concentration of 1 mg / mL) and Tris-HCl buffer of different pH values ​​(pH 3, 4, 5, 6, 7.4, 8) were mixed and incubated to react. The final reaction solution volume was 300 μL and the final concentration of EP was 30 μg / mL. The reaction was carried out at 30°C for 15 minutes. After the reaction was completed, the reaction systems of different pH values ​​were colorimetrically measured. The UV absorbance was measured at a wavelength of 485 nm using a UV spectrophotometer. The results are shown in Table 1. Figure 6 a. The results showed that Cu-MB exhibited good UV signal response at pH 6-8, especially at pH 7.4, where it exhibited the best UV signal response.

[0068] (2) Effect of different temperatures on UV signal response

[0069] 20 μL of EP solution (prepared with ultrapure water), 40 μL of Cu-MB dispersion (dispersed in ultrapure water, initial concentration 1 mg / mL), and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated for a final reaction volume of 300 μL, with a final EP concentration of 30 μg / mL. The reaction was carried out at 10°C, 20°C, 30°C, 37°C, 45°C, and 60°C for 15 minutes. After the reaction, the reaction systems at different temperatures were colorimetrically analyzed using a UV spectrophotometer to measure the UV absorbance at a wavelength of 485 nm. The results are shown in Table 1. Figure 6 b. The results show that Cu-MB exhibits good UV signal response at temperatures between 20 and 60°C, and especially exhibits the best UV signal response at 37°C.

[0070] (3) Effects of different times on UV signal response

[0071] 20 μL of EP solution (prepared with ultrapure water), 40 μL of Cu-MB dispersion (dispersed in ultrapure water, initial concentration 1 mg / mL), and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated. The final reaction volume was 300 μL, and the final EP concentration was 30 μg / mL. The reaction was carried out at 37°C for 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, and 25 minutes. After the reaction, the reaction systems with different reaction times were colorimetrically measured using a UV spectrophotometer at a wavelength of 485 nm. The results are shown in Figure 2. Figure 6 c. The results show that the UV signal response reaches its peak after 10 minutes of incubation. As the incubation time increases, the UV signal response no longer increases. Therefore, the incubation time can be 10 to 25 minutes, preferably 10 minutes.

[0072] (4) Effect of different concentrations on UV signal response

[0073] 20 μL of EP solution (prepared with ultrapure water), various concentrations of Cu-MB dispersion (dispersed in ultrapure water), and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated. The final reaction volume was 300 μL, the final concentration of EP was 30 μg / mL, and the final concentrations of Cu-MB were 33 μg / mL, 66 μg / mL, 100 μg / mL, 133 μg / mL, and 166 μg / mL. The reaction was carried out at 37°C for 10 minutes. After the reaction, the reaction systems with different Cu-MB concentrations were colorimetrically measured using a UV spectrophotometer at a wavelength of 485 nm. The results are shown in Table 2. Figure 6 d. The results show that the ideal UV signal response is achieved when the final Cu-MB concentration in the system is 100 μg / mL. Therefore, the final Cu-MB concentration in the system can be 100-166 μg / mL, preferably 100 μg / mL.

[0074] Example 3

[0075] (1) Effect of different pH on fluorescence signal response

[0076] 20 μL of EP solution (prepared in ultrapure water), 40 μL of Cu-MB dispersion (dispersed in ultrapure water, initial concentration 1 mg / mL), and Tris-HCl buffer at different pH values ​​(pH 3, 4, 5, 6, 7.4, 8) were mixed and incubated. The final reaction volume was 300 μL, and the final EP concentration was 30 μg / mL. The reaction was incubated at 30°C for 15 minutes. After the reaction, the fluorescence intensity of the reaction system at different pH values ​​was measured using a fluorescence spectrophotometer at a wavelength of 435 nm. The results are shown in Figure 2. Figure 7 a. The results showed that Cu-MB exhibited a good fluorescence signal response at pH 7-8, especially at pH 7.4, which showed the best fluorescence signal response.

[0077] (2) Effects of different temperatures on fluorescence signal response

[0078] 20 μL of EP solution (prepared with ultrapure water), 40 μL of Cu-MB dispersion (dispersed in ultrapure water, initial concentration 1 mg / mL), and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated. The final reaction volume was 300 μL, and the final EP concentration was 30 μg / mL. The reaction was carried out at 10°C, 20°C, 30°C, 37°C, 45°C, and 60°C for 15 minutes. After the reaction, the fluorescence of the reaction system at different temperatures was measured using a fluorescence spectrophotometer at a wavelength of 435 nm. The results are shown in Figure 2. Figure 7 b. The results show that Cu-MB exhibits good fluorescence signal response at temperatures between 35 and 60°C, and especially exhibits the best fluorescence signal response at 37°C.

[0079] (3) Effects of different times on fluorescence signal response

[0080] 20 μL of EP solution (prepared in ultrapure water), 40 μL of Cu-MB dispersion (dispersed in ultrapure water, initial concentration 1 mg / mL), and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated. The final reaction volume was 300 μL, and the final EP concentration was 30 μg / mL. The reaction was carried out at 37°C for 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, and 25 minutes. After the reaction, the fluorescence intensity of the reaction system at different reaction times was measured using a fluorescence spectrophotometer at a wavelength of 435 nm. The results are shown in Figure 2. Figure 7 c. The results show that the UV signal response reaches the strongest fluorescence signal response after 10 minutes of incubation. As the incubation time increases, the fluorescence signal response no longer increases. Therefore, the incubation time can be 10 to 25 minutes, preferably 10 minutes.

[0081] (4) Effects of different concentrations on fluorescence signal response

[0082] 20 μL of EP solution (prepared with ultrapure water), various concentrations of Cu-MB dispersion (dispersed in ultrapure water), and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated. The final reaction volume was 300 μL, the final concentration of EP was 30 μg / mL, and the final concentrations of Cu-MB were 33 μg / mL, 66 μg / mL, 100 μg / mL, 133 μg / mL, and 166 μg / mL. The reaction was incubated at 37°C for 10 minutes. After completion of the reaction, the fluorescence intensity of the reaction systems with different Cu-MB concentrations was measured at a wavelength of 435 nm using a fluorescence spectrophotometer. The results are shown in Figure 2. Figure 7The results showed that when the final concentration of Cu-MB in the system was 100-140 μg / mL, it showed good fluorescence signal response, especially when the concentration was 100 μg / mL, the strongest fluorescence signal response was achieved.

[0083] Example 4

[0084] The effects of different ratios of 2-aminoterephthalic acid (BDC-NH2) and 2-methylimidazole (2-MI) on the activity of Cu-MB laccase were investigated.

[0085] Construction of bifunctional copper-based nanozyme Cu-MB: 29 mg of copper nitrate and 10.9 mg, 27.2 mg, and 43.5 mg of 2-aminoterephthalic acid were dissolved in N,N-dimethylformamide (DMF) to obtain solution A; 19.7 mg, 12.3 mg, and 4.9 mg of 2-methylimidazole were dissolved in a mixed solution of ethanol and DMF to obtain solution B; solution A was slowly added to solution B under continuous stirring. In the reaction system, the final concentration of 2-aminoterephthalic acid was 1.25 mM, and the molar ratios of 2-aminoterephthalic acid, 2-methylimidazole, and copper nitrate were 0.8:0.2:0.4, 0.5:0.5:0.4, 0.2:0.8:0.2, the volume ratio of ethanol and DMF is 1:2; the reaction system is transferred to an autoclave, heated at 140°C in an oven for 5 hours, naturally cooled, and centrifuged at 12000 rpm for 6 minutes to obtain a precipitate, which is washed with ethanol and an aqueous solution to obtain a bifunctional nanoenzyme copper-based Cu-MB, which is dispersed in ultrapure water to obtain a Cu-MB dispersion with a concentration of 1 mg / mL.

[0086] The laccase activity of Cu-MB was tested using a conventional substrate for natural laccase (i.e., a colorimetric reaction between 2,4-dichlorophenol and 4-aminoantipyrine). 20 μL of Cu-MB dispersion (initial concentration: 1 mg / mL), 20 μL of 2,4-dichlorophenol (prepared with ultrapure water, initial concentration: 1 mg / mL), 20 μL of 4-aminoantipyrine (prepared with ultrapure water, initial concentration: 1 mg / mL), and Tris-HCl buffer (50 mM, pH 7.4) were mixed to a final reaction volume of 300 μL. The reaction was carried out at 37°C for 30 minutes. The UV absorbance was measured at a wavelength of 510 nm using a UV spectrophotometer, and the relative enzyme activity was calculated (the calculation of relative enzyme activity was based on the highest enzyme activity as 100%). The results are shown in Table 1. Figure 8 When the molar ratio of 2-aminoterephthalic acid (BDC-NH2), 2-methylimidazole (2-MI) and copper nitrate was (0.8-0.5):(0.2-0.5):0.4, Cu-MB showed excellent laccase activity, especially when the molar ratio was 0.5:0.5:0.4, the enzyme activity was optimal.

[0087] Example 5

[0088] In order to quantify the catalytic efficiency and affinity of the materials, the double reciprocal method was used to calculate the steady-state kinetic parameters, including the Michaelis constant (K m ). K m The lower the value, the higher the affinity of the mimetic enzyme to the substrate.

[0089] The preparation method of Cu-(BDC-NH2) is as follows: copper nitrate and 2-aminoterephthalic acid are dissolved in N,N-dimethylformamide (DMF) to obtain solution A; ethanol and DMF are mixed to obtain solution B; solution A is slowly added to solution B under constant stirring, the final concentration of 2-aminoterephthalic acid in the reaction system is 1.25mM, the molar concentration ratio of 2-aminoterephthalic acid and copper nitrate is 0.5:0.4, and the volume ratio of ethanol and DMF is 1:2; the reaction system is transferred to an autoclave, heated at 140°C in an oven for 5 hours, cooled naturally, and centrifuged at 12000rpm for 6 minutes to obtain a precipitate, which is washed with ethanol and water to obtain nanoenzyme Cu-(BDC-NH2), which is dispersed in ultrapure water.

[0090] The preparation method of Cu-MI is as follows: dissolve copper nitrate in N,N-dimethylformamide (DMF) to obtain solution A; dissolve 2-methylimidazole in a mixed solution of ethanol and DMF to obtain solution B; slowly add solution A to solution B under constant stirring, the final concentration of 2-methylimidazole in the reaction system is 1.25mM, the molar concentration ratio of 2-methylimidazole and copper nitrate is 0.5:0.4, and the volume ratio of ethanol and DMF is 1:2; transfer the reaction system to an autoclave, heat at 140°C in an oven for 5 hours, cool naturally, and centrifuge at 12000rpm for 6 minutes to obtain a precipitate, wash it with ethanol and water to obtain nanoenzyme Cu-MI, and disperse it in ultrapure water.

[0091] Comparison of different enzyme activities: Cu-MB (Example 1 step (a)), Cu-(BDC-NH2) and Cu-MI of the same concentration (all dispersed in ultrapure water, the final concentration in the final reaction system is 200 μg / mL) were added to different concentrations of 2,4-dichlorophenol solution (prepared with ultrapure water), 45 μL of 4-aminoantipyrine solution (prepared with ultrapure water, initial concentration is 1 mg / mL) and Tris-HCl buffer (50 mM, pH 7.4) and mixed. The final reaction system volume was 300 μL, and the final concentrations of 2,4-dichlorophenol were 0.06, 0.12, 0.24, 0.36, 0.49 and 0.61 mM, respectively; the reaction was carried out at room temperature for 20 min, and the ultraviolet absorption value was measured at a wavelength of 510 nm using an ultraviolet spectrophotometer. The reaction rate value was obtained using each absorption value and substituted into 1 / V=(Km / V max )(1 / [S])+1 / V max The double reciprocal curve is obtained, and the Michaelis constant K is obtained using the intercept and slope. m .

[0092] Figure 9 a, b, and c are the Michaelis-Menten curves of the catalytic oxidation of 2,4-dichlorophenol by Cu-MB, Cu-(BDC-NH2), and Cu-MI at room temperature and pH = 7.4, respectively. Figure 9 d, e, and f are the Michaelis-Menten curves of Cu-MB, Cu-(BDC-NH2), and Cu-MI, respectively. K m The values ​​were 0.0898mM, 0.095mM, and 0.258mM, respectively. This indicates that Cu-MB has a high affinity for 2,4-dichlorophenol.

[0093] Example 6

[0094] A method for visual detection of Cu-MB functionalized hydrogels, comprising the following steps:

[0095] Step (a), synthesis of Cu-MB: same as step (a) of Example 1, to obtain a Cu-MB dispersion with a concentration of 1 mg / mL;

[0096] Step (b), Preparation of Cu-MB-functionalized hydrogel: 75 mg of agarose powder was added to 5 mL of Tris-HCl buffer solution (pH 7.4, 50 mM), and the agarose was dissolved using 700 W microwave heating to obtain an agarose solution. Subsequently, the agarose solution was heated in a 40°C water bath. 3 mL of the 40°C agarose solution was taken and 375 μL of the Cu-MB dispersion was added. The resulting mixed solution (final Cu-MB concentration was approximately 0.1 mg / mL) was added to the cap of a centrifuge tube, cooled to room temperature, and the hydrogel was removed.

[0097] Step (c), visualization detection based on hydrogel: Figure 10a. 30 μL of EP solution of different concentrations (prepared with ultrapure water, 5, 10, 20, 30, 40, 50 μg / mL) was dropped onto the hydrogel and reacted at room temperature. The color of the hydrogel under natural light was observed, and it was found that it gradually turned orange with the increase of EP concentration. At the same time, the fluorescence under 365 nm ultraviolet light was observed, and it was found that the fluorescence gradually weakened with the increase of EP concentration. Based on the different colors corresponding to different EP concentrations, color cards under natural light and 365 nm ultraviolet light were constructed respectively. The color images under natural light and 365 nm ultraviolet light were recorded using a color recognition application (color recognition), and the colors were converted into RGB values. A colorimetric standard curve was established with the concentration of EP as the horizontal axis and the ratio of the red channel to the blue channel R / B under natural light as the vertical axis: y = 0.10388x + 0.83747, R 2 =0.996; with the concentration of EP as the horizontal axis and the ratio of the green channel to the blue channel G / B under 365nm ultraviolet light as the vertical axis, a fluorescence standard curve was established: y=0.00485x+0.00831, R 2 =0.999;

[0098] Step (d), treating the sample to be tested with an unknown EP concentration according to step (c), and qualitatively analyzing the sample to be tested based on a colorimetric card; according to step (e), respectively measuring the RGB values ​​of the sample to be tested with an unknown EP concentration under natural light and under a 365nm ultraviolet lamp, obtaining the red channel / blue channel ratio R / B under natural light and the green channel / blue channel ratio G / B under a 365nm ultraviolet lamp, substituting the R / B under natural light into the colorimetric standard curve of step (e) to obtain the EP concentration in the sample to be tested, or substituting the G / B under a 365nm ultraviolet lamp into the fluorescence standard curve of step (e) to obtain the EP concentration in the sample to be tested.

[0099] Application Example 1

[0100] Normal human plasma samples were assayed. Sample pretreatment and testing were performed according to step (c) of Example 1, with 5 μg / mL, 10 μg / mL, and 30 μg / mL added to the plasma. The colorimetric and fluorescence signals were measured according to the methods of Example 1, and the EP standard curve established in Example 1 was substituted to obtain the EP concentration in the sample. Each sample was assayed three times, and the average value was taken. The RSD and recovery were calculated and shown in Table 1.

[0101] Table 1. Recovery of EP in actual samples (n=3)

[0102]

[0103] While the embodiments of the present invention have been described above, the above description is intended to be exemplary, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance Cu-MB, characterized by: The steps include: Step (a), constructing a bifunctional copper-based nanozyme Cu-MB: dissolving copper nitrate and 2-aminoterephthalic acid in N,N-dimethylformamide to obtain solution A; dissolving 2-methylimidazole in a mixed solution of ethanol and DMF to obtain solution B; adding solution A to solution B, in the reaction system, the molar ratio of 2-aminoterephthalic acid, 2-methylimidazole and copper nitrate is (0.8-0.2):(0.2-0.8):0.4, and the volume ratio of ethanol and DMF is (1-2):(2-1), heating reaction, the heating reaction temperature is 130-160 ° C, the heating reaction time is 3-6 hours, after the reaction is completed, cooling, centrifugation, and the precipitate is washed with ethanol and water to obtain a bifunctional copper-based nanozyme Cu-MB; Step (b), constructing a colorimetric-fluorescence dual-mode adrenaline sensor: mixing different concentrations of adrenaline and Cu-MB, incubating the reaction, and after the reaction, measuring the ultraviolet absorption curve of the sample at a wavelength of 350-650 nm, with the concentration of EP as the horizontal axis and the absorbance value at 485 nm as the vertical axis, to establish an ultraviolet standard curve of EP; measuring the fluorescence emission curve of the sample at a wavelength of 380-600 nm at an excitation wavelength of 350 nm, with the concentration of EP as the horizontal axis and the fluorescence intensity value at 435 nm as the vertical axis, to establish a fluorescence standard curve of EP; Step (c), sample detection: According to step (b), the maximum ultraviolet absorbance value and fluorescence intensity value of the sample to be tested are measured, and the maximum ultraviolet absorbance value is substituted into the ultraviolet standard curve of EP in step (b) to obtain the EP concentration in the sample to be tested, or the fluorescence intensity value is substituted into the fluorescence standard curve of EP in step (b) to obtain the EP concentration in the sample to be tested.

2. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 1, characterized in that: In step (a), the molar ratio of 2-aminoterephthalic acid, 2-methylimidazole and copper nitrate is (0.8-0.5):(0.2-0.5):0.

4.

3. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 2, characterized in that: In step (a), the molar ratio of 2-aminoterephthalic acid, 2-methylimidazole and copper nitrate is 0.5:0.5:0.

4.

4. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 2, characterized in that: In step (a), in the reaction system, the volume ratio of ethanol to DMF is 1:

2.

5. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 1, characterized in that: In step (a), the heating reaction temperature is 140° C. and the heating reaction time is 5 h.

6. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 1, characterized in that: In step (b), a Cu-MB dispersion and EP solutions of different concentrations were prepared using ultrapure water, and 20 to 30 μL of EP solutions of different concentrations, 30 to 40 μL of Cu-MB dispersion, and Tris-HCl buffer solution were mixed to obtain an incubation system. The final volume of the incubation system was 250 to 350 μL.

7. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 1 or 6, characterized in that: In step (b), the concentration of the Cu-MB dispersion is 1 mg / mL.

8. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 6, characterized in that: In step (b), in the incubation system, the final concentration of Cu-MB is 33 to 166 μg / mL; in the incubation system, the final concentration of EP is 5 to 50 μg / mL; The Tris-HCl buffer solution is a Tris-HCl buffer solution with a pH of 3 to 8; The incubation reaction temperature is 20 to 60° C.; the incubation reaction time is 3 to 25 minutes; The final volume of the incubation system is 250-350 μL.

9. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 8, characterized in that: In step (b), the final concentration of Cu-MB in the incubation system is 100-166 μg / mL.

10. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 9, characterized in that: In step (b), the final concentration of Cu-MB in the incubation system is 100-140 μg / mL.

11. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 10, characterized in that: In step (b), the final concentration of Cu-MB in the incubation system is 100 μg / mL.

12. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 8, characterized in that: In step (b), the Tris-HCl buffer solution is a Tris-HCl buffer solution with a pH of 7 to 8.

13. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 12, characterized in that: In step (b), the Tris-HCl buffer solution is a 50 mM Tris-HCl buffer solution with a pH of 7.

4.

14. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 8, characterized in that: In step (b), the incubation reaction temperature is 35-45° C.; the incubation reaction time is 10-25 minutes.

15. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 14, characterized in that: In step (b), the incubation reaction temperature is 37° C. and the incubation reaction time is 10 minutes.

16. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 8, characterized in that: In step (b), the final volume of the incubation system is 300 μL.

17. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 1, characterized in that: In step (c), 20 to 30 μL of the sample to be tested, 30 to 40 μL of the Cu-MB dispersion, and a Tris-HCl buffer solution are mixed to obtain an incubation system, and the final volume of the incubation system is 250 to 350 μL; an incubation reaction is performed. After the reaction is completed, the absorbance value at 485 nm and the fluorescence intensity value at 435 nm are respectively measured, and the absorbance value at 485 nm is substituted into the ultraviolet standard curve of EP in step (b) to obtain the EP concentration in the sample to be tested. The fluorescence intensity value at 435 nm is substituted into the fluorescence standard curve of EP in step (b) to obtain the EP concentration in the sample to be tested.

18. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 17, characterized in that: In step (c), the final concentration of Cu-MB in the incubation system is 33 to 166 μg / mL; The Tris-HCl buffer solution is a Tris-HCl buffer solution with a pH of 3 to 8; The incubation reaction temperature is 20 to 60° C.; the incubation reaction time is 3 to 25 minutes.

19. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 18, characterized in that: In step (c), the final concentration of Cu-MB in the incubation system is 100-166 μg / mL.

20. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 19, characterized in that: In step (c), the final concentration of Cu-MB in the incubation system is 100-140 μg / mL.

21. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 20, characterized in that: In step (c), the final concentration of Cu-MB in the incubation system is 100 μg / mL.

22. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 18, characterized in that: In step (c), the Tris-HCl buffer solution is a Tris-HCl buffer solution with a pH of 7 to 8.

23. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 22, characterized in that: In step (c), the Tris-HCl buffer solution is a 50 mM Tris-HCl buffer solution with a pH of 7.

4.

24. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 18, characterized in that: In step (c), the incubation reaction temperature is 35-45° C.; the incubation reaction time is 10-25 minutes.

25. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 24, characterized in that: In step (c), the incubation reaction temperature is 37° C. and the incubation reaction time is 10 minutes.

26. A method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance Cu-MB, characterized in that: include: Step (1), constructing a bifunctional copper-based nanozyme Cu-MB: dissolving copper nitrate and 2-aminoterephthalic acid in N,N-dimethylformamide to obtain solution A; 2-Methylimidazole was dissolved in a mixed solution of ethanol and DMF to obtain solution B; solution A was added to solution B; in the reaction system, the molar ratio of 2-aminoterephthalic acid, 2-methylimidazole and copper nitrate was (0.8-0.2):(0.2-0.8):0.4, and the volume ratio of ethanol and DMF was (1-2):(2-1); the reaction was heated at a temperature of 130-160°C for 3-6 hours. After the reaction was completed, the reaction was cooled and centrifuged, and the precipitate was washed with ethanol and water to obtain a bifunctional copper-based nanozyme Cu-MB; Step (2), preparing a hydrogel: using a Tris-HCl buffer solution to prepare an agarose solution, according to the volume ratio of the agarose solution to the Cu-MB dispersion of 8:1, the agarose solution at 40-50° C. and the Cu-MB dispersion were mixed to obtain a mixed solution, the mixed solution was dripped into a mold, and cooled to room temperature to obtain a hydrogel; Step (3), visualization detection based on hydrogel: EP solutions of different concentrations are dripped onto the hydrogel, reacted at room temperature, and colorimetric cards under natural light and 365nm ultraviolet light are constructed based on the different colors corresponding to different EP concentrations; a color recognition application is used to record the color images under natural light and 365nm ultraviolet light, and the colors are converted into RGB values; a colorimetric standard curve is established with the concentration of EP as the horizontal coordinate and the ratio of the red channel / blue channel R / B under natural light as the vertical coordinate; a fluorescence standard curve is established with the concentration of EP as the horizontal coordinate and the ratio of the green channel / blue channel G / B under 365nm ultraviolet light as the vertical coordinate; Step (4), processing the sample to be tested with unknown EP concentration according to step (3), and qualitatively analyzing the sample to be tested based on the colorimetric card; measuring the RGB value of the sample to be tested with unknown EP concentration under natural light and 365nm ultraviolet light according to step (3), obtaining the ratio R / B of the red channel / blue channel under natural light and the ratio G / B of the green channel / blue channel under 365nm ultraviolet light, substituting the R / B under natural light into the colorimetric standard curve of step (3) to obtain the EP concentration in the sample to be tested, or substituting the G / B under 365nm ultraviolet light into the fluorescence standard curve of step (3) to obtain the EP concentration in the sample to be tested.

27. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 26, characterized in that: In step (1), the molar ratio of 2-aminoterephthalic acid, 2-methylimidazole and copper nitrate is (0.8-0.5):(0.2-0.5):0.

4.

28. The method for detecting epinephrine based on a colorimetric-fluorescent dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 27, characterized in that: In step (1), the molar ratio of 2-aminoterephthalic acid, 2-methylimidazole and copper nitrate is 0.5:0.5:0.

4.

29. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 26, characterized in that: In step (1), in the reaction system, the volume ratio of ethanol to DMF is 1:

2.

30. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 26, characterized in that: In step (1), the heating reaction temperature is 140° C.; the heating reaction time is 5 h.

31. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 26, characterized in that: In step (2), the final concentration of the agarose is 5 to 20 mg / mL; in the mixed solution, the final concentration of Cu-MB is 0.05 mg / mL to 0.5 mg / mL; The Tris-HCl buffer solution is a Tris-HCl buffer solution with a pH of 3 to 8.

32. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 31, characterized in that: In step (2), the Tris-HCl buffer solution is a 50 mM Tris-HCl buffer solution with a pH of 7.

4.

33. The method for detecting epinephrine based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance of Cu-MB according to claim 26, characterized in that: In step (3), the amount of the EP solution is 25-35 µL; the concentration of the EP solution is 5-50 µg / mL.

Images