Method for detecting epinephrine based on colorimetric-fluorescent dual-mode sensor of Cu-MB with high laccase activity and high fluorescence property

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of analysis and detection, and relates to a method for detecting adrenaline based on a bifunctional copper-based nanozyme (Cu-MB) with high laccase activity and high fluorescence performance, and specifically relates to a method for detecting adrenaline using a colorimetric-fluorescence dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance, or a method for visually detecting adrenaline using a hydrogel based on Cu-MB with high laccase activity and high fluorescence performance and a smart phone. Background Art

[0002] Laccase is the most commonly used green catalyst, which can promote the oxidation of a variety of phenolic compounds and effectively remove pollutants. However, due to the inherent defects of laccase, including susceptibility to denaturation due to the influence of the surrounding environment and the high cost of purification process, its practical application is greatly hindered. At present, nanozymes that can mimic enzyme activity have aroused great interest among researchers due to their high stability, low toxicity and low cost. However, it has been observed that most of the developed nanozymes exhibit activities similar to peroxidase (POD) and oxidase (OXD). Therefore, the development of nanozymes with laccase-like activities can increase the diversity of nanozymes and expand their application prospects in analytical chemistry. In recent years, some researchers have designed nanozymes by mimicking the active sites of natural enzymes, making them have similar or even stronger catalytic activities 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 nanozymes with chemical structures similar to enzymes.

[0003] Currently, most laccase-like activities observed in nanozymes are limited to the construction of analytical sensors using a single colorimetric signal. Single-mode sensors are susceptible to external influences and operator errors, leading to erroneous results. It is worth noting that dual-mode sensors have the ability to self-verify and self-calibrate by collecting different signals, which can obtain more accurate detection results. The development of bifunctional nanozymes can achieve multimodal analysis of targets. This enzyme combines enzyme-like catalytic activity and fluorescence properties, thereby improving the accuracy of detection. Therefore, bifunctional nanozymes are currently an important direction for the development and research of nanozyme biosensors.

[0004] In the central nervous system (CNS), epinephrine (EP), as a catecholamine neurotransmitter, is secreted by the adrenal medulla and has the functions of activating sympathetic nerves, increasing blood pressure, enhancing cardiac contractility, and promoting muscle activity. EP can not only be used to treat a variety of diseases, such as arrhythmias, bronchial asthma, and allergic reactions, but changes in its levels can also be used to predict diseases of the central nervous system, angina pectoris, and brain damage. Therefore, in order to facilitate disease diagnosis and drug analysis, EP must be quantified. However, the current methods for measuring EP levels are costly, time-consuming, and limited by the need to use sophisticated instruments, which makes operation difficult and prevents rapid real-time detection. Therefore, it is crucial to use a convenient, portable, and visual method to detect trace amounts of EP. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of existing detection methods such as low sensitivity, time-consuming detection, high cost and complicated steps. A new nanozyme Cu-MB is synthesized, a colorimetric and fluorescence sensing system is established using Cu-MB, and this new dual-mode analysis system is used for the detection of EP.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

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

[0008] 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, after the addition is complete, heating to react, after the reaction is completed, naturally cooling, centrifuging, and washing the precipitate with ethanol and water to obtain a bifunctional copper-based nanozyme Cu-MB;

[0009] Step (b), constructing a colorimetric-fluorescence dual-mode adrenaline (EP) sensor: mixing different concentrations of adrenaline (EP) and Cu-MB to obtain an incubation system, and performing an incubation reaction. After the reaction is completed, measuring the ultraviolet absorption curve of the sample at a wavelength of 350 to 650 nm, taking the concentration of EP as the horizontal coordinate and the absorbance value at 485 nm as the vertical coordinate, and establishing an ultraviolet standard curve of EP; measuring the fluorescence emission curve of the sample at a wavelength of 380 to 600 nm at an excitation wavelength of 350 nm, taking the concentration of EP as the horizontal coordinate, the absorbance value at 485 nm and the fluorescence intensity value at 435 nm as the vertical coordinate, and establishing a fluorescence standard curve of EP;

[0010] Step (c), sample detection: according to step (b), the maximum ultraviolet absorbance value and fluorescence intensity value of the sample to be tested with unknown EP concentration 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.

[0011] In step (a), the molar ratio of 2-aminoterephthalic acid, 2-methylimidazole 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 to DMF is (1-2):(2-1), preferably 1:2.

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

[0014] The heating time is 3 to 6 hours, preferably 5 hours.

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

[0016] In step (b), preferably, ultrapure water is used to prepare Cu-MB dispersion and EP solutions of different concentrations, respectively, and 20 to 30 μL of EP solutions of different concentrations, 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, and an incubation reaction is performed. After the reaction is completed, the ultraviolet absorption curve of the sample is measured at a wavelength of 350 to 650 nm, and the ultraviolet standard curve of EP is established with the concentration of EP as the horizontal coordinate and the absorbance value at 485 nm as the vertical coordinate; the fluorescence emission curve of the sample at a wavelength of 380 to 600 nm is measured at an excitation wavelength of 350 nm, and the ultraviolet standard curve of EP is established with the absorbance value at 485 nm and the concentration of EP as the horizontal coordinate, and the fluorescence standard curve of EP is established with the fluorescence intensity value at 435 nm as the vertical coordinate and the concentration of EP as the horizontal coordinate.

[0017] The concentration of the Cu-MB dispersion is 1 mg / 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, and more preferably 37°C; the incubation reaction time is 3-25 minutes, preferably 10-25 minutes, and 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, and 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; an incubation reaction is performed, 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 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.

[0026] The incubation reaction temperature is 20-60°C, preferably 35-45°C, and more preferably 37°C; the incubation reaction time 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 using a colorimetric-fluorescence dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance according to the present invention, the method further includes:

[0028] Step (d), 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 being 8:1, mixing the agarose solution at 40 to 50° C. and the Cu-MB dispersion to obtain a mixed solution, dripping the mixed solution into a mold, and cooling to room temperature to obtain a hydrogel;

[0029] Step (e), visual detection based on hydrogel: EP solutions of different concentrations are respectively added dropwise onto the hydrogel, reacted at room temperature, and colorimetric cards under natural light and 365nm ultraviolet light are respectively constructed based on different colors corresponding to different EP concentrations; color images under natural light and 365nm ultraviolet light are respectively recorded using a color recognition application, 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;

[0030] Step (f), treating the sample to be tested with unknown EP concentration according to step (e), 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 under 365nm ultraviolet light according to step (e), 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 (e) 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 (e) to obtain the EP concentration in the sample to be tested.

[0031] In step (d), preferably, the agarose solution is prepared by adding agarose powder into Tris-HCl buffer and heating by microwave to dissolve the agarose.

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

[0033] The final concentration of the agarose is 5-20 mg / mL, preferably 15 mg / mL; in the mixed solution, the final concentration of Cu-MB is 0.05 mg / mL-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, an agarose solution at a temperature of 40° C. and a Cu-MB Cu-MB dispersion are mixed to obtain a mixed solution.

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

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

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

[0039] The sample to be tested is a normal human plasma sample. Pretreatment of the sample to be tested: 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 the protein in the plasma and eliminate its interference, centrifuged at 12000rpm for 15 minutes, the supernatant is taken, and a 1mM NaOH solution is added to adjust the pH value to 7.4.

[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 the detection of fluorescence mode. 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 different collected signals 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 adding different concentrations of EP into the detection system constructed in Example 1.

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

[0050] Figure 5 The fluorescence standard curve of adding different concentrations of EP into the detection system constructed in Example 1.

[0051] Figure 6 is the influence of different conditions on the colorimetric sensing system; among them, a is the influence of different pH on the ultraviolet signal response, b is the influence of different incubation temperatures on the ultraviolet signal response, c is the influence of different incubation times on the ultraviolet signal response, and d is the influence 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 The results of laccase activity test of Cu-MB prepared with different ratios of 2-aminoterephthalic acid and 2-methylimidazole.

[0054] Fig. 9It is a comparison 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] Fig.10 This is a visualization detection diagram of EP by the hydrogel detection system constructed in Example 6; wherein, a is a schematic diagram of the visualization 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 by combining 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 here.

[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 has stronger laccase activity.

[0058] Example 1

[0059] A method for detecting adrenaline based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance Cu-MB, the steps are as follows:

[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 under constant 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 to DMF is 1:2; transferring the reaction system to an autoclave, heating at 140°C in an oven for 5 hours, cooling the reaction solution naturally, and centrifuging at 12000 rpm for 6 minutes to obtain a precipitate, washing the precipitate with ethanol and water 4 times, and dispersing the bifunctional copper-based nanozyme Cu-MB 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 adrenaline (EP) sensor: using ultrapure water 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; incubating the reaction at 37°C for 10 minutes, and after the reaction was completed, measuring the ultraviolet absorption curve of the sample at a wavelength of 350-650 nm ( Figure 2 ), with the absorbance at 485nm 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, the 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 measured respectively, and 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, and 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 12000rpm for 15 minutes, the supernatant was taken, and NaOH solution (1mM) was added to adjust the pH value 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 EP solution (prepared with ultrapure water), 40 μL Cu-MB dispersion (dispersed in ultrapure water, initial concentration of 1 mg / mL) and Tris-HCl buffer of different pH (pH 3, 4, 5, 6, 7.4, 8) were mixed and incubated for reaction. 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 with different pH values ​​were colorimetrically compared. The ultraviolet absorption value was measured at a wavelength of 485 nm using an ultraviolet spectrophotometer. The results are shown in Figure 6 a. The results show that Cu-MB exhibits good UV signal response at pH 6-8, especially at pH 7.4, where it exhibits the best UV signal response.

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

[0069] 20 μL EP solution (prepared with ultrapure water), 40 μL Cu-MB dispersion (dispersed in ultrapure water, initial concentration of 1 mg / mL) and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated for reaction. The final reaction solution volume was 300 μL, and the final concentration of EP 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 reaction system at different temperatures was colorimetrically compared. The ultraviolet absorption value was measured at a wavelength of 485 nm using an ultraviolet spectrophotometer. The results are shown in Figure 6 b. The results show that Cu-MB exhibits good UV signal response at temperatures ranging from 20 to 60°C, especially at 37°C, where it exhibits the best UV signal response.

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

[0071] 20 μL EP solution (prepared with ultrapure water), 40 μL Cu-MB dispersion (dispersed in ultrapure water, initial concentration of 1 mg / mL) and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated for reaction. The final reaction solution volume was 300 μL, and the final concentration of EP 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 compared. The ultraviolet absorption value was measured at a wavelength of 485 nm using an ultraviolet spectrophotometer. The results are shown in Figure 6 c. The results show that the UV signal response can reach the strongest UV signal response after incubation for 10 minutes, and the UV signal response no longer increases with the increase of incubation time. 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 EP solution (prepared with ultrapure water), different concentrations of Cu-MB dispersion (dispersed in ultrapure water) and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated for reaction. The final reaction solution 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 system with different Cu-MB concentrations was colorimetrically compared. The ultraviolet absorption value was measured at a wavelength of 485 nm using an ultraviolet spectrophotometer. The results are shown in Figure 6 d. The results show that when the final concentration of Cu-MB in the system is 100 μg / mL, the UV signal response can reach the ideal UV signal response. Therefore, the final concentration of Cu-MB 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 EP solution (prepared with ultrapure water), 40 μL Cu-MB dispersion (dispersed in ultrapure water, initial concentration of 1 mg / mL) and Tris-HCl buffer of different pH (pH 3, 4, 5, 6, 7.4, 8) were mixed and incubated for reaction. 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, the fluorescence of the reaction system with different pH was measured. The fluorescence intensity value was measured at a wavelength of 435 nm using a fluorescence spectrophotometer. The results are shown in Figure 7 a. The results show that Cu-MB exhibits a good fluorescence signal response at pH 7-8, especially at pH 7.4, it exhibits the best fluorescence signal response.

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

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

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

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

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

[0082] 20 μL EP solution (prepared with ultrapure water), different concentrations of Cu-MB dispersion (dispersed in ultrapure water) and Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated for reaction. The final reaction solution 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 system with different Cu-MB concentrations was subjected to fluorescence measurement. The fluorescence intensity value was measured at a wavelength of 435 nm using a fluorescence spectrophotometer. The results are shown in Figure 7d. The 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 to 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 12000rpm for 6 minutes to obtain a precipitate, and the precipitate is washed with ethanol and an aqueous solution to obtain a bifunctional nanozyme 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 by the conventional substrate of natural laccase (i.e., the color reaction of 2,4-dichlorophenol and 4-aminoantipyrine). 20 μL of Cu-MB dispersion (initial concentration of 1 mg / mL), 20 μL of 2,4-dichlorophenol (prepared with ultrapure water, initial concentration of 1 mg / mL), 20 μL of 4-aminoantipyrine (prepared with ultrapure water, initial concentration of 1 mg / mL) and Tris-HCl buffer (50 mM, pH 7.4) were mixed, and the final reaction solution volume was 300 μL. The reaction was carried out at 37°C for 30 minutes, and the ultraviolet absorption value was measured at a wavelength of 510 nm using an ultraviolet spectrophotometer to calculate the relative enzyme activity (the calculation of relative enzyme activity is based on the highest enzyme activity as 100%). The results are shown in 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] 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 ). 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: dissolve copper nitrate and 2-aminoterephthalic acid in N,N-dimethylformamide (DMF) to obtain solution A; mix ethanol and DMF to obtain solution B; slowly add solution A 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; 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 the precipitate with ethanol and water to obtain nanozyme Cu-(BDC-NH2), and disperse it 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, centrifuge at 12000rpm for 6 minutes to obtain a precipitate, wash it with ethanol and water to obtain nanozyme Cu-MI, and disperse it in ultrapure water.

[0091] Comparison of different enzyme activities: Take the same concentration of Cu-MB (step (a) of Example 1), Cu-(BDC-NH2) and Cu-MI (all dispersed in ultrapure water, the final concentration in the final reaction system is 200 μg / mL), add different concentrations of 2,4-dichlorophenol solution (prepared with ultrapure water), 45 μL of 4-aminoantipyrine solution (prepared with ultrapure water, the initial concentration is 1 mg / mL) and Tris-HCl buffer (50 mM, pH 7.4) and mix, the final reaction system volume is 300 μL, and the final concentration of 2,4-dichlorophenol is 0.06, 0.12, 0.24, 0.36, 0.49, 0.61 mM respectively; react at room temperature for 20 min, use an ultraviolet spectrophotometer to measure the ultraviolet absorption value at a wavelength of 510 nm, use each absorption value to obtain the reaction rate value, substitute it 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] Fig. 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. Fig. 9 d, e, and f are the Michaelis-Menten curves of Cu-MB, Cu-(BDC-NH2), and Cu-MI, respectively. m The values ​​are 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, the steps are as follows:

[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 by 700 W microwave heating to obtain an agarose solution, and then heated in a 40° C. water bath, 3 mL of 40° C. agarose solution was taken, and 375 μL of Cu-MB dispersion was added; the obtained mixed solution (the final concentration of Cu-MB was about 0.1 mg / mL) was added to the lid of a centrifuge tube, cooled to room temperature, and the hydrogel was obtained, and taken out;

[0097] Step (c), visual detection based on hydrogel: Fig.10a. 30 μL of EP solution of different concentrations (prepared with ultrapure water, 5, 10, 20, 30, 40, 50 μg / mL) was dripped 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, colorimetric 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 respectively 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 coordinate and the ratio of the red channel / blue channel R / B under natural light as the vertical coordinate: 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 / 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 unknown EP concentration according to step (c), and qualitatively analyzing the sample to be tested based on the colorimetric card; according to step (e), respectively measuring the RGB values ​​of the sample to be tested with unknown EP concentration under natural light and under 365nm ultraviolet light, 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 (e) 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 (e) to obtain the EP concentration in the sample to be tested.

[0099] Application Example 1

[0100] Normal human plasma samples were measured. Sample pretreatment and detection were performed according to step (c) of Example 1, and 5 μg / mL, 10 μg / mL, and 30 μg / mL were added to the plasma, respectively. The colorimetric and fluorescent signals were measured according to the method 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 measured 3 times, and the average value was taken. The RSD and recovery rate were calculated and shown in Table 1.

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

[0102]

[0103] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes 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 adrenaline based on a colorimetric-fluorescence dual-mode sensor with high laccase activity and high fluorescence performance Cu-MB, characterized in that: 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, heating to react, cooling after the reaction, centrifuging, and washing the precipitate 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 is completed, measuring the ultraviolet absorption curve of the sample at a wavelength of 350-650nm, taking the concentration of EP as the horizontal coordinate and the absorbance value at 485nm as the vertical coordinate, and establishing an ultraviolet standard curve of EP; measuring the fluorescence emission curve of the sample at a wavelength of 380-600nm at an excitation wavelength of 350nm, taking the concentration of EP as the horizontal coordinate and the fluorescence intensity value at 435nm as the vertical coordinate, and establishing a fluorescence standard curve of EP; Step (c), sample detection: according to step (b), the maximum ultraviolet absorbance value and the 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 adrenaline based on the colorimetric-fluorescence dual-mode sensor of Cu-MB with high laccase activity and high fluorescence performance according to claim 1, characterized in that: In step (a), the molar ratio of 2-aminoterephthalic acid, 2-methylimidazole and copper nitrate is (0.8-0.2):(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; The volume ratio of ethanol to DMF is (1-2):(2-1), preferably 1:

2. The reaction temperature is 130-160°C, preferably 140°C; The heating time is 3 to 6 hours, preferably 5 hours.

3. The method for detecting adrenaline based on the colorimetric-fluorescence dual-mode sensor of Cu-MB with high laccase activity and high fluorescence performance according to claim 1, characterized in that: In step (b), ultrapure water is used to prepare Cu-MB dispersion and EP solutions of different concentrations, 20 to 30 μL of EP solutions of different concentrations, 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.

4. The method for detecting adrenaline using a colorimetric-fluorescence dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance according to claim 1 or 3, characterized in that: In step (b), the concentration of the Cu-MB dispersion is 1 mg / mL.

5. The method for detecting adrenaline using a colorimetric-fluorescence dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance according to claim 1 or 3, characterized in that: In step (b), in the incubation system, the final concentration of Cu-MB is 33 to 166 μg / mL, preferably 100 to 166 μg / mL, more preferably 100 to 140 μg / mL, and most preferably 100 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, 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; 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; The final volume of the incubation system is 250-350 μL, preferably 300 μL.

6. The method for detecting adrenaline using a colorimetric-fluorescence dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance 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 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; an incubation reaction is performed, 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.

7. The method for detecting adrenaline using a colorimetric-fluorescence dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance according to claim 6, characterized in that: In step (c), in the incubation system, the final concentration of Cu-MB is 33 to 166 μg / mL, preferably 100 to 166 μg / mL, more preferably 100 to 140 μg / mL, and most preferably 100 μg / mL; 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; The incubation reaction temperature is 20-60°C, preferably 35-45°C, and more preferably 37°C; the incubation reaction time is 3-25 minutes, preferably 10-25 minutes, and more preferably 10 minutes.

8. The method for detecting adrenaline using a colorimetric-fluorescence dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance according to claim 1, characterized in that: Also includes: Step (d), 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 being 8:1, mixing the agarose solution at 40 to 50° C. and the Cu-MB dispersion to obtain a mixed solution, dripping the mixed solution into a mold, and cooling to room temperature to obtain a hydrogel; Step (e), visual detection based on hydrogel: EP solutions of different concentrations are respectively added dropwise onto the hydrogel, reacted at room temperature, and colorimetric cards under natural light and 365nm ultraviolet light are respectively constructed based on different colors corresponding to different EP concentrations; color images recorded under natural light and 365nm ultraviolet light are respectively recorded using a color recognition application, 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 (f), treating the sample to be tested with unknown EP concentration according to step (e), 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 under 365nm ultraviolet light according to step (e), 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 (e) 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 (e) to obtain the EP concentration in the sample to be tested.

9. The method for detecting adrenaline using a colorimetric-fluorescence dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance according to claim 8, characterized in that: In step (d), the final concentration of 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, preferably a Tris-HCl buffer solution with a pH of 7.4 and 50 mM.

10. The method for detecting adrenaline using a colorimetric-fluorescence dual-mode sensor based on Cu-MB with high laccase activity and high fluorescence performance according to claim 8, characterized in that: In step (e), the dosage of the EP solution is 25-35 μL; the concentration of the EP solution is 5-50 μg / mL.

Images