Method for detecting oxytetracycline based on fluorescent nano-enzyme e-Cu-BN with high laccase-like activity

Abstract

The invention discloses a method for detecting oxytetracycline based on fluorescent nano-enzyme e-Cu-BN with high laccase-like activity. The method comprises the following steps: synthesizing the fluorescent nano-enzyme e-Cu-BN; establishing an oxytetracycline colorimetric sensor: establishing an oxytetracycline ultraviolet standard curve by taking the log oxytetracycline concentration as an abscissa and the absorbance value of the sample at 510nm as an ordinate; and measuring the absorbance value of the sample to be detected at 510nm, and substituting the absorbance value into the oxytetracycline ultraviolet standard curve to obtain the oxytetracycline concentration in the sample to be detected. Establishing a high-sensitivity oxytetracycline fluorescence sensor: establishing an oxytetracycline fluorescence standard curve by taking the concentration of oxytetracycline as an abscissa and the fluorescence intensity value of a sample at 430nm as an ordinate; and measuring the fluorescence intensity value of a to-be-detected sample with unknown oxytetracycline concentration at 430nm, and substituting the fluorescence intensity value into the oxytetracycline fluorescence standard curve to obtain the oxytetracycline concentration in the to-be-detected sample. The method disclosed by the invention can be used for simply, quickly and sensitively detecting oxytetracycline.

Description

Technical Field

[0001] The present invention belongs to the field of analytical detection, and relates to an ammonia-etched laccase-like mimetic enzyme with fluorescence characteristics, a preparation method thereof, and an application thereof in detecting oxytetracycline (OTC); more specifically, it relates to a highly sensitive colorimetric / fluorescent dual-mode sensor based on a fluorescent nanozyme e-Cu-BN with high laccase-like activity or a method for visually detecting oxytetracycline by means of a hydrogel detection platform. Background Art

[0002] Oxytetracycline (OTC) is a derivative of tetracycline antibiotics and can inhibit the protein synthesis of many bacteria and microorganisms. OTC is commonly found in feed for promoting animal growth and preventing animal diseases. However, due to the abuse of antibiotics, OTC residues also exist in daily foods such as milk, honey, and meat. Long-term consumption can cause allergic reactions, carcinogenesis, teratogenesis, and mutagenesis, endangering human health. Moreover, the abuse of antibiotics will also lead to the emergence of multi-drug resistant bacteria, causing an irreversible situation.

[0003] Currently, the established OTC detection methods include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), surface enhanced Raman scattering (SERS), lateral flow immunoassay (LFIC), lateral flow assay (LFA), and electrochemical method. However, most of these methods require complex analytical instruments, long operation times, and sometimes the connection of carrier proteins, which makes the operability of the experiment very limited. Therefore, there is an urgent need for a time-saving and cost-effective method for detecting OTC.

[0004] Laccase is a multi-copper oxidase and is called a green catalyst because it does not produce H2O2 during the process of catalyzing the reduction of toxic phenolic pollutants into water. Laccase is widely used in the fields of food, water quality optimization, lignin degradation, and fermentation. However, due to the disadvantages of natural enzymes such as poor stability, easy denaturation and inactivation, high price, and difficult preservation, it will bring many inconveniences to experimental operations. Therefore, researchers use nanozymes as laccase mimics. According to the structure design of natural laccase, Cu elements are combined with ligands such as amino acids and nucleotides, and Cu-BH, LM-nanozyme, ATP-Cu, GMP-Cu, and Cu-adenine are successfully synthesized. However, most laccase mimetic enzymes do not have fluorescence characteristics and can only perform single-mode detection.

[0005] Due to the simplicity, strong operability, and high sensitivity of fluorescence and colorimetric methods, more and more researchers have started to use these methods to detect antibiotics. Compared with single-mode detection methods, dual-mode detection methods have a wider range of applications and can meet the needs of more analysis occasions. In addition, the colorimetric and fluorescence signals confirm each other, essentially providing built-in correction and reducing environmental interference. In contrast, the dual-mode strategy not only provides multiple methods for actual detection needs but also enables the mutual verification of quantitative measurement results, improving the reliability of data. Summary of the Invention

[0006] The object of the present invention is to overcome the disadvantages of low sensitivity, time-consuming detection, high cost, and cumbersome steps of existing detection methods. A new nanozyme e-Cu-BN was synthesized, and a colorimetric and fluorescence sensing system was established using e-Cu-BN, and this new type of dual-mode analysis system was used for OTC. The present invention provides a method for detecting oxytetracycline using a highly sensitive colorimetric / fluorescence dual-mode sensor based on a fluorescent nanozyme e-Cu-BN with high laccase-like activity, which can detect OTC simply, quickly, intuitively, and highly sensitively.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A fluorescent nanozyme e-Cu-BN with high laccase-like activity, which uses copper nitrate, 2-aminoterephthalic acid, and 4,4'-bipyridine as reaction raw materials, N,N-dimethylformamide and ethanol as solvents, and is prepared by a solvothermal method to obtain a precursor Cu-BN. The precursor Cu-BN is dispersed in water to obtain a Cu-BN dispersion, and then ammonia water is added for etching to obtain the fluorescent nanozyme e-Cu-BN.

[0009] The molar ratio of the copper nitrate to the 2-aminoterephthalic acid is 0.995:1 to 1:1; the molar ratio of the copper nitrate to the 4,4'-bipyridine is 1:1 to 1:2, preferably 1:1.5.

[0010] The volume ratio of the N,N-dimethylformamide (DMF) to the ethanol is 1:1 to 3:1, preferably 2:1.

[0011] The reaction temperature of the solvothermal method is 120 to 160 °C, preferably 140 °C; the reaction time of the solvothermal method is 5 to 12 hours, preferably 6 hours.

[0012] After the solvothermal reaction is completed, centrifuge, wash the precipitate with ultrapure water, and dry it; wherein, the centrifugal speed for washing is 10000 rpm; the drying temperature is 60 °C.

[0013] The mass fraction of the ammonia water is 25%; the concentration of Cu-BN in the Cu-BN dispersion is 2 mg / mL; the volume ratio of the Cu-BN dispersion to the ammonia water is 10:1 to 10000:1, preferably 1000:1; the etching temperature is room temperature, and the etching time is 5 to 30 minutes, preferably 15 minutes. The present invention uses ammonia water for etching at room temperature, with simple operation and a short reaction time.

[0014] The method for ammonia water etching is as follows: Cu-BN is uniformly dispersed in ultrapure water to prepare a dispersion, ammonia water is added, and it is stirred at room temperature for 15 minutes. During the stirring process, it can be observed that the dark green solution gradually turns gray. After the etching is completed, it is centrifuged and washed to obtain e-Cu-BN.

[0015] Another object of the present invention is to provide a method for detecting oxytetracycline using a highly sensitive colorimetric / fluorescent dual-mode sensor based on a fluorescent nanozyme e-Cu-BN with high laccase-like activity, including the following steps:

[0016] Step (a), synthesizing the fluorescent nanozyme e-Cu-BN: Using copper nitrate, aminoterephthalic acid, and 4,4'-bipyridine as reaction raw materials, and N,N-dimethylformamide and ethanol as solvents, a precursor Cu-BN is prepared by a solvothermal method. The precursor Cu-BN is dispersed in water, and then ammonia water is added for etching to obtain the fluorescent nanozyme e-Cu-BN;

[0017] Step (b), constructing a highly sensitive oxytetracycline colorimetric sensor: Mixing an oxytetracycline solution, an e-Cu-BN solution, a 2,4-dichlorophenol solution, a 4-aminoantipyrine solution, and a buffer solution to obtain a detection system containing different concentrations of oxytetracycline, incubating and reacting to obtain a sample; measuring the ultraviolet absorption curve of the sample at a wavelength of 400 - 650 nm, using log 土霉素的浓度 as the abscissa and the absorbance value of the sample at 510 nm as the ordinate to establish an oxytetracycline ultraviolet standard curve;

[0018] Or mixing an oxytetracycline solution, an e-Cu-BN solution, a 2,4-dichlorophenol solution, a 4-aminoantipyrine solution, and a buffer solution to obtain a detection system containing different concentrations of oxytetracycline, incubating and reacting to obtain a sample; replacing the oxytetracycline solution with an equal volume of ultrapure water to obtain a blank control sample; measuring the ultraviolet absorption curves of the sample and the blank control sample at a wavelength of 400 - 650 nm respectively, using log 土霉素的浓度 as the abscissa and the difference in absorbance at 510 nm between the blank control sample and the sample as the ordinate to establish an oxytetracycline ultraviolet standard curve;

[0019] Step (c), sample detection: Measure the absorbance value of the test sample with an unknown oxytetracycline concentration at 510 nm according to step (b), and substitute it into the oxytetracycline ultraviolet standard curve in step (b) to obtain the oxytetracycline concentration in the test sample;

[0020] Or measure the absorbance values of the test sample with an unknown oxytetracycline concentration and the blank control sample at 510 nm according to step (d), obtain the absorbance difference between the blank control sample and the test sample at 510 nm, and substitute it into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the test sample;

[0021] Step (d), construct a highly sensitive oxytetracycline fluorescence sensor: Mix the oxytetracycline solution, e-Cu-BN solution and buffer solution to obtain a detection system containing oxytetracycline at different concentrations, incubate the reaction to obtain a sample; Measure the fluorescence emission curve of the sample at wavelengths from 400 to 550 nm under the excitation of 340 nm, and establish an oxytetracycline fluorescence standard curve with the oxytetracycline concentration as the abscissa and the fluorescence intensity value of the sample at 430 nm as the ordinate;

[0022] Or mix the oxytetracycline solution, e-Cu-BN solution and buffer solution to obtain a detection system containing oxytetracycline at different concentrations, incubate the reaction to obtain a sample; Replace the oxytetracycline solution with an equal volume of ultrapure water to obtain a blank control sample; Measure the fluorescence emission curves of the sample and the blank control sample at wavelengths from 400 to 550 nm under the excitation of 340 nm respectively, and establish an oxytetracycline fluorescence standard curve with the oxytetracycline concentration as the abscissa and the fluorescence intensity difference (△FL) between the blank control sample and the sample at 430 nm as the ordinate;

[0023] Step (e), sample detection: Measure the fluorescence intensity value of the test sample with an unknown oxytetracycline concentration at 430 nm according to step (d), and substitute it into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the test sample;

[0024] Or measure the fluorescence intensity values of the test sample with an unknown oxytetracycline concentration and the blank control sample at 430 nm according to step (d), obtain the fluorescence intensity difference between the blank control sample and the test sample at 430 nm, and substitute it into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the test sample.

[0025] In step (b), in the detection system, the final concentration of oxytetracycline is 2.5 - 150 μM; the final concentration of e-Cu-BN is 200 - 400 μg / mL, preferably 150 μg / mL; the final concentration of 2,4-dichlorophenol is 150 - 200 μg / mL, preferably 167 μg / mL, and the final concentration of 4-aminoantipyrine is 150 - 200 μg / mL, preferably 167 μg / mL; the final volume of the detection system is 200 - 400 μL, preferably 300 μL.

[0026] According to the specific embodiments of the present invention, oxytetracycline with different concentrations in equal volumes is added to the system, and the final concentrations of oxytetracycline are 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 120 μM, and 150 μM.

[0027] The buffer solution is 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 3.0 - 8.0, preferably 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 7.4 and a concentration of 20 mM.

[0028] The oxytetracycline solution can be prepared with water or buffer solution; the e-Cu-BN solution can be prepared with water or buffer solution; the 2,4-dichlorophenol solution can be prepared with water or buffer solution; the 4-aminoantipyrine solution can be prepared with water or buffer solution.

[0029] When the oxytetracycline solution, e-Cu-BN solution, 2,4-dichlorophenol solution, and 4-aminoantipyrine solution are all prepared with water, the buffer solution accounts for 1 / 5 - 3 / 5 of the total volume of the detection volume, preferably 2 / 5.

[0030] Specifically, the detection system is as follows: different concentrations of oxytetracycline solution, e-Cu-BN solution, 2,4-dichlorophenol solution, and 4-aminoantipyrine solution are prepared with ultrapure water respectively. 30 μL of oxytetracycline solution with different concentrations is added to the system composed of 50 μL of e-Cu-BN solution, 50 μL of 2,4-dichlorophenol solution, and 50 μL of 4-aminoantipyrine solution, and 2-(N-morpholino)ethanesulfonic acid buffer solution is added until the final volume of the detection system is 300 μL.

[0031] The temperature of the incubation reaction is 20 - 70 °C, and the time of the incubation reaction is 5 - 50 min.

[0032] Preferably, the temperature of the incubation reaction is 35 - 40 °C, and the time of the incubation reaction is 20 - 30 min.

[0033] More preferably, the temperature of the incubation reaction is 37 °C, and the time of the incubation reaction is 20 min.

[0034] In step (c), the sample detection is as follows: mix the sample to be tested, e-Cu-BN solution, 2,4-dichlorophenol solution, 4-aminoantipyrine solution and buffer solution to obtain a detection system, incubate and react to obtain a sample; measure the ultraviolet absorption curve of the sample at a wavelength of 400-650 nm, obtain the maximum ultraviolet absorbance value of the sample at 510 nm, and substitute it into the oxytetracycline ultraviolet standard curve in step (b) to obtain the oxytetracycline concentration in the sample to be tested.

[0035] In the said detection system, the final concentration of e-Cu-BN is 200-400 μg / mL, preferably 150 μg / mL; the final concentration of 2,4-dichlorophenol is 150-200 μg / mL, preferably 167 μg / mL, and the final concentration of 4-aminoantipyrine is 150-200 μg / mL, preferably 167 μg / mL; the final volume of the said detection system is 200-400 μL, preferably 300 μL.

[0036] The said buffer solution is 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 3.0-8.0, preferably 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 7.4 and a concentration of 20 mM.

[0037] The temperature of the said incubation reaction is 20-70 °C, and the time of the said incubation reaction is 5-50 min.

[0038] Preferably, the temperature of the said incubation reaction is 35-40 °C, and the time of the said incubation reaction is 20-30 min.

[0039] More preferably, the temperature of the said incubation reaction is 37 °C, and the time of the said incubation reaction is 20 min.

[0040] Specifically, the said sample detection is as follows: prepare e-Cu-BN solution, 2,4-dichlorophenol solution and 4-aminoantipyrine solution respectively with ultrapure water; add 30 μL of the sample to be tested into a system composed of 50 μL of e-Cu-BN solution, 50 μL of 2,4-dichlorophenol solution and 50 μL of 4-aminoantipyrine solution, add 2-(N-morpholino)ethanesulfonic acid buffer solution until the final volume of the detection system is 300 μL to obtain a detection system, incubate and react to obtain a sample; measure the ultraviolet absorption curve of the sample at a wavelength of 400-650 nm, obtain the maximum ultraviolet absorbance value of the sample at 510 nm, and substitute it into the oxytetracycline ultraviolet standard curve in step (b) to obtain the oxytetracycline concentration in the sample to be tested.

[0041] In step (d), in the detection system, the final concentration of oxytetracycline is 2.5 - 150 μM; the final concentration of e-Cu-BN is 200 - 400 μg / mL, preferably 150 μg / mL; the final volume of the detection system is 200 - 400 μL, preferably 300 μL.

[0042] The buffer solution is 2-(N-morpholino)ethanesulfonic acid buffer solution with pH 3.0 - 8.0, preferably 2-(N-morpholino)ethanesulfonic acid buffer solution with pH 7.4 and 20 mM.

[0043] The oxytetracycline solution can be prepared with water or buffer solution; the e-Cu-BN solution can be prepared with water or buffer solution.

[0044] When both the oxytetracycline solution and the e-Cu-BN solution are prepared with water, the buffer solution accounts for 7 / 10 - 8 / 10 of the total volume of the detection volume.

[0045] Specifically, the detection system is as follows: Prepare oxytetracycline solutions and e-Cu-BN solutions with different concentrations using ultrapure water respectively; add 30 μL of oxytetracycline solutions with different concentrations into 50 μL of e-Cu-BN solution, and add 2-(N-morpholino)ethanesulfonic acid buffer solution until the final volume of the detection system is 300 μL.

[0046] The temperature of the incubation reaction is room temperature, and the time of the incubation reaction is 0.5 - 20 min, preferably 2 min.

[0047] In step (e), for the sample detection: Mix the sample to be tested, the e-Cu-BN solution and the buffer solution to obtain a detection system, carry out an incubation reaction to obtain a sample; measure the fluorescence emission curve of the sample at a wavelength of 400 - 550 nm under the excitation of 340 nm, obtain the fluorescence intensity value of the sample at 430 nm, and substitute it into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the sample to be tested;

[0048] Or mix the oxytetracycline solution, the e-Cu-BN solution and the buffer solution to obtain a detection system, carry out an incubation reaction to obtain a sample; replace the oxytetracycline solution with an equal volume of ultrapure water to obtain a blank control sample; measure the fluorescence emission curves of the sample and the blank control sample at a wavelength of 400 - 550 nm under the excitation of 340 nm respectively, obtain the fluorescence intensity difference (△FL) between the blank control sample and the sample at 430 nm, and substitute it into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the sample to be tested.

[0049] In the detection system, the final concentration of e-Cu-BN is 200 - 400 μg / mL, preferably 150 μg / mL; the final volume of the detection system is 200 - 400 μL, preferably 300 μL.

[0050] The buffer solution is 2-(N-morpholino)ethanesulfonic acid buffer solution with pH 3.0 - 8.0, preferably 2-(N-morpholino)ethanesulfonic acid buffer solution with pH 7.4 and 20 mM.

[0051] The e-Cu-BN solution can be prepared with water or buffer solution.

[0052] When the e-Cu-BN solution is prepared with water, the buffer solution accounts for 7 / 10 - 8 / 10 of the total volume of the detection volume.

[0053] Specifically, for the sample detection: prepare the e-Cu-BN solution with ultrapure water; add 30 μL of the sample to be tested into 50 μL of the e-Cu-BN solution, add 2-(N-morpholino)ethanesulfonic acid buffer solution until the final volume of the detection system is 300 μL, incubate at room temperature for 2 minutes to obtain a sample; measure the fluorescence intensity value of the sample at 430 nm, substitute the fluorescence intensity value into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the sample to be tested; or add 30 μL of the sample to be tested into 50 μL of the e-Cu-BN solution, add 2-(N-morpholino)ethanesulfonic acid buffer solution until the final volume of the detection system is 300 μL, incubate at room temperature for 2 minutes to obtain a sample; replace the oxytetracycline solution with an equal volume of ultrapure water to obtain a blank control sample; measure the difference in fluorescence intensity at 430 nm between the blank control sample and the sample, and substitute it into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the sample to be tested.

[0054] The temperature of the incubation reaction is room temperature, and the time of the incubation reaction is 0.5 - 20 min, preferably 2 min.

[0055] As a further preferred technical solution of the method for detecting oxytetracycline by the highly sensitive colorimetric / fluorescent dual-mode sensor based on the high laccase-like activity fluorescent nanozyme e-Cu-BN of the present invention, it further includes:

[0056] Step (f), Visual detection based on the hydrogel platform: Disperse agarose in 2-(N-morpholino)ethanesulfonic acid buffer and dissolve it by microwave heating to obtain a gel liquid; use water or buffer solution as the solvent, mix e-Cu-BN, 2,4-dichlorophenol, and 4-aminoantipyrine in a centrifuge tube, then add the gel liquid and quickly invert to obtain a hydrogel detection platform; drop equal volumes of oxytetracycline solutions with different concentrations on the hydrogel detection platform to obtain a reaction matrix, react at room temperature, establish a colorimetric card based on different colors corresponding to different oxytetracycline concentrations, use a color recognition application program to record the colorimetric pictures, convert the colors into RGB values, obtain the ratio R / G of the red channel to the green channel, and establish a standard curve with the oxytetracycline concentration as the abscissa and the ratio R / G of the red channel to the green channel as the ordinate;

[0057] Step (g), Sample detection: Drop the sample to be tested on the hydrogel detection platform and react at room temperature, qualitatively analyze the sample to be tested according to the colorimetric card in step (c); use a color recognition application program to record the colorimetric pictures, convert the colors into RGB values, obtain the ratio R / G of the red channel to the green channel, substitute the ratio R / G of the red channel to the green channel into the standard curve in step (c) to obtain the oxytetracycline concentration in the sample to be tested.

[0058] In step (f), the concentration of the gel liquid is 10 mg / mL. The buffer solution is 2-(N-morpholino)ethanesulfonic acid buffer with a pH of 3.0 - 8.0, preferably 2-(N-morpholino)ethanesulfonic acid buffer with a pH of 7.4 and a concentration of 20 mM.

[0059] In the reaction matrix, the final concentration of e-Cu-BN is 200 - 400 μg / mL, preferably 150 μg / mL; the final concentration of 2,4-dichlorophenol is 150 - 200 μg / mL, preferably 167 μg / mL; the final concentration of 4-aminoantipyrine is 150 - 200 μg / mL, preferably 167 μg / mL.

[0060] According to the specific embodiments of the present invention, the final concentrations of oxytetracycline in the reaction matrix are: 20 μM, 50 μM, 80 μM, 100 μM, 120 μM, 150 μM.

[0061] The reaction time at room temperature is 5 - 50 minutes, preferably 40 minutes.

[0062] Specifically, for the visual detection based on the hydrogel platform: Prepare e-Cu-BN solution, 50 μL of 2,4-dichlorophenol solution, and 4-aminoantipyrine solution using ultrapure water respectively. Add 50 μL of e-Cu-BN solution, 50 μL of 2,4-dichlorophenol solution, and 50 μL of 4-aminoantipyrine solution into a centrifuge tube, then add 120 μL of gel liquid at about 50 °C, and quickly invert it so that the mixed solution is transferred from the bottom of the centrifuge tube to the lid, and a spherical hydrogel detection platform is obtained at room temperature.

[0063] For the color comparison card described above: Under natural light, as the concentration of oxytetracycline increases, the color of the hydrogel is red and shows a trend of changing from dark to light.

[0064] The color recognition application program is "RGB Color detector".

[0065] In step (g), for the sample detection: Drop 30 μL of the sample to be tested on the hydrogel detection platform, react at room temperature for 40 minutes, and qualitatively analyze the sample to be tested according to the color comparison card in step (c); Use the color recognition application program to record the color comparison picture, convert the color into RGB values, obtain the ratio R / G of the red channel to the green channel, and substitute the ratio R / G of the red channel to the green channel into the standard curve in step (c) to obtain the oxytetracycline concentration in the sample to be tested.

[0066] In the reaction matrix described above, the final concentration of e-Cu-BN is 200 - 400 μg / mL, preferably 150 μg / mL; the final concentration of 2,4-dichlorophenol is 150 - 200 μg / mL, preferably 167 μg / mL; the final concentration of 4-aminoantipyrine is 150 - 200 μg / mL, preferably 167 μg / mL.

[0067] The time for the reaction at room temperature is 5 - 50 minutes, preferably 40 minutes.

[0068] The sample to be tested is a food sample or an environmental sample; the food sample is milk; the environmental sample is lake water.

[0069] Pretreatment of the sample to be tested: First, centrifuge 10 mL of lake water and take the supernatant, then filter it with a 0.45 μM filter membrane to obtain the supernatant as the sample solution to be tested. Put 1 mL of milk into a centrifuge tube, mix it with 4 mL of acetonitrile, stir for 5 minutes and then centrifuge, then filter with a 0.45 μM filter membrane to remove the protein precipitate, vacuum dry the obtained solution, and dissolve it with 10 mL of water after drying to obtain the sample solution to be tested.

[0070] The detection mechanism of the method of the present invention ( Figure 1):The laccase-like activity of e-Cu-BN is enhanced after etching with ammonia water, catalyzing the oxidation of 2,4-dichlorophenol. The product complexes with the chromogenic reagent 4-aminoantipyrine to form a red quinoline dye (QI), which has a strong ultraviolet absorption at 510 nm. When oxytetracycline is introduced, the ultraviolet absorbance decreases; meanwhile, oxytetracycline can quench the fluorescence of e-Cu-BN.

[0071] Compared with the prior art, the present invention has the following remarkable advantages:

[0072] The fluorescent nanozyme e-Cu-BN of the present invention has stronger laccase-like activity than Cu-BN.

[0073] The colorimetric / fluorescent dual-mode sensor for detecting oxytetracycline based on the fluorescent nanozyme e-Cu-BN of the present invention has higher accuracy and can apply different signals collected to reduce the incidence of false positive and negative results.

[0074] The present invention establishes a smartphone-assisted hydrogel detection platform based on the fluorescent nanozyme e-Cu-BN. Through the color recognition software on the smartphone, the color change is converted into specific RGB values, making the detection results visual, and the real-time evaluation and on-site inspection of food safety can be realized. Description of the Drawings

[0075] Figure 1 It is the schematic diagram of the highly sensitive colorimetric / fluorescent dual-mode sensor for detecting oxytetracycline based on the fluorescent nanozyme e-Cu-BN with high laccase-like activity of the present invention.

[0076] Figure 2 It is the preparation flow chart of the nanozyme e-Cu-BN of the present invention.

[0077] Figure 3 It is the ultraviolet absorption curve and ultraviolet standard curve of different concentrations of oxytetracycline in the detection system constructed in Example 1. Among them, A is the ultraviolet absorption curve and B is the ultraviolet standard curve.

[0078] Figure 4 It is the fluorescence curve of oxytetracycline and the fluorescence standard curve of oxytetracycline in the detection system constructed in Example 1. Among them, A is the fluorescence curve and B is the fluorescence standard curve.

[0079] Figure 5 It is the ultraviolet absorption curve of different concentrations of 2,4-dichlorophenol in the laccase and e-Cu-BN system constructed in Example 2.

[0080] Figure 6 It is the standard curve graph of different concentrations of 2,4-dichlorophenol in the laccase and e-Cu-BN system constructed in Example 2.

[0081] Figure 7Investigation results of the enzymatic activity and fluorescence spectra of Cu-BN prepared from copper nitrate, amino terephthalic acid, and 4,4'-bipyridine in different ratios; among them, A is the ultraviolet absorption curve and B is the fluorescence curve.

[0082] Figure 8 Effect of different types of buffer solutions on the detection of oxytetracycline by the sensing system.

[0083] Figure 9 Effect of different conditions on the enzymatic activity and fluorescence response of the sensing system; among them, Figure 9 A shows the effect of different incubation times on the enzymatic activity of the oxytetracycline + e-Cu-BN + 2,4-dichlorophenol + 4-aminoantipyrine system, Figure 9 B shows the effect of different incubation temperatures on the enzymatic activity of the oxytetracycline + e-Cu-BN + 2,4-dichlorophenol + 4-aminoantipyrine system, Figure 9 C shows the effect of different pH values on the enzymatic activity of the oxytetracycline + e-Cu-BN + 2,4-dichlorophenol + 4-aminoantipyrine system, Figure 9 D shows the effect of different incubation times on the fluorescence response of the oxytetracycline + e-Cu-BN system.

[0084] Figure 10 Comparison diagram of the selectivity and anti-interference ability of the detection system constructed in Example 1 for oxytetracycline; among them, A is the experimental results of the selectivity and anti-interference ability of the colorimetric sensing system, and B is the experimental results of the selectivity and anti-interference ability of the fluorescence sensing system.

[0085] Figure 11 Visual detection diagram of the detection system constructed in Example 9 for oxytetracycline and standard curve diagram linearly related to the concentration. Detailed implementation manners

[0086] The technical solution of the present invention will be described in more detail by combining the accompanying drawings and specific implementation manners. Although the following are the preferred specific implementation manners 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 implementation manners described herein.

[0087] The inventors studied the properties of the fluorescent nanozyme e-Cu-BN by colorimetric and fluorescence methods. In addition, the catalytic reaction degrees of e-Cu-BN and laccase under the same conditions were compared, indicating that e-Cu-BN has strong laccase-like activity.

[0088] Example 1

[0089] A highly sensitive colorimetric / fluorescent dual-mode sensor based on the fluorescent nanozyme e-Cu-BN with high laccase-like activity for detecting oxytetracycline, the steps are as follows:

[0090] Step (a), synthesize the fluorescent nanozyme e-Cu-BN: As Figure 2 shown, 24 mg of Cu(NO3)2·3H2O and 18 mg of 2-aminoterephthalic acid (NH2-BDC) were mixed and added to 4 mL of DMF to obtain solution A. 23.4 mg of 4,4'-bipyridine (Bpy) was dissolved in a mixed solvent of 4 mL of absolute ethanol and 4 mL of DMF to obtain solution B. Solution A and solution B were mixed and stirred at room temperature for 10 minutes. The solution was transferred to a reaction kettle and reacted at 140 °C for 6 hours. Centrifuged at 10000 rpm for 10 minutes, and the precipitate was washed three times with ultrapure water. The washing conditions were centrifuged at 10000 rpm for 10 minutes and dried in vacuum at 60 °C to obtain the precursor Cu-BN. Etch with 25% ammonia water by mass fraction to prepare e-Cu-BN: Disperse 20 mg of the precursor Cu-BN powder into 10 mL of water, add 10 μL of 25% ammonia water by mass fraction, stir at room temperature for 15 min. It can be observed that the dark green solution gradually turns gray during the stirring process. The precipitate was washed three times with ultrapure water. The washing conditions were centrifuged at 10000 rpm for 10 minutes and dried in vacuum at 60 °C to obtain the fluorescent nanozyme e-Cu-BN, which was stored in a 4 °C refrigerator;

[0091] Step (b), construct a highly sensitive oxytetracycline colorimetric sensor: Add 30 μL of oxytetracycline solutions with different concentrations (prepared with ultrapure water) to a system composed of 50 μL of e-Cu-BN solution (concentration 0.9 mg / mL, prepared with ultrapure water), 50 μL of 2,4-dichlorophenol solution (concentration 1 mg / mL, prepared with ultrapure water), and 50 μL of 4-aminoantipyrine solution (concentration 1 mg / mL, prepared with ultrapure water). Add 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer, 20 mM, pH 7.4) to make the final volume of the detection system 300 μL. The final concentrations of oxytetracycline in the detection system were: 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 120 μM, 150 μM. The final concentration of e-Cu-BN was 0.15 mg / mL. Incubate and react at 37 °C for 20 minutes to obtain samples;

[0092] After the incubation reaction, measure the ultraviolet absorption curve of the sample at wavelengths from 400 to 550 nm ( Figure 3 A), with the logarithm value of the oxytetracycline concentration (log 土霉素的浓度 , that is, logc) as the abscissa (X), and the absorbance value (A) of the sample containing different concentrations of oxytetracycline at 510 nm as the ordinate (Y), establish the oxytetracycline ultraviolet standard curve ( Figure 3 B): Y = 0.1952X + 0.622, R 2 = 0.9975;

[0093] Step (c), sample detection: Add 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) to a system composed of 30 μL of the sample to be tested, 50 μL of e-Cu-BN solution (concentration 0.9 mg / mL, prepared with ultrapure water), 50 μL of 2,4-dichlorophenol solution (concentration 1 mg / mL, prepared with ultrapure water), and 50 μL of 4-aminoantipyrine solution (concentration 1 mg / mL, prepared with ultrapure water) until the final volume of the detection system is 300 μL. Incubate at 37 °C for 20 minutes to obtain a sample. After the incubation reaction, measure the maximum ultraviolet absorbance value of the sample at 510 nm, and substitute the maximum ultraviolet absorbance value into the oxytetracycline ultraviolet standard curve in step (b) to obtain the oxytetracycline concentration in the sample to be tested;

[0094] Step (d), constructing a highly sensitive oxytetracycline fluorescence sensor: Add 30 μL of oxytetracycline solutions with different concentrations (prepared with ultrapure water) to 50 μL of e-Cu-BN solution (concentration 0.9 mg / mL, prepared with ultrapure water), add 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) until the final volume of the detection system is 300 μL. The final concentrations of oxytetracycline in the detection system are: 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 120 μM, 150 μM, and the final concentration of e-Cu-BN is 0.15 mg / mL. React at room temperature for 2 minutes to obtain a sample;

[0095] After the reaction, measure the fluorescence emission curve of the sample at wavelengths from 400 to 650 nm under an excitation light of 340 nm ( Figure 4 A),; Establish an oxytetracycline fluorescence standard curve ( Figure 4 B): Y = -8.7856X + 2406.5943, R 2 = 0.9913;

[0096] Step (e), sample detection: Add 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) to 30 μL of the sample to be tested and 50 μL of e-Cu-BN solution (concentration 0.9 mg / mL, prepared with ultrapure water) until the final volume of the detection system is 300 μL. React at room temperature for 2 minutes to obtain a sample. After the reaction, measure the fluorescence intensity value of the sample at 430 nm, and substitute the fluorescence intensity value into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the sample to be tested;

[0097] Among them, the preparation of the test samples in steps (c) and (e): Centrifuge 10 mL of lake water and take the supernatant, filter it with a 0.45 μM filter membrane to obtain the supernatant as the test sample. Put 1 mL of milk into a centrifuge tube, mix it with 4 mL of acetonitrile, stir for 5 minutes, then centrifuge at a speed of 1000 rpm for 10 minutes, take the supernatant, filter it with a 0.45 μM filter membrane to remove the protein precipitate, vacuum-dry the obtained solution at 60 °C, and then dissolve it with 10 mL of ultrapure water to obtain the test sample.

[0098] Example 2

[0099] 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 ) and the maximum initial velocity value V max . The lower the K m value, the higher the affinity between the mimetic enzyme and the matrix. The larger the V max value, the higher the saturation reaction rate.

[0100] Comparison of different enzyme activities: Prepare natural laccase solution (300 mg / mL) and e-Cu-BN solution (90 μg / mL) with ultrapure water respectively. Take 50 μL of natural laccase solution (300 mg / mL) and e-Cu-BN solution (90 μg / mL) respectively, and add 50 μL of 4-aminoantipyrine solution (1 mg / mL, prepared with ultrapure water) and 50 μL of 2,4-dichlorophenol solution with different concentrations (prepared with ultrapure water). Add 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) to make the final volume of the detection system 300 μL, so that the final concentrations of 2,4-dichlorophenol are 0.061, 0.123, 0.245, 0.368, 0.491, 0.92 mM respectively; incubate and react at 37 °C for 30 min. After the incubation reaction ends, measure the absorbance value of the solution at 510 nm with an enzyme-labeling instrument. Calculate the reaction rate value from each absorbance value, and substitute it into 1 / V = (K m / V max )(1 / [S]) + 1 / V max to obtain the double-reciprocal curve, and calculate the Michaelis constant K m and the maximum initial velocity V max using the intercept and slope.

[0101] Figure 5 are the Michaelis curves of laccase and e-Cu-BN at 37 °C and pH = 7.4, Figure 6 are the Michaelis-Menten curves of laccase and e-Cu-BN. The V max values of e-Cu-BN and laccase are 2.2 μM / min and 0.36 μM / min respectively, and the V maxThe value is greater than that of laccase; the K of e-Cu-BN and laccase m are 0.13 mM and 0.30 mM respectively, and the K of e-Cu-BN m value is less than that of laccase. This indicates that e-Cu-BN has a higher catalytic efficiency for 2,4-dichlorophenol.

[0102] Example 3

[0103] Investigate the effects of Cu-BN prepared with different ratios of copper nitrate, aminoterephthalic acid, and 4,4'-bipyridine on the enzymatic activity and fluorescence spectrum.

[0104] Construct the material Cu-BN: Mix 24 mg of Cu(NO3)2·3H2O and 18 mg of 2-aminoterephthalic acid (NH2-BDC) and add them to 4 mL of DMF to obtain solution A. Dissolve 15.6 mg, 23.4 mg, and 31.2 mg of 4,4'-bipyridine (Bpy) in a mixed solvent of 4 mL of ethanol and 4 mL of DMF respectively to obtain solution B. Mix solution A and solution B (the molar ratios of Cu(NO3)2·3H2O, 2-aminoterephthalic acid, and 4,4'-bipyridine are 1:1:1, 1:1:1.5, and 1:1:2 respectively), and stir at room temperature for 10 minutes; transfer the solution to a reaction kettle, react at 140 °C for 6 hours, centrifuge at a speed of 1000 rpm for 10 minutes, wash the precipitate three times with ultrapure water, and the washing condition is to centrifuge at a speed of 10000 rpm for 10 minutes, dry in vacuum at 60 °C, and store in a 4 °C refrigerator.

[0105] Construct a colorimetric sensor with reference to Example 2: Add 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) to a system composed of 50 μL of Cu-BN dispersion (concentration 0.9 mg / mL, prepared with ultrapure water), 50 μL of 2,4-dichlorophenol solution (concentration 1 mg / mL, prepared with ultrapure water), and 50 μL of 4-aminoantipyrine solution (concentration 1 mg / mL, prepared with ultrapure water) until the volume of the detection system is 300 μL. Incubate and react at a temperature of 37 °C for 30 min, perform colorimetry on the reaction systems of Cu-BN prepared with different ratios of copper nitrate, aminoterephthalic acid, and 4,4'-bipyridine, and use an ultraviolet spectrophotometer to measure the ultraviolet absorption value at a wavelength of 510 nm.

[0106] Add 50 μL of Cu-BN dispersion (concentration 0.9 mg / mL, prepared with ultrapure water) to 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) until the volume of the detection system is 300 μL, and measure the fluorescence intensity value (F) of the reaction systems of Cu-BN prepared with different ratios of copper nitrate, aminoterephthalic acid, and 4,4'-bipyridine at 430 nm.

[0107] The results are shown in Figure 7 (The calculation of the relative enzyme activity of the present invention takes the highest enzyme activity as 100%, the same below). Cu-BN prepared according to the molar ratios of Cu(NO3)2·3H2O, 2-aminoterephthalic acid and 4,4'-bipyridine of 1:1:1, 1:1:1.5, and 1:1:2 all have good enzyme activity and fluorescence response. In particular, Cu-BN prepared according to the molar ratio of Cu(NO3)2·3H2O, 2-aminoterephthalic acid and 4,4'-bipyridine of 1:1:1.5 has the most excellent enzyme activity and fluorescence response.

[0108] Example 5

[0109] Based on the colorimetric sensor constructed in step (b) of Example 1, the effects of different types of buffer solutions on the enzyme activity of the oxytetracycline + e-Cu-BN + 2,4-dichlorophenol + 4-aminoantipyrine system were investigated. Tris-HCl buffer solution (20 mM, pH 7.4), 2-(N-morpholino)ethanesulfonic acid buffer solution (20 mM, pH 7.4), and phosphate buffer solution (20 mM, pH 7.4) were used respectively.

[0110] Add 30 μL of oxytetracycline solution (prepared with ultrapure water) to a system composed of 50 μL of e-Cu-BN solution (concentration 0.9 mg / mL, prepared with ultrapure water), 50 μL of 2,4-dichlorophenol solution (concentration 1 mg / mL, prepared with ultrapure water), and 50 μL of 4-aminoantipyrine solution (concentration 1 mg / mL, prepared with ultrapure water). Add Tris-HCl buffer solution, 2-(N-morpholino)ethanesulfonic acid buffer solution, and phosphate buffer solution (all 20 mM, pH 7.4) respectively until the final volume of the detection system is 300 μL. The final concentration of oxytetracycline in the detection system is 150 μM. Incubate and react at 37 °C for 20 minutes to obtain the sample. In addition, use 30 μL of ultrapure water to replace the oxytetracycline solution as a blank control. After the incubation reaction is completed, measure the ultraviolet absorption curve of the sample at a wavelength of 510 nm. The experimental results are shown in Figure 8 , indicating that the detection result of oxytetracycline in 2-(N-morpholino)ethanesulfonic acid buffer solution is the best.

[0111] Example 6

[0112] Based on the colorimetric / fluorescent dual-mode sensor constructed in step (b) of Example 1, the effects of different temperatures, different times, and different pH values on the enzyme activity of the oxytetracycline + e-Cu-BN + 2,4-dichlorophenol + 4-aminoantipyrine system and the effect of different times on the fluorescence response of oxytetracycline + e-Cu-BN were investigated respectively.

[0113] (1) Effect of different incubation times on the enzyme activity of the system

[0114] Add 30 μL of oxytetracycline solution (prepared with ultrapure water) and 30 μL of ultrapure water into a system composed of 50 μL of e-Cu-BN solution (concentration 0.9 mg / mL, prepared with ultrapure water), 50 μL of 2,4-dichlorophenol solution (concentration 1 mg / mL, prepared with ultrapure water), and 50 μL of 4-aminoantipyrine solution (concentration 1 mg / mL, prepared with ultrapure water). Then add 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) until the final volume of the detection system is 300 μL. The final concentration of oxytetracycline in the detection system is 150 μM. Incubate and react at 37 °C for 5, 10, 15, 20, 40, and 50 minutes respectively to obtain samples. Additionally, use 30 μL of ultrapure water to replace 30 μL of oxytetracycline solution as a blank control. After the incubation reaction ends, measure the ultraviolet absorption curve of the samples at a wavelength of 510 nm to obtain the absorbance values of the samples at 510 nm (denoted as A) and the absorbance value of the blank control at 510 nm (denoted as A0), and calculate A0 - A( Figure 9 A).

[0115] (2) Effect of different incubation temperatures on the enzyme activity of the system

[0116] Add 30 μL of oxytetracycline solution (prepared with ultrapure water) and 30 μL of ultrapure water into a system composed of 50 μL of e-Cu-BN solution (concentration 0.9 mg / mL, prepared with ultrapure water), 50 μL of 2,4-dichlorophenol solution (concentration 1 mg / mL, prepared with ultrapure water), and 50 μL of 4-aminoantipyrine solution (concentration 1 mg / mL, prepared with ultrapure water). Then add 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) until the final volume of the detection system is 300 μL. The final concentration of oxytetracycline in the detection system is 150 μM. Incubate and react at 20, 30, 37, 50, 60, and 70 °C for 20 minutes respectively to obtain samples. Additionally, use 30 μL of ultrapure water to replace 30 μL of oxytetracycline solution as a blank control. After the incubation reaction ends, measure the ultraviolet absorption curve of the samples at a wavelength of 510 nm to obtain the absorbance values of the samples at 510 nm (denoted as A) and the absorbance value of the blank control at 510 nm (denoted as A0), and calculate A0 - A( Figure 9 B).

[0117] (3) Effect of different incubation pH on the enzyme activity and fluorescence response of the system

[0118] 30 μL of oxytetracycline (prepared with ultrapure water) and 30 μL of ultrapure water were respectively added into a system composed of 50 μL of e-Cu-BN solution (concentration: 0.9 mg / mL, prepared with ultrapure water), 50 μL of 2,4-dichlorophenol solution (concentration: 1 mg / mL, prepared with ultrapure water), and 50 μL of 4-aminoantipyrine solution (concentration: 1 mg / mL, prepared with ultrapure water). 2-(N-morpholino)ethanesulfonic acid buffer solution with different pH values (pH 3, 4, 5, 6, 7.4, 8) was added until the final volume of the detection system was 300 μL. The final concentration of oxytetracycline in the detection system was 150 μM. The reaction was incubated at 37 °C for 20 minutes to obtain samples. Additionally, 30 μL of ultrapure water was used to replace 30 μL of the oxytetracycline solution as a blank control. After the incubation reaction ended, the ultraviolet absorption curves of the two samples were measured at a wavelength of 510 nm, and the absorbance values of the samples at 510 nm at different incubation temperatures (denoted as A) and the absorbance value of the blank control at 510 nm (denoted as A0) were obtained, and A0 - A was calculated. Figure 9 C), and the fluorescence emission curves of the samples at wavelengths from 400 to 650 nm were measured under an excitation light of 340 nm. The fluorescence intensity values of the samples at 430 nm at different incubation temperatures (denoted as F) and the fluorescence intensity value of the blank control at 430 nm (denoted as F0) were obtained, and F0 - F was calculated.

[0119] (4) Influence of different incubation times on the fluorescence intensity of the system

[0120] 30 μL of oxytetracycline solution (prepared with ultrapure water) and 30 μL of ultrapure water were respectively added into 50 μL of e-Cu-BN solution (concentration: 0.9 mg / mL, prepared with ultrapure water). 2-(N-morpholino)ethanesulfonic acid buffer solution (20 mM, pH 7.4) was added until the volume of the detection system was 300 μL. The final concentration of oxytetracycline in the detection system was 50 μM. The reaction was carried out at room temperature for 2, 4, 6, 8, and 10 minutes respectively to obtain samples. After the reaction ended, the fluorescence intensity values of the samples at 430 nm at different reaction times (denoted as F) and the fluorescence intensity value of the blank control at 430 nm (denoted as F0) were obtained, and F0 - F was calculated. Figure 9 D).

[0121] It can be seen from the results that when the pH of the detection system is 3 - 8, the incubation reaction temperature is 20 - 70 °C, and the incubation reaction time is 5 - 50 min, the colorimetric response of e-Cu-BN to oxytetracycline is relatively good. When the pH of the detection system is 7 - 7.4, the incubation reaction temperature is 35 - 40 °C, and the incubation reaction time is 20 - 30 min, the colorimetric response of e-Cu-BN to oxytetracycline is relatively better. Especially when the pH of the detection system is 7.4, the incubation reaction temperature is 37 °C, and the incubation reaction time is 20 min, the colorimetric response of e-Cu-BN to oxytetracycline is the best. When the reaction time of the detection system is 0.5 - 20 min, the fluorescence response of e-Cu-BN to oxytetracycline is relatively good. Especially when the reaction time of the detection system is 2 min, the fluorescence response of e-Cu-BN to oxytetracycline is the best.

[0122] Example 7

[0123] Selectivity and anti-interference experiments of oxytetracycline

[0124] Examine the selectivity and anti-interference of the colorimetric / fluorescent dual-mode sensor system constructed in Example 1 to Mg 2+ , Zn 2+ , K + , Na + , phenylalanine (Phe), arginine (Arg), histidine (His), chloramphenicol (Chl), L-lysine (Lys). Obtain the fluorescence intensity value of the sample at 430 nm (denoted as F), the fluorescence intensity value of the blank control at 430 nm (denoted as F0), and calculate △FL = F0 - F; obtain the absorbance value of the sample at 510 nm (denoted as A), the absorbance value of the blank control at 510 nm (denoted as A0), and calculate △A = A0 - A. Among them, the final concentration of oxytetracycline in the selectivity experiment is 50 μM, and the final concentration of other interfering substances is 100 μM; the final concentration of oxytetracycline in the anti-interference experiment is 50 μM, and the final concentration of other interfering substances is 50 μM.

[0125] The results of the selectivity experiment and the anti-interference experiment are shown in Figure 10 A, indicating that the highly sensitive colorimetric sensor based on the fluorescent nanozyme e-Cu-BN with high laccase-like activity has good selectivity and anti-interference to oxytetracycline.

[0126] The results of the selectivity experiment and the anti-interference experiment are shown in Figure 10 B, indicating that the highly sensitive fluorescent sensor based on the fluorescent nanozyme e-Cu-BN with high laccase-like activity has good selectivity and anti-interference to oxytetracycline.

[0127] Example 8

[0128] Detection limit (LOD) of the colorimetric sensor: In a system composed of 50 μL of e-Cu-BN solution (step (a) of Example 1, with a concentration of 0.9 mg / mL, prepared with ultrapure water), 50 μL of 2,4-dichlorophenol solution (concentration of 1 mg / mL, prepared with ultrapure water), and 50 μL of 4-aminoantipyrine solution (concentration of 1 mg / mL, prepared with ultrapure water), 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) was added until the volume of the detection system was 300 μL. The reaction was incubated at 37 °C for 20 min, and 20 parallel groups were made. After the incubation reaction ended, the ultraviolet absorption curve of the sample was measured at a wavelength of 510 nm.

[0129] Detection limit (LOD) of the fluorescence sensor: 50 μL of e-Cu-BN solution (concentration of 0.9 mg / mL, prepared with ultrapure water) was mixed with 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 7.4) until the volume of the detection system was 300 μL. The reaction was carried out at room temperature for 2 min, and 20 parallel groups were made. After the reaction ended, the fluorescence emission curve of the sample at wavelengths of 400 - 650 nm was measured under an excitation light of 340 nm.

[0130] The LOD of colorimetry calculated according to the formula LOD = 3σ / m is 0.43 μM; where σ is the standard deviation of the blank, and m is the slope of the linear equation (Y = 0.1952X + 0.622, X is log (土霉素的浓度) , and Y is the absorbance value of oxytetracycline at different concentrations at 510 nm).

[0131] The LOD of fluorescence is 0.32 μM; where σ is the standard deviation of the blank, and m is the slope of the linear equation (Y = -8.7856X + 2406.5943, R 2 = 0.9913, X is the concentration of oxytetracycline, and Y is the fluorescence intensity value of the sample at 430 nm).

[0132] Example 9

[0133] A method for visual detection based on an e-Cu-BN hydrogel platform

[0134] Step (a), synthesize e-Cu-BN: same as step (a) of Example 1;

[0135] Step (b), Preparation of the e-Cu-BN-based hydrogel platform: Disperse 50 mg of agarose in 5 mL of 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 7.4, and heat it in a microwave (power 700 W) for 1 minute to dissolve; quickly place the obtained gel liquid in a water bath at 50 °C to keep it warm and prevent solidification. First, add 50 μL of e-Cu-BN solution (step (a) of Example 1, with a concentration of 0.9 mg / mL, prepared with ultrapure water), 50 μL of 2,4-dichlorophenol solution (concentration of 1 mg / mL, prepared with ultrapure water), and 50 μL of 4-aminoantipyrine solution (concentration of 1 mg / mL, prepared with ultrapure water) into a centrifuge tube, then add 120 μL of gel liquid at about 50 °C, quickly invert it so that the mixed solution transfers from the bottom of the centrifuge tube to the lid, and obtain a spherical hydrogel detection platform at room temperature;

[0136] Step (c), Visual detection of the e-Cu-BN-based hydrogel detection platform: Drop 30 μL of oxytetracycline solutions with different concentrations on the hydrogel detection platform to obtain a reaction matrix. The final concentrations of oxytetracycline in the reaction matrix are: 20 μM, 50 μM, 80 μM, 100 μM, 120 μM, 150 μM. React at room temperature for 40 minutes, and observe the color change under natural light. As the concentration of oxytetracycline increases, the color of the hydrogel is red and shows a trend of changing from deep to light; establish a colorimetric card based on different colors corresponding to different oxytetracycline concentrations; use the color recognition application "RGB Color detector" to record the colorimetric pictures, convert the color into RGB values, obtain the ratio R / G of the red channel to the green channel, use the concentration of oxytetracycline as the abscissa and the ratio R / G of the red channel to the green channel as the ordinate to establish a standard curve: Y = 0.0036X + 1.526, R 2 = 0.9851;

[0137] Step (d), Sample detection: Drop 30 μL of the sample to be tested on the hydrogel detection platform to obtain a reaction matrix, react at room temperature for 40 minutes, and qualitatively analyze the sample to be tested according to the colorimetric card in step (c); use the color recognition application "RGB Color detector" to record the colorimetric pictures, convert the color into RGB values, obtain the ratio R / G of the red channel to the green channel, substitute the ratio R / G of the red channel to the green channel into the standard curve in step (c), and obtain the oxytetracycline concentration in the sample to be tested.

[0138] Among them, the preparation of the sample to be tested is the same as that in Example 1.

[0139] Application Example 1

[0140] Take the actual samples of lake water and milk, and perform sample pretreatment according to step (c) of Example 1 to obtain the samples to be tested. Add oxytetracycline standard products to the samples to be tested so that their final concentrations are 20, 50, and 100 μM respectively. Measure the colorimetric and fluorescence signals according to the method of Example 1, and substitute them into the oxytetracycline ultraviolet standard curve and oxytetracycline fluorescence standard curve established in Example 1 to obtain the oxytetracycline concentration in the samples; each sample is measured 3 times repeatedly, and the average value is taken. Calculate the RSD and recovery rate as shown in Table 1. It shows that the colorimetric / fluorescence dual-mode sensor for detecting oxytetracycline has good accuracy and the feasibility of analyzing actual samples.

[0141] Table 1. Oxytetracycline spike recovery rate in actual samples (n = 3)

[0142]

[0143] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A fluorescent nanozyme e-Cu-BN with high laccase-like activity, characterized in that: It uses copper nitrate, 2-aminoterephthalic acid, and 4,4'-bipyridine as reaction raw materials, N,N-dimethylformamide and ethanol as solvents, and prepares the precursor Cu-BN by a solvothermal method. The precursor Cu-BN is dispersed in water to obtain a Cu-BN dispersion, and then ammonia water is added for etching to obtain the fluorescent nanozyme e-Cu-BN.

2. The fluorescent nanozyme e-Cu-BN with high laccase-like activity according to claim 1, characterized in that: The molar ratio of copper nitrate to 2-aminoterephthalic acid is 0.995:1 to 1:1; the molar ratio of copper nitrate to 4,4'-bipyridine is 1:1 to 1:2, preferably 1:1.5; the volume ratio of N,N-dimethylformamide to ethanol is 1:1 to 3:1, preferably 2:

1.

3. The fluorescent nanozyme e-Cu-BN with high laccase-like activity according to claim 1, characterized in that: The reaction temperature of the solvothermal method is 120 - 160 °C, preferably 140 °C; the reaction time of the solvothermal method is 5 - 12 hours, preferably 6 hours; the mass fraction of the ammonia water is 25%; the concentration of Cu-BN in the Cu-BN dispersion is 2 mg / mL; the volume ratio of the Cu-BN dispersion to ammonia water is 10:1 to 10000:1, preferably 1000:1; the etching temperature is room temperature; the etching time is 5 - 30 minutes, preferably 15 minutes.

4. A method for detecting oxytetracycline by a highly sensitive colorimetric / fluorescent dual-mode sensor based on the fluorescent nanozyme e-Cu-BN with high laccase-like activity as described in claim 1, characterized in that: It includes the following steps: Step (a), synthesize the fluorescent nanozyme e-Cu-BN; Step (b), constructing a highly sensitive oxytetracycline colorimetric sensor: Mix an oxytetracycline solution, an e-Cu-BN solution, a 2,4-dichlorophenol solution, a 4-aminoantipyrine solution, and a buffer solution to obtain a detection system containing oxytetracycline at different concentrations, incubate the reaction to obtain a sample; measure the ultraviolet absorption curve of the sample at a wavelength of 400-650 nm, and use log 土霉素的浓度 as the abscissa and the absorbance value of the sample at 510 nm as the ordinate to establish an oxytetracycline ultraviolet standard curve; Or mix the oxytetracycline solution, e-Cu-BN solution, 2,4-dichlorophenol solution, 4-aminoantipyrine solution, and buffer solution to obtain a detection system containing different concentrations of oxytetracycline, incubate the reaction to obtain a sample; Replace the oxytetracycline solution with an equal volume of ultrapure water to obtain a blank control sample; measure the ultraviolet absorption curves of the sample and the blank control sample at wavelengths of 400-650 nm respectively, and use log 土霉素的浓度 as the abscissa and the difference in absorbance between the blank control sample and the sample at 510 nm as the ordinate to establish an oxytetracycline ultraviolet standard curve; Step (c), sample detection: Measure the absorbance value of the test sample with an unknown oxytetracycline concentration at 510 nm according to step (b), and substitute it into the oxytetracycline ultraviolet standard curve in step (b) to obtain the oxytetracycline concentration in the test sample; Or measure the absorbance values of the test sample with an unknown oxytetracycline concentration and the blank control sample at 510 nm according to step (d), obtain the absorbance difference between the blank control sample and the test sample at 510 nm, and substitute it into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the test sample; Step (d), construct a highly sensitive oxytetracycline fluorescence sensor: Mix the oxytetracycline solution, e-Cu-BN solution, and buffer solution to obtain a detection system containing different concentrations of oxytetracycline, incubate the reaction to obtain a sample; Measure the fluorescence emission curve of the sample at wavelengths of 400 - 550 nm under the excitation of 340 nm, use the oxytetracycline concentration as the abscissa and the fluorescence intensity value of the sample at 430 nm as the ordinate to establish an oxytetracycline fluorescence standard curve; Or mix the oxytetracycline solution, e-Cu-BN solution, and buffer solution to obtain a detection system containing different concentrations of oxytetracycline, incubate the reaction to obtain a sample; Replace the oxytetracycline solution with an equal volume of ultrapure water to obtain a blank control sample; Measure the fluorescence emission curves of the sample and the blank control sample at wavelengths of 400 - 550 nm under the excitation of 340 nm respectively, use the oxytetracycline concentration as the abscissa and the fluorescence intensity difference between the blank control sample and the sample at 430 nm as the ordinate to establish an oxytetracycline fluorescence standard curve; Step (e), sample detection: Measure the fluorescence intensity value of the test sample with an unknown oxytetracycline concentration at 430 nm according to step (d), and substitute it into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the test sample; Or measure the fluorescence intensity values of the test sample with an unknown oxytetracycline concentration and the blank control sample at 430 nm according to step (d), obtain the difference in fluorescence intensity between the blank control sample and the test sample at 430 nm, and substitute it into the oxytetracycline fluorescence standard curve in step (d) to obtain the oxytetracycline concentration in the test sample.

5. A method for detecting oxytetracycline by a highly sensitive colorimetric / fluorescent dual-mode sensor of a fluorescent nanozyme e-Cu-BN with high laccase-like activity according to claim 4, characterized in that: In step (b), in the detection system, the final concentration of oxytetracycline is 2.5 - 150 μM; the final concentration of e-Cu-BN is 200 - 400 μg / mL, preferably 150 μg / mL; the final concentration of 2,4-dichlorophenol is 150 - 200 μg / mL, preferably 167 μg / mL, and the final concentration of 4-aminoantipyrine is 150 - 200 μg / mL, preferably 167 μg / mL; the final volume of the detection system is 200 - 400 μL, preferably 300 μL; the buffer solution is 2-(N-morpholino)ethanesulfonic acid buffer solution with pH 3.0 - 8.0, preferably 2-(N-morpholino)ethanesulfonic acid buffer solution with pH 7.4 and 20 mM; The oxytetracycline solution is prepared with water or buffer solution; the e-Cu-BN solution is prepared with water or buffer solution; the 2,4-dichlorophenol solution is prepared with water or buffer solution; the 4-aminoantipyrine solution is prepared with water or buffer solution.

6. The method for detecting oxytetracycline by a highly sensitive colorimetric / fluorescent dual-mode sensor of the fluorescent nanozyme e-Cu-BN with high laccase-like activity according to claim 4, characterized in that: In step (b), the temperature of the incubation reaction is 20 - 70 °C, and the time of the incubation reaction is 5 - 50 min; preferably, the temperature of the incubation reaction is 35 - 40 °C, and the time of the incubation reaction is 20 - 30 min; more preferably, the temperature of the incubation reaction is 37 °C, and the time of the incubation reaction is 20 min.

7. A method for detecting oxytetracycline by a highly sensitive colorimetric / fluorescent dual-mode sensor of a fluorescent nanozyme e-Cu-BN with high laccase-like activity according to claim 4, characterized in that: In step (d), in the detection system, the final concentration of oxytetracycline is 2.5 - 150 μM; the final concentration of e-Cu-BN is 200 - 400 μg / mL, preferably 150 μg / mL; the final volume of the detection system is 200 - 400 μL, preferably 300 μL; the buffer solution is 2-(N-morpholino)ethanesulfonic acid buffer solution with pH 3.0 - 8.0, preferably 2-(N-morpholino)ethanesulfonic acid buffer solution with pH 7.4 and 20 mM; the oxytetracycline solution is prepared with water or buffer solution; the e-Cu-BN solution is prepared with water or buffer solution; the temperature of the incubation reaction is room temperature, and the time of the incubation reaction is 0.5 - 20 min, preferably 2 min.

8. A method for detecting oxytetracycline by a highly sensitive colorimetric / fluorescent dual-mode sensor of a fluorescent nanozyme e-Cu-BN with high laccase-like activity according to claim 4, characterized in that: In step (d), the temperature of the incubation reaction is room temperature, and the time of the incubation reaction is 0.5 - 20 min, preferably 2 min.

9. A method for detecting oxytetracycline by a highly sensitive colorimetric / fluorescent dual-mode sensor of a fluorescent nanozyme e-Cu-BN with high laccase-like activity according to claim 4, characterized in that: Comprising: Step (f), Visual detection based on the hydrogel platform: Dissolve agarose in 2-(N-morpholino)ethanesulfonic acid buffer by microwave heating to obtain a gel liquid; using water or buffer solution as the solvent, mix e-Cu-BN, 2,4-dichlorophenol, and 4-aminoantipyrine in a centrifuge tube, then add the gel liquid and quickly invert to obtain a hydrogel detection platform; drop equal volumes of oxytetracycline solutions with different concentrations on the hydrogel detection platform to obtain a reaction matrix, react at room temperature, establish a colorimetric card based on different oxytetracycline concentrations corresponding to different colors, use a color recognition application to record the colorimetric pictures, convert the colors into RGB values, obtain the ratio R / G of the red channel to the green channel, and establish a standard curve with the oxytetracycline concentration as the abscissa and the ratio R / G of the red channel to the green channel as the ordinate; Step (g), Sample detection: Drop the sample to be detected on the hydrogel detection platform and react at room temperature, qualitatively analyze the sample to be detected according to the colorimetric card in step (c); use a color recognition application to record the colorimetric pictures, convert the colors into RGB values, obtain the ratio R / G of the red channel to the green channel, substitute the ratio R / G of the red channel to the green channel into the standard curve in step (c) to obtain the oxytetracycline concentration in the sample to be detected.

10. A method for detecting oxytetracycline using a highly sensitive colorimetric / fluorescent dual-mode sensor based on the fluorescent nanozyme e-Cu-BN with high laccase-like activity according to claim 9, characterized in that: In step (f), the buffer solution is 2-(N-morpholino)ethanesulfonic acid buffer with a pH of 3.0 - 8.0, preferably 2-(N-morpholino)ethanesulfonic acid buffer with a pH of 7.4 and a concentration of 20 mM; In the reaction matrix, the final concentration of e-Cu-BN is 200 - 400 μg / mL, preferably 150 μg / mL; the final concentration of 2,4-dichlorophenol is 150 - 200 μg / mL, preferably 167 μg / mL; the final concentration of 4-aminoantipyrine is 150 - 200 μg / mL, preferably 167 μg / mL; The reaction time at room temperature is 5 - 50 minutes, preferably 40 minutes.

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