Method for detecting beta-lactam antibiotic residues based on laccase-like active nano-enzyme Cu-MOF dual mode

Abstract

A method for dual-mode detection of beta-lactam antibiotic residues based on laccase-like activity nano-enzyme Cu-MOF is characterized in that a copper metal organic framework (Cu-MOF) with laccase-like activity and blue fluorescence is prepared by adopting a hydrothermal method, color change is generated in the process of oxidizing o-phenylenediamine (OPD) to generate a yellow product 2, 3-diaminophenazine (DAP), and the color change is changed in the process of oxidizing o-phenylenediamine (OPD) to generate the yellow product 2, 3-diaminophenazine (DAP). And dual-mode signal output of colorimetric and ratiometric fluorescence is realized. When the BLs exist, the BLs and OPD are competitively combined with Cu-MOF active sites, generation of DAP is inhibited, a colorimetric signal is weakened, blue fluorescence is recovered, and therefore high-sensitivity and high-selectivity detection of the BLs is achieved. According to the invention, a portable on-site quantitative detection platform is established by further combining smartphone image acquisition and RGB analysis technologies, is successfully applied to rapid and accurate detection of BLs residues in animal-derived foods such as milk and pork, and has the advantages of simplicity and convenience in operation, low cost, on-site applicability and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food safety detection, and specifically relates to a method for detecting beta-lactam antibiotic residues based on laccase-like activity nanoscale enzyme Cu-MOF dual-mode. BACKGROUND

[0002] With the continuous growth of people's demand for animal-derived food, the food safety problem caused by antibiotic residues has attracted widespread attention. Among them, beta-lactam antibiotics (BLs) have become one of the widely used broad-spectrum antibiotics due to their mechanism of inhibiting the catalytic activity of bacterial transpeptidase and blocking the synthesis of bacterial cell walls to achieve antibacterial effect. However, excessive use can cause serious allergic reactions and side effects, and even lead to the formation and spread of microbial drug resistance. Therefore, it is of great significance to develop efficient and convenient analysis techniques for on-site rapid detection of BLs residues to ensure the safety of animal-derived food.

[0003] At present, the detection methods of BLs mainly include high-performance liquid chromatography, mass spectrometry and immunoassay. Although these methods have high accuracy, they have problems such as expensive equipment, complex operation, long time-consuming, etc., which are difficult to meet the needs of on-site rapid detection. In recent years, nanoscale enzymes have shown good application prospects in the field of rapid detection due to their high stability and low cost. However, existing nanoscale enzyme detection methods mostly rely on single signal output, which is easily affected by environmental interference and affects the detection accuracy. SUMMARY

[0004] The purpose of the present application is to provide a method for detecting beta-lactam antibiotic residues based on laccase-like activity nanoscale enzyme Cu-MOF dual-mode, which realizes the rapid and visual detection of BLs in animal-derived food.

[0005] To achieve the above object and other related objects, the technical scheme provided by the present application is as follows: a method for detecting beta-lactam antibiotic residues based on laccase-like activity nanoscale enzyme Cu-MOF dual-mode, comprising the following steps:

[0006] S1: Mix Cu-MOF nanoscale enzyme, o-phenylenediamine and the sample containing amoxicillin to be detected, transfer the reaction product to a UV dish after reaction, measure the absorbance value A of the reaction product at 418 nm with a UV spectrophotometer, then capture the colorimetric signal with a smart phone, and perform image RGB analysis with ImageJ software to obtain the B / G value of AMX;

[0007] S2: mixing Cu-MOF nanoszyme, o-phenylenediamine and the sample to be tested containing amoxicillin, after reaction, transferring the reaction product to a fluorescence dish, measuring the fluorescence intensity of the reaction product at 425 nm and 550 nm under 365 nm excitation wavelength by a fluorescence spectrophotometer, and obtaining the fluorescence intensity ratio F 425 nm / F 550 nm Then, the fluorescence signal is captured by using a smart phone, and image RGB analysis is performed by using ImageJ software, and the B / G value of AMX is obtained.

[0008] S3: substituting the absorbance value A and the B / G value obtained in step S1 into the colorimetric linear equation constructed based on the standard samples containing different concentrations of AMX, and respectively calculating the concentration of AMX in the sample.

[0009] S4: substituting the fluorescence intensity ratio F 425 nm / F 550 nm and the B / G value obtained in step S2 into the fluorescence linear equation constructed based on the standard samples containing different concentrations of AMX, and respectively calculating the concentration of AMX in the sample.

[0010] The preferred technical solution is that the reaction in step S1 and step S2 is mixed in HEPES buffer.

[0011] The preferred technical solution is that the construction method of the colorimetric linear equation is that, under normal temperature conditions, Cu-MOF nanoszyme, o-phenylenediamine and amoxicillin standard samples with different concentration gradients are added to HEPES buffer, and mixed by stirring, after reaction, the reaction product is transferred to an ultraviolet dish, the absorbance value A of the reaction product at 418 nm is measured by using an ultraviolet spectrophotometer, then the colorimetric signal is captured by using a smart phone, and image RGB analysis is performed by using ImageJ software, and the B / G value of AMX is obtained; the absorbance value A, the B / G value and the concentration of the standard sample measured by using the standard samples with different concentrations of AMX are used to construct a colorimetric linear regression equation A = XC AMX + y, B / G = XC AMX + y, wherein C AMX is the concentration of the standard sample.

[0012] The preferred technical solution is that the construction method of the fluorescence linear equation is that, under normal temperature conditions, Cu-MOF nanoszyme, o-phenylenediamine and amoxicillin standard samples with different concentrations are added to HEPES buffer, and mixed by stirring, after reaction for a period of time, the reaction product is transferred to a fluorescence dish, and the fluorescence intensity ratio F 425 nm / F 550 nmThen the fluorescence signal was captured using a smart phone under the irradiation of a 365 nm ultraviolet lamp, and the image RGB analysis was performed using ImageJ software to obtain the B / G value of AMX; the fluorescence intensity ratio F 425 nm / F 550 nm , B / G value and the concentration of the standard sample to construct a fluorescence linear regression equation F 425 nm / F 550 nm = XC AMX + y, B / G = XC AMX + y, wherein C AMX is the concentration of the standard sample.

[0013] Preferably, the pH value of the HEPES buffer is 8.3-8.7; the concentration of the Cu-MOF nanozyme is 18.0-22.0 μg / mL; the concentration of the o-phenylenediamine is 8.0-12.0 mM; the total volume of the mixed solution is 150.0-250.0 μL; the reaction time is 20.0-50.0 min, and the reaction temperature is 30.0-80.0 ℃.

[0014] Preferably, the colorimetric linear regression equation is A = -0.03989C AMX + 2.19926, B / G = 0.00655C AMX + 0.58509.

[0015] Preferably, the fluorescence linear regression equation is F 425 nm / F 550 nm = 0.05042C AMX +0.65818, B / G = 0.01056C AMX + 0.41305.

[0016] To achieve the above object and other related objects, the technical solutions of the present application are as follows: a preparation method of a nanozyme Cu-MOF, comprising: adding copper chloride dihydrate and 2-amino terephthalic acid into N,N-dimethylformamide to obtain solution A; at the same time, adding 2-methyl imidazole into a mixed solution composed of ethanol and N,N-dimethylformamide to obtain solution B; at room temperature, after solution A is added into solution B, the mixture is transferred into a polytetrafluoroethylene reaction kettle and heated at 120.0-150.0 °C; after cooling to room temperature, the precipitate is collected by centrifugation at a speed of 8000-12000 r / min, and then washed with water and ethanol alternately; and the nanozyme Cu-MOF is obtained by freeze-drying under vacuum.

[0017] Advantages

[0018] Compared with the prior art, the application has the advantages that:

[0019] 1、The application innovatively combines colorimetric and ratio fluorescence detection modes. Colorimetric method provides intuitive visual judgment, which is convenient for on-site preliminary screening; ratio fluorescence method effectively eliminates environmental interference through self-calibration, and has higher sensitivity. The two signals verify each other, effectively avoiding false positives / false negatives, making the detection result more accurate and reliable.

[0020] 2、The method used in the application shows high selectivity to β-lactam antibiotics and strong anti-interference ability. The detection limit is as low as 0.052 μM, which can meet the effectiveness in actual application.

[0021] 3、The method used in the application is integrated with a smart phone platform to develop a portable detection scheme, which can complete the whole process analysis from sample to result within 30.0 min, realizing the transition from complex laboratory analysis to on-site rapid and visual detection.

[0022] 4、The Cu-MOF nanometer enzyme used in the application is simple to synthesize and low in cost, and the catalytic process of the laccase-like enzyme is free of toxic hydrogen peroxide, and the byproduct is only water, which is safe and environmentally friendly. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 (A - B) are TEM images of Cu-MOF, and (C - F) are element mapping images of Cu, C, O and N elements in Cu-MOF.

[0024] Figure 2 (A) is the hydration particle size of Cu-MOF, and (B) is the infrared spectrum of Cu-MOF.

[0025] Figure 3 (A) is the different excitation wavelength spectrum of Cu-MOF, and (B) is the time stability of Cu-MOF.

[0026] Figure 4 (A) is the pH value, (B) is the temperature, (C) is the reaction time of Cu-MOF + OPD before and after the addition of AMX, and (D) is the influence of reaction time on the absorbance difference at 418 nm before and after the addition of AMX.

[0027] Figure 5 (A) is the pH value, (B) is the temperature, (C) is the reaction time of Cu-MOF + OPD before and after the addition of AMX, and (D) is the influence of reaction time on the absorbance difference at 418 nm before and after the addition of AMX.

[0028] Figure 6 The colorimetric / fluorescent standard curve containing different concentrations of amoxicillin standard, and the standard curve of RGB signal (B / G) in the corresponding mode.

[0029] Figure 7 For the selective verification of Cu-MOF detection of amoxicillin (AMX), 1-14 are blank, Zn 2+ , Mg 2+ , Glu, Gly, KAN, AK, TAP, CAP, STR, VAN, AMX, PG and AMP, respectively.

[0030] Figure 8 The detection flow chart of the present application DETAILED DESCRIPTION

[0031] The embodiments of the present application are described below through specific examples, and those skilled in the art can understand other advantages and effects of the present application from the content disclosed in the examples.

[0032] Please refer to Figures 1-8 . It should be understood that the structure, proportion, size, etc. shown in the drawings attached to the present specification are only used to cooperate with the content disclosed in the specification for understanding and reading by those skilled in the art, and do not define the limiting conditions for the implementation of the present application, so they do not have technical substantive significance. Any modification of the structure, change of the proportion relationship or adjustment of the size should be within the scope of the technical content disclosed in the present application without affecting the effects and purposes that can be achieved by the present application. The following examples are provided to better understand the present application, but not to limit the present application. The experimental materials used in the following examples are commercially available from conventional consumables and biochemical reagent stores unless otherwise specified.

[0033] Some reagents used in the embodiments of the present application:

[0034] 2-aminoterephthalic acid (NH2-BDC), o-phenylenediamine (OPD), 2,4-dichlorophenol (2,4-DP), 4-aminoantipyrine (4-AP), N,N-dimethylformamide (DMF), ampicillin (AMP), penicillin G (PG), amikacin sulfate (AK), thiamphenicol (TAP), and Amoxicillin (AMX) (Shanghai McLean Biochemical Science and Technology Co., Ltd.); 2-methylimidazole (2-MI), glutamate (Glu), glycine (Gly), trichloroacetic acid, zinc chloride dihydrate (ZnCl2·2H2O), magnesium chloride hexahydrate (MgCl2·6H2O), and CuCl2·2H2O (Shanghai Aladdin Biochemical Technology Co., Ltd.); kanamycin (KAN), chloramphenicol (CAP), streptomycin (STR), and vancomycin (VAN) (Shanghai Yuan Ye Biological Technology Co., Ltd.). The experimental water is deionized water.

[0035] Example 1: Synthesis of Cu-MOF nanozyme

[0036] A method for preparing a nanozyme Cu-MOF, the method comprising:

[0037] Preparation of Cu-MOF: copper chloride dihydrate and 2-aminoterephthalic acid were dissolved in N,N-dimethylformamide to obtain solution A; at the same time, 2-methylimidazole was dissolved in a mixed solution of ethanol and N,N-dimethylformamide (volume ratio 1:1) to obtain solution B. After slowly dropping A into B at room temperature, it was transferred into a polytetrafluoroethylene reaction kettle and heated at 140.0 °C. After cooling to room temperature, the precipitate was collected by centrifugation at a speed of 10000 r / min, washed with water and ethanol alternately for 3 times, and freeze-dried under vacuum to obtain the nanozyme Cu-MOF.

[0038] Preferably, the present embodiment discloses a method for preparing a nanozyme Cu-MOF, the method comprising:

[0039] Cu-MOF. Solution A was prepared by dissolving 58.0 mg of CuCl2·2H2O and 54.4 mg of NH2-BDC in 8.0 mL of DMF; at the same time, solution B was prepared by dissolving 24.6 mg of 2-MI in 16.0 mL of a mixture of ethanol and DMF (volume ratio 1:1). Solution A was slowly added to solution B at room temperature. After stirring at 200.0 r / min for 15.0 min, the mixture was transferred to a 50.0 mL Teflon reactor and heated at 140.0 °C for 8.0 h. After cooling to room temperature, the precipitate was collected by centrifugation at 10,000 r / min for 10.0 min, washed with water and ethanol three times alternately, and freeze-dried under vacuum to obtain fluorescent nanoscale enzyme Cu-MOF. The characterization of the synthesized Cu-MOF is shown in Figure 1 、 Figure 2 .

[0040] Example 2: Optimal excitation wavelength and stability of Cu-MOF nanoscale enzyme

[0041] With HEPES as the reaction buffer, the fluorescence intensity at a wavelength of 335-375 nm was measured by a fluorescence spectrophotometer after adding Cu-MOF to a fluorescence dish. The results are shown in Figure 3 A, indicating that the optimal excitation wavelength of the Cu-MOF nanoscale enzyme is 365 nm.

[0042] With HEPES as the buffer, a dispersion of the Cu-MOF nanoscale enzyme was prepared, and the sample was stored in the dark at 4.0 °C. At fixed time intervals (every three days), a sample was taken out, and the fluorescence intensity at 425 nm was measured at the optimal excitation wavelength (365 nm). The results are shown in Figure 3 B, indicating that the Cu-MOF has good time stability.

[0043] In the above verification steps, the concentration of the added Cu-MOF nanoscale enzyme was 20.0 μg / mL; and the pH of the added HEPES buffer was 8.3-8.7.

[0044] Example 3: Optimization of optimal conditions for Cu-MOF detection of amoxicillin (AMX)

[0045] In order to optimize the analytical performance of Cu-MOF, the pH value, temperature, and time experimental parameters in the amoxicillin (AMX) detection system were tested in detail.

[0046] The performance of the system in the pH value range of 7.0-9.5 was explored, with HEPES as the reaction buffer. The results are shown in Figure 4As shown in Figure A, when the pH value is in the range of 7.0 to 8.5, the absorbance of the reaction system at 418 nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 8.5, the absorbance decreases with further increases. Therefore, 8.5 was selected as the optimal pH value for detecting the AMX system.

[0047] The optimal temperature for this system was investigated, and HEPES was selected as the reaction buffer. The results are as follows: Figure 4 As shown in Figure B, the absorbance of the reaction system is highest at 50.0 °C as the temperature changes. Therefore, 50.0 °C was chosen as the reaction temperature for detecting the AMX system.

[0048] The reaction time of this system was investigated, and HEPES was selected as the reaction buffer. The results are as follows: Figure 4 As shown in C and D, the absorbance of the reaction system at 418 nm remained essentially unchanged after 30.0 min as time changed. Therefore, 30.0 min was chosen as the reaction time for detecting the AMX system.

[0049] Based on the pH, temperature, and time used to detect AMX, the optimal conditions for the system were selected as pH 8.5, 50.0 ℃, and 30.0 min.

[0050] Example 4: Feasibility verification of Cu-MOF detection of amoxicillin (AMX)

[0051] The following five reaction systems were designed: (1) Cu-MOF, (2) Cu-MOF + 2,4-DP, (3) Cu-MOF + 4-AP, (4) 2,4-DP + 4-AP, and (5) Cu-MOF + 2,4-DP + 4-AP. HEPES was used as the reaction buffer. After complete reaction, the mixture was transferred to a UV / fluorescent dish, and the absorbance / fluorescence intensity was measured using a UV / spectrophotometer. The results are shown in [reference needed]. Figure 5 The results indicate that AMX can inhibit the oxidation of OPD by Cu-MOF and cause a corresponding color change. This demonstrates the feasibility of the detection system proposed in this invention.

[0052] In the above verification steps, the concentration of Cu-MOF nanozyme was 20.0 μg / mL; the OPD concentration was 10.0 mM; the AMX concentration was 30.0 μM; the pH of the added HEPES buffer was 8.3-8.7; the mixing and reaction time was 30.0 min; and the reaction temperature was 50.0 ℃.

[0053] Example 5: Detection of amoxicillin standards at different concentrations using Cu-MOF dual mode

[0054] Different concentration gradients of AMX solutions (0.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, and 35.0 μM) were reacted with 20.0 μg / mL Cu-MOF nanozyme and 10.0 mM OPD in HEPES buffer for 30.0 min. After the reaction, the reaction solution was transferred to a UV-Vis dish, and the absorbance (A) at 418 nm was measured using a UV spectrophotometer. The sample was dropped onto a filter paper strip, and the fluorescence signal was captured using a smartphone. ImageJ software was used for RGB analysis to obtain the B / G value. The results showed that the absorbance at 418 nm decreased with increasing AMX concentration.

[0055] Different concentration gradients of AMX solutions (including 0.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, and 35.0 μM) were reacted with 20.0 μg / mL Cu-MOF nanozyme and 10.0 mM OPD in HEPES buffer for 30.0 min. After the reaction, the reaction solution was transferred to a fluorescence dish, and the fluorescence intensity ratio F at 425 nm and 550 nm under a 365 nm excitation wavelength was measured using a fluorescence spectrophotometer. 425 nm / F 550 nm The sample was dropped onto a filter paper strip, irradiated with a 365 nm UV lamp, and the fluorescence signal was captured using a smartphone. ImageJ software was used for RGB image analysis to obtain the B / G value. The results showed that as the concentration of AMX increased, the fluorescence intensity ratio F... 425 nm / F 550 nm Increase.

[0056] Therefore, as Figure 6 As shown, a colorimetric linear regression equation A = -0.03989C was constructed using the absorbance values, B / G values, and standard concentrations measured with AMX standards at different concentration gradients. AMX + 2.19926, B / G = 0.00655C AMX +0.58509, where C AMX At the standard concentration, the concentration of AMX showed a linear relationship with absorbance A in the range of 0.34–35.0 μM, with a detection limit of 0.101 μM. The fluorescence linear regression equation was F1. 425 nm / F 550 nm = 0.05042C AMX + 0.65818, B / G = 0.01056C AMX + 0.41305, where C AMX The concentration of standard AMX is expressed as the ratio of AMX concentration to fluorescence intensity, F. 425 nm / F 550 nmIt exhibits linearity in the range of 0.17–35.0 μM, with a detection limit of 0.052 μM.

[0057] In the above steps, the concentration of Cu-MOF nanozyme was 20.0 μg / mL; the OPD concentration was 10.0 mM; the pH of the added HEPES buffer was 8.3-8.7; the mixing and reaction time was 30.0 min; and the reaction temperature was 50.0 ℃.

[0058] Example 6: Selectivity test for AMX detection in Cu-MOF system

[0059] To evaluate the selectivity of Cu-MOF + OPD based systems for AMX, some representative contents were used as proof of concept, including blank, Zn 2+ Mg 2+ Glu, Gly, KAN, AK, TAP, CAP, STR, VAN, AMX, PG, and AMP were added. Cu-MOF nanozymes and OPD were reacted with the above substances in HEPES buffer at room temperature; after thorough mixing and reaction for 30.0 min, the mixture was transferred to a UV / fluorescent dish. The absorbance value (A) at 418 nm and the ratio (F) of fluorescence intensity at 425 nm and 550 nm under 365 nm excitation were measured. 425 nm / F 550 nm The result is as follows Figure 7 As shown, only the absorbance value A of AMX decreased significantly, and the fluorescence intensity ratio F... 425 nm / F 550 nm An increase occurred, indicating that the detection system has good selectivity for AMX.

[0060] In the above steps, the concentration of Cu-MOF nanozyme was 20.0 μg / mL; the OPD concentration was 10.0 mM; the concentration of all added substances was 35.0 μM; the pH of the HEPES buffer was 8.3-8.7; the mixing and reaction time was 30.0 min; and the reaction temperature was 50.0 ℃.

[0061] Example 7: Validation of Cu-MOF in the detection of AMX in real samples

[0062] To verify the sensing performance of Cu-MOF AMX detection in practical sample applications, pork was selected as a representative sample. The pork in this test was randomly sampled from Hefei, China. 5.0 g of pork was chopped and mixed with 15.0 mL of 5% trichloroacetic acid. After sonication for 20.0 min, the mixture was centrifuged at 8000 r / min for 5.0 min. The supernatant was filtered through a 0.22 μm microporous membrane and retained for further analysis. Then, 0.0, 15.0, and 25.0 μM AMX were added respectively. After adjusting the pH to 8.5 with HEPES buffer, the mixture was added to a 20.0 μg / mL Cu-MOF + 10.0 mmol / L OPD system and reacted at 50.0 ℃ for 30.0 min. 50.0 μL of the solution was evenly added to dry filter paper, and colorimetric / fluorescence images were captured using a smartphone. ImageJ software was used for RGB analysis to obtain the B / G value. The obtained B / G values ​​were substituted into the corresponding linear equation, and the measured AMX concentration results are shown in Table 1.

[0063] To verify the sensing performance of Cu-MOF AMX detection in practical sample applications, beef was selected as a representative sample. The beef in this test was randomly sampled from Hefei, China. 5.0 g of beef was chopped and mixed with 15.0 mL of 5% trichloroacetic acid. After sonication for 20.0 min, the mixture was centrifuged at 8000 r / min for 5.0 min. The supernatant was filtered through a 0.22 μm microporous membrane and retained for further analysis. Then, 0.0, 15.0, and 25.0 μM AMX were added respectively. After adjusting the pH to 8.5 using HEPES buffer, the mixture was added to a 20.0 μg / mL Cu-MOF + 10.0 mmol / L OPD system and reacted at 50.0 ℃ for 30.0 min. 50.0 μL of the reaction solution was evenly added to dry filter paper, and colorimetric / fluorescence images were captured using a smartphone. ImageJ software was used for RGB analysis to obtain the B / G value. The obtained B / G values ​​were substituted into the corresponding linear equation, and the measured AMX concentration results are shown in Table 1.

[0064] Table 1 shows the detection of AMX in actual samples by Cu-MOF, with the actual samples being pork and beef.

[0065]

[0066] The results show that the reaction system of the present invention can be applied to the detection of actual samples, and the recovery rate is between 94.18% and 106.42%, with a relative standard deviation (RSD) of less than 5.0%.

[0067] In summary, this invention discloses a smartphone-assisted colorimetric / fluorescence dual-mode detection platform that utilizes the laccase activity of Cu-MOF to achieve visualized detection of antibiotic residues (BLs). The Cu-MOF + OPD system exhibits concentration-dependent color and fluorescence intensity changes in BLs, which can be captured by a smartphone and converted into RGB values ​​for analysis using ImageJ software. The linear detection ranges of this method in the colorimetric and fluorescence modes are 0.34–30.0 μM and 0.17–25.0 μM, respectively, with detection limits of 0.101 μM and 0.052 μM. The application of this method in pork and beef samples validates its reliability and accuracy, and it has potential reference and application value for the development of visualized detection of antibiotic residues in food.

[0068] Example 8: A method for detecting β-lactam antibiotic residues based on Cu-MOF dual-mode laccase-like nanozymes.

[0069] A method for preparing Cu-MOF nanozymes for on-site visualization and dual-mode detection of amoxicillin (AMX) residues, the method comprising:

[0070] Preparation of Cu-MOF: Copper chloride dihydrate and 2-aminoterephthalic acid were dissolved in N,N-dimethylformamide to obtain solution A; simultaneously, 2-methylimidazole was dissolved in a mixed solution of ethanol and N,N-dimethylformamide (volume ratio 1:1) to obtain solution B. At room temperature, solution A was slowly added dropwise to solution B, and the mixture was transferred to a polytetrafluoroethylene reactor and heated at 140.0 °C. After cooling to room temperature, the precipitate was collected by centrifugation at 10000 r / min, washed three times alternately with water and ethanol, and then freeze-dried under vacuum to obtain the nanozyme Cu-MOF.

[0071] In one embodiment, the present invention discloses a method for preparing Cu-MOF nanozymes for on-site visualization and dual-mode detection of amoxicillin (AMX) residues, the method comprising:

[0072] 58.0 mg CuCl2·2H2O and 54.4 mg NH2-BDC were dissolved in 8.0 mL DMF to obtain solution A; simultaneously, 24.6 mg 2-MI was dissolved in 16.0 mL of a mixed solution of ethanol and DMF (volume ratio 1:1) to obtain solution B. At room temperature, solution A was slowly added dropwise to solution B, and the mixture was stirred at 200.0 r / min for 15.0 min. The mixture was then transferred to a 50.0 mL polytetrafluoroethylene reactor and heated at 140.0 °C for 8.0 h. After cooling to room temperature, the precipitate was collected by centrifugation at 10000 r / min for 10.0 min, washed three times alternately with water and ethanol, and then freeze-dried under vacuum to obtain the fluorescent nanozyme Cu-MOF.

[0073] In one aspect, the present invention discloses a method for on-site visual dual-mode detection of amoxicillin (AMX) residues, the method comprising the following steps:

[0074] S1: The Cu-MOF nanozyme, o-phenylenediamine (OPD) and amoxicillin (AMX) sample were mixed and reacted for a period of time. The mixture was then transferred to a UV dish and the absorbance value A of the reactants at 418 nm was measured using a UV spectrophotometer. The colorimetric signal was then captured using a smartphone and the image RGB analysis was performed using ImageJ software to obtain the B / G value of AMX.

[0075] S2: The Cu-MOF nanozyme, o-phenylenediamine (OPD), and amoxicillin (AMX) were mixed and reacted for a period of time. The mixture was then transferred to a fluorescence dish, and the ratio F of the fluorescence intensity of the reactants at 425 nm and 550 nm under a 365 nm excitation wavelength was measured using a fluorescence spectrophotometer. 425 nm / F 550 nm Then, the fluorescence signal was captured using a smartphone, and the image RGB analysis was performed using ImageJ software to obtain the B / G value of AMX;

[0076] S3: Substitute the absorbance values ​​A and B / G values ​​obtained in step S1 into the colorimetric linear equations constructed based on AMX standards with different concentrations, and calculate the AMX concentration in the sample respectively;

[0077] S4: The fluorescence intensity ratio F obtained in step S2 425 nm / F 550 nm The B / G values ​​were substituted into the fluorescence linear equations constructed based on AMX standards with different concentrations to calculate the AMX concentration in the samples.

[0078] In one embodiment, the method for constructing the colorimetric linear regression equation for amoxicillin (AMX) is as follows: Under room temperature conditions, Cu-MOF nanozyme, o-phenylenediamine (OPD), and amoxicillin (AMX) of different concentrations are added to HEPES buffer, thoroughly stirred and mixed, and after reacting for a period of time, transferred to a UV dish. The absorbance value A of the reactants at 418 nm is measured using a UV spectrophotometer. Then, the colorimetric signal is captured using a smartphone, and the image RGB analysis is performed using ImageJ software to obtain the B / G value of AMX. The colorimetric linear regression equation A = XC is constructed using the absorbance values ​​A and B / G values ​​measured from different concentrations of AMX standards and the concentration of the standards. AMX + y、B / G= XC AMX + y, where C AMX This refers to the concentration of the standard.

[0079] In one embodiment, the amoxicillin (AMX) fluorescence linear regression equation is constructed as follows: At room temperature, Cu-MOF nanozyme, o-phenylenediamine (OPD), and different concentrations of amoxicillin (AMX) are added to HEPES buffer, thoroughly mixed, and after reacting for a period of time, transferred to a fluorescence dish. The ratio F of the fluorescence intensity of the reactants at 425 nm and 550 nm under a 365 nm excitation wavelength is measured using a fluorescence spectrophotometer. 425 nm / F 550 nm Then, fluorescence signals were captured using a smartphone under 365 nm UV light irradiation, and the B / G values ​​of AMX were obtained by RGB analysis of the images using ImageJ software; the fluorescence intensity ratio F was measured using AMX standards at different concentration gradients. 425 nm / F 550 nm A fluorescence linear regression equation F was constructed using the B / G value and the concentration of the standard. 425 nm / F 550 nm = XC AMX + y、B / G= XC AMX + y, where C AMX This refers to the concentration of the standard.

[0080] In one aspect, this invention discloses a method for on-site visual dual-mode detection of amoxicillin (AMX) residues, comprising the following steps:

[0081] S1: Determination of absorbance at 418 nm for reaction systems of different concentrations of AMX standards. A: Under room temperature conditions, Cu-MOF nanozyme, o-phenylenediamine (OPD), and different concentrations of AMX were added to HEPES buffer and reacted thoroughly. The mixture was reacted at 50.0 ℃ for 30.0 min, and then transferred to a UV dish. The absorbance at 418 nm was then measured using a UV spectrophotometer and recorded as A. Subsequently, the sample was dropped onto a filter paper strip, and the colorimetric signal was captured using a smartphone. ImageJ software was used for RGB analysis of the image to obtain the B / G value of AMX.

[0082] S2: Determine the ratio F of fluorescence intensity at 425 nm and 550 nm for reaction systems of AMX standards with different concentration gradients at an excitation wavelength of 365 nm. 425 nm / F 550 nm At room temperature, Cu-MOF nanozyme, o-phenylenediamine (OPD), and different concentrations of AMX were added to HEPES buffer and reacted thoroughly. The mixture was incubated at 50.0 °C for 30.0 min, and then transferred to a fluorescence dish. The fluorescence intensity ratio F at 425 nm and 550 nm under a 365 nm excitation wavelength was then measured using a fluorescence spectrophotometer. 425 nm / F 550 nmThe value is denoted as F. The sample is then dropped onto a filter paper strip, excited by 365 nm ultraviolet light, and the fluorescence signal is captured using a smartphone. ImageJ software is then used for RGB analysis to obtain the B / G value of the AMX.

[0083] S3: Take the sample to be tested, repeat S1 to obtain the absorbance value and B / G value of the sample, substitute them into the colorimetric linear regression equation, and obtain the AMX concentration in the sample to be tested.

[0084] S4: Take the sample to be tested and repeat S2 to obtain the fluorescence intensity ratio F of the sample. 425 nm / F 550 nm The B / G value is substituted into the fluorescence linear regression equation to obtain the AMX concentration in the sample to be tested.

[0085] In one embodiment: in steps S1 and S2, the pH of the HEPES buffer is 8.3-8.7; the concentration of Cu-MOF nanozyme is 20.0 μg / mL; the OPD concentration is 10.0 Mm; the total volume of the mixed solution is 200.0 μL; the reaction time is 30.0 min; and the temperature is 50.0 ℃.

[0086] In one embodiment: In S3, the colorimetric linear regression equation is A = -0.03989C AMX + 2.19926, B / G = 0.00655C AMX + 0.58509;

[0087] In one embodiment: In S4, the fluorescence linear regression equation is F 425 nm / F 550 nm = 0.05042C AMX +0.65818, B / G = 0.01056C AMX + 0.41305.

[0088] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A method for detecting β-lactam antibiotic residues based on a Cu-MOF dual-mode laccase-like nanozyme, characterized in that: Includes the following steps: S1: The Cu-MOF nanozyme, o-phenylenediamine and the test sample containing amoxicillin were mixed. After the reaction, the reactants were transferred to a UV dish. The absorbance value A of the reactants at 418 nm was measured using a UV spectrophotometer. Then, the colorimetric signal was captured using a smartphone and the image RGB analysis was performed using ImageJ software to obtain the B / G value of AMX. S2: Cu-MOF nanozyme, o-phenylenediamine, and the test sample containing amoxicillin were mixed. After the reaction, the reactants were transferred to a fluorescence dish, and the fluorescence intensity of the reactants at 425 nm and 550 nm under a 365 nm excitation wavelength was measured using a fluorescence spectrophotometer. The fluorescence intensity ratio F was obtained. 425 nm / F 550 nm Then, the fluorescence signal was captured using a smartphone, and the image RGB analysis was performed using ImageJ software to obtain the B / G value of AMX; S3: Substitute the absorbance values ​​A and B / G values ​​obtained in step S1 into the colorimetric linear equations constructed based on AMX standards with different concentrations, and calculate the AMX concentration in the sample respectively; S4: The fluorescence intensity ratio F obtained in step S2 425 nm / F 550 nm The B / G values ​​were substituted into the fluorescence linear equations constructed based on AMX standards with different concentrations to calculate the AMX concentration in the samples.

2. The method for detecting β-lactam antibiotic residues based on Cu-MOF dual-mode laccase-like nanoenzymes according to claim 1, characterized in that: The reactions in steps S1 and S2 are mixed and reacted in HEPES buffer.

3. The method for detecting β-lactam antibiotic residues based on Cu-MOF dual-mode laccase-like nanozymes according to claim 1, characterized in that: The method for constructing the colorimetric linear equation is as follows: Under room temperature conditions, Cu-MOF nanozyme, o-phenylenediamine, and amoxicillin standards at different concentration gradients are added to HEPES buffer, stirred and mixed, and after reaction, the reactants are transferred to a UV dish. The absorbance value A of the reactants at 418 nm is measured using a UV spectrophotometer. Then, the colorimetric signal is captured using a smartphone, and the image RGB analysis is performed using ImageJ software to obtain the B / G value of AMX. The colorimetric linear regression equation A = XC is constructed using the absorbance values ​​A, B / G values ​​measured with different concentrations of AMX standards and the concentration of the standards. AMX + y、B / G=XC AMX + y, where C AMX This refers to the concentration of the standard.

4. The method for detecting β-lactam antibiotic residues based on Cu-MOF dual-mode laccase-like nanozymes according to claim 1, characterized in that: The method for constructing the fluorescence linear equation is as follows: Under room temperature conditions, Cu-MOF nanozyme, o-phenylenediamine, and amoxicillin standards of different concentrations are added to HEPES buffer, thoroughly stirred and mixed, and after reacting for a period of time, transferred to a fluorescence dish. The ratio F of the fluorescence intensity of the reactants at 425 nm and 550 nm under a 365 nm excitation wavelength is measured using a fluorescence spectrophotometer. 425 nm / F 550 nm Then, fluorescence signals were captured using a smartphone under 365 nm UV light irradiation, and the B / G values ​​of AMX were obtained by RGB analysis of the images using ImageJ software. The fluorescence intensity ratio F was measured using AMX standards at different concentration gradients. 425 nm / F 550 nm A fluorescence linear regression equation F was constructed using the B / G value and the concentration of the standard. 425 nm / F 550 nm =XC AMX + y、B / G= XC AMX + y, where C AMX This refers to the concentration of the standard.

5. The method for detecting β-lactam antibiotic residues based on Cu-MOF dual-mode laccase-like nanozymes according to any one of claims 2-4, characterized in that: The pH of the HEPES buffer solution was 8.3-8.7; the concentration of Cu-MOF nanozyme was 18.0-22.0 μg / mL; the concentration of o-phenylenediamine was 8.0-12.0 mM; the total volume of the mixed solution was 150.0-250.0 μL; the reaction time was 20.0-50.0 min; and the reaction temperature was 30.0-80.0℃.

6. The method for detecting β-lactam antibiotic residues based on Cu-MOF dual-mode laccase-like nanozymes according to claim 3, characterized in that: The colorimetric linear regression equation is A = -0.03989C. AMX + 2.19926, B / G = 0.00655C AMX + 0.58509.

7. The method for detecting β-lactam antibiotic residues based on Cu-MOF dual-mode laccase-like nanozymes according to claim 4, characterized in that: The fluorescence linear regression equation is F 425 nm / F 550 nm = 0.05042C AMX + 0.65818, B / G = 0.01056C AMX + 0.41305.

8. A method for preparing Cu-MOF nanozymes, characterized in that: include: Copper chloride dihydrate and 2-aminoterephthalic acid were added to N,N-dimethylformamide to obtain solution A; simultaneously, 2-methylimidazole was added to a mixed solution of ethanol and N,N-dimethylformamide to obtain solution B; at room temperature, solution A was added to solution B, and the mixture was transferred to a polytetrafluoroethylene reactor and heated at 120.0-150.0 °C; after cooling to room temperature, the precipitate was collected by centrifugation at 8000-12000 r / min, and washed alternately with water and ethanol; the precipitate was then freeze-dried under vacuum to obtain the nanozyme Cu-MOF.

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