Multi-antibiotic colorimetric array detection method for controlling Cu-HOF double-enzyme activity through light driving

Abstract

The invention discloses a multi-antibiotic colorimetric array detection method for controlling Cu-HOF double enzyme activity through light driving. According to the colorimetric sensing array, a four-channel sensing unit is constructed by two light response mimic enzyme activities and two illumination times (6min and 9min). Under the assistance of a neural network and linear discriminant analysis, the colorimetric sensing array can realize high-sensitivity, rapid and simultaneous detection of various antibiotics, the sensitivity and stability of the sensor are improved, the detection time is shortened, the operation steps are simplified, the detection cost is reduced, the detection range is expanded, and the reliability is high; the method has important research significance in the fields of food safety and environmental monitoring.

Description

Technical Field

[0001] This invention relates to antibiotic detection methods, specifically to a colorimetric array detection method for multiple antibiotics that uses light-driven control of Cu-HOF dual enzyme activity. Background Technology

[0002] Antibiotics are important drugs for preventing or treating bacterial infections. Due to their low cost and strong antibacterial effects, they are widely used in various fields such as human medicine, aquaculture, animal husbandry, and animal husbandry. However, with the widespread use of antibiotics, problems such as food contamination, water pollution, and bacterial resistance caused by antibiotic residues occur frequently. These antibiotic residues seriously threaten human health and the ecological environment. Long-term consumption of food containing antibiotic residues may disrupt normal metabolism and alter the microbial community, leading to hearing loss, weakened immunity, diarrhea, kidney disease, allergic reactions, and other direct threats to human safety. Furthermore, the continuous release of antibiotics into the environment leads to chemical pollution and promotes the generation and spread of antibiotic-resistant genes and antibiotic-resistant bacteria, causing long-term damage to the ecological environment and reducing the therapeutic potential of antibiotics against human and animal pathogens. Therefore, strengthening the monitoring of antibiotic residues in food and the environment is essential.

[0003] Currently, the main methods for detecting antibiotics include: 1. Chromatography. Chromatography has the advantages of high sensitivity, strong anti-interference ability, and high detection accuracy. However, due to the limitations of instrumental analysis methods, such as expensive equipment, complex operation, and time consumption, its use outside the laboratory is restricted. 2. Bio / chemical sensors. Bio / chemical sensor detection methods have shorter detection times and can be reused, but their sensitivity and anti-interference ability need to be improved. Most of these techniques can only detect one antibiotic. In addition, the signal stability of sensor detection methods based on simulated enzyme activity is easily affected by dynamic reaction processes. Since the detection of multiple antibiotics still faces challenges, it is necessary to develop new detection methods for the simultaneous, high-throughput, and stable detection of multiple antibiotics in real samples. Summary of the Invention

[0004] The purpose of this invention is to provide a colorimetric array detection method for multiple antibiotics based on light-driven control of Cu-HOF dual enzyme activity. This method overcomes the shortcomings of existing technologies and utilizes the differences among various antibiotics to achieve simultaneous detection of multiple antibiotics. This method offers advantages such as high throughput, rapid detection, high sensitivity, strong anti-interference ability, and good stability.

[0005] The technical solution of this invention is: a colorimetric array detection method for multiple antibiotics based on light-driven control of Cu-HOF dual enzyme activity. This method utilizes the light-responsive laccase and oxidase activities of Cu-HOF and the photocatalytic time to construct a colorimetric array sensor. The colorimetric array sensor is then used to detect different types and concentrations of antibiotic samples to obtain colorimetric data. Data analysis is performed, and a standard curve is plotted. Finally, the colorimetric array sensor is used for actual sample detection, and quantitative detection of multiple antibiotics is achieved through standard curve comparison.

[0006] The aforementioned detection method is performed according to the following steps:

[0007] (1) Preparation method of Cu-HOF: Take 0.18-0.22g of Pluronic F-127, 0.04-0.06g of melamine and 0.18-0.22mL of tricresylbenzene and disperse them in 15-25mL of 45-55% ethanol solution. Stir in a water bath at 50-70℃, and add 35-45mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene while stirring. After stirring for 0.4-0.6 hours, let it stand for 1.5-2.5 hours, take it out, stir at room temperature for 0.4-0.6 hours and cool. After cooling to room temperature, wash the powder 2-4 times with ultrapure water (9-11 mL each time), then wash 2-4 times with anhydrous ethanol (9-11 mL each time). After washing, dry the powder overnight in an oven at 55-65°C. The resulting powder is HOF. Disperse 95-105 mg of HOF evenly in 18-25 mL of 45-55% ethanol solution and sonicate. While sonicating, add 10 mmol L... -1 1.8-2.2 mL of copper nitrate trihydrate was sonicated for 25-35 min, then removed and washed. The mixture was first washed 2-4 times by centrifugation with ultrapure water, using 9-11 mL of ultrapure water each time, and then washed 2-4 times by centrifugation with anhydrous ethanol, using 9-11 mL of anhydrous ethanol each time. After washing, the mixture was placed in an oven at 55-65℃ and dried overnight to obtain Cu-HOF.

[0008] (2) Preparation of antibiotic standards: Tetracycline, chlortetracycline, oxytetracycline, streptomycin, kanamycin, and vancomycin were prepared into antibiotic standards with concentrations of 20 μM, 15 μM, 10 μM, 5 μM, 1 μM, and 0.5 μM respectively using ultrapure water; thus, the following six antibiotic standards with different concentrations were obtained:

[0009] 20μM oxycycline standard, 15μM oxycycline standard, 10μM oxycycline standard, 5μM oxycycline standard, 1μM oxycycline standard and 0.5μM oxycycline standard;

[0010] 20μM chlortetracycline standard, 15μM chlortetracycline standard, 10μM chlortetracycline standard, 5μM chlortetracycline standard, 1μM chlortetracycline standard and 0.5μM chlortetracycline standard;

[0011] 20μM oxytetracycline standard, 15μM oxytetracycline standard, 10μM oxytetracycline standard, 5μM oxytetracycline standard, 1μM oxytetracycline standard and 0.5μM oxytetracycline standard;

[0012] 20μM streptomycin standard, 15μM streptomycin standard, 10μM streptomycin standard, 5μM streptomycin standard, 1μM streptomycin standard and 0.5μM streptomycin standard;

[0013] 20μM kanamycin standard, 15μM kanamycin standard, 10μM kanamycin standard, 5μM kanamycin standard, 1μM kanamycin standard and 0.5μM kanamycin standard;

[0014] 20μM vancomycin standard, 15μM vancomycin standard, 10μM vancomycin standard, 5μM vancomycin standard, 1μM vancomycin standard and 0.5μM vancomycin standard;

[0015] (3) Preparation of Component 1: Take 20 μL of each of the six different concentrations of oxytetracycline standard, chlortetracycline standard, oxytetracycline standard, streptomycin standard, kanamycin standard and vancomycin standard from step (2), and react them with 0.8-1.1 mg·mL⁻¹ of each standard. -1 20 μL of Cu-HOF was mixed and incubated, and then 3.8–4.2 mg / mL was added at room temperature. -1 2,4-Dichlorophenol 9-11 μL, 3.8-4.2 mg·mL -1 9-11 μL of 4-aminoantipyrine and 120-160 μL of PBS buffer, wherein the 10 mM PBS buffer has a pH of 8, constitute 36 groups of different types and concentrations of element 1, as follows:

[0016] 20μM-Cyclocycline element 1, 15μM-Cyclocycline element 1, 10μM-Cyclocycline element 1, 5μM-Cyclocycline element 1, 1μM-Cyclocycline element 1, 0.5μM-Cyclocycline element 1;

[0017] 20μM-chlortetracycline element 1, 15μM-chlortetracycline element 1, 10μM-chlortetracycline element 1, 5μM-chlortetracycline element 1, 1μM-chlortetracycline element 1, 0.5μM-chlortetracycline element 1;

[0018] 20μM-oxytetracycline element 1, 15μM-oxytetracycline element 1, 10μM-oxytetracycline element 1, 5μM-oxytetracycline element 1, 1μM-oxytetracycline element 1, 0.5μM-oxytetracycline element 1;

[0019] 20μM streptomycin element 1, 15μM streptomycin element 1, 10μM streptomycin element 1, 5μM streptomycin element 1, 1μM streptomycin element 1, 0.5μM streptomycin element 1;

[0020] 20μM-Kanamycin element 1, 15μM-Kanamycin element 1, 10μM-Kanamycin element 1, 5μM-Kanamycin element 1, 1μM-Kanamycin element 1, 0.5μM-Kanamycin element 1;

[0021] 20μM vancomycin element 1, 15μM vancomycin element 1, 10μM vancomycin element 1, 5μM vancomycin element 1, 1μM vancomycin element 1, 0.5μM vancomycin element 1;

[0022] (4) Preparation of Component 2: Take 20 μL of each of the six different concentrations of tebuconazole standard, chlortetracycline standard, oxytetracycline standard, streptomycin standard, kanamycin standard and vancomycin standard from step (2), and first react them with 0.5-0.7 mg·mL⁻¹. -1 18-22 μL of Cu-HOF was mixed and incubated, and then 18-22 μL of 10 mM 3,3,5,5-tetramethylbenzidine and 120-160 μL of 0.1 M NaAc-HAc buffer were added at room temperature. The pH of the 0.1 M NaAc-HAc buffer was 3.5-4.5. This resulted in 36 groups of different types and concentrations of element 2, as follows:

[0023] 20 μM oxycycline element 2, 15 μM oxycycline element 2, 10 μM oxycycline element 2, 5 μM oxycycline element 2, 1 μM oxycycline element 2 and 0.5 μM oxycycline element 2;

[0024] 20μM-chlortetracycline element 2, 15μM-chlortetracycline element 2, 10μM-chlortetracycline element 2, 5μM-chlortetracycline element 2, 1μM-chlortetracycline element 2 and 0.5μM-chlortetracycline element 2;

[0025] 20 μM oxytetracycline element 2, 15 μM oxytetracycline element 2, 10 μM oxytetracycline element 2, 5 μM oxytetracycline element 2, 1 μM oxytetracycline element 2 and 0.5 μM oxytetracycline element 2;

[0026] 20 μM streptomycin element 2, 15 μM streptomycin element 2, 10 μM streptomycin element 2, 5 μM streptomycin element 2, 1 μM streptomycin element 2 and 0.5 μM streptomycin element 2;

[0027] 20 μM kanamycin element 2, 15 μM kanamycin element 2, 10 μM kanamycin element 2, 5 μM kanamycin element 2, 1 μM kanamycin element 2 and 0.5 μM kanamycin element 2;

[0028] 20 μM vancomycin element 2, 15 μM vancomycin element 2, 10 μM vancomycin element 2, 5 μM vancomycin element 2, 1 μM vancomycin element 2 and 0.5 μM vancomycin element 2;

[0029] (5) Construction of colorimetric array sensor: The 36 different types and concentrations of element 1 in step (3) and the 36 different types and concentrations of element 2 in step (4) are irradiated under blue LED light respectively. The reaction signals at 6 minutes and 9 minutes of irradiation are taken to complete the construction of colorimetric array sensor and obtain 4 signal sensing units with 2 elements × 2 light irradiation times for different concentrations and types of antibiotic standards.

[0030] (6) Plotting the standard curve: Neural network analysis and linear discriminant analysis were performed on different types and concentrations of antibiotics, and the data of their first influencing factor were analyzed and processed to obtain the linear relationship equation between concentration and Factor 1, and the standard curve was plotted.

[0031] (7) Sample testing: The actual samples were tested using the spiked recovery method.

[0032] In step (1) above, the preparation method of Cu-HOF is as follows: 0.2g of Pluronic F-127, 0.05g of melamine and 0.2mL of thiol are dispersed in 20mL of 50% ethanol solution and stirred in a water bath at 60℃. While stirring, 40mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene is added. After stirring for 0.5 hours, it is left to stand for 2.0 hours, taken out, stirred at room temperature for 0.5 hours and cooled to room temperature. It is first washed three times with ultrapure water by centrifugation, with 10mL of ultrapure water used each time, and then washed three times with anhydrous ethanol by centrifugation, with 10mL of anhydrous ethanol used each time. After washing, it is placed in an oven at 60℃ and dried overnight. The powder obtained is HOF. 100mg of HOF is uniformly dispersed in 20mL of 50% ethanol solution and sonicated. 10mmol L is added under sonication. -12 mL of copper nitrate trihydrate was sonicated for 30 min, then removed and washed. The mixture was first washed three times by centrifugation with 10 mL of ultrapure water each time, and then washed three times by centrifugation with 10 mL of anhydrous ethanol each time. After washing, the mixture was placed in an oven at 60 °C and dried overnight to obtain Cu-HOF.

[0033] In step (3) above, the preparation of element 1 is as follows: 20 μL of each of the six different concentrations of oxytetracycline standard, chlortetracycline standard, oxytetracycline standard, streptomycin standard, kanamycin standard and vancomycin standard from step (2) are taken and reacted with 0.9 mg·mL⁻¹ of chlortetracycline standard. -1 20 μL of Cu-HOF was mixed and incubated at 40 °C for 30 minutes, and then 4 mg·mL⁻¹ was added to each solution at room temperature. -1 10 μL, 4 mg·mL of 2,4-dichlorophenol -1 10 μL of 4-aminoantipyrine and 140 μL of PBS buffer, wherein the 10 μL PBS buffer has a pH of 8, constitute 36 groups of different types and concentrations of element 1.

[0034] In step (4) above, the preparation of element 2 involves taking 20 μL of each of the six different concentrations of tebuconazole, chlortetracycline, oxytetracycline, streptomycin, kanamycin, and vancomycin standards from step (2), and first reacting them with 0.6 mg·mL⁻¹ of chlortetracycline. -1 20 μL of Cu-HOF was mixed and incubated at 40 °C for 30 minutes. Then, 20 μL of 10 mM 3,3,5,5-tetramethylbenzidine and 140 μL of 0.1 M NaAc-HAc buffer were added at room temperature. The pH of the 0.1 M NaAc-HAc buffer was 4. This resulted in 36 groups of elements with different types and concentrations.

[0035] In the aforementioned step (5), the absorption wavelength of the acquisition signal of element 1 is 503nm, and the absorption wavelengths of the acquisition signal of element 2 are 357nm and 658nm.

[0036] In step (7) above, the actual sample includes river water, honey, chicken or milk.

[0037] In step (7) above, the absorption wavelengths of the acquisition signals used to detect the actual sample are 503nm, 357nm and 658nm.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] This invention constructs a light-driven Cu-HOF colorimetric array sensor that uses photoresponsive laccase and oxidase activities to control the catalytic reaction process. This enables highly sensitive and rapid simultaneous detection of multiple antibiotics, improves the sensor's sensitivity and stability, shortens detection time, simplifies operation steps, reduces detection costs, expands the detection range, and has high reliability. It has significant research value in the fields of food safety and environmental monitoring.

[0040] The working principle of this invention is as follows: Cu-HOF possesses photoresponsive laccase and oxidase activities. Under blue light irradiation, Cu-HOF catalyzes the oxidation of the substrate 2,4-DP to generate colorless quinone compounds. These colorless quinone compounds further combine with 4-AAP to generate a red product, causing the sensing solution to appear red. Under blue light irradiation, Cu-HOF catalyzes the oxidation of the substrate TMB to generate blue and yellow products, causing the sensing solution to appear green. Upon addition of the target antibiotic, the photoresponsive laccase activity of Cu-HOF decreases while the oxidase activity increases, resulting in a lighter red and a deeper green in the reaction solution. By utilizing the differences in antibiotic activity to construct a colorimetric sensor array, and combining it with a neural network model and LDA analysis, the detection of multiple antibiotics and their mixtures can be achieved.

[0041] In summary, traditional bio / chemical sensing technologies can only detect a single analyte. This invention enables the simultaneous detection of multiple antibiotics and their mixtures, optimizes the operation steps, shortens the detection time, reduces experimental costs, improves sensitivity and stability, expands the detection range, and yields more accurate and reliable experimental results. It can be applied to the rapid detection of antibiotics in actual food and water samples. Attached Figure Description

[0042] Figure 1 Schematic diagram of the feasibility of using Cu-HOF photoresponsive enzyme mimicry to detect antibiotics (A shows Cu-HOF with photoresponsive laccase activity; B shows Cu-HOF with photoresponsive oxidase activity; C shows the response of different antibiotics to the photoresponsive enzyme mimicry activity of Cu-HOF).

[0043] Figure 2 A light-driven Cu-HOF colorimetric array sensor that uses photoresponsive laccase and oxidase activities to control the catalytic reaction process;

[0044] Figure 3 LDA graphs of different concentrations of antibiotics (A = 0.5 μM; B = 10 μM; C = 20 μM);

[0045] Figure 4: 20μM antibiotic mixture LDA (A is different ratios of CTC:KAN (0:4, 1:3, 2:2, 3:1, 4:0); B is different ratios of OTC:KAN (0:4, 1:3, 2:2, 3:1, 4:0); C is an elemental antibiotic and an equimolar mixture of antibiotics);

[0046] Figure 5 : Schematic diagram of a neural network model (the output layer has 4 neurons, there is 1 hidden layer with 20 neurons, and the output layer has 46 neurons);

[0047] Figure 6 Linear relationship between different antibiotic concentrations and Factor 1 (A: Doxycycline (DOX); B: Chlortetracycline (CTC); C: Oxytetracycline (OTC); D: Streptomycin (STR); E: Kanamycin (KAN); F: Vancomycin (VAN));

[0048] Figure 7 LDA plot of antibiotics and interfering substances, where interfering substances include aspartic acid (Asp), glutamate (Glu), glucose, and metal salt ions (Na). + K + and Cl - ) and penicillin G (PNG) and amoxicillin (AMX);

[0049] Figure 8 LDA graphs of different antibiotics in actual samples (A is honey; B is river water; C is chicken; D is milk). Detailed Implementation

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0051] Unless otherwise specified, all reagents used in the following examples are commercially available.

[0052] Example 1: Preparation method of Cu-HOF

[0053] Disperse 0.2 g Pluronic F-127 (poloxam 407), 0.05 g melamine, and 0.2 mL tricresylbenzene in 20 mL of ethanol solution (V 水 :V 乙醇In a 1:1 ratio, 40 mg of H4TBAPy (1,3,6,8-tetrakis(4-carboxyphenyl)pyrene) was added while stirring in a 60°C water bath. After stirring for 0.5 hours, the mixture was allowed to stand for 2.0 hours. The mixture was then removed, stirred at room temperature for 0.5 hours, and cooled to room temperature. It was first washed three times with 10 mL of ultrapure water by centrifugation, and then washed three times with 10 mL of anhydrous ethanol by centrifugation. After washing, the mixture was dried overnight in a 60°C oven. The resulting powder was HOF. 100 mg of HOF was uniformly dispersed in 20 mL of 50% ethanol solution and sonicated. 10 mmol L⁻¹ was added while sonicating. -1 2 mL of copper nitrate trihydrate was sonicated for 30 min, then removed and washed. The mixture was first washed three times by centrifugation with 10 mL of ultrapure water each time, and then washed three times by centrifugation with 10 mL of anhydrous ethanol each time. After washing, the mixture was placed in an oven at 60 °C and dried overnight to obtain Cu-HOF.

[0054] Example 2: Feasibility of using Cu-HOF photoresponsive enzyme mimicry for antibiotic detection

[0055] Cu-HOF exhibits photoresponsive laccase and oxidase activities. It can only catalyze 2,4-DP (2,4-dichlorophenol) and TMB (3,3,5,5-tetramethylbenzidine) under blue light, showing characteristic peak signals at 503 nm, 367 nm, and 658 nm. Different antibiotics produce different signals in response to the photoresponsive laccase and oxidase activities of Cu-HOF, see [link to relevant documentation]. Figure 1 .

[0056] Example 3: Preparation of antibiotic standards

[0057] Antibiotic standards of 20 μM, 15 μM, 10 μM, 5 μM, 1 μM, and 0.5 μM were prepared using ultrapure water from oxytetracycline, chlortetracycline, oxytetracycline, streptomycin, kanamycin, and vancomycin, respectively; thus, the following six antibiotic standards of different concentrations were obtained:

[0058] 20μM oxycycline standard, 15μM oxycycline standard, 10μM oxycycline standard, 5μM oxycycline standard, 1μM oxycycline standard and 0.5μM oxycycline standard;

[0059] 20μM chlortetracycline standard, 15μM chlortetracycline standard, 10μM chlortetracycline standard, 5μM chlortetracycline standard, 1μM chlortetracycline standard and 0.5μM chlortetracycline standard;

[0060] 20μM oxytetracycline standard, 15μM oxytetracycline standard, 10μM oxytetracycline standard, 5μM oxytetracycline standard, 1μM oxytetracycline standard and 0.5μM oxytetracycline standard;

[0061] 20μM streptomycin standard, 15μM streptomycin standard, 10μM streptomycin standard, 5μM streptomycin standard, 1μM streptomycin standard and 0.5μM streptomycin standard;

[0062] 20μM kanamycin standard, 15μM kanamycin standard, 10μM kanamycin standard, 5μM kanamycin standard, 1μM kanamycin standard and 0.5μM kanamycin standard;

[0063] 20μM vancomycin standard, 15μM vancomycin standard, 10μM vancomycin standard, 5μM vancomycin standard, 1μM vancomycin standard and 0.5μM vancomycin standard;

[0064] Example 4: Sensing Condition Optimization

[0065] Let A be the sensing signal for the Cu-HOF light-responsive laccase and oxidase activities. 元件1 and A 元件2 ,in:

[0066] A 元件1 =(A1-A0) / A0

[0067] In the formula A 元件1 A1 represents the absorbance of the control sample at 503 nm; A0 represents the absorbance of the blank sample at 503 nm.

[0068] A 元件2 = (A3-A2) / A2+(A5-A4) / A4

[0069] In the formula A 元件2 A3 represents the absorbance of the control sample at 367 nm; A2 represents the absorbance of the blank sample at 367 nm; A5 represents the absorbance of the control sample at 658 nm; A4 represents the absorbance of the blank sample at 658 nm.

[0070] To ensure sensor signal A 元件1 and A 元件2 To achieve optimal catalytic performance, the reaction conditions were optimized using a controlled variable method. A 元件1 Experimental conditions included the pH of the PBS buffer (6.5–8.5) and the concentration of Cu-HOF (60–100 μg / mL). -1 The incubation time (10–50 minutes) and light exposure time (6–10 minutes) are also considered. Experimental conditions for element A2 include the pH of the NaAc-HAc buffer (3–7) and the Cu-HOF concentration (60–100 μg / mL). -1), incubation time (10-50 minutes), and light exposure time (6-10 minutes).

[0071] (1)A 元件1 and A 元件2 The optimal pH value for the reaction was investigated by changing the pH of the buffer solution (from 6.5 to 8.5). 元件1 First, select a Cu-HOF concentration of 100 μg·mL. -1 Add 10 μM KAN and incubate for 30 minutes at 40°C. Then add 2,4-DP at a concentration of 200 μg / mL. -1 The concentration of 4-AAP was 200 μg·mL. - The reaction was carried out at room temperature and exposed to light for 10 minutes. The pH of the buffer solution was changed (from 6.5 to 8.5) to explore the optimal pH value for the reaction.

[0072] A 元件2 The concentration of Cu-HOF was chosen to be 100 μg·mL. -1 The TMB concentration is 1 mM and the CTC concentration is 10 μM. Also, first take 100 μg / mL. -1 Cu-HOF was incubated with 10 μM CTC for 30 minutes at 40 °C. Then, 1 mM TMB and NaAc-HAc buffer were added at room temperature and the reaction was carried out under light for 10 minutes. The pH of the buffer solution was changed (3-7) to explore the optimal pH value for the reaction.

[0073] Add 20 μL KAN and 20 μL Cu-HOF to the wells of the enzyme-labeled product, incubate at 40°C for 30 minutes, then add 10 μL 2,4-DP, 10 μL 4-AAP, and 140 μL PBS buffer. Incubate under light at room temperature for 10 minutes, then measure the absorbance using a full-wavelength microplate reader to calculate A. 元件1 ;

[0074] Add 20 μL CTC and 20 μL Cu-HOF to the wells of the enzyme-labeled enzyme, incubate at 40°C for 30 minutes, then add 20 μL TMB and 140 μL NaAc-HAc buffer and react under light at room temperature for 10 minutes. Measure the absorbance using a full-wavelength microplate reader to calculate A. 元件2 Data analysis revealed that when the pH of the buffer solution was 8, A... 元件1 The value is highest when the pH of the buffer solution is 4. 元件2 The value is the largest, therefore the sensing effect is best under this condition.

[0075] (2)A 元件1 and A 元件2 Change the Cu-HOF concentration (60–100 μg·mL) -1 Investigating the optimal Cu-HOF concentration for the reaction

[0076] A 元件1 The incubation time for Cu-HOF and 10 μM KAN was 30 minutes at 40 °C. The PBS buffer solution was selected with a pH of 8 and a 2,4-DP (2,4-dichlorophenol) concentration of 200 μg / mL. -1 The concentration of 4-AAP (4-aminoantipyrine) was 200 μg·mL. -1 React at room temperature, then under light for 10 minutes;

[0077] A 元件2 The incubation time for Cu-HOF and 10 μM CTC was 30 minutes, and the incubation temperature was 40 °C. The pH of the NaAc-HAc buffer solution was 4, the TMB concentration was 1 mM, and the reaction was carried out at room temperature with light irradiation for 10 minutes.

[0078] Add 20 μL KAN and 20 μL Cu-HOF to the wells of the enzyme-labeled product, incubate at 40°C for 30 minutes, then add 10 μL 2,4-DP, 10 μL 4-AAP, and 140 μL PBS buffer. Incubate under light at room temperature for 10 minutes, then measure the absorbance using a full-wavelength microplate reader to calculate A. 元件1 ;

[0079] Add 20 μL CTC and 20 μL Cu-HOF to the wells of the enzyme-labeled microplate, incubate at 40°C for 30 minutes, then add 20 μL TMB and 140 μL NaAc-HAc buffer and irradiate under light at room temperature for 10 minutes. Measure the absorbance using a full-wavelength microplate reader to calculate A. 元件2 Analysis of the data showed that when the Cu-HOF concentration was 90 μg·mL... -1 Time A 元件1 The value is highest when the Cu-HOF concentration is 60 μg·mL. -1 Time A 元件2 The value is the largest, therefore the sensing effect is best under this condition.

[0080] (3)A 元件1 and A 元件2 To explore the optimal incubation time for the response, the incubation period was varied (10–50 minutes).

[0081] A 元件1 The Cu-HOF concentration was selected as 90 μg·mL. -1 Add 10 μM KAN, incubate at 40 °C, and then select PBS buffer solution with pH 8 and 2,4-DP concentration of 200 μg·mL⁻¹. -1 The concentration of 4-AAP was 200 μg·mL. -1The reaction was carried out at room temperature and under light for 10 minutes. The incubation time was changed (10, 20, 30, 40, 50 minutes) to explore the optimal incubation time.

[0082] A 元件2 The Cu-HOF concentration was selected as 60 μg·mL. -1 Add 10 μM CTC, incubate at 40 °C, with the NaAc-HAc buffer solution at pH 4 and the Cu-HOF concentration at 60 μg·mL. -1 The optimal incubation time for the reaction was investigated by changing the incubation time (10, 20, 30, 40, and 50 minutes) with a TMB concentration of 1 mM, a reaction temperature of room temperature, and light exposure for 10 minutes.

[0083] Add 20 μL KAN and 20 μL Cu-HOF to the wells of the enzyme-labeled product, and incubate at 40°C for 10, 20, 30, 40, and 50 minutes, respectively. Then add 10 μL 2,4-DP, 10 μL 4-AAP, and 140 μL PBS buffer. Incubate under light at room temperature for 10 minutes, and then measure the absorbance using a full-wavelength microplate reader to calculate A. 元件1 ;

[0084] Add 20 μL CTC and 20 μL Cu-HOF to the wells of the enzyme-labeled enzyme, and incubate at 40 °C for 10, 20, 30, 40, and 50 minutes, respectively. Then add 20 μL TMB and 140 μL NaAc-HAc buffer and irradiate under light at room temperature for 10 minutes. Measure the absorbance using a full-wavelength microplate reader to calculate A. 元件2 Data analysis revealed that when the incubation time was 40 minutes, A... 元件1 The value is highest when the incubation time is 30 minutes. 元件2 To maximize the value and improve sensing efficiency, an incubation time of 30 minutes was chosen as A. 元件1 A 元件2 Incubation time.

[0085] (4)A 元件1 and A 元件2 To investigate the optimal light exposure time for the reaction, the light exposure time was varied from 6 to 10 minutes.

[0086] A 元件1 The Cu-HOF concentration was selected as 90 μg·mL. -1 Add 10 μM KAN and incubate for 30 minutes at 40 °C. Then, use PBS buffer solution with a pH of 8 and a 2,4-DP concentration of 200 μg / mL. -1 The concentration of 4-AAP was 200 μg·mL. -1 The reaction temperature was room temperature. The reaction time was varied by 6-10 minutes to explore the optimal light exposure time.

[0087] A 元件2 The Cu-HOF concentration was selected as 60 μg·mL. -1 Add 10 μM CTC and incubate for 30 minutes at 40°C. Then, select NaAc-HAc buffer solution with pH 4, TMB concentration of 1 mM, and reaction temperature at room temperature. Change the light exposure time (6-10 minutes) to explore the optimal light exposure time for the reaction.

[0088] Add 20 μL KAN and 20 μL Cu-HOF to the wells of the enzyme-labeled product, incubate at 40°C for 30 minutes, then add 10 μL 2,4-DP, 10 μL 4-AAP, and 140 μL PBS buffer. Incubate under light for 6, 7, 8, 9, and 10 minutes at room temperature, respectively. Measure the absorbance using a full-wavelength microplate reader to calculate A. 元件1 ;

[0089] Add 20 μL CTC and 20 μL Cu-HOF to the wells of the enzyme-labeled product, incubate at 40°C for 30 minutes, then add 20 μL TMB and 140 μL NaAc-HAc buffer. Incubate under light at room temperature for 6, 7, 8, 9, and 10 minutes. Measure the absorbance using a full-wavelength microplate reader to calculate A. 元件2 Data analysis revealed that when the illumination time was 9 minutes, A... 元件1 The value is maximum when the illumination time is 6 minutes. 元件2 The value is the largest, therefore, 6 minutes and 9 minutes of illumination time are chosen as A. 元件1 A 元件2 The illumination time, denoted as A1 for the four signal sensing units. 6min A1 9min A2 6min A2 9min .

[0090] Example 5: Confirmation of the fabrication methods for sensing element 1 and sensing element 2

[0091] Preparation of Component 1: Take 20 μL of each of the six different concentrations of oxytetracycline standard, chlortetracycline standard, oxytetracycline standard, streptomycin standard, kanamycin standard and vancomycin standard from Example 3, and react them with 0.9 mg·mL⁻¹ of each standard. -1 20 μL of Cu-HOF was mixed and incubated at 40 °C for 30 minutes, and then 4 mg·mL⁻¹ was added to each solution at room temperature. -1 2,4-DP 10 μL, 4 mg·mL -1 10 μL of 4-AAP and 140 μL of PBS buffer, wherein the 10 mM PBS buffer has a pH of 8, constitute 36 groups of different types and concentrations of element 1, as follows:

[0092] 20μM-Cyclocycline element 1, 15μM-Cyclocycline element 1, 10μM-Cyclocycline element 1, 5μM-Cyclocycline element 1, 1μM-Cyclocycline element 1, 0.5μM-Cyclocycline element 1;

[0093] 20μM-chlortetracycline element 1, 15μM-chlortetracycline element 1, 10μM-chlortetracycline element 1, 5μM-chlortetracycline element 1, 1μM-chlortetracycline element 1, 0.5μM-chlortetracycline element 1;

[0094] 20μM-oxytetracycline element 1, 15μM-oxytetracycline element 1, 10μM-oxytetracycline element 1, 5μM-oxytetracycline element 1, 1μM-oxytetracycline element 1, 0.5μM-oxytetracycline element 1;

[0095] 20μM streptomycin element 1, 15μM streptomycin element 1, 10μM streptomycin element 1, 5μM streptomycin element 1, 1μM streptomycin element 1, 0.5μM streptomycin element 1;

[0096] 20μM-Kanamycin element 1, 15μM-Kanamycin element 1, 10μM-Kanamycin element 1, 5μM-Kanamycin element 1, 1μM-Kanamycin element 1, 0.5μM-Kanamycin element 1;

[0097] 20μM vancomycin element 1, 15μM vancomycin element 1, 10μM vancomycin element 1, 5μM vancomycin element 1, 1μM vancomycin element 1, 0.5μM vancomycin element 1;

[0098] (4) Preparation of Component 2: Take 20 μL of each of the six different concentrations of tebuconazole standard, chlortetracycline standard, oxytetracycline standard, streptomycin standard, kanamycin standard and vancomycin standard from Example 3, and first react them with 0.6 mg·mL⁻¹. -1 20 μL of Cu-HOF was mixed and incubated at 40 °C for 30 minutes. Then, 20 μL of 10 mM 3,3,5,5-tetramethylbenzidine and 140 μL of 0.1 M NaAc-HAc buffer (pH = 4) were added at room temperature to form 36 different types and concentrations of element 2, as follows:

[0099] 20 μM oxycycline element 2, 15 μM oxycycline element 2, 10 μM oxycycline element 2, 5 μM oxycycline element 2, 1 μM oxycycline element 2 and 0.5 μM oxycycline element 2;

[0100] 20μM-chlortetracycline element 2, 15μM-chlortetracycline element 2, 10μM-chlortetracycline element 2, 5μM-chlortetracycline element 2, 1μM-chlortetracycline element 2 and 0.5μM-chlortetracycline element 2;

[0101] 20 μM oxytetracycline element 2, 15 μM oxytetracycline element 2, 10 μM oxytetracycline element 2, 5 μM oxytetracycline element 2, 1 μM oxytetracycline element 2 and 0.5 μM oxytetracycline element 2;

[0102] 20 μM streptomycin element 2, 15 μM streptomycin element 2, 10 μM streptomycin element 2, 5 μM streptomycin element 2, 1 μM streptomycin element 2 and 0.5 μM streptomycin element 2;

[0103] 20 μM kanamycin element 2, 15 μM kanamycin element 2, 10 μM kanamycin element 2, 5 μM kanamycin element 2, 1 μM kanamycin element 2 and 0.5 μM kanamycin element 2;

[0104] 20 μM vancomycin element 2, 15 μM vancomycin element 2, 10 μM vancomycin element 2, 5 μM vancomycin element 2, 1 μM vancomycin element 2 and 0.5 μM vancomycin element 2;

[0105] Example 6: Construction of a colorimetric sensor array

[0106] A four-channel signal sensing unit colorimetric sensing array was constructed using the photoresponsive laccase and oxidase activities of Cu-HOF and two illumination times. Figure 2 This colorimetric sensor array consists of four signal sensing units, comprising two mimic enzymes and two light exposure times. It can effectively identify antibiotics at different concentration levels. Figure 3 ).

[0107] The absorption wavelength of the acquisition signal of component 1 is 503nm, and the absorption wavelengths of the acquisition signal of component 2 are 357nm and 658nm.

[0108] To further verify the recognition performance of the colorimetric sensor array, an experiment was conducted on antibiotic mixtures. The total concentration of antibiotics was maintained at 20 μM, and the constructed colorimetric sensor array was used to distinguish between binary and ternary antibiotic mixtures. First, mixtures containing two antibiotics in different proportions were identified, and the results are as follows: Figure 4 As shown in AB, these antibiotic mixtures with different proportions were well classified. Then, mixtures of six antibiotics with the same molar ratio, along with their individual elements, were tested; the 12 samples were well classified and did not overlap. Figure 4C). The above results demonstrate that the constructed colorimetric sensor array can accurately identify complex antibiotic mixtures.

[0109] Subsequently, an artificial neural network was used to classify and identify 46 different levels of antibiotics and their mixtures. First, a model with one hidden layer and 20 nodes was built. Then, the dataset was divided into 70% training data and 30% test data for model training. Figure 5 (The meaning of the numerical designations is shown in Table 1). The training accuracy of this artificial neural network model is 99.4%, and the testing accuracy is 97.1%, demonstrating good recognition performance. Combining the constructed colorimetric array with the neural network may hopefully help identify unknown antibiotics and their mixtures.

[0110] Example 7: Plotting Standard Curves

[0111] The collected data matrix (2 response signals × 2 illumination periods × 6 antibiotic concentrations × 5 replicates) was processed using LDA to obtain Factor1 as the ordinate and antibiotic concentration as the abscissa to plot a standard curve. A regression equation was then established, as follows: Figure 6 As shown. The regression equations are as follows: When the concentration of doxycycline is 0.5 μM ≤ x ≤ 20 μM, the regression equation for the relationship between its concentration and the change in Factor1 is: y = 7.16191x - 58.40555; When the concentration of chlortetracycline is 0.5 μM ≤ x ≤ 20 μM, the regression equation for the relationship between its concentration and the change in Factor1 is: y = 6.54497x - 57.82932; When the concentration of oxy ...7.16191x - 58.40555; When M≤x≤20μM, the regression equation for the relationship between its concentration and the change in Factor1 is: y=5.54946x-48.30462; when the streptomycin concentration is 0.5μM≤x≤20μM, the regression equation for the relationship between its concentration and the change in Factor1 is: y=3.04054x-25.56352; when the kanamycin concentration is 0.5μM≤x≤20μM, the regression equation for the relationship between its concentration and the change in Factor1 is: y=-4.62399x+41.5507; when the vancomycin concentration is 0.5μM≤x≤20μM, the regression equation for the relationship between its concentration and the change in Factor1 is: y=2.90196x-24.05973.

[0112] The linear range of all six antibiotics was 0.5-20 μM. The limits of detection (LOD) for doxycycline were 2.454 nM, for chlortetracycline 2.681 nM, for oxytetracycline 3.162 nM, for streptomycin 5.772 nM, for kanamycin 3.795 nM, and for vancomycin 6.048 nM. This indicates that the invention has the characteristics of good stability and high sensitivity.

[0113] Example 6: Anti-interference capability of colorimetric sensor array

[0114] To verify the anti-interference performance of the colorimetric sensor array, several common coexisting interfering substances in food were identified, including aspartic acid (Asp), glutamic acid (Glu), glucose, and metal salt ions (Na). + K + and Cl - The target antibiotics, including penicillin G (PNG) and amoxicillin (AMX), were well clustered into 6 groups and separated from each other. Interference substances were classified as a blank group, indicating that the identification of antibiotics is not affected by other substances, thus verifying the feasibility of using the sensor array for antibiotic identification in complex environments. Figure 7 ).

[0115] Example 7: Actual Sample Detection

[0116] The spiked recovery method was used to test the actual samples. Six 1 mM·kg⁻¹ spiking solutions were added to samples of honey, river water, chicken, and milk, respectively. -1 Antibiotics (doxycycline (DOX), chlortetracycline (CTC), oxytetracycline (OTC), streptomycin (STR), kanamycin (KAN), vancomycin (VAN)) were used. The absorbance of the test samples and blank samples was measured after 6 min and 9 min of illumination. The absorption wavelengths for the detection were 503 nm, 357 nm, and 658 nm. A1 was calculated. 6min A1 9min A2 6min A2 9min These values ​​are then fed into a neural network model to determine the antibiotic type. LDA processing is used to obtain Factor 1, which is then used in a linear regression equation of antibiotic concentration and the change in Factor 1 to obtain the type and concentration of antibiotics in the sample.

[0117] The constructed colorimetric sensor array exhibits high reliability and can be used for honey ( Figure 8 A) River water ( Figure 8 B), Chicken ( Figure 8 C) and milk ( Figure 8 D) Detection of antibiotics in the sample.

[0118] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

[0119] Table 1. 46 classes of antibiotics and mixtures

[0120]

[0121]

Claims

1. A method for detecting multiple antibiotics using a colorimetric array based on light-driven control of Cu-HOF dual enzyme activity, characterized in that: A colorimetric array sensor was constructed using the photoresponsive laccase and oxidase activities and photocatalytic time of Cu-HOF. The colorimetric array sensor was then used to detect different types and concentrations of antibiotic samples to obtain colorimetric data, which were then analyzed to plot a standard curve. Finally, the colorimetric array sensor was used for actual sample detection, and quantitative detection of multiple antibiotics was achieved by comparing with the standard curve.

2. The method for detecting multiple antibiotics using a colorimetric array based on light-driven control of Cu-HOF dual enzyme activity according to claim 1, characterized in that: The detection method is performed according to the following steps: (1) Preparation method of Cu-HOF: Take 0.18-0.22g of Pluronic F-127, 0.04-0.06g of melamine and 0.18-0.22mL of thiol and disperse them in 15-25mL of 45-55% ethanol solution. Stir in a water bath at 50-70°C, and add 35-45mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene while stirring. After stirring for 0.4-0.6 hours, let it stand for 1.5-2.5 hours, take it out, stir at room temperature for 0.4-0.6 hours and cool. After cooling to room temperature, wash the powder 2-4 times with ultrapure water (9-11 mL each time), then wash 2-4 times with anhydrous ethanol (9-11 mL each time). After washing, dry the powder overnight in an oven at 55-65°C. The resulting powder is HOF. Disperse 95-105 mg of HOF evenly in 18-25 mL of 45-55% ethanol solution and sonicate. While sonicating, add 10 mmol L... −1 1.8-2.2 mL of copper nitrate trihydrate was sonicated for 25-35 min, then removed and washed. The mixture was first washed 2-4 times by centrifugation with ultrapure water, using 9-11 mL of ultrapure water each time, and then washed 2-4 times by centrifugation with anhydrous ethanol, using 9-11 mL of anhydrous ethanol each time. After washing, the mixture was placed in an oven at 55-65°C and dried overnight to obtain Cu-HOF. (2) Preparation of antibiotic standards: Tetracycline, chlortetracycline, oxytetracycline, streptomycin, kanamycin and vancomycin were prepared into antibiotic standards with concentrations of 20µM, 15µM, 10µM, 5µM, 1µM and 0.5µM respectively using ultrapure water; thus, the following 6 different concentrations of antibiotic standards were obtained: 20µM oxycycline standard, 15µM oxycycline standard, 10µM oxycycline standard, 5µM oxycycline standard, 1µM oxycycline standard and 0.5µM oxycycline standard; 20µM chlortetracycline standard, 15µM chlortetracycline standard, 10µM chlortetracycline standard, 5µM chlortetracycline standard, 1µM chlortetracycline standard and 0.5µM chlortetracycline standard; 20µM oxytetracycline standard, 15µM oxytetracycline standard, 10µM oxytetracycline standard, 5µM oxytetracycline standard, 1µM oxytetracycline standard and 0.5µM oxytetracycline standard; 20µM streptomycin standard, 15µM streptomycin standard, 10µM streptomycin standard, 5µM streptomycin standard, 1µM streptomycin standard and 0.5µM streptomycin standard; 20µM kanamycin standard, 15µM kanamycin standard, 10µM kanamycin standard, 5µM kanamycin standard, 1µM kanamycin standard and 0.5µM kanamycin standard; 20µM vancomycin standard, 15µM vancomycin standard, 10µM vancomycin standard, 5µM vancomycin standard, 1µM vancomycin standard and 0.5µM vancomycin standard; (3) Preparation of Component 1: Take 20 µL of each of the six different concentrations of tebuconazole standard, chlortetracycline standard, oxytetracycline standard, streptomycin standard, kanamycin standard and vancomycin standard from step (2), and react them with 0.8-1.1 mg·mL⁻¹ of each standard. −1 20 µL of Cu-HOF was mixed and incubated, and then 3.8–4.2 mg·mL⁻¹ was added at room temperature. −1 2,4-Dichlorophenol 9-11 µL, 3.8-4.2 mg·mL −1 9-11 µL of 4-aminoantipyrine and 120-160 µL of PBS buffer, wherein the 10 mM PBS buffer has a pH of 8, constitute 36 groups of different types and concentrations of element 1, as follows: 20µM-Cyclocycline element 1, 15µM-Cyclocycline element 1, 10µM-Cyclocycline element 1, 5µM-Cyclocycline element 1, 1µM-Cyclocycline element 1, 0.5µM-Cyclocycline element 1; 20µM-chlortetracycline element 1, 15µM-chlortetracycline element 1, 10µM-chlortetracycline element 1, 5µM-chlortetracycline element 1, 1µM-chlortetracycline element 1, 0.5µM-chlortetracycline element 1; 20µM-oxytetracycline element 1, 15µM-oxytetracycline element 1, 10µM-oxytetracycline element 1, 5µM-oxytetracycline element 1, 1µM-oxytetracycline element 1, 0.5µM-oxytetracycline element 1; 20µM streptomycin element 1, 15µM streptomycin element 1, 10µM streptomycin element 1, 5µM streptomycin element 1, 1µM streptomycin element 1, 0.5µM streptomycin element 1; 20µM-kanamycin element 1, 15µM-kanamycin element 1, 10µM-kanamycin element 1, 5µM-kanamycin element 1, 1µM-kanamycin element 1, 0.5µM-kanamycin element 1; 20µM vancomycin element 1, 15µM vancomycin element 1, 10µM vancomycin element 1, 5µM vancomycin element 1, 1µM vancomycin element 1, 0.5µM vancomycin element 1; (4) Preparation of Component 2: Take 20 µL of each of the six different concentrations of tebuconazole standard, chlortetracycline standard, oxytetracycline standard, streptomycin standard, kanamycin standard and vancomycin standard from step (2), and first react them with 0.5-0.7 mg·mL⁻¹. −1 18-22 µL of Cu-HOF was mixed and incubated, and then 18-22 µL of 10 mM 3,3ʹ,5,5ʹ-tetramethylbenzidine and 120-160 µL of 0.1 M NaAc-HAc buffer were added at room temperature. The pH of the 0.1 M NaAc-HAc buffer was 3.5-4.

5. This resulted in 36 groups of different types and concentrations of element 2, as follows: 20µM-oxycycline element 2, 15µM-oxycycline element 2, 10µM-oxycycline element 2, 5µM-oxycycline element 2, 1µM-oxycycline element 2 and 0.5µM-oxycycline element 2; 20µM-chlortetracycline element 2, 15µM-chlortetracycline element 2, 10µM-chlortetracycline element 2, 5µM-chlortetracycline element 2, 1µM-chlortetracycline element 2 and 0.5µM-chlortetracycline element 2; 20µM-oxytetracycline element 2, 15µM-oxytetracycline element 2, 10µM-oxytetracycline element 2, 5µM-oxytetracycline element 2, 1µM-oxytetracycline element 2 and 0.5µM-oxytetracycline element 2; 20µM streptomycin element 2, 15µM streptomycin element 2, 10µM streptomycin element 2, 5µM streptomycin element 2, 1µM streptomycin element 2 and 0.5µM streptomycin element 2; 20µM-kanamycin element 2, 15µM-kanamycin element 2, 10µM-kanamycin element 2, 5µM-kanamycin element 2, 1µM-kanamycin element 2 and 0.5µM-kanamycin element 2; 20µM vancomycin element 2, 15µM vancomycin element 2, 10µM vancomycin element 2, 5µM vancomycin element 2, 1µM vancomycin element 2 and 0.5µM vancomycin element 2; (5) Construction of colorimetric array sensor: The 36 different types and concentrations of element 1 in step (3) and the 36 different types and concentrations of element 2 in step (4) are irradiated under blue LED light respectively. The reaction signals at 6 minutes and 9 minutes of irradiation are taken to complete the construction of colorimetric array sensor and obtain 4 signal sensing units with 2 elements × 2 light irradiation times for different concentrations and types of antibiotic standards. (6) Plotting the standard curve: Neural network analysis and linear discriminant analysis were performed on different types and concentrations of antibiotics, and the data of their first influencing factor were analyzed and processed to obtain the linear relationship equation between concentration and Factor 1, and the standard curve was plotted. (7) Sample testing: The actual samples were tested using the spiked recovery method.

3. The method for detecting multiple antibiotics using a colorimetric array based on light-driven control of Cu-HOF dual enzyme activity according to claim 2, characterized in that: The preparation method of Cu-HOF in step (1) is as follows: Take 0.2g of Pluronic F-127, 0.05g of melamine and 0.2mL of thiol and disperse them in 20mL of 50% ethanol solution. Stir in a water bath at 60°C. While stirring, add 40mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene. After stirring for 0.5 hours, let it stand for 2.0 hours. Take it out and stir at room temperature for 0.5 hours. Cool to room temperature. First, wash it three times with ultrapure water by centrifugation, with 10mL of ultrapure water each time. Then wash it three times with anhydrous ethanol by centrifugation, with 10mL of anhydrous ethanol each time. After washing, put it in an oven at 60°C and dry it overnight. The powder obtained is HOF. Disperse 100mg of HOF evenly in 20mL of 50% ethanol solution and sonicate. Add 10mmol L under sonication. −1 2 mL of copper nitrate trihydrate was sonicated for 30 min, then removed and washed. The mixture was first washed three times by centrifugation with 10 mL of ultrapure water each time, and then washed three times by centrifugation with 10 mL of anhydrous ethanol each time. After washing, the mixture was placed in a 60°C oven and dried overnight to obtain Cu-HOF.

4. The method for detecting multiple antibiotics using a colorimetric array based on light-driven control of Cu-HOF dual enzyme activity according to claim 2, characterized in that: In step (3), 20 µL of each of the six different concentrations of tebuconazole, chlortetracycline, oxytetracycline, streptomycin, kanamycin, and vancomycin standards from step (2) are taken and reacted with 0.9 mg·mL⁻¹ of each standard. −1 20 µL of Cu-HOF was mixed and incubated at 40°C for 30 minutes, and then 4 mg·mL⁻¹ was added to each solution at room temperature. −1 2,4-Dichlorophenol 10µL, 4mg·mL −1 10 µL of 4-aminoantipyrine and 140 µL of PBS buffer, wherein the 10 mM PBS buffer has a pH of 8, constitute 36 groups of different types and concentrations of element 1.

5. The method for detecting multiple antibiotics using a colorimetric array based on light-driven control of Cu-HOF dual enzyme activity according to claim 2, characterized in that: In step (4), 20 µL of each of the six different concentrations of oxytetracycline standard, chlortetracycline standard, oxytetracycline standard, streptomycin standard, kanamycin standard and vancomycin standard from step (2) are taken and first reacted with 0.6 mg·mL⁻¹ of each standard. −1 20 µL of Cu-HOF was mixed and incubated at 40°C for 30 minutes. Then, 20 µL of 10 mM 3,3ʹ,5,5ʹ-tetramethylbenzidine and 140 µL of 0.1 M NaAc-HAc buffer were added at room temperature. The pH of the 0.1 M NaAc-HAc buffer was 4. This resulted in 36 groups of elements with different types and concentrations.

6. The method for detecting multiple antibiotics using a colorimetric array based on light-driven control of Cu-HOF dual enzyme activity according to claim 2, characterized in that: In step (5), the absorption wavelength of the acquisition signal of element 1 is 503nm, and the absorption wavelengths of the acquisition signal of element 2 are 357nm and 658nm.

7. The method for detecting multiple antibiotics using a colorimetric array based on light-driven control of Cu-HOF dual enzyme activity according to claim 2, characterized in that: In step (7), the actual sample includes river water, honey, chicken, or milk.

8. The method for detecting multiple antibiotics using a colorimetric array based on light-driven control of Cu-HOF dual enzyme activity according to claim 2, characterized in that: In step (7), the absorption wavelengths of the acquisition signals used to detect the actual sample are 503nm, 357nm, and 658nm.

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