Preparation of iron-copper bimetallic nano-enzyme and application of iron-copper bimetallic nano-enzyme in detection of tetracycline antibiotics in food

Abstract

The invention provides preparation of an iron-copper bimetallic nano-enzyme and application of the iron-copper bimetallic nano-enzyme in detection of tetracycline antibiotics in food. The nano enzyme is synthesized by a simple coordination-assisted polymerization assembly method, and has excellent colorimetric performance (peroxidase-like and laccase-like activity) and fluorescence performance. The three-channel colorimetric fluorescent sensing array constructed on the basis of the performance can realize accurate recognition of five tetracycline antibiotics (tetracycline, oxytetracycline, doxycycline, minocycline and metacycline), and can efficiently distinguish binary and ternary mixed tetracycline antibiotics in different proportions at the same time. In order to break through equipment and cost limitations of a traditional detection method, the invention also develops a portable detection device with a built-in test paper sheet, and realizes rapid and field detection of tetracycline antibiotics in actual samples such as milk, chicken farm sewage and the like in combination with smartphone photographing and Image J software analysis, and has a good actual application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of nanotechnology and analytical detection technology, specifically relating to the preparation of an iron-copper bimetallic nanozyme and its application in the detection of tetracycline antibiotics in food. Background Technology

[0002] Tetracycline antibiotics are widely used in food production, particularly in livestock and aquaculture, due to their broad-spectrum antibacterial properties and low cost. They are frequently used to treat and prevent animal diseases. However, some producers lack awareness of proper drug use, leading to issues such as overdosing, exceeding withdrawal periods, and even illegal additives. This results in tetracycline antibiotic residues in meat, eggs, dairy products, and aquatic products, which then enter the consumer market through the food supply chain, posing a potential threat to food safety and human health. Therefore, establishing effective tetracycline antibiotic detection technologies is of significant practical importance.

[0003] Currently, the main methods for detecting tetracycline antibiotics include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), capillary electrophoresis, and enzyme-linked immunosorbent assay (ELISA). While these methods offer high accuracy, they often suffer from drawbacks such as high instrument costs, cumbersome sample pretreatment, and long detection times, making them unsuitable for rapid on-site screening. Furthermore, most of these methods operate on a single signal response model, hindering the accurate differentiation of multiple tetracycline antibiotics and failing to address the complex detection scenarios where multiple tetracycline antibiotic residues coexist in food.

[0004] Nanozymes, as artificial enzymes that combine the unique physicochemical properties of nanomaterials with the catalytic activity of natural enzymes, have become a research hotspot in the field of analytical detection due to their advantages such as simple preparation, high stability, and tunable catalytic activity. Bimetallic nanozymes, as an important branch of nanozymes, can effectively regulate the electronic structure of materials and increase the number of active sites through the synergistic effect of two metal elements, significantly improving catalytic efficiency. Among them, iron-copper bimetallic nanozymes, due to the excellent synergistic effect between the two metals, possess high catalytic activity, high selectivity, and high stability, making them ideal materials for constructing detection systems. Their performance can also be flexibly controlled through preparation conditions, better adapting to the detection needs of target analytes.

[0005] Multi-channel sensor array technology offers an effective solution to the challenge of accurately distinguishing between multiple targets. This technology constructs a multi-channel sensor array and utilizes the differences in interaction between different sensing units and the target to generate a characteristic "fingerprint" signal, enabling efficient and accurate detection and differentiation of multiple targets. Compared to single-signal detection modes, this technology combines high throughput, high discrimination, and strong anti-interference capabilities, making it suitable for detection scenarios where multiple targets coexist in complex matrices.

[0006] This invention synthesizes an iron-copper bimetallic nanozyme (FeCu nanozyme), which possesses excellent colorimetric properties (laccase-like activity and peroxidase-like activity) and fluorescence properties. Based on these properties, a three-channel colorimetric fluorescence sensing array was constructed. When five tetracycline antibiotics (tetracycline (TC), oxytetracycline (OTC), doxycycline (DOX), minocycline (MINO), and methacycline (MET)) interact with the FeCu nanozyme, they trigger colorimetric and fluorescence signals of varying intensities due to differences in their molecular structures, thus forming their own unique "signal fingerprints." Through the acquisition and pattern recognition analysis of multidimensional signals, the accurate differentiation and quantitative detection of the five tetracycline antibiotics can be rapidly achieved, effectively solving the technical problem of traditional methods' difficulty in distinguishing similar antibiotics. To overcome the limitations of traditional detection methods, which are expensive and cumbersome, this invention also develops a portable detection device with built-in test strips. Combined with smartphone photography and ImageJ software analysis, it can achieve rapid, on-site detection of tetracycline antibiotics in actual samples such as milk and chicken farm wastewater. This method is simple to operate, low in cost, and highly efficient, and has good prospects for practical application. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting tetracycline antibiotics in food based on a colorimetric fluorescence sensing array constructed using iron-copper bimetallic nanozymes. This method solves the problems of existing detection methods that rely on large instruments, are cumbersome to operate, and cannot accurately distinguish between similar antibiotics, thus enabling rapid, convenient, sensitive detection and accurate differentiation of tetracycline antibiotics in food.

[0008] The objective of this invention is achieved through the following solution:

[0009] The preparation of a FeCu nanozyme and its application in the detection of tetracycline antibiotics in food are characterized by comprising the following steps:

[0010] A. Preparation of FeCu nanozymes;

[0011] B. Optimize reaction conditions based on the colorimetric properties (two enzyme activities, peroxidase-like activity and laccase-like activity) and fluorescence properties of FeCu nanozymes.

[0012] C. Construct a three-channel colorimetric fluorescence sensing array by utilizing the colorimetric properties (two enzyme activities, peroxidase-like activity and laccase-like activity) and fluorescence properties of FeCu nanozymes.

[0013] D. To detect and differentiate five common tetracycline antibiotics (TC, OTC, DOX, MINO, MET) found in food;

[0014] E. Construct a portable detection device to detect tetracycline antibiotics in actual samples (milk, chicken farm wastewater).

[0015] In step A, the preparation is specifically as follows: a simple coordination-assisted polymerization assembly method is used for synthesis. First, equal volumes of ethanol and distilled water are added to a round-bottom flask and mixed thoroughly. Then, 1 g of Pluronic F127, 1 mL of 1,3,5-trimethylbenzene (TMBE), and 0.75 g of dopamine hydrochloride (DA) are added to the system and stirred thoroughly. Then, ferrous ammonium sulfate hexahydrate ((NH4)2Fe(SO4)2·6H2O) and copper ammonium sulfate hexahydrate ((NH4)2Cu(SO4)2·6H2O) with a molar concentration of 1:1 are dissolved in the above solution. Subsequently, a certain amount of ammonia water is added dropwise to the solution, and after reacting for 3 hours, the FeCu / PDA / F127 composite material is obtained. The collected product is centrifuged, washed, and freeze-dried, and then calcined at 350 °C for 3 hours and 800 °C for 5 hours under nitrogen to obtain the FeCu nanozyme.

[0016] Step B begins with the optimization of colorimetric performance. Parameters affecting the activity of FeCu nanozymes and peroxidases, such as pH, substrate concentration, and reaction time, were optimized to obtain the optimal reaction conditions. The optimized conditions were: pH range 2.5-7; reaction time range 1-10 min; hydrogen peroxide (H₂O₂) concentration range 0.2-1.8 mmol / L; 3,3',5,5'-tetramethylbenzidine (TMB) concentration range 0.1-1 mmol / L, and the UV absorption spectra at 500-800 nm were recorded. Next, the pH and reaction temperature affecting the activity of FeCu nanozymes and laccases were optimized. The optimized conditions were: pH range 6-7; reaction temperature range 40-90 ℃. The absorbance values ​​at the characteristic wavelengths were recorded using a UV-Vis spectrophotometer. Following this, the fluorescence performance was optimized, focusing on the main factors affecting fluorescence performance: pH and reaction time. The optimized conditions were: pH range 3-8; reaction time range 30-300 s, and the fluorescence intensity values ​​were recorded.

[0017] In step C, two enzyme activities (peroxidase-like activity and laccase-like activity) and fluorescence properties were selected to construct a three-channel colorimetric fluorescence sensing array. The array consisted of three channels × five tetracycline antibiotics to be tested × five parallel experiments. The reaction system for the three channels was as follows: Channel 1 (laccase-like colorimetric channel): 2-morpholine ethanesulfonic acid buffer (MES buffer) + FeCu nanozyme + tetracycline antibiotics + 2,4-dichlorophenol (2,4-DP) + 4-aminobitriptyline (4-AP); Channel 2 (peroxidase-like colorimetric channel): acetate-sodium acetate buffer (HAc-NaAc buffer) + FeCu bimetallic nanozyme + tetracycline antibiotics + H2O2 + TMB; Channel 3 (fluorescence channel): phosphate buffer (PBS buffer) + FeCu bimetallic nanozyme + tetracycline antibiotics.

[0018] In step D, the constructed colorimetric fluorescence sensor array is used to detect and differentiate single tetracycline antibiotics of different concentrations, different types of tetracycline antibiotics, and mixed tetracycline antibiotics of different proportions in binary / ternary ratios.

[0019] In step E, the tetracycline antibiotic content in actual samples (spikeped milk and spiked chicken farm wastewater) is detected using a portable detection device. After the test strip loaded with functional material reacts with the actual sample, it is placed in the device to take a picture. The grayscale value is extracted using ImageJ software and substituted into the standard curve to obtain the concentration of tetracycline antibiotics in the actual sample.

[0020] The beneficial effects of this invention are:

[0021] (1) A three-channel colorimetric fluorescence sensor array integrating two colorimetric signals and one fluorescence signal was constructed, which broke through the limitations of single signal detection technology, which cannot effectively distinguish, has poor selectivity and low quantitative accuracy, and can achieve efficient differentiation and accurate quantification of tetracycline antibiotics.

[0022] (2) A portable detection device was constructed. It does not require large precision instruments, is easy to operate and can detect quickly. It can realize the on-site convenient detection of tetracycline antibiotics in actual samples and has good practical application value. Attached Figure Description

[0023] Figure 1 This is a scanning electron microscope image of the FeCu nanozyme.

[0024] Figure 2 The X-ray electron spectrum of FeCu nanozymes;

[0025] Figure 3 X-ray diffraction pattern of FeCu nanozyme;

[0026] Figure 4 Raman spectrum of FeCu nanozyme;

[0027] Figure 5 Results of optimized reaction conditions for FeCu nanozymes;

[0028] Figure 6 Standard curves for five tetracycline antibiotics under peroxidase-like activity assay mode;

[0029] Figure 7 Standard curves for five tetracycline antibiotics under laccase-like enzyme activity assay mode;

[0030] Figure 8 Standard curves for five tetracycline antibiotics under fluorescence detection mode;

[0031] Figure 9 Linear discriminant analysis of different concentrations of tetracycline antibiotics by a colorimetric fluorescence sensing array;

[0032] Figure 10 This is a linear discriminant analysis diagram of different types of tetracycline antibiotics using a colorimetric fluorescence sensing array.

[0033] Figure 11 Linear discriminant analysis of binary and ternary mixed tetracycline antibiotics by a colorimetric fluorescence sensing array;

[0034] Figure 12 This is a standard curve showing the relationship between oxytetracycline concentration and grayscale value of the test strip. Detailed Implementation

[0035] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0036] Example 1:

[0037] A simple coordination-assisted polymerization assembly method was used for synthesis. First, equal volumes of ethanol and distilled water were added to a round-bottom flask and mixed thoroughly. Then, 1 g of Pluronic F127, 1 mL of TMBE, and 0.75 g of DA were added to the system and stirred thoroughly. Next, (NH4)2Fe(SO4)2·6H2O and (NH4)2Cu(SO4)2·6H2O with a molar ratio of 1:1 were dissolved in the above solution. Subsequently, a certain amount of ammonia water was added dropwise to the solution, and after reacting for 3 hours, the FeCu / PDA / F127 composite material was obtained. The collected product was centrifuged, washed, and freeze-dried, and then calcined at 350 °C for 3 hours and 800 °C for 5 hours under nitrogen to obtain the nanozyme. Figure 1 As shown, the FeCu nanozyme exhibits a uniform spherical morphology with a diameter of approximately 100 nm.

[0038] Example 2:

[0039] The morphology of FeCu nanozymes was determined by scanning electron microscopy at 200 kV; X-ray electron spectra were measured using Al Kα radiation to obtain the elemental composition and valence states of the FeCu nanozymes; X-ray diffraction patterns were determined using X-ray powder diffraction to identify the phases and structure of the FeCu nanozymes; and the Raman spectrum of the FeCu nanozymes was measured using a 532 nm laser source. Figure 2 A. The results show that the FeCu nanozyme contains Fe, Cu, C, N, and O elements. High-resolution energy dispersive spectroscopy of C 1s (…) Figure 2 B) showed 283.6 eV (sp 2), 284.8 eV (sp 3 The high-resolution energy spectrum of N 1s shows three peaks at 289.1 eV (C=O / CN). Figure 2 C) can be deconvolved into four components, corresponding to the pyridine nitrogen peak, MN bond peak, graphitic nitrogen peak, and nitrogen oxide peak, respectively, indicating the formation of metal-N coordination in the material. Because the iron and copper loadings are low, the high-resolution energy dispersive spectroscopy (EDS) of Fe 2p is... Figure 2 D) and high-resolution energy dispersive spectra of Cu 2p ( Figure 2 E) shows relatively weak Fe 2p and Cu 2p peaks, at 710.80 eV (Fe 2p) and 2p, respectively. 3 / 2 ), 723.30 eV (Fe 2p 1 / 2 ), 931 eV (Cu 2p 3 / 2 ), 950.4 eV (Cu 2p 1 / 2 Peak. (e.g.) Figure 3 Two characteristic peaks appear in the 20-30° and 40-50° ranges, attributed to the (002) and (101) crystal planes of graphitic carbon, respectively. No crystalline Fe or crystalline Cu was observed in the X-ray diffraction pattern. Figure 4 Raman spectra at 1358 cm⁻¹ -1 (Defect zone) and 1600 cm -1 There are two representative peaks at the (graphite band), which indicates that there are a large number of defects and disordered non-graphitized carbon in the material, while the loading of Fe and Cu increases the graphitization degree of the material.

[0040] Example 3:

[0041] First, the colorimetric performance was optimized. The main factors affecting the peroxidase activity of FeCu nanozymes include pH, TMB concentration, H₂O₂ concentration, and reaction time. These factors were optimized. Using an HAc-NaAc buffer solution + FeCu nanozyme + TMB + H₂O₂ system, the peroxidase-like activity of FeCu nanozymes was measured under different pH (2.5-7), H₂O₂ concentration (0.2-1.8 mmol / L), TMB concentration (0.1-1 mmol / L), and reaction time (1-10 min) to determine the optimal reaction conditions. The results are as follows: Figure 5 A. Nanozymes exhibit optimal peroxidase-like activity in an acidic environment at pH 3.5; therefore, pH 3.5 is the optimal pH for nanozymes to exert their maximum activity. Secondly, the concentrations of TMB and H2O2 were optimized. For example... Figure 5For B and 5C, the nanozyme activity showed a trend of first increasing and then decreasing with increasing TMB and H2O2 concentrations. During this process, the oxidation of TMB changed from rapid to slow, and the optimal activity was observed at TMB and H2O2 concentrations of 0.9 mmol / L and 1.4 mmol / L, respectively. Finally, the reaction time was optimized. Figure 5 D) When the reaction time is 9 min, the absorbance of the system is at its maximum, reaching the optimal response time.

[0042] The pH and reaction temperature affecting the laccase-like activity of FeCu nanozymes were optimized. The laccase-like activity of FeCu nanozymes was measured using a MES buffer solution + FeCu nanozyme + 2,4-DP + 4-AP system at different pH values ​​(6.0-7.0) and reaction temperatures (40-90 °C) to determine the optimal pH and reaction temperature. The results are as follows: Figure 5 For E-5F, the absorbance of the system reached its peak at pH 6.6, indicating the highest catalytic efficiency. The optimal reaction temperature for laccase was also optimized. Results showed that within the range of 30-90 °C, the absorbance value initially increased and then decreased with increasing temperature. The absorbance at the characteristic wavelength tended to reach its highest value during incubation at 80 °C; therefore, 80 °C was determined as the optimal incubation temperature. However, to balance antibiotic activity and reaction rate, a reaction temperature of 50 °C was selected for subsequent laccase-like detection modes.

[0043] Next, the fluorescence performance was optimized. The pH and reaction time, which affected the fluorescence performance, were optimized. The fluorescence intensity was measured at different pH values ​​(3-8) and reaction times (30-300 s) in a PBS buffer solution + FeCu nanozyme + antibiotic system. The results (5G-5H) showed that the FeCu nanozyme exhibited the optimal fluorescence quenching rate at pH=6 and a reaction time of 2 min, under which it demonstrated the greatest fluorescence performance.

[0044] Example 4:

[0045] A colorimetric fluorescence sensing array was constructed based on the fluorescence and colorimetric properties (peroxidase-like activity and laccase-like activity) of nanozymes to achieve the detection and differentiation of five common tetracycline antibiotics (TC, OTC, DOX, MINO, and MET) in food.

[0046] First, the detection and differentiation of tetracycline antibiotics at different concentrations were performed. The first channel utilized laccase activity in a system of MES buffer + FeCu nanozyme + different concentrations of tetracycline antibiotics + 2,4-DP + 4-AP. After the reaction, the system was vortexed to mix thoroughly, and 200 μL was transferred to a 96-well microplate, with absorbance measured at 510 nm. The second channel utilized peroxidase activity in a system of HAc-NaAc buffer + FeCu nanozyme + different concentrations of tetracycline antibiotics + TMB + H2O2. Absorbance was then measured at 652 nm. The third channel involved a reaction in a system of PBS buffer + FeCu nanozyme + different concentrations of tetracycline antibiotics, followed by measurement of the corresponding fluorescence intensity. Based on the different combinations of detection channels and antibiotics, five linear calibration curves were obtained for each mode, each corresponding to a specific antibiotic. Figure 6 (Peroxidase-like enzyme activity detection mode) Figure 7 (Lacase-like enzyme activity detection mode) Figure 8 As shown in the fluorescence detection mode, the relative absorbance and relative fluorescence intensity values ​​exhibit a good linear relationship with the concentrations of TC, OTC, DOX, MINO, and MET. Subsequently, based on the established linear detection ranges for various antibiotics, the sensor array's ability to accurately distinguish between different concentrations of antibiotics was further investigated. Figure 7 As shown, five tetracycline antibiotics (TC ( ) Figure 9 A), OTC ( Figure 9 B), DOX Figure 9 C), MINO ( Figure 9 D), MET Figure 9 E) The response data points at their respective concentrations all formed clearly defined, non-overlapping independent clusters, and the data from the five parallel experiments within each cluster showed high aggregation. These results indicate that the constructed detection array can effectively identify tetracycline antibiotics at different concentrations.

[0047] Secondly, the detection and differentiation of different types of tetracycline antibiotics. Channel 1: Reactions were carried out in a system of MES buffer + FeCu nanozyme + different types of tetracycline antibiotics + 2,4-DP + 4-AP, and the absorbance at 510 nm was measured. Each experiment was repeated five times. Channel 2: TC, OTC, DOX, MINO, and MET were added to a system of HAc-NaAc buffer + FeCu nanozyme + TMB + H2O2, respectively. The system was then vortexed to mix thoroughly, and 200 μL was transferred to a 96-well microplate. The absorbance at 652 nm was then measured using a microplate reader. Each experiment was repeated five times. Channel 3: Reactions were carried out in a system of PBS buffer + FeCu nanozyme + different types of tetracycline antibiotics, and the corresponding fluorescence intensity was measured. Finally, the data were processed using SPSS software to obtain the linear discriminant analysis (LDA) plot. Figure 10 As shown, the response signals of the five tetracycline antibiotics all clustered into five well-defined and closely distributed independent clusters. All data points for the same type of tetracycline antibiotic were highly clustered in the same region, while the clusters of different types of tetracycline antibiotics were separated and did not overlap significantly. This result fully demonstrates that the designed three-channel sensor array can achieve efficient and accurate identification of different types of tetracycline antibiotics.

[0048] To further demonstrate the performance of the sensing array, binary mixtures (TC:OTC = 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10) and ternary mixtures (TC:OTC:DOX = 1:2:7, 2:2:6, 1:5:4, 3:4:3, 2:3:5) with a total concentration of 10 μmol / L were added to the reaction system. After the reaction, the absorbance and corresponding fluorescence intensity of the three-channel sensing array at specific wavelengths were measured. As shown in the figure, binary mixtures with different ratios (TC:OTC:DOX = 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10) were added to the reaction system. Figure 11 A) Ternary hybrid system ( Figure 11 B) All clusters formed distinct and independent clusters, with no overlap in the response data clusters of various mixtures. This result fully confirms that the constructed three-channel sensor array has excellent distinguishing sensitivity for mixed tetracycline antibiotic systems.

[0049] Example 5:

[0050] This portable testing device was designed and 3D modeled, then manufactured using 3D printing. The device is equipped with LED supplemental lights on both sides and has an internal slot for pull-out test strips. Using oxytetracycline as a representative tetracycline antibiotic, the detection effect of the portable testing device on actual samples was evaluated. Test strips loaded with functional materials were reacted with spiked milk and spiked chicken farm wastewater samples, respectively. The test strips were then placed inside the device, and images were acquired through the viewfinder. The images were imported into ImageJ software to extract grayscale values, which were then substituted into a standard curve. Figure 12 The concentration of oxytetracycline in the sample can be calculated using methods A and B. The results are shown in Table 1. The results obtained by the portable device detection method (test strip method) and the HPLC method are highly consistent, and the spiked recovery rate is within a reasonable range. This result fully demonstrates that the detection method constructed in this study has both high accuracy and practicality, and can meet the needs of rapid on-site detection of tetracycline antibiotics in complex real-world samples.

[0051] Table 1. Summary of the determination of oxytetracycline content in milk and chicken farm wastewater using high performance liquid chromatography (HPLC) and test strip methods (peroxidase-like activity (POD) method and laccase-like activity (La) method).

[0052]

Claims

1. A bimetallic iron-copper nanozyme, characterized in that: The preparation method is as follows: First, equal volumes of ethanol and distilled water were added to a round-bottom flask and mixed thoroughly. Then, 1 g of Pluronic F127, 1 mL of 1,3,5-trimethylbenzene (TMBE), and 0.75 g of dopamine hydrochloride (DA) were added to the system and stirred thoroughly. Next, ferrous ammonium sulfate hexahydrate ((NH4)2Fe(SO4)2·6H2O) and copper ammonium sulfate hexahydrate ((NH4)2Cu(SO4)2·6H2O) with a molar ratio of 1:1 were dissolved in the above solution. Subsequently, a certain amount of ammonia water was added dropwise to the solution, and after reacting for 3 hours, the FeCu / PDA / F127 composite material was obtained. After centrifuging, washing, and freeze-drying the collected product, it was calcined at 350 °C for 3 hours and then at 800 °C for 5 hours under nitrogen to obtain the FeCu nanozyme.

2. The application of the iron-copper bimetallic nanozyme according to claim 1 in the detection of tetracycline antibiotics in food, characterized in that: Utilizing the colorimetric and fluorescence properties of FeCu bimetallic nanozymes, a three-channel colorimetric fluorescence sensing array was constructed to detect and differentiate five common tetracycline antibiotics in food. Simultaneously, a portable detection device was constructed to achieve rapid and accurate detection of tetracycline antibiotics in actual samples.

3. The application according to claim 2, characterized in that, The three-channel colorimetric fluorescence sensing array consists of a peroxidase-like colorimetric channel, a laccase-like colorimetric channel, and a fluorescence channel. The array is configured as three channels × five tetracycline antibiotics to be tested × five parallel experiments. The five tetracycline antibiotics are tetracycline, oxytetracycline, doxycycline, minocycline, and methacycline.

4. The application according to claim 3, characterized in that, The three-channel reaction system is as follows: First channel, laccase-like colorimetric channel: 2-morpholine ethanesulfonic acid buffer (MES buffer) + FeCu nanozyme + tetracycline antibiotics + 2,4-dichlorophenol (2,4-DP) + 4-aminobitriptyline (4-AP); Second channel, peroxidase-like colorimetric channel: acetate-sodium acetate buffer (HAc-NaAc buffer) + FeCu bimetallic nanozyme + tetracycline antibiotics + hydrogen peroxide (H2O2) + 3,3',5,5'-tetramethylbenzidine (TMB); Third channel, fluorescence channel: phosphate buffer (PBS buffer) + FeCu bimetallic nanozyme + tetracycline antibiotics.

5. The application according to claim 2, characterized in that, The detection and differentiation include the detection and differentiation of different concentrations of single tetracycline antibiotics, different types of tetracycline antibiotics, and different proportions of binary / ternary mixed tetracycline antibiotics.

6. The application according to claim 2, characterized in that, The portable detection device is manufactured by 3D printing after design and 3D modeling. The main body of the device is a cuboid box with a reserved viewport, which contains a pull-out test strip carrier component. LED light sources are installed at both ends of the box, and the grooves on the pull-out component can accurately fix the sensor test strip.

7. The application according to claim 2, characterized in that, The detection process of the portable detection device is as follows: after reacting the test strip loaded with FeCu nanozyme with the actual sample, it is placed in the portable detection device to take a picture, and the gray value of the image is extracted by ImageJ software. The gray value is then substituted into the standard curve to calculate the concentration of tetracycline antibiotics in the actual sample. The actual sample is milk and chicken farm sewage.

Images