Method for colorimetric chemiluminescence detection of tetracycline antibiotics by using K-C3N4Cu and Fe nano-enzyme under neutral condition

Abstract

The invention discloses a method for colorimetric / chemiluminiscence detection of tetracycline antibiotics by using K-C3N4 / Cu, Fe nano-enzyme under a neutral condition, according to the method, a novel K-doped g-C3N4 composite material (K-C3N4 / Cu, Fe) assembled by copper and iron co-doped carbon dots (Cu, Fe-CDs) is synthesized by a hydrothermal method and a thermal polymerization method, and Cu and Fe-CDs are used as effective conductive bridges. K-C3N4 / Cu and Fe show excellent peroxidase-like (POD) activity when the pH value is 7, H2O2 is catalyzed to be decomposed to generate O2 and OH active oxygen free radicals, and the reduction of absorbance and CL intensity of oxABTS at 416 nm is caused by the inhibition. According to the invention, principal component analysis (PCA) is utilized to distinguish different TC, and the method has the characteristics of high sensitivity, strong specificity, simple operation, rapidness and the like.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, specifically to a method for colorimetric / chemiluminescent detection of tetracycline antibiotics using K-C3N4 / Cu,Fe nanozymes under neutral conditions. Background Technology

[0002] Antibiotic pollution poses a threat to human health and has become a widespread global concern. Tetracycline antibiotics (TCs) are a class of broad-spectrum antibiotics, mainly including tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC). Due to their excellent antibacterial activity, they are commonly used in the treatment of human diseases and in animal husbandry. However, the overuse of these antibiotics can lead to a series of side effects and the development of antibiotic resistance in humans, resulting in high residual levels in the aquatic environment, posing a serious threat to the environment and public health. Therefore, developing accurate and specific detection methods for monitoring tetracycline antibiotics is of great significance. Currently, routine analytical methods for TCs include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), gas chromatography, capillary electrophoresis (CE), and electrochemical techniques. These methods generally have high sensitivity and accuracy, but they have some limitations, such as expensive instruments, complex sample pretreatment, and time-consuming procedures. In recent years, nanozymes have attracted increasing attention as sensors for TC detection due to their advantages of simple preparation, low cost, and high stability.

[0003] In recent years, dual-mode sensors have attracted widespread attention due to their advantages such as wide detection linear range, high accuracy, and strong application flexibility. Dual-mode sensors combine two signal transduction channels into one technology. Chemiluminescence (CL) is the phenomenon where excited-state molecules produced by chemical reactions return to their ground state and emit light. Due to its high sensitivity and wide detection linear range, CL has been widely used for the detection of ions, small molecules, and nucleic acids. Colorimetric methods are simple and convenient, mainly relying on color changes during the detection process, making them suitable for on-site analysis. This invention combines these two methods to construct a colorimetric-chemiluminescence dual-mode system for TCs detection, providing an effective strategy for more convenient and accurate TCs detection. Summary of the Invention

[0004] This invention discloses a method for colorimetric / chemiluminescent detection of tetracycline antibiotics using K-C3N4 / Cu,Fe nanozymes under neutral conditions. This method synthesizes a novel K-doped g-C3N4 composite material (K-C3N4 / Cu,Fe) assembled from copper-iron co-doped carbon dots (Cu,Fe-CDs) via hydrothermal and thermal polymerization methods. Cu,Fe-CDs act as an effective conductive bridge, facilitating efficient space charge separation and carrier transfer. K-C3N4 / Cu,Fe exhibits excellent peroxidase-like (POD) activity at pH 7, catalyzing the decomposition of H2O2 to produce •O2. − Reactive oxygen species (•OH) initiate the chemiluminescence (CL) of luminol and oxidize ABTS to a green product (oxABTS) with an absorption peak at 416 nm. When tetracycline antibiotics are introduced, the activity of POD-like compounds is inhibited due to the consumption of •OH and the reduction of available catalytic active sites. This inhibition leads to a decrease in the absorbance of oxABTS at 416 nm and the intensity of CL. Based on this result, this invention develops a simple and sensitive colorimetric / chemiluminescence dual-mode sensor for TCs, utilizing principal component analysis (PCA) to distinguish different TCs. The method features high sensitivity, high specificity, simple operation, and speed.

[0005] This invention relates to a method for the colorimetric / chemiluminescent detection of tetracycline antibiotics using K-C3N4 / Cu,Fe nanozymes under neutral conditions, comprising the following steps: Includes the following steps: (1) Mix 3.00-3.50 g of melamine and 1.00-1.50 g of KCl, then grind with agate slurry for 10-15 min, transfer the mixture to a corundum boat, and calcine at 550 ℃ for 4-5 h under nitrogen atmosphere at a heating rate of 5 ℃ / min. After cooling to room temperature, the material is ground into powder. 1.00-1.50 g of the ground powder is ground together with 3.50-4.50 g of KSCN and placed in a covered corundum boat. The powder is heated at 400 °C for 1-2 h in a nitrogen atmosphere at a heating rate of 5 °C / min, and then heated at 500 °C for 30-40 min. The collected powder is dispersed in 40-50 mL of 1-2 mol / L HNO3 and then magnetically stirred at room temperature for 2-3 h. The powder is washed 2-3 times with ethanol and dried in a vacuum oven to obtain K-C3N4. (2) Disperse 0.10-0.20 g tartaric acid, 0.10-0.20 g L-tryptophan, 0.30-0.50 g FeCl3 and 0.30-0.50 g CuCl2 in 20-30 mL of deionized water by ultrasonication. Transfer the mixture to a polytetrafluoroethylene container and heat at 160 °C for 10-12 h. After natural cooling, centrifuge and filter through a 0.22 μm filter membrane. Collect the filtrate to obtain Cu,Fe-CDs. (3) Mix 8-10 mg of K-C3N4 with 4-5 mL of Cu,Fe-CDs and 2-3 mL of deionized water, sonicate for 5-10 min, and then stir for 60-90 min to obtain K-C3N4 / Cu,Fe; (4) After mixing K-C3N4 / Cu,Fe nanozyme solution, 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid solution and H2O2, add tetracycline antibiotic solutions of different concentrations and pH 7.2 PIPES buffer solution. After mixing, let stand for 5-10 min and measure absorbance at 417 nm wavelength to determine the linear relationship between tetracycline antibiotic concentration and absorbance value ΔA and obtain regression equation, where ΔA=A-A0, A and A0 are the absorbance values ​​of the reaction system without tetracycline antibiotic and with tetracycline antibiotic, respectively. (5) After mixing K-C3N4 / Cu,Fe nanozyme solution, luminol solution, and H2O2, add tetracycline antibiotic solutions of different concentrations and pH 11.0 NaHCO3-Na2CO3 buffer solution. After reacting for 5-10 min, measure the chemiluminescence intensity ΔI at 458 nm using an enzyme-linked immunosorbent assay (ELISA) reader. CL Determine the relationship between tetracycline antibiotic concentration and ΔI CL The linear relationship is used to obtain the regression equation, where ΔI CL = I-I0, where I and I0 are the chemiluminescence intensities of the reaction systems without tetracycline antibiotics and with tetracycline antibiotics, respectively; (6) Following the steps in (4) or (5), measure ΔA and ΔI of the sample solution to be tested. CL Substitute the values ​​into the regression equation to obtain the concentration of tetracycline antibiotics in the sample solution.

[0006] The tetracycline antibiotics mentioned include one of tetracycline, oxytetracycline, and chlortetracycline.

[0007] The concentration of the K-C3N4 / Cu,Fe nanozyme solution is 1.5 mg / mL, and the amount added is 5-30 μL; the concentration of the 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid solution is 5 mmol / L, and the amount added is 10-15 μL; the concentration of the luminol solution is 50 mmol / L, and the amount added is 80-90 μL; the concentration of the H2O2 is 20 mmol / L, and the amount added is 20-50 μL.

[0008] The advantages of this invention are: 1. Compared with K-C3N4 or Cu,Fe-CDs alone, the K-C3N4 / Cu,Fe composite material prepared in this invention exhibits significantly enhanced catalytic activity. This is because Cu,Fe-CDs modulate the enzyme-like activity of K-C3N4, thereby improving and synergistically enhancing the catalytic performance in the K-C3N4 / Cu,Fe hybrid system. Furthermore, the prepared K-C3N4 / Cu,Fe composite material demonstrates excellent POD-like activity at pH 7. This not only overcomes the limitation that traditional nanozymes with POD-like activity typically require acidic reaction conditions, but also makes it potentially applicable in catalytic reactions in luminol systems based on alkaline conditions.

[0009] 2. TCs can inhibit the catalytic activity of K-C3N4 / Cu,Fe and reduce the UV signal and CL intensity of ox-ABTS. On the one hand, the degradation of TCs consumes the •OH generated in the ABTS / Luminol system, reducing the concentration of •OH participating in the reaction; on the other hand, TCs may form complexes with K-C3N4 / Cu,Fe, blocking the catalytic active sites of nanozymes, thereby inhibiting the colorimetric / chemiluminescent reaction mediated by K-C3N4 / Cu,Fe.

[0010] 3. The established colorimetric and chemiluminescent methods for detecting tetracycline antibiotics exhibit a wide linear range, with limits of detection of 0.025 mg / kg-0.081 mg / kg and 0.014 mg / kg-0.097 mg / kg, respectively, which are 6-10 times lower than the limits of detection (0.15 mg / kg-0.65 mg / kg) in GB 5009.116-2003. The established methods have been successfully applied to the detection of tetracycline antibiotic residues in milk and meat products. The spiked method successfully detected tetracycline antibiotics in samples, with recoveries of 84.1%–106.2%. The detection system provided by this invention has high detection accuracy. Attached Figure Description

[0011] Figure 1 The image shows a TEM image of the synthesized K-C3N4 / Cu,Fe in Example 1. Figure 2The image shows the XRD pattern of K-C3N4 / Cu,Fe synthesized in Example 1. Figure 3 The image shows the FT-IR spectrum of K-C3N4 / Cu,Fe synthesized in Example 1. Figure 4 This is a high-resolution XPS Cu 2p image of K-C3N4 / Cu,Fe synthesized in Example 1; Figure 5 This is a high-resolution XPS Fe 2p image of the synthesized K-C3N4 / Cu,Fe in Example 1; Figure 6 shows the UV-Vis absorption spectra of ABTS, K-C3N4 / Cu,Fe, ABTS + K-C3N4 / Cu,Fe, and ABTS + H2O2 + K-C3N4 / Cu,Fe in Example 1. Figure 7 shows the CL spectra of the reaction systems K-C3N4 / Cu,Fe+H2O2+luminol and potassium ferricyanide+H2O2+luminol in Example 1; Figure 8 The Michaelis-Menten kinetics curves for the oxidation of (a, b) ABTS and (c, d) H2O2 by K-C3N4 / Cu,Fe and K-C3N4 / Cu,Fe + OTC in Example 1 are shown. Figure 9 This is the colorimetric linear regression equation for OTC in Example 1; Figure 10 This is the linear regression equation for CL in OTC in Example 1; Figure 11 The results show the impact of interfering substances on the TC detection system. Detailed Implementation

[0012] The technical solution of the present invention will be described in further detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0013] Example 1: Determination of tetracycline antibiotic residues in aquatic product samples 1. Mix 3.00 g of melamine and 1.00 g of KCl, then grind with agate slurry for 10 min. Transfer the mixture to a corundum boat and calcine at 550 °C for 4 h under nitrogen atmosphere at a heating rate of 5 °C / min. After cooling to room temperature, grind the material into powder. Take 1.00 g of the ground powder and grind it together with 3.50 g of KSCN, and place it in a covered corundum boat. Heat the powder at 400 °C for 1 h under nitrogen atmosphere at a heating rate of 5 °C / min, then heat it again at 500 °C for 30 min. Disperse the collected powder in 50 mL of 1 mol / L HNO3, and then magnetically stir at room temperature for 2 h. Wash the obtained powder twice with ethanol and dry it in a vacuum oven to obtain K-C3N4.

[0014] 2. Disperse 0.10 g tartaric acid, 0.10 g L-tryptophan, 0.30 g FeCl3, and 0.30 g CuCl2 ultrasonically in 20 mL of deionized water. Transfer the mixture to a polytetrafluoroethylene (PTFE) container and heat at 160 °C for 10 h. After natural cooling, centrifuge at 10,000 rpm for 10 min. Filter the supernatant through a 0.22 μm filter membrane and collect the filtrate to obtain Cu,Fe-CDs.

[0015] 3. Mix 8 mg of K-C3N4 with 4 mL of Cu,Fe-CDs and 2 mL of deionized water, sonicate for 5 min, then stir for 60-90 min to obtain K-C3N4 / Cu,Fe; analyze the prepared K-C3N4 / Cu,Fe nanozyme using transmission electron microscopy (TEM). Figure 1 As shown in figure a, the synthesized K-C3N4 / Cu,Fe nanozymes exhibit a typical stacked two-dimensional layered structure, as shown in the high-resolution TEM (HR-TEM) image (). Figure 1 As shown in b), the lattice spacing of Cu,Fe-CDs in K-C3N4 / Cu,Fe is 0.23 nm, corresponding to the (100) crystal plane of graphitic carbon. The X-ray diffraction (XRD) pattern is shown below. Figure 2 As shown, K-C3N4 in 2 θ = 28.1° shows a relatively strong diffraction peak, corresponding to the (002) crystal plane. In contrast, compared with pure K-C3N4 and Cu,Fe-CDs, the diffraction peak of K-C3N4 / Cu,Fe nanozyme has changed to some extent, indicating that certain structural changes have occurred during the formation of the composite material. Figure 3 The FTIR spectra of K-C3N4, Cu,Fe-CDs, and K-C3N4 / Cu,Fe are shown. In the FTIR spectrum of K-C3N4 / Cu,Fe, the peak wavelengths are 3385, 3172, and 2192 cm⁻¹. -1The absorption peaks at 1607, 1547, and 1405 cm⁻¹ are attributed to the stretching vibrations of the OH, NH, and C≡N groups, respectively. -1 The peaks at 1326 and 1052 cm⁻¹ are attributed to stretching vibrations of C=O, C=N, and C=C, respectively. -1 The peaks at 568 cm⁻¹ correspond to the stretching vibrations of CN and CO, respectively. -1 The peak at the specified position is attributed to the Fe-O stretching vibration. The FTIR spectrum of K-C3N4 / Cu,Fe is similar to that of Cu,Fe-CDs, indicating that the composite material still contains abundant hydroxyl and amino groups after Cu,Fe-CDs are combined with K-C3N4 nanosheets, confirming the successful synthesis of K-C3N4 / Cu,Fe. X-ray photoelectron spectroscopy (XPS) analysis was used to determine the composition, valence state, and binding energy of the elements present in K-C3N4 / Cu,Fe. Figure 4 In the K-C3N4 / Cu,Fe spectrum, the Cu 2p peaks at 954.3, 952.2, 934.5, and 932.4 eV are attributed to Cu, respectively. 2+ 2p 1 / 2 Cu + 2p 1 / 2 Cu 2+ 2p 3 / 2 and Cu + 2p 3 / 2 The Fe 2p spectrum of K-C3N4 / Cu,Fe is as follows: Figure 5 As shown, it was decomposed into four peaks located at 727.7, 724.5, 713.9, and 711.2 eV, which were attributed to Fe. 3+ 2p 1 / 2 Fe 2+ 2p 1 / 2 Fe 3+ 2p 3 / 2 and Fe 2+ 2p 3 / 2 Characteristic peaks indicate that Cu in K-C3N4 / Cu,Fe 2+ / Cu + and Fe 3+ / Fe 2+ Coexistence.

[0016] 4. Evaluation of peroxidase activity of K-C3N4 / Cu,Fe nanozymes: 100 μL of 5 mmol / L ABTS was added, along with 100 μL of 50 mmol / L H2O2 and 50 μL of 1.5 mg / mL K-C3N4 / Cu,Fe nanozymes. The volume was then adjusted to 3 mL with pH 7.2 PIPES buffer, shaken well, and allowed to stand for 10 min. The absorbance was measured at 417 nm. Figure 6The UV absorption spectrum of the catalytic oxidation of ABTS is shown, indicating that ABTS is only catalytically oxidized in the presence of K-C3N4 / Cu,Fe, ABTS, and H2O2. 200 μL of 50 mmol / L luminol was added, along with 200 μL of 50 mmol / L H2O2, 100 μL each of 1.5 mg / mL K-C3N4 / Cu,Fe nanozyme or K3[Fe(CN)6], and then diluted to 3 mL with 0.1 mmol / L pH 11.0 NaHCO3-Na2CO3 buffer solution. The mixture was shaken well and allowed to stand for 5 min. Figure 7 It can be seen that the K-C3N4 / Cu,Fe system oxidizes luminol more effectively than K3[Fe(CN)6], exhibiting stronger CL strength.

[0017] This embodiment also included the determination of Michaelis catalytic kinetic parameters, the results of which are shown below. Figure 8 (ad) and Table 1, in which Figure 8 The Michaelis constants of K-C3N4 / Cu,Fe nanozymes for substrates ABTS and H2O2 are shown. K m The concentrations were 0.246 mmol / L and 3.879 mmol / L, respectively, and the reaction rate constants were 7.397 × 10⁻⁶. -6 mol / L·s and 6.468×10 -6 mol / L·s, after adding OTC, the concentrations of ABTS and H2O2 K m The concentrations were 0.543 mmol / L and 5.530 mmol / L, respectively, with reaction rate constants of 5.072 × 10⁻⁶. -6 mol / L·s and 5.322×10 -6 The concentration of mol / L·s indicates that the addition of OTC significantly inhibited the affinity and reaction rate between the K-C3N4 / Cu,Fe nanozyme and the substrates ABTS and H2O2.

[0018]

[0019] 4. Preparation of colorimetric working curves for tetracycline antibiotics Add 10 μL of 5 mmol / L ABTS, 10 μL of 50 mmol / L H2O2, and 10 μL of 1.5 mg / mL K-C3N4 / Cu,Fe nanozyme to a 96-well ELISA plate. Then add 20 μL of tetracycline (OTC), oxytetracycline (TC), or chlortetracycline (CTC) solutions of different concentrations. Make up the total volume to 300 μL with pH 7.2 PIPES buffer, mix well, and measure ΔA at 417 nm. ΔA = A - A0, where A and A0 are the absorbance values ​​of the reaction system before and after adding tetracycline, respectively. Plot a standard curve with tetracycline concentration on the x-axis and ΔA on the y-axis. The regression equation, correlation coefficient, relative standard deviation, and linear range are shown in Table 2. The results are shown in Table 2. Figure 9 .

[0020] 5. Preparation of working curves for tetracycline antibiotics (CL) Add 50 μL of 50 mmol / L luminol, 30 μL of 50 mmol / L H2O2, and 40 μL of 1.5 mg / mL K-C3N4 / Cu,Fe nanozyme to a 96-well microplate. Then add 20 μL of tetracycline (OTC), oxytetracycline (TC), or chlortetracycline (CTC) solutions of different concentrations. Make up the total volume to 300 μL with 0.1 mmol / L pH 11.0 NaHCO3-Na2CO3 buffer solution, mix well, and measure ΔI at 458 nm. CL Intensity, ΔI CL = I-I0, where I and I0 are the chemiluminescence intensities of the reaction system without and with tetracycline, respectively. The tetracycline concentration is plotted on the x-axis. ΔI CL Using the vertical axis as the ordinate, a standard curve was plotted, and the regression equation, correlation coefficient, relative standard deviation, linear range, etc., were obtained (see Table 2). The results are shown below. Figure 10 .

[0021]

[0022] 6. Method Specificity Study: Tetracycline antibiotics were mixed with other antibiotics, and the effects of coexisting antibiotics and metal ions on the tetracycline antibiotics in the above detection system were detected. The concentration of tetracycline antibiotics was 10 mg / kg, and the concentration of the interfering substances was 50 mg / kg. Figure 11 These are commonly used antibiotics (metronidazole MNZ, penicillin sodium PEN, clarithromycin CLR, chloramphenicol CAP, cephalexin LEX, azithromycin AZM, roxithromycin ROX, sulfamethoxazole SMZ), and metal ions (Ca). 2+ Mg 2 + K + Na+ The study investigated the effects of amino acids (glutamic acid Glu, L-arginine L-Arg, L-serine L-Ser, and cysteine ​​L-Cys) on the detection system of tetracycline antibiotics. The results showed that only tetracycline antibiotics had a good inhibitory effect on the K-C3N4 / Cu,Fe nanozyme catalytic colorimetric and CL systems. This result indicates that the K-C3N4 / Cu,Fe nanozyme has good selective specificity for tetracycline antibiotics.

[0023] 7. Determination of tetracycline antibiotic residues in aquatic product samples (1) Sample preparation: Accurately weigh 5 g of homogenized fish sample (accurate to 0.001 g), place it in a 50 mL polyethylene centrifuge tube, and add 20 mL, 20 mL, and 10 mL of 0.1 mol·L⁻¹ solution in sequence. -1 The sample was extracted three times with Na2EDTA-Mclvaine buffer solution in an ice-water bath for 10 min each time, followed by vortex extraction for 3 min. Then, it was centrifuged at 10,000 rpm for 10 min. The supernatants were combined and the volume was adjusted to 25 mL to obtain the sample solution to be tested. (2) Sample determination: Add 10 μL of 5 mmol / L ABTS, 10 μL of 50 mmol / L H2O2, and 10 μL of 1.5 mg / mL K-C3N4 / Cu,Fe nanozyme to a 96-well microplate, then add 100 μL of sample extraction buffer and replenish to a total volume of 300 μL with pH 7.2 PIPES buffer. Mix well and measure ΔA at 417 nm. Substitute ΔA into the regression equation of step 5. Tetracycline antibiotics were not detected in the fish sample. Simultaneously, add 50 μL of 50 mmol / L luminol, 30 μL of 50 mmol / L H2O2, and 40 μL of 1.5 mg / mL K-C3N4 / Cu,Fe nanozyme to a 96-well microplate, then add 100 μL of sample extraction buffer and measure with 0.1 mmol / L pH 11.0 buffer. The NaHCO3-Na2CO3 buffer solution was added to a total volume of 300 μL, mixed well, and ΔI was measured at a wavelength of 458 nm. CL Intensity, ΔI CL Substituting into the regression equation of step 5, tetracycline antibiotics were not detected in the fish samples.

[0024] Example 2: Tetracycline antibiotic residues in milk samples 1. Mix 3.50 g of melamine and 1.50 g of KCl, then grind with agate slurry for 15 min. Transfer the mixture to a corundum boat and calcine at 550 °C for 5 h under nitrogen atmosphere at a heating rate of 5 °C / min. After cooling to room temperature, grind the material into powder. Take 1.50 g of the ground powder and grind it together with 4.50 g of KSCN, and place it in a covered corundum boat. Heat the powder at 400 °C for 2 h under nitrogen atmosphere at a heating rate of 5 °C / min, then heat it again at 500 °C for 40 min. Disperse the collected powder in 40 mL of 2 mol / L HNO3, and then magnetically stir at room temperature for 3 h. Wash the obtained powder three times with ethanol and dry it in a vacuum oven to obtain K-C3N4.

[0025] 2. Disperse 0.20 g tartaric acid, 0.20 g L-tryptophan, 0.50 g FeCl3, and 0.50 g CuCl2 ultrasonically in 30 mL of deionized water. Transfer the mixture to a polytetrafluoroethylene (PTFE) container and heat at 160 °C for 12 h. After natural cooling, centrifuge at 8000 rpm for 15 min. Filter the supernatant through a 0.22 μm filter membrane and collect the filtrate to obtain Cu,Fe-CDs.

[0026] 3. Mix 10 mg of K-C3N4 with 5 mL of Cu,Fe-CDs and 3 mL of deionized water, sonicate for 10 min, and then stir for 90 min to obtain K-C3N4 / Cu,Fe.

[0027] 4. Preparation of colorimetric working curves for tetracycline antibiotics: Same as in Example 1.

[0028] 5. Preparation of working curves for tetracycline antibiotics: Same as in Example 1.

[0029] 6. Determination of tetracycline antibiotic residues in milk samples (1) Sample preparation: Take 2.0 mL of milk sample, dilute it to 20 mL with 0.01 mol / L PBS (pH 7.4), then add 2 mL of chloroform and 2 mL of 10% trichloroacetic acid, which can precipitate proteins and dissolve organic matter such as fats. Sonicate for 30 min, then centrifuge at 12000 rpm for 10 min, collect the supernatant, filter it three times with a 0.45 μm ultrafiltration membrane to obtain the sample solution to be tested; (2) Determination of tetracycline antibiotic residues in milk samples: Same as in Example 1, tetracycline antibiotics were not detected in milk samples.

[0030] Example 3: Determination of Tetracycline Antibiotic Residues in Honey 1. Preparation of K-C3N4 / Cu,Fe nanozymes: Same as in Example 1; 2. Preparation of colorimetric working curves for tetracycline antibiotics: Same as in Example 1; 3. Preparation of working curves for tetracycline antibiotics (CL): Same as in Example 1; 4. Determination of tetracycline antibiotic residues in honey (1) Sample preparation: Same as in Example 2; (2) Determination of tetracycline antibiotic residues in honey: Same as in Example 1, tetracycline antibiotic residues were not detected in honey.

[0031] Example 4: Determination of tetracycline antibiotic residues in pork samples 1. Preparation of K-C3N4 / Cu,Fe nanozymes: Same as in Example 1; 2. Preparation of colorimetric working curves for tetracycline antibiotics: Same as in Example 1; 3. Preparation of working curves for tetracycline antibiotics (CL): Same as in Example 1; 4. Determination of tetracycline antibiotic residues in pork (1) Sample preparation: Same as in Example 2; (2) Determination of tetracycline antibiotic residues in pork: Same as in Example 1, tetracycline antibiotic residues were not detected in pork.

[0032] (1) Processing of pork samples: Same as in Example 2; (2) Determination of tetracycline antibiotic residues in pork: Same as in Example 1, tetracycline antibiotic residues were not detected in pork.

[0033] (3) Recovery and precision experiments: Two different concentrations of OA and Tyr standard solutions were added to the above samples respectively; each concentration was measured in parallel 3 times, the spiked recovery rate was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 3. The spiked recovery rates of OA and Tyr were found to be 83.9% to 108.8%, and the RSD was 2.01% to 4.90%. This method has good accuracy and precision.

[0034]

[0035] The method for determining tetracycline antibiotic residues established in this invention has higher sensitivity, is rapid and simple to use, has a short processing time, low processing cost, and is easy to operate. It does not require large-scale instruments and equipment, and has strong advantages in practical detection.

Claims

1. A method for colorimetric / chemiluminescent detection of tetracycline antibiotics using K-C3N4 / Cu,Fe nanozymes under neutral conditions, characterized in that... Includes the following steps: (1) Mix 3.00-3.50 g of melamine and 1.00-1.50 g of KCl, then grind with agate slurry for 10-15 min, transfer the mixture to a corundum boat, and calcine at 550 ℃ for 4-5 h under nitrogen atmosphere at a heating rate of 5 ℃ / min. After cooling to room temperature, the material is ground into powder. 1.00-1.50 g of the ground powder is ground together with 3.50-4.50 g of KSCN and placed in a covered corundum boat. The powder is heated at 400 °C for 1-2 h in a nitrogen atmosphere at a heating rate of 5 °C / min, and then heated at 500 °C for 30-40 min. The collected powder is dispersed in 40-50 mL of 1-2 mol / L HNO3 and then magnetically stirred at room temperature for 2-3 h. The powder is washed 2-3 times with ethanol and dried in a vacuum oven to obtain K-C3N4. (2) Disperse 0.10-0.20 g tartaric acid, 0.10-0.20 g L-tryptophan, 0.30-0.50 g FeCl3 and 0.30-0.50 g CuCl2 in 20-30 mL of deionized water by ultrasonication. Transfer the mixture to a polytetrafluoroethylene container and heat at 160 °C for 10-12 h. After natural cooling, centrifuge and filter through a 0.22 μm filter membrane. Collect the filtrate to obtain Cu,Fe-CDs. (3) Mix 8-10 mg of K-C3N4 with 4-5 mL of Cu,Fe-CDs and 2-3 mL of deionized water, sonicate for 5-10 min, and then stir for 60-90 min to obtain K-C3N4 / Cu,Fe; (4) After mixing K-C3N4 / Cu,Fe nanozyme solution, 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid solution and H2O2, add tetracycline antibiotic solutions of different concentrations and pH 7.2 PIPES buffer solution. After mixing, let stand for 5-10 min and measure absorbance at 417 nm wavelength to determine the linear relationship between tetracycline antibiotic concentration and absorbance value ΔA and obtain regression equation, where ΔA=A-A0, A and A0 are the absorbance values ​​of the reaction system without tetracycline antibiotic and with tetracycline antibiotic, respectively. (5) After mixing K-C3N4 / Cu,Fe nanozyme solution, luminol solution, and H2O2, add tetracycline antibiotic solutions of different concentrations and pH 11.0 NaHCO3-Na2CO3 buffer solution. After reacting for 5-10 min, measure the chemiluminescence intensity ΔI at 458 nm using an enzyme-linked immunosorbent assay (ELISA) reader. CL Determine the relationship between tetracycline antibiotic concentration and ΔI CL The linear relationship is used to obtain the regression equation, where ΔI CL = I-I0, where I and I0 are the chemiluminescence intensities of the reaction systems without tetracycline antibiotics and with tetracycline antibiotics, respectively; (6) Following the steps in (4) or (5), measure ΔA and ΔI of the sample solution to be tested. CL Substitute the values ​​into the regression equation to obtain the concentration of tetracycline antibiotics in the sample solution.

2. The method according to claim 1, characterized in that: The tetracycline antibiotics mentioned include one of tetracycline, oxytetracycline, and chlortetracycline.

3. The method according to claim 1, characterized in that: The concentration of K-C3N4 / Cu,Fe nanozyme solution is 1.5 mg / mL, and the amount added is 5-30 μL; the concentration of 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid solution is 5 mmol / L, and the amount added is 10-15 μL; the concentration of luminol solution is 50 mmol / L, and the amount added is 80-90 μL; the concentration of H2O2 is 20 mmol / L, and the amount added is 20-50 μL.

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