A visual detection probe of tetracycline antibiotics, a distinguishing and quantitative detection method and application of the probe in food detection

Abstract

The application discloses a tetracycline antibiotic visual detection probe, a distinguishing and quantitative detection method and application of the probe in food detection, and belongs to the technical field of antibiotic detection. The detection probe is bio-MOF-1-XO, which is prepared by compounding bio-MOF-1 and dimethyl phenol orange (XO) in an alkaline Tris buffer solution. The probe is placed in an alkaline system, and under excitation of 350-380 nm, the probe can produce characteristic fluorescent color, realizes rapid visual distinguishing of four tetracycline antibiotics (TCs), i.e. tetracycline, oxytetracycline, doxycycline and chlortetracycline, and can collect fluorescent spectra or extract fluorescent image RGB parameters to establish a standard curve, and realizes high-sensitivity quantitative detection. The probe has the advantages of simple preparation, rapid detection, good visual effect and strong anti-interference ability, can simultaneously complete distinguishing and quantification of multiple TCs in a single system, can be used for antibiotic residue detection in animal-derived food such as honey and pork, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic detection technology, specifically a visual detection probe for tetracycline antibiotics, a method for distinguishing and quantifying detection, and the application of the probe in food detection. Background Technology

[0002] Tetracycline antibiotics (TCs) are a class of broad-spectrum antibiotics with a naphthalene core. Commonly used types include tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DC). TCs, OTCs, and CTCs are natural antibiotics, while DCs are semi-synthetic antibiotics. TCs are widely used in livestock and aquaculture to prevent bacterial infections and promote animal growth. However, improper use can easily lead to high residues in animal-derived foods, threatening food safety. my country has established clear limits for TC residues in animal-derived foods: the maximum residue limits for TCs, OTCs, and CTCs are 200–1200 μg / kg. –1 DC with a longer half-life has a relatively low maximum residue limit, ranging from 10 to 600 μg kg. –1 Given the differences in maximum residue limits for different TCs in animal-derived foods, developing methods capable of distinguishing and quantifying multiple TCs is of significant practical importance.

[0003] Currently, there are many methods for detecting total toxic substances (TCs), mainly including high-performance liquid chromatography (HPLC), surface-enhanced Raman scattering (SERS), electrochemical sensing, colorimetry, and fluorescence methods. Among these, HPLC is the traditional method for TC detection. While it offers high accuracy and sensitivity, it suffers from drawbacks such as high cost, long processing time, complex operation, and high operator skill requirements. Compared to HPLC, fluorescence methods offer advantages such as ease of operation, low cost, and high visualization. The detection principle of fluorescence methods involves regulating the fluorescence signal output through the interaction between the target analyte and the fluorescent probe, such as changing the emission peak position or intensity, thereby achieving the identification of the target analyte. However, current fluorescence methods still have significant limitations in TC detection. For four structurally similar TCs, it is difficult to simultaneously achieve specific identification, visual differentiation, and accurate quantitative detection in a single system.

[0004] Based on this, the development of a fluorescent probe and detection method that is easy to operate, has good visualization effects, and can simultaneously achieve rapid differentiation and quantitative detection of TC, OTC, CTC and DC is of great practical significance for ensuring the quality and safety of animal-derived foods. Summary of the Invention

[0005] The purpose of this invention is to provide a visual detection probe for tetracycline antibiotics, a method for distinguishing and quantifying them, and the application of this probe in food testing. The probe is composed of a metal-organic framework material bio-MOF-1 and xylenol orange (XO) to form bio-MOF-1-XO. When suspended in an alkaline solution, the four types of TCs are detected and distinguished through different fluorescence color changes. As the antibiotic concentration increases, the fluorescence intensity of the probe gradually increases and the color change becomes more obvious. There is a good non-linear relationship between concentration and visualization change, and the detection limit is low, which can realize the quantitative detection of TCs, thereby meeting the needs of distinguishing and detecting TC residues in animal-derived foods.

[0006] The objective of this invention is achieved through the following technical solution: A visual detection probe for TCs is prepared by combining bio–MOF–1 and XO in an alkaline Tris buffer solution, denoted as bio–MOF–1–XO.

[0007] As some possible embodiments of this application, the alkaline Tris buffer solution has a pH of 8–10 and a concentration of 5–20 mM.

[0008] As some possible embodiments of this application, the bio–MOF–1 is prepared by reacting adenine, 4,4'-biphenyldicarboxylic acid and Zn(Ac)2·2H2O in DMF and nitric acid aqueous solution under reflux at 120–140 °C for 12–36 h.

[0009] Furthermore, to achieve the above objectives, this application also provides a method for distinguishing and quantitatively detecting TCs, comprising the following steps: (1) Place the bio–MOF–1–XO probe in an alkaline Tris buffer solution to obtain the probe solution; (2) Add the test sample containing TCs to the probe solution, and the test mixture is obtained after the reaction; (3) Observe the fluorescence color of the test mixture under 350–380 nm excitation to achieve visual differentiation of TCs; at the same time, collect fluorescence spectra or extract RGB parameters of fluorescence images to achieve quantitative detection.

[0010] As one of the possible implementation methods of this application, in step (2), the sample to be tested is an animal-derived food, including honey and pork.

[0011] As some possible implementations of this application, in step (2), the reaction temperature is 20–30 °C and the reaction time is 30–60 min.

[0012] As some possible implementations of this application, in step (2), the TCs include TC, OTC, DC, and CTC; the fluorescent colors that can be visually distinguished are: TC is yellow, OTC is green, DC is orange-yellow, and CTC is purple-red.

[0013] As some possible implementations of this application, in step (3), the quantitative detection linear ranges of TC, OTC, DC, and CTC are 0.3–35.0 μM, 0.4–35.0 μM, 0.4–35.0 μM, and 0.3–35.0 μM, respectively, and the detection limits are 25.6 nM, 35.1 nM, 42.5 nM, and 24.1 nM, respectively.

[0014] As some possible implementations of this application, in step (3), the quantitative detection is achieved by establishing a standard curve between the RGB parameters and the antibiotic concentration; wherein TC, OTC, and DC are the sum of the G and R channel values, and CTC is the sum of the R, G, and B channel values.

[0015] Furthermore, to achieve the above objectives, this application also provides the application of visual detection probes in food testing.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides for the first time a bio–MOF–1–XO-based visual detection probe for TCs. The probe is constructed from a metal-organic framework material bio–MOF–1–XO and XO composite. When suspended in an alkaline solution, it can achieve rapid visual differentiation of four TCs—tetracycline, oxytetracycline, chlortetracycline, and doxycycline—through characteristic fluorescence color changes. This effectively solves the technical problem that existing fluorescence methods are unable to simultaneously and specifically identify, differentiate, and quantify TCs with highly similar structures.

[0017] 2. As the concentration of TCs increases, the fluorescence intensity of the probe of this invention gradually increases and the color change becomes more obvious. There is a good non-linear relationship between antibiotic concentration and visual changes. Moreover, the detection limit is low and the linear range is suitable. Combined with the RGB parameters of a smartphone, it can achieve highly sensitive and accurate quantitative detection of four types of TCs.

[0018] 3. The probe and detection method of this invention are simple to operate, have good visualization effects, and strong anti-interference ability. They maintain excellent selectivity even in the presence of interfering substances such as inorganic ions, amino acids, and other antibiotics. They can be directly applied to the differentiation and quantitative detection of TCs residues in animal-derived foods such as honey and pork. The detection results have high recovery rate, good repeatability, and are accurate and reliable, providing an efficient and practical new detection method for ensuring the quality and safety of animal-derived foods. Attached Figure Description

[0019] Figure 1 Material structure, spectrum and morphology characterization diagrams; among which, (A) experimental and simulated PXRD spectra of bio–MOF–1 and PXRD spectra of bio–MOF–1 and bio–MOF–1–XO; (B) absorption spectra of XO before and after adding bio–MOF–1; (C) SEM image of bio–MOF–1; (D) SEM image of bio–MOF–1–XO; Figure 2 Fluorescence spectrum and CIE chromaticity diagram of the probe at pH 9; (A) Fluorescence spectrum of bio–MOF–1–XO before and after the addition of TCs at pH 9; (B) CIE chromaticity diagram of bio–MOF–1–XO after the addition of TCs at pH 9. Figure 3 Fluorescence spectra of probes under different concentrations of tetracycline antibiotics at pH 9; among them, the fluorescence spectra of probes under (A) TC, (B) OTC, (C) DC, and (D) CTC are shown. Figure 4 : Fluorescence images and quantitative standard curves of the probes; among them, fluorescence images of the probes under the action of TC, (B) OTC, (C) DC, and (D) CTC; standard curves between the RGB values ​​and the content of fluorescence images of TC, (F) OTC, (G) DC, and (H) CTC. Figure 5 Selectivity and anti-interference performance of probes against tetracycline antibiotics; among them, the selectivity and anti-interference performance of (A and B)TC, (C and D)OTC, (E and F)DC, and (G and H)CTC in the presence of potential interfering substances in animal-derived foods (assessed based on fluorescence images and RGB values). Figure 6 Identification of various TCs in animal-derived foods. Detailed Implementation

[0020] 1. Experimental Section 1.1 Materials and Instruments NaF, NaCl, NaBr, KI, KMnO4, Na2CO3, Na2SO4, Na2C2O4, Na2HPO4, Na2S2O8, Zn(Ac)2·2H2O, GaCl3, LiNO3, NaNO3, KNO3, Mg(NO3)2·6H2O, Ca(NO3)2·4H2O, Fe(NO3)3·9H2O, Sr(NO3)2、Cr(NO3)3·9H2O、 Al(NO3)3·9H2O, L-alanine (L-Ala), L-lysine (L-Lys), DL-phenylalanine (DL-Phe), L-cysteine ​​(L-Cst), DL-aspartic acid (DL-Asp), L-glutamic acid (L-Glu), L-tyrosine (L-Tyr), DL-methionine (DL-Met), metronidazole (MDZ), thiamphenicol (TAP), roxithromycin (ROX), clarithromycin (CLA), ornidazole (ODZ), sulfathiazole (STZ), sulfamethoxazole (SMZ), sulfamethoxypyrimidine (SMM), sulfadiazine (SM2), tris(hydroxymethyl)aminomethane (Tris), adenine, 4,4'-biphenylcarboxylic acid, chlortetracycline hydrochloride, tetracycline hydrochloride, doxycycline hydrochloride, and oxytetracycline hydrochloride were all purchased from Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Trifluoroacetic acid (TFA, 99) wt N,N-dimethylformamide (DMF, 99.5%), HgSO4, and NaAc·3H2O were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). wt %), nitric acid (HNO3, 65–68 wt %), hydrochloric acid (36–38%) wt %), ethanol (99.7%) wt %), methanol (99.5%) wt %) and acetonitrile (99.9%) wt %) was purchased from Xilong Scientific Co., Ltd. (Guangdong, China).

[0021] All the above materials are of analytical grade and can be used without purification. Acetonitrile (HPLC grade, 99.99%) wt %) and TFA (HPLC grade, 99.99%) wt The solution (%) was purchased from Xilong Scientific Co., Ltd. (Guangdong, China). Ultrapure water was used in the experiment. Tris buffer solution (10 mM) was selected to maintain the pH value within the range of 7-9.

[0022] UV-Vis absorption spectra were measured using a UV-2700 UV-Vis spectrometer (Shimadzu, Japan). Fluorescence spectra were recorded using an RF-6000 fluorescence spectrometer (Shimadzu, Japan). The positions and intensities of diffraction peaks in the materials were measured using a Miniflex 600 powder X-ray diffractometer (PXRD, Rigaku, Japan). The morphology of the materials was measured using an S-3000 scanning electron microscope (SEM, Hitachi, Japan). TCs in the actual samples were determined using an Agilent 1200 high-performance liquid chromatograph (HPLC, Agilent, USA) equipped with a UV-Vis absorption detector. Chromatographic analysis of TCs was performed on a C18 column (150 mm × 4.6 mm, 5 μm) at 30 °C, with the mobile phase consisting of TFA (0.08%). φ ) and acetonitrile (70 / 30, v / v The solution consists of an aqueous solution of [amount], with a mobile phase flow rate of 1.0 mL / min. –1 The determination was performed with a detector wavelength of 350 nm and a sample injection volume of 10 μL.

[0023] 1.2 Preparation of bio–MOF–1-XO materials Adenine (0.27 g, 2 mmol) and 4,4'-biphenyl dicarboxylic acid (0.97 g, 4 mmol) were dispersed in 40 mL of DMF, and Zn(Ac)₂·2H₂O (1.32 g, 6 mmol) was dispersed in 120 mL of DMF. The three solutions were mixed in a 500 mL round-bottom flask, and then 16 mL of DMF and 16 mL of HNO₃ aqueous solution (2 M) were added. The mixture was refluxed and stirred at 130 °C for 24 h. After the reaction was complete, the mixture was cooled to room temperature. The precipitate was collected by centrifugation (9000 rpm, 3 min), washed once with 16 mL of DMF, and twice with 16 mL of ethanol to remove impurities. After drying at 60 °C, a white powder bio–MOF–1 was obtained, with a yield of 92.80%.

[0024] Prepare an aqueous solution of bio–MOF–1 by dispersing bio–MOF–1 in Tris buffer (pH 9, 10 mM). Disperse bio–MOF–1 (4 mg / mL) in Tris buffer to prepare an aqueous solution. –1 An aqueous solution of bio–MOF–1 (200 μL) and XO (1 mM, 30 μL) was mixed in Tris buffer (pH 9, 10 mM, 1770 μL). The bio–MOF–1 and XO interact at pH 9 to form an alkaline aqueous solution of bio–MOF–1–XO.

[0025] 1.3 Detection of TCs To distinguish TCs, bio–MOF–1 (4 mg / mL) was used. –1 An aqueous solution of XO (1 mM, 30 μL) and TCs (pH 9, 10 mM, 1730 μL) was mixed in Tris buffer (pH 9, 10 mM, 1730 μL). An aqueous solution of TCs (2 mM, 40 μL) was added to the probe solution, and the mixture was reacted at 25 °C for 50 min. Subsequently, fluorescence spectra were measured using a fluorescence spectrometer under 365 nm excitation, and photographs were taken under a 365 nm lamp. RGB parameters were extracted from the fluorescence photographs using a smartphone app called Color Picker.

[0026] To quantify TCs, bio–MOF–1 (4 mg mL) was used. –1 Aqueous solutions of TCs (200 μL) and XO (1 mM, 30 μL) were mixed in Tris buffer (pH 9, 10 mM, 1730 μL) to form a probe solution. Aqueous solutions of each TC (40 μL) were added to the probe solution, and the mixture was reacted at 25 °C for 50 min. Subsequently, fluorescence spectra were measured using a fluorescence spectrometer under 365 nm excitation, and photographs were taken under a 365 nm lamp. RGB parameters were extracted from the fluorescence photographs using a smartphone app called Color Picker.

[0027] The substances that may be present in animal-derived foods mainly include cations and anions (Li). + Na + K + Ca 2+ Mg 2+ 、Sr 2 + Ga 3+ Cr 3+ Al 3+ Fe 3+ F – Cl – ,Br – I – Ac – MnO4 – CO3 2– SO4 2– C2O4 2– HPO4 2– and S2O8 2–The study included amino acids (sarcosine (SA), L-alanine (L-Ala), L-lysine (L-Lys), DL-phenylalanine (DL-Phe), L-cysteine ​​(L-Cst), DL-aspartic acid (L-Asp), L-glutamic acid (L-Glu), L-tyrosine (L-Tyr), and DL-methionine (DL-Met)) and drug molecules (ornidazole (ODZ), metronidazole (MDZ), thiamphenicol (TAP), roxithromycin (ROX), clarithromycin (CLA), sulfathiazole (STZ), sulfamethoxazole (SMZ), sulfamethoxypyrimidine (SMM), and sulfadiazine (SM2)). To investigate the material's response to TCs, cations and anions, amino acids, and drug molecules, bio-MOF-1 was dispersed in Tris buffer (pH 9, 10 mM) to prepare an aqueous solution of bio-MOF-1. Then, bio-MOF-1 (4 mg / mL) was... –1 Aqueous solutions of bio–MOF–1 (200 μL) and XO (1 mM, 30 μL) were mixed in Tris buffer (pH 9, 10 mM, 1730 μL). Subsequently, aqueous solutions (2 mM, 40 μL) of potentially interfering animal-derived food matrices, such as TCs, anions, cations, amino acids, and drug molecules, were added to the probe solution, and the reaction was carried out at 25 °C for 50 min. Images were captured under a 365 nm light, and RGB parameters were extracted from the fluorescence images using a smartphone app called ColorPicker. To investigate the effects of anions, cations, amino acids, and drug molecules on TC recognition, bio–MOF–1 (4 mg / mL) was mixed with XO. –1 An aqueous solution of XO (1 mM, 30 μL) and 200 μL of amino acids and 1 mM of TCs was mixed in Tris buffer (pH 9, 10 mM, 1690 μL). Then, an aqueous solution of anions, cations, amino acids and drug molecules (2 mM, 40 μL) was added, followed by an aqueous solution of each TC (2 mM, 40 μL). The mixture was reacted at 25 °C for 50 min, and photographs were taken under a 365 nm light. The RGB parameters were extracted from the fluorescence photographs using a smartphone app called Color Picker.

[0028] 1.4 Actual Sample Testing Honey was purchased from a local supermarket in Haikou, China, and pretreated as follows: 1 g of honey was dissolved in 1 mL of water. The diluted honey was centrifuged at 9000 rpm for 5 min, and the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane for analysis using a probe. Pork was purchased from a local supermarket in Haikou, China, and pretreated according to the following steps: To extract TCs, 1 g of pork was cut into small pieces and mixed with a solution containing 70% methanol. φThe extract was prepared by homogenizing the honey and pork in an aqueous solution (5 mL). After homogenization for 10 min, the mixture was placed in an ultrasonic bath for 1 h and centrifuged at 9000 rpm for 3 min to collect the extract. To remove proteins and lipids, the extract (1 mL) was mixed with acetonitrile (1.5 mL). After homogenization for 30 min, the mixture was centrifuged at 9000 rpm for 3 min to collect the supernatant. The supernatant was filtered through a 0.22 μm organic phase filter membrane for TCs analysis. To prepare spiked honey and pork, different concentrations of TCs were added before pretreatment of the honey and pork. The treated samples were tested according to the procedures described in Section 1.3 for quantitative RGB analysis of TCs.

[0029] 2 Results and Discussion 2.1 Characteristics of the material ( Figure 1 ) Figure 1 As shown in Figure A, the consistency between experimental and simulated PXRD data for bio-MOF-1 confirms the successful construction of bio-MOF-1. SEM characterization reveals that the prepared bio-MOF-1 particles exhibit a polyhedral shape, such as... Figure 1 As shown in C. XO is a common metal indicator. Its structure contains carboxyl, amino, and hydroxyl groups. After metal coordination, the absorption band of XO redshifts from 430 nm to 570 nm. Therefore, the Zn in bio–MOF–1 2+ The application of materials can be expanded by coordinating with XO. For example... Figure 1 As shown in Figure B, XO has an absorption band at 430 nm. After adding bio–MOF–1, the absorption band of XO redshifts to 570 nm. This phenomenon indicates that XO reacts with Zn in bio–MOF–1. 2+ Coordination. For example... Figure 1 As shown in Figure A, bio–MOF–1 and bio–MOF–1–XO have the same diffraction peaks, confirming that the addition of XO preserves the crystal structure of bio–MOF–1. (See SEM image). Figure 1 D), bio–MOF–1–XO retains the polyhedral particle shape of bio–MOF–1, which proves that the introduction of XO preserves the morphology of bio–MOF–1.

[0030] 2.2 Differentiation of TCs ( Figure 2 ) like Figure 2 As shown in Figure A, the fluorescence emission spectra of bio–MOF–1–XO before and after the addition of TCs were investigated. For bio–MOF–1–XO, the fluorescence signals of the R (625 nm), G (525 nm), and B (425 nm) channels were in the off state. Figure 2A). After adding TC, OTC, and CTC to bio–MOF–1–XO, the fluorescence signals of the R (625 nm) and G (525 nm) channels were activated. Figure 2 A). However, for TC, OTC, and DC, the fluorescence intensity order of the R (625 nm) and G (525 nm) channels is R ≈ G, R>G, and RC, respectively. <G( Figure 2 A). After adding CTC to bio–MOF–1–XO, the fluorescence signals of the R (625 nm), G (525 nm), and B (425 nm) channels were activated. Figure 2 A). It can be observed that, for CTC, the fluorescence intensity order of R (625 nm), G (525 nm), and B (425 nm) is B>R>G ( Figure 2 A). The fluorescence emission spectrum of bio–MOF–1–XO after adding TC was converted into a CIE chromaticity diagram to calculate the corresponding fluorescence color ( Figure 2 B). In the presence of TC, OTC, DC, and CTC, the CIE chromaticity coordinates of the probe are (0.36179, 0.4105), (0.32675, 0.4004), (0.39394, 0.4014), and (0.20564, 0.10222), respectively, assigned to yellow, green, orange-yellow, and magenta. Figure 2 B). Further fluorescence images of the probe after adding TCs were generated. The probe showed no fluorescence emission, but in the presence of TC, OTC, DC, and CTC, the probe exhibited yellow, green, orange-yellow, and purple-red fluorescence emission, respectively. Figure 2 A), which is consistent with the simulation results of the CIE chromaticity diagram ( Figure 2 B).

[0031] The above results demonstrate that bio–MOF–1–XO can rapidly distinguish multiple TCs in alkaline solution by fluorescence color.

[0032] 2.3 Quantization of TCs ( Figure 3 and Figure 4 ) The fluorescence emission spectra of the probe before and after the addition of TCs were studied. Figure 5 After adding TC, OTC, and DC to bio–MOF–1–XO at pH 9, the fluorescence signals of the R (625 nm) and G (525 nm) channels were activated and gradually increased with increasing concentration. Figure 3 A–C). In the presence of CTC, the fluorescence signals of the probe's R (625 nm), G (525 nm), and B (425 nm) channels are activated and gradually increase with increasing concentration. Figure 3D). For the probe, increasing the concentration of TCs leads to a proportional enhancement of the RGB fluorescence signal, which is directly reflected in the visually observable change in fluorescence intensity. Fluorescence images of the probe were taken after adding different concentrations of TCs, as shown... Figure 4 As shown, the probe hardly emits light, but yellow, green, orange-yellow, and purple-red emission of the probe were observed in the presence of TC, OTC, DC, and CTC, respectively. The yellow, green, orange-yellow, and purple-red fluorescence emission of the probe increased with increasing concentrations of TC, OTC, DC, and CTC, respectively. Figure 4 (A–D). Therefore, the probe enables the quantitative detection of TCs by fluorescence intensity.

[0033] To improve quantification accuracy, RGB parameters were extracted from the fluorescence image using the Color Picker app. As the concentration of TCs increased, the RGB values ​​were proportional to the luminescence intensity of the RGB components in the fluorescence image. Therefore, a quantitative standard curve could be established between the RGB values ​​and the TC concentration. TC, OTC, and DC increased the fluorescence signal of the probe in the G (525 nm) and R (625 nm) channels. Figure 2 A) A quantitative standard curve was established between the sum of G and R values ​​and the concentrations of TC (0.3–35.0 μM), OTC (0.4–35.0 μM), or DC (0.4–35.0 μM). Figure 4 E–G). Similarly, due to the presence of CTC, the fluorescence signal in the R (625 nm), G (525 nm), and B (425 nm) channels of the probe is enhanced ( Figure 2 Therefore, a quantitative standard curve was established between the sum of R, G, and B values ​​and the CTC concentration (0.3–35.0 μM). Figure 4 H). The limit of detection (LOD) for TCs was further calculated, and all standard curves for TC quantification exhibited non-linear characteristics. Figure 4 E–H). According to the literature Analytical and Bioanalytical Chemistry, 2011, 401(9): 2881–2889, the LOD of the nonlinear standard curve is calculated using formula 3. σ / k Confirmed, among which σ Indicates the standard deviation of the blank measurement. k This represents the slope of the tangent line at the lowest concentration on the standard curve. Based on this calculation, the LOD of the probe for TC, OTC, DC, and CTC was determined to be 25.6 nM, 35.1 nM, 42.5 nM, and 24.1 nM, respectively.

[0034] 2.4 TCs identification and anti-interference capability The selectivity and anti-interference ability of probes against TCs, which are potential interfering substances in animal-derived foods, were investigated. Figure 5 ), including inorganic ions (Li ions) + Na + K + Ca 2+ Mg 2+ 、Sr 2+ Ga 3+ Cr 3+ Al 3+ Fe 3+ F – Cl – ,Br – I – Ac – MnO4 – CO3 2– SO4 2– C2O4 2– HPO4 2– and S2O8 2– Biomolecules (SA, L-Ala, L-Lys, DL-Phe, L-Cst, DL-Asp, L-Glu, L-Tyr, and DL-Met) and other drug compounds (ODZ, MDZ, TAP, ROX, CLA, STZ, SMZ, SMM, and SM2). For example... Figure 5 A, Figure 5 C Figure 5 E and Figure 5 As shown in G, the probe showed no fluorescence. The addition of TC, OTC, DC, and CTC resulted in different fluorescence responses, exhibiting yellow, green, orange-yellow, and purple-red fluorescence emission, respectively. In contrast, no fluorescence emission was observed when potential interfering substances were introduced. Figure 5 A, Figure 5 C Figure 5 E and Figure 5 These results clearly demonstrate the probe's excellent selectivity for TCs. Figure 5 A, Figure 5 C Figure 5 E and Figure 5 As shown in G, the probe maintains its characteristic fluorescence response (yellow for TC, green for OTC, orange-yellow for DC, and magenta for CTC) in the presence of potential interfering substances. These observations confirm that the probe has excellent anti-interference capabilities in TC detection. To achieve a rigorous evaluation of selectivity and anti-interference capabilities, a smartphone application called Color Picker was used to extract RGB parameters from the fluorescence images. The RGB values ​​were used to quantitatively assess the probe's selectivity and anti-interference capabilities. Figure 5 B Figure 5 D、 Figure 5 F and Figure 5 The results in H consistently demonstrate that the RGB-value-based evaluation method confirms that the probe exhibits excellent selectivity and anti-interference capabilities for TC detection.

[0035] 2.5 Detection of TCs in animal-derived foods ( Figure 6 ) At pH 9, bio–MOF–1–XO is used to distinguish various TCs in animal-derived foods. For example... Figure 6 As shown, when the probe was mixed with honey / pork samples containing TC, OTC, DC, or CTC, yellow, green, orange-yellow, or purple-red fluorescence emission was observed. These results indicate that bio–MOF–1–XO can distinguish multiple TCs in animal-derived foods at pH 9 and can be applied to the quantification of various TCs in animal-derived foods. At pH 9, bio–MOF–1–XO achieved recoveries of TCs in honey and pork ranging from 91.1% to 104.9%, with relative standard deviations (RSDs) of TCs within the range of 0.7% to 1.5% (Table 1). Furthermore, the reliability of this method was verified by high-performance liquid chromatography (HPLC), with recoveries of TCs in honey and pork ranging from 95.8% to 105.1% and RSDs of TCs within the range of 0.3% to 2.1% (Table 1). These results demonstrate that bio–MOF–1–XO can accurately quantify multiple TCs in animal-derived foods at pH 9.

[0036] Table 1. Analysis results of TCs in animal-derived foods at pH 9 ( n = 3) a Not detected.

Claims

1. A visual detection probe for tetracycline antibiotics, characterized in that, It is obtained by recombination of bio–MOF–1 and XO in alkaline Tris buffer solution, and is denoted as bio–MOF–1–XO.

2. The visual detection probe according to claim 1, characterized in that, The alkaline Tris buffer solution has a pH of 9–10 and a concentration of 5–20 mM.

3. The visual detection probe according to claim 1, characterized in that, The bio–MOF–1 was prepared by reacting adenine, 4,4'-biphenyldicarboxylic acid and Zn(Ac)2・2H2O in DMF and nitric acid aqueous solution under reflux at 120–140 °C for 12–36 h.

4. A method for differentiating and quantifying tetracycline antibiotics, characterized in that, Includes the following steps: (1) The bio-MOF-1-XO probe according to any one of claims 1–3 is placed in an alkaline Tris buffer solution to obtain a probe solution; (2) Add the test sample containing TCs to the probe solution, and the test mixture is obtained after the reaction; (3) Observe the fluorescence color of the test mixture under 350–380 nm excitation to achieve visual differentiation of TCs; at the same time, extract the RGB parameters of the fluorescence image to achieve quantitative detection.

5. The method for differentiating and quantifying tetracycline antibiotics according to claim 4, characterized in that, In step (2), the reaction temperature is 20–30 ℃ and the reaction time is 30–70 min.

6. The method for differentiating and quantitatively detecting tetracycline antibiotics according to claim 4, characterized in that, In step (2), the TCs include TC, OTC, DC, and CTC; the fluorescence colors that can be visually distinguished are: TC is yellow, OTC is green, DC is orange-yellow, and CTC is purple-red.

7. The method for differentiating and quantitatively detecting tetracycline antibiotics according to claim 4, characterized in that, In step (3), the linear ranges for quantitative detection of TC, OTC, DC and CTC are 0.3–35.0 μM, 0.4–35.0 μM, 0.4–35.0 μM and 0.3–35.0 μM, respectively, and the limits of detection are 25.6 nM, 35.1 nM, 42.5 nM and 24.1 nM, respectively.

8. The method for differentiating and quantitatively detecting tetracycline antibiotics according to claim 4, characterized in that, In step (3), the quantitative detection is achieved by establishing a standard curve between the RGB parameters and the antibiotic concentration; wherein TC, OTC, and DC are the sum of the G and R channel values, and CTC is the sum of the R, G, and B channel values.

9. The application of the visual detection probe according to any one of claims 1–3 in food detection.

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