Me-based 2+ Method for detecting tetracycline antibiotics using MA-IPA-SA hydrogel film

Abstract

The application relates to a Me 2+ The method for detecting tetracycline antibiotics by using the MA-IPA@SA hydrogel film comprises the following steps: S1, in-situ synthesis of a MA-IPA@SA hydrogel by using melamine MA and isophthalic acid IPA as hydrogen bond active ligands and sodium alginate SA as a carrier; Me 2+ Crosslinking SA forms a porous structure hydrogel film to obtain the Me 2+ MA-IPA@SA hydrogel film; S2, reaction of the Me 2+ MA-IPA@SA hydrogel film with a TCs standard solution for more than 6 min; S3, placing the Me 2+ MA-IPA@SA hydrogel film into a UV analyzer, and analyzing the RGB value of the fluorescence color by using software; a standard working curve is established by using the fluorescence ratio as a TCs solution concentration response signal; and S4, the same method is used to process a sample to be detected, and the TCs concentration in the solution is quantified by combining the standard working curve.

Description

Technical Field

[0001] This invention relates to the field of antibiotic detection technology, specifically to a method based on Me 2+ A method for detecting tetracycline antibiotics using @MA-IPA@SA hydrogel membranes. Background Technology

[0002] Tetracycline antibiotics (TCs) are a class of broad-spectrum antibiotics produced by Streptomyces, including tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX). Currently, TCs are widely used to treat bacterial infections in animals and humans, or as feed additives to promote livestock growth, due to their low cost, simple synthesis, and good therapeutic effects. It is estimated that the global annual consumption of TCs reaches 100,000 to 200,000 tons. Excessive use of TCs inevitably leads to residues in animal products, which accumulate through the food chain, causing various health problems in humans, such as liver damage, allergic reactions, and increased drug resistance. Therefore, developing a simple, rapid, and sensitive point-of-care testing (POCT) method for TCs is of great significance.

[0003] Currently, established methods for TC detection mainly include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), electrochemical methods, capillary electrophoresis (CE), and enzyme-linked immunosorbent assay (ELISA). While these methods offer high accuracy and sensitivity, they are mostly expensive, cumbersome, and time-consuming, requiring specialized techniques and personnel. The inherent limitations of traditional detection techniques restrict their further application in TC point-of-care testing (POCT), especially in remote areas with insufficient laboratory resources. Fluorescence analysis, due to its ease of operation, high sensitivity, low cost, and real-time detection capabilities, is increasingly used for TC detection. Various fluorescent nanomaterials, such as metal-organic frameworks (MOFs), quantum dots (QDs), upconversion fluorescent nanoparticles (UCNPs), and metal nanoparticles, have been used for TC detection. However, the complex synthesis processes of these fluorescent nanomaterials often involve the use of organic solvents and metal elements, which can cause secondary pollution. Therefore, developing simple, low-toxicity, and environmentally friendly fluorescent nanomaterials is particularly important.

[0004] Hydrogen-bonded organic frameworks (HOFs) are novel porous crystalline materials self-assembled from organic building blocks through hydrogen bonding, electrostatic interactions, π-π superposition, and van der Waals interactions. Compared to MOFs and covalent organic frameworks (COFs), HOFs possess superior properties such as mild synthesis conditions, ordered pore structures, easy regeneration, good biocompatibility, and low toxicity. They hold broad application prospects in adsorption separation, fluorescence sensing, catalysis, proton conduction, and biomedicine. Due to the unique physicochemical properties of their inherent fluorescent emitting units, some HOFs, such as HOF-20, HOF-1111, and Lumi-HOF@Tb, have been used as fluorescent probes in sensing research. However, fluorescence sensing research using HOFs is still in its early stages, and the relatively weak non-covalent interactions between HOFs lead to low stability, limiting their applications. Furthermore, most hydrogel-based analytical devices typically detect targets through a single signal response, which is susceptible to background, environmental, and instrument-related factors, resulting in low detection sensitivity. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method based on Me 2+ The method for detecting tetracycline antibiotics using @MA-IPA@SA hydrogel membranes overcomes the disadvantage of poor stability of HOF nanomaterials. At the same time, the ratio fluorescence sensing technology with self-calibration function solves the problem that the detection of targets by relying solely on a single signal response is easily affected by background, environment and instrument, resulting in low sensitivity.

[0007] (II) Technical Solution

[0008] Based on Me 2+ The method for detecting tetracycline antibiotics using @MA-IPA@SA hydrogel membranes includes:

[0009] S1. Using melamine (MA) and isophthalic acid (IPA) as hydrogen-bonding active ligands and sodium alginate (SA) as a carrier, MA-IPA@SA hydrogel was synthesized in situ. MA-IPA binds to SA through hydrogen bonding and electrostatic interaction.

[0010] Then use divalent metal ions Me 2+ Crosslinking MA-IPA@SA hydrogels forms a porous hydrogel membrane, which encapsulates and immobilizes MA-IPA to obtain Me. 2+ @MA-IPA@SA hydrogel membrane; the divalent metal ions Me 2+ For Ca 2+ Zn 2+ Or Ba 2+ ;

[0011] S2, Take the Me prepared in step S1 2+ The @MA-IPA@SA hydrogel membrane was reacted with solutions of tetracycline antibiotics (TCs) of different known concentrations for more than 6 minutes, and then removed to obtain drug-loaded Me. 2+ @MA-IPA@SA hydrogel membrane; during the reaction, TCs permeate through Me 2+ @MA-IPA@SA hydrogel membrane and the internal Me 2+ Fluorescent complexes are produced;

[0012] S3, Me carrying the drug 2+ The @MA-IPA@SA hydrogel membrane was placed in a dark-box UV analyzer, and its fluorescence color under ultraviolet light was digitally photographed and recorded. The digital photograph was analyzed using software to obtain the RGB values. The drug loading Me was calculated using a ratio fluorescence sensing method. 2+ The RGB values ​​of the response fluorescence of the @MA-IPA@SA hydrogel membrane compared to the blank Me 2+ @MA-IPA@SA hydrogel membrane fluorescence + drug loading Me 2+ The ratio of the sum of the RGB values ​​of the response fluorescence of the @MA-IPA@SA hydrogel membrane was used as the TCs solution concentration response signal to establish a standard working curve.

[0013] S4. Process the TCs solution of unknown concentration to be tested using the same method as S2-S3, analyze and calculate the fluorescence ratio, and quantify the TCs concentration in the TCs solution of unknown concentration to be tested by combining the standard working curve.

[0014] According to a preferred embodiment of the present invention, in S1, the divalent metal ion Me 2+ For Ca 2+ .

[0015] According to a preferred embodiment of the present invention, S1 includes the following steps:

[0016] Step 1: Add isophthalic acid (IPA) to water, heat to no more than 90°C and stir until IPA is completely dissolved to obtain an IPA dispersion;

[0017] Step 2: Add melamine (MA) to water, heat to no more than 90°C and stir until MA is completely dissolved to obtain an MA solution;

[0018] Step 3: Mix sodium alginate (SA) powder with melamine (MA) solution and stir continuously to obtain MA-SA hydrogel suspension; while stirring, add the IPA dispersion from Step 1 dropwise into the MA-SA hydrogel suspension, and continue stirring after the addition is complete to obtain MA-IPA@SA hydrogel; wherein, MA-IPA is bound to SA through hydrogen bonding and electrostatic interaction.

[0019] Step 4: Transfer the MA-IPA@SA hydrogel to a mold, add calcium salt solution to soak the MA-IPA@SA hydrogel in the mold overnight, so that SA cross-links under the action of calcium ions to form a porous sodium alginate membrane. The porous sodium alginate membrane encapsulates MA-IPA inside, resulting in a Ca@MA-IPA@SA hydrogel membrane, which is then cleaned and set aside.

[0020] According to a preferred embodiment of the present invention, in step 1, the concentration of the IPA dispersion is 0.06 mmol / L-0.07 mmol / L; in step 2, the concentration of the MA solution is 0.06 mmol / L-0.07 mmol / L; the molar ratio of IPA to MA in the IPA dispersion and the MA solution is 0.95-1.05:0.95-1.05; preferably 1:1.

[0021] According to a preferred embodiment of the present invention, in step 3, the amount of sodium alginate (SA) powder used is 1.90-2.2g of sodium alginate (SA) powder added for every 1 mmol of melamine (MA).

[0022] In step 3, the IPA dispersion from step 1 is added dropwise to the MA-SA hydrogel suspension while being vigorously stirred; after the addition is complete, stirring continues for 3-3.5 hours to ensure the reaction is fully completed.

[0023] According to a preferred embodiment of the present invention, in step 4, the calcium salt solution is a calcium nitrate solution, a calcium chloride solution, or a calcium lactate solution, preferably a calcium chloride solution; the concentration of the calcium salt solution is 0.2-0.35 mmol / mL; the overnight soaking is performed at 4°C overnight. Finally, the Ca@MA-IPA@SA hydrogel membrane is washed with plenty of water to remove excess impurities, including impurities not encapsulated and fixed by the SA membrane.

[0024] According to a preferred embodiment of the present invention, in S2, the concentrations of the plurality of TCs solutions with different known concentrations are distributed in the range of 0.01-1000 μg / mL; following the method of steps S2-S3, each TCs solution sample is performed in 3 parallel steps, and the average value is taken to form a standard working curve.

[0025] According to a preferred embodiment of the present invention, in S2, the specific wavelength of ultraviolet light is 365nm.

[0026] According to a preferred embodiment of the present invention, when Me in S1 2+ For Ca 2+When the blank Ca@MA-IPA@SA hydrogel membrane that had not reacted with the TCs solution showed obvious blue fluorescence under ultraviolet light, it showed green room temperature phosphorescence (RTP) after the ultraviolet light was turned off. After reacting with the TCs solution, the Ca@MA-IPA@SA hydrogel membrane showed green fluorescence, and as the concentration of TCs in the TCs solution increased, the Ca@MA-IPA@SA hydrogel membrane showed an obvious change in fluorescence color from blue to green. In step S3, the Ca@MA-IPA@SA hydrogel membrane that had reacted with the TCs solution was taken out and placed in a darkroom UV analyzer. The fluorescence color of the hydrogel membrane under ultraviolet light was digitally photographed and recorded. The digital photo was analyzed using ImageJ software to obtain the RGB values. The ratio of G / (G+B) was used as the TCs solution concentration response signal to create a standard working curve.

[0027] It should be noted that when Me 2+ Zn 2+ Or Ba 2+ When the blank Zn@MA-IPA@SA hydrogel membrane or Ba@MA-IPA@SA hydrogel membrane, which has not been soaked in TCs solution, exhibits blue fluorescence under ultraviolet light, the fluorescence originates from MA-IPA. However, the drug-loaded hydrogel membrane with TCs adsorbed may not appear green (depending on the type of metal ion), and specific analysis is required for each case. For example, if the blank Zn@MA-IPA@SA is blue, while the drug-loaded hydrogel membrane is red, and the red color becomes more pronounced with higher TCs concentrations, then the final fluorescence ratio calculation method is as follows: digitally photograph and record the fluorescence color of the hydrogel membrane under ultraviolet light, analyze the digital image using ImageJ software to obtain the RGB values; use the R / (R+B) ratio as the TCs solution concentration response signal. Although the specific methods for calculating the fluorescence ratio differ, they can all refer to the above method and do not depart from the scope of this invention.

[0028] According to a preferred embodiment of the present invention, S3 is implemented based on a novel smartphone integrated ratio fluorescence sensing platform; specifically, a smartphone is used as a fluorescence analyzer, and the fluorescence color of the hydrogel under 365nm ultraviolet light is recorded by taking a digital photo with the smartphone, and the RGB value is obtained by analyzing the digital photo with ImageJ software; a ratio fluorescence sensing method is used to create a standard working curve by using the fluorescence ratio as the TCs solution concentration response signal.

[0029] (III) Beneficial Effects

[0030] The technical effects of this invention include:

[0031] (1) This invention encapsulates and fixes MA-IPA (HOFs) inside a porous sodium alginate membrane (SA membrane), overcoming the poor stability of MA-IPA nanomaterials. Simultaneously, the analyte molecule TCs can diffuse freely within the SA membrane, allowing TCs to form fluorescent complexes with metal ions. Therefore, the SA membrane does not affect the detection of the analyte molecule. MA-IPA is fixed to SA through hydrogen bonding and electrostatic interactions, making it less prone to loss. Furthermore, sodium alginate (SA) is a natural polymer extracted from seaweed or brown algae. Its porous membrane possesses advantages such as negligible fluorescence emission and background color, transparency to visible light, high drug loading capacity, good biocompatibility, and non-toxicity. This invention is the first to apply Ca@MA-IPA@SA hydrogel membrane to the detection of TCs, showing broad application prospects in on-site food safety monitoring.

[0032] (2) This invention employs ratio fluorescence sensing technology with self-calibration function. Compared with existing technologies that detect targets through a single signal response, this invention is less susceptible to the influence of background, environment, and instrument, thereby improving detection sensitivity. Self-calibration using two different wavelengths helps improve the accuracy of the results. This ratio fluorescence sensing strategy has good sensitivity, selectivity, and portability, and can detect TC, OTC, and CTC within 6 minutes, with detection limits of 5.1, 7.7, and 32.7 ng / mL, respectively.

[0033] (3) In a preferred embodiment of the present invention, the quantitative detection of TCs is realized based on a novel smartphone integrated ratio fluorescence sensing platform. Unlike most existing technologies that rely on HOFs detection in the laboratory, the present invention utilizes a novel smartphone integrated ratio fluorescence sensing platform, which does not require expensive equipment and can be used for rapid and on-site detection of TCs. It can quickly and on-site determine the concentration of TCs in milk and pork samples and has good practical application value. Attached Figure Description

[0034] Figure 1 SEM images of MA-IPA, SA, MA-IPA@SA and Ca@MA-IPA@SA hydrogel membranes.

[0035] Figure 2 (A) and (B) are the fluorescence spectra of MA-IPA and Ca@MA-IPA@SA hydrogel films, respectively; Figure 2 (C) and (D) are the time-resolved fluorescence decay and fitting curves of MA-IPA and Ca@MA-IPA@SA hydrogel films, respectively. Figure 2 (E) and (F) are the time-resolved phosphorescence decay and fitting curves of MA-IPA and Ca@MA-IPA@SA hydrogel films, respectively.

[0036] Figure 3 The correlation curves between the fluorescence ratio of G / (G+B) and the logarithms of the concentrations of TC, OTC, and CTC in the standard sample are shown.

[0037] Figure 4 (A) shows the changes in the fluorescence ratio G / (G+B) of streptomycin (STR), florfenicol (FF), chloramphenicol (CAP), gentamicin (GS), kanamycin (KAN), doxycycline (DOX), oxytetracycline (OTC), chlortetracycline (CTC), and a mixture of these antibiotics and TC on the Ca@MA-IPA@SA hydrogel membrane (Blank); (B) shows the changes in the fluorescence ratio G / (G+B) of streptomycin (STR), florfenicol (FF), chloramphenicol (CAP), gentamicin (GS), kanamycin (KAN), doxycycline (DOX), oxytetracycline (OTC), chlortetracycline (CTC), and a mixture of these antibiotics and TC on the Ca@MA-IPA@SA hydrogel membrane (Blank); + K + Mg 2+ Cl - CO3 2- The changes in the fluorescence ratio G / (G+B) of the interfering substance and representative amino acids (glutamic acid (Glu), cysteine ​​(Cys), histidine (His), glycine (Gly), lysine (Lys)) and the fluorescence ratio of the mixed solution of interfering substances and TC were studied.

[0038] Figure 5 This is a comparison of the recovery rate and standard deviation obtained when using the detection method of the present invention and ELISA to quantitatively determine TCs in spiked milk and pork samples in Example 4. Detailed Implementation

[0039] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] The reagents used in the embodiments of this invention are described below:

[0041] Melamine (MA), isophthalic acid (IPA), sodium alginate (SA), tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), doxycycline (DOX), and trichloroacetic acid were purchased from Shanghai Maclean Biochemical Co., Ltd. (Shanghai, China). Streptomycin (STR), florfenicol (FF), chloramphenicol (CAP), gentamicin (GS), kanamycin (KAN), glutamic acid (Glu), cysteine ​​(Cys), histidine (His), glycine (Gly), and lysine (Lys) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing). Calcium chloride (CaCl2), sodium chloride (NaCl), magnesium chloride (MgCl2), potassium chloride (KCl), and sodium carbonate (Na2CO3) were also purchased. 3)Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Aladdin Holding Group Co., Ltd. (Beijing). The TC enzyme-linked immunosorbent assay (ELISA) kit was purchased from Beacon Analytical Systems, USA. Unless otherwise specified, all chemicals and reagents are analytical reagent (AR) grade and require no further purification before use. In the following examples, fluorescence ratio analysis was performed on a ratio fluorescence sensing platform integrated into a smartphone.

[0042] Example 1

[0043] In this embodiment, a Ca@MA-IPA@SA hydrogel membrane was synthesized using the following method:

[0044] (1) MA-IPA@SA hydrogel was prepared by in-situ synthesis. First, IPA (0.1662 g, 0.001 mol) was added to 15 mL of H2O and stirred in an oil bath at 70 °C for 1 h to obtain an IPA dispersion. Then, MA (0.1262 g, 0.001 mol) was mixed with 15 mL of H2O and added to a 100 mL three-necked flask. The mixture was stirred and heated in an oil bath at 70 °C until the powder was completely dissolved. Subsequently, sodium alginate powder (SA) (2.0 g) was mixed with the MA solution and stirred continuously to obtain an MA-SA hydrogel suspension. At the same time, under vigorous stirring, the above IPA dispersion was added dropwise to the MA-SA hydrogel suspension, and stirring was continued for 3 h to obtain a white MA-IPA@SA hydrogel.

[0045] (2) A Ca@MA-IPA@SA hydrogel membrane was prepared by crosslinking sodium alginate (SA) with calcium salts. A CaCl2 solution was prepared by dissolving 0.3 g of solid CaCl2 in 10 mL of H2O. The MA-IPA@SA hydrogel was then dropped into a circular plastic mold. The CaCl2 solution was then added and the membrane was soaked overnight at 4°C to form the hydrogel membrane. Finally, the Ca@MA-IPA@SA hydrogel membrane was washed thoroughly with plenty of water to remove excess elements.

[0046] Comparative Example 1

[0047] This example demonstrates the synthesis of MA-IPA (HOFs) material. The synthesis method is as follows: Melamine (MA) (0.1262 g, 0.001 mol) was mixed with 30 mL of H2O and added to a round-bottom flask. The mixture was stirred in a 70°C oil bath until the MA powder was completely dissolved. Then, isophthalic acid (IPA) (0.1662 g, 0.001 mol) was added to the above solution, and the mixture was stirred in a 70°C oil bath for 3 hours. After the reaction was completed, the solution was cooled to room temperature, and the sample was washed three times with H2O to separate excess reactants. Finally, the product was dried overnight in a 60°C vacuum oven to obtain pure white needle-like MA-IPA crystals.

[0048] Product performance characterization:

[0049] 1. The morphology and structure of MA-IPA (prepared in Comparative Example 1), SA, MA-IPA@SA (prepared in step (1) of Example 1), and Ca@MA-IPA@SA hydrogel membrane (prepared in Example 1) were characterized using scanning electron microscopy (SEM). SEM images of each sample are shown below. Figure 1 As shown.

[0050] Figure 1 Image (A) is a SEM image of MA-IPA; the image illustrates that MA-IPA was synthesized based on the hydrogen bonding interaction between MA and IPA, exhibiting a needle-like crystal structure. Figure 1 (B) is the SEM image of SA; the illustration shows that the SEM image of the freeze-dried SA film shows a multipolar cross-linked network structure, which is beneficial for the loading of functional materials and the free diffusion of target molecules. Figure 1 (C) is the SEM image of MA-IPA@SA; the figure shows that MA-IPA@SA hydrogel was prepared by in-situ synthesis and the SEM image shows that MA-IPA is uniformly distributed in the SA hydrogel network framework. Figure 1 (D) is a SEM image of the Ca@MA-IPA@SA hydrogel membrane; the illustration shows the interaction between SA and Ca. 2+ Ionic crosslinking forms a Ca@MA-IPA@SA hydrogel membrane with a flat surface.

[0051] 2. Testing the photoluminescence properties of Ca@MA-IPA@SA hydrogel membranes

[0052] The fluorescence spectra of MA-IPA (prepared in Comparative Example 1) and Ca@MA-IPA@SA hydrogel films (prepared in Example 1) were analyzed under 300 nm excitation. Figure 2 As shown in (A), the blue emission at 352 nm and green emission at 470 nm of MA-IPA are attributed to fluorescence (S1→S0 transition) and phosphorescence (T1→S0 transition), respectively. Under ambient conditions, MA-IPA powder exhibits significant blue fluorescence under 365 nm UV light and significant green phosphorescence when the UV light is turned off. For comparison, see [reference needed]. Figure 2 As shown in (B): the fluorescence spectrum of the Ca@MA-IPA@SA hydrogel film did not change significantly, and MA-IPA retained its photoluminescence properties after being combined with SA hydrogel. However, according to time-resolved spectral measurements... Figure 2As shown in (C) and (D), the fluorescence lifetime of the Ca@MA-IPA@SA hydrogel film (2.54 ns) is higher than that of MA-IPA (1.87 ns). These results indicate that the free movement of MA-IPA in the hydrogel is restricted, suppressing non-radiative relaxation pathways and further improving the fluorescence lifetime. Both MA-IPA and Ca@MA-IPA@SA hydrogel films also exhibit ultralong RTP, with a phosphorescence lifetime of 1.60 s (see Figure 1). Figure 2 (E) and (F)).

[0053] Example 2

[0054] This embodiment uses the Ca@MA-IPA@SA hydrogel membrane prepared above and detects TCs standards based on ratio fluorescence sensing to verify the accuracy and sensitivity of the detection method of the present invention.

[0055] The Ca@MA-IPA@SA hydrogel membrane was immersed in 200 μL of TC aqueous solutions of different concentrations (0.01-1000 μg / mL) for 6 min, and then the hydrogel was removed. During the immersion process, TCs in the solution dispersed into the porous SA and formed fluorescent complexes with calcium ions. The hydrogel was placed in a darkroom UV analyzer (using a smartphone as the fluorescence analyzer), and the fluorescence color change of the hydrogel under ultraviolet light (365 nm) was recorded using the smartphone camera. The digital photos were analyzed using ImageJ software to obtain the red-green-blue (RGB) values, and the G / (G+B) value was calculated as the response signal of TC concentration to plot a standard working curve. Each gradient concentration (0, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, 1000 μg / mL) was measured three times, and the average value was used to plot the standard working curve.

[0056] like Figure 3 As shown in (A), under 365 nm ultraviolet light irradiation, the fluorescence color changed significantly from blue to green with increasing TC concentration. Furthermore, the G / (G+B) ratio increased with increasing TC concentration, and the G / (G+B) ratio showed a good linear correlation with the logarithm of TC concentration in the range of 0.01-1000 μg / mL. The linear fit of the plotted and calibrated curve was Y = 0.0445X + 0.4565, with a correlation coefficient R0. 2 =0.9956. The limit of detection was calculated to be 5.1 ng / mL (11.5 nM) based on three times the standard deviation (SD) of 10 blank measurements.

[0057] Using the same method, the Ca@MA-IPA@SA hydrogel membrane was immersed in 200 μL of OTC aqueous solutions of different concentrations (0.01-1000 μg / mL) for 6 min, and then the hydrogel was removed for detection. Each concentration gradient (0, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, 1000 μg / mL) was measured three times, and the average value was used to plot a standard working curve. Figure 3 As shown in (B), the G / (G+B) value is linearly proportional to the logarithm of the OTC concentration in the range of 0.01–1000 μg / mL. The detection limit for OTC concentration is 7.7 ng / mL (16.7 nM).

[0058] Using the same method, the Ca@MA-IPA@SA hydrogel membrane was immersed in 200 μL of CTC aqueous solutions of different concentrations (0.05-1000 μg / mL) for 6 min, and then the hydrogel was removed for detection. Each concentration gradient (0, 0.05 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, 1000 μg / mL) was measured three times, and the average value was used to plot a standard working curve. Figure 3 As shown in (C), the G / (G+B) value is linearly proportional to the logarithm of the CTC concentration in the range of 0.05–1000 μg / mL. The detection limit for CTC concentration is 32.7 ng / mL (68.3 nM).

[0059] Example 3

[0060] This embodiment uses the Ca@MA-IPA@SA hydrogel membrane prepared above and detects TC containing multiple interfering substances based on ratio fluorescence sensing to verify the specificity of the detection method of the present invention.

[0061] Interfering substances include different types of antibiotics (oxytetracycline (OTC), chlortetracycline (CTC), doxycycline (DOX), streptomycin (STR), florfenicol (FF), chloramphenicol (CAP), gentamicin (GS), and kanamycin (KAN)), and common inorganic ions (Na+). + K + Mg 2+ Cl - CO3 2- The specificity of the method was validated using glutamic acid (Glu), cysteine ​​(Cys), histidine (His), glycine (Gly), and lysine (Lys). Interfering substances (100 μg / mL) and mixtures thereof with TC (1 μg / mL) at different ratios were analyzed under the same conditions. The detection method is described in Example 2. The validation results are as follows: Figure 4 As shown.

[0062] like Figure 4 As shown in (A), no significant change in fluorescence color occurred when different types of other antibiotics were added to the solution containing the Ca@MA-IPA@SA hydrogel membrane. Only after the addition of oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX) did the fluorescence color change from blue to green, and the fluorescence ratio G / (G+B) increased significantly, indicating that this method has good selectivity for TCs.

[0063] Simultaneously, inorganic ions and various amino acids, such as TC-loaded Ca@MA-IPA@SA hydrogel membrane, were added to the solution. Figure 4 As shown in (B), when high concentrations of interfering substances (inorganic ions and amino acids) coexist with TC, the G / (G+B) value hardly changes compared to when TC exists alone, and the effect of the coexisting substances on the fluorescence color of the hydrogel is negligible. The aforementioned experimental results demonstrate that the detection method of this invention has high specificity for TCs and the detection results are stable.

[0064] Example 4

[0065] This embodiment demonstrates the application of the detection method of the present invention to actual samples. The fresh milk and pork samples used in the experiment were purchased from a local supermarket.

[0066] Add 5 mL of milk to 5 mL of 3% (v / v) trichloroacetic acid and sonicate for 20 min to remove protein precipitate. Centrifuge at 12000 rpm for 10 min, then filter the supernatant through a 0.22 μm membrane to remove lipids. The processing method for pork samples is roughly the same as that for milk samples: grind 2 g of pork sample, add it to 4 mL of 3% (w / v) trichloroacetic acid, and vortex for 10 min. Centrifuge at 12000 rpm for 10 min, then filter the supernatant through a 0.22 μm membrane to collect the supernatant. Finally, adjust the pH of the treated milk and pork samples to neutral using NaOH.

[0067] Samples were spiked with four different concentrations of TC (0.05 μg / mL, 0.5 μg / mL, 5 μg / mL, and 50 μg / mL), and detected using the detection method of this invention (see Example 2 for specific procedures) and the ELISA kit, respectively. The ELISA kit can accurately detect TC in the concentration range of 0.3-9.6 ng / mL. Therefore, samples should be gradually diluted when performing ELISA testing.

[0068] Experimental results are as follows Figure 5As shown, the recovery rates of the detection methods of the present invention are significantly higher than those of the ELISA kits. Meanwhile, the RSD (standard deviation) of the detection methods of the present invention is generally smaller than that of the ELISA kits, indicating that the quantitative detection results of the present invention are more accurate and have higher stability compared to the ELISA kits.

[0069] Experimental results show that the detection method proposed in this invention has high accuracy and sensitivity for detecting trace amounts of TCs in samples. In summary, this invention successfully proposes a ratio fluorescence sensing platform based on Ca@MA-IPA@SA hydrogel membrane for the detection of TCs. In this invention, MA-IPA serves as a fluorescence internal reference, and Ca... 2+ As a specific recognition unit for TCs, the fluorescence color of the Ca@MA-IPA@SA hydrogel membrane changes from blue to green as the TC concentration increases. TC concentration can be quantitatively detected using a ratio fluorescence sensing platform integrated into a smartphone, without the need for any complex instruments. This method has advantages such as simple operation, rapid reaction, high sensitivity, and strong selectivity, and has successfully determined the TC concentration in milk and pork samples, demonstrating good practical application value.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Based on Me 2+ A method for detecting tetracycline antibiotics using @MA-IPA@SA hydrogel membranes, characterized in that... It includes: S1. Using melamine (MA) and isophthalic acid (IPA) as hydrogen-bonding active ligands and sodium alginate (SA) as a carrier, MA-IPA@SA hydrogels were synthesized in situ. MA-IPA binds to SA through hydrogen bonding and electrostatic interactions; then, divalent metal ions (Me) were used... 2+ Crosslinking MA-IPA@SA hydrogels forms a porous hydrogel membrane, which encapsulates and immobilizes MA-IPA to obtain Me. 2+ @MA-IPA@SA hydrogel membrane; the divalent metal ions Me 2+ For Ca 2+ Zn 2+ Or Ba 2+ ; S2, Take the Me prepared in step S1 2+ The @MA-IPA@SA hydrogel membrane was reacted with solutions of tetracycline antibiotics (TCs) of different known concentrations for more than 6 minutes, and then removed to obtain drug-loaded Me. 2+ @MA-IPA@SA hydrogel membrane; during the reaction, TCs permeate through Me 2+ @MA-IPA@SA hydrogel membrane and the internal Me 2+ Fluorescent complexes are produced; S3, Me carrying the drug 2+ The @MA-IPA@SA hydrogel membrane was placed in a dark-box UV analyzer, and its fluorescence color under ultraviolet light was digitally photographed and recorded. The digital photographs were analyzed using software to obtain RGB values. The drug loading capacity (Me) was calculated using a ratio fluorescence sensing method. 2+ The RGB values ​​of the response fluorescence of the @MA-IPA@SA hydrogel membrane compared to the blank Me 2+ @MA-IPA@SA hydrogel membrane fluorescence + drug loading Me 2+ The ratio of the sum of the RGB values ​​of the response fluorescence of the @MA-IPA@SA hydrogel membrane was used as the TCs solution concentration response signal to establish a standard working curve. S4. Process the TCs solution of unknown concentration to be tested using the same method as S2-S3, analyze and calculate the fluorescence ratio, and quantify the TCs concentration in the TCs solution of unknown concentration to be tested by combining the standard working curve.

2. The method according to claim 1, characterized in that, In S1, the divalent metal ion Me 2+ For Ca 2+ .

3. The method according to claim 1, characterized in that, S1 includes the following steps: Step 1: Add isophthalic acid (IPA) to water, heat to no more than 90°C and stir until IPA is completely dissolved to obtain an IPA dispersion; Step 2: Add melamine (MA) to water, heat to no more than 90°C and stir until MA is completely dissolved to obtain an MA solution; Step 3: Mix sodium alginate (SA) powder with melamine (MA) solution and stir continuously to obtain MA-SA hydrogel suspension; while stirring, add the IPA dispersion from Step 1 dropwise into the MA-SA hydrogel suspension, and continue stirring after the addition is complete to obtain MA-IPA@SA hydrogel; wherein, MA-IPA is bound to SA through hydrogen bonding and electrostatic interaction. Step 4: Transfer the MA-IPA@SA hydrogel to a mold, add calcium salt solution to soak the MA-IPA@SA hydrogel in the mold overnight, so that SA cross-links under the action of calcium ions to form a porous sodium alginate membrane. The porous sodium alginate membrane encapsulates MA-IPA inside, resulting in a Ca@MA-IPA@SA hydrogel membrane, which is then cleaned and set aside.

4. The method according to claim 3, characterized in that, In step 1, the concentration of the IPA dispersion is 0.06 mmol / L-0.07 mmol / L; in step 2, the concentration of the MA solution is 0.06 mmol / L-0.07 mmol / L; the molar ratio of IPA to MA in the IPA dispersion and MA solution is 0.95-1.05:0.95-1.05; in step 3, the amount of sodium alginate (SA) powder used is 1.90-2.2 g of sodium alginate (SA) powder added for every 1 mmol of melamine (MA).

5. The method according to claim 3, characterized in that, In step 4, the calcium salt solution is calcium nitrate solution, calcium chloride solution, or calcium lactate solution; the concentration of the calcium salt solution is 0.2-0.35 mmol / mL; overnight soaking is performed at 4°C.

6. The method according to claim 1, characterized in that, In S2, the concentrations of the multiple known TCs solutions are distributed in the range of 0.01-1000 μg / mL; following the method in steps S2-S3, each TCs solution sample is performed in triplicate, and the average value is used to create a standard working curve.

7. The method according to claim 1, characterized in that, In S2, the ultraviolet light wavelength is 365nm.

8. The method according to claim 1, characterized in that, When Me in S1 2+ For Ca 2+ In step S3, the Ca@MA-IPA@SA hydrogel membrane that has reacted with the TCs solution is taken out and placed in a darkroom UV analyzer. The fluorescence color of the hydrogel membrane under ultraviolet light is recorded by digital photography. The digital photo is analyzed using ImageJ software to obtain the RGB values. The ratio of G / (G+B) is used as the TCs solution concentration response signal to create a standard working curve.

9. The method according to claim 1, characterized in that, S3 is based on a new smartphone integrated ratio fluorescence sensing platform.

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