Kanamycin sulfate identification fluorescence sensor and kanamycin sulfate detection method

Abstract

The invention provides a kanamycin sulfate recognition fluorescence sensor and a kanamycin sulfate detection method. According to the invention, the inhibition effect of kanamycin sulfate (KAN) on the activity of MOF-808-Al acetylcholin esterase (AChE) is combined with a sulfydryl response fluorescent metal nanocluster, and a method for detecting the KAN is developed. The MOF-808-Al shows AChE-like activity and is used for catalyzing the decomposition of acetylthiocholine into thiocholine; and the KAN can be used for inhibiting the AChE-like activity of the MOF-808-Al. Thiocholine attacks a metal core of a green emission gold nanocluster (Au NCs) by forming an Au-S bond, so that fluorescence quenching is caused. The blue luminous copper nanocluster (Cu NCs) is well protected by the template and the ligand, and the fluorescence of the blue luminous copper nanocluster (Cu NCs) has higher tolerance to thiocholine. The KAN is quantitatively detected by analyzing the evolution of green and blue light-emitting metal nanoparticles.

Description

Technical Field

[0001] The present invention relates to the field of veterinary drug kanamycin sulfate residue analysis and detection, and specifically to a fluorescence sensor for kanamycin sulfate recognition based on MOF inhibition reaction and thiol-responsive metal nanoclusters and a method for detecting kanamycin sulfate. Background Art

[0002] Kanamycin sulfate (KAN) is a type of drug composed of amino sugars linked to amino cycloalcohols by glycosidic bonds. KAN exhibits broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. Due to its high stability, low price, and excellent antibacterial activity, it is widely used in human and veterinary treatments. With the frequent and large-scale use of KAN, drug residues are released into food samples and the environment, and these residues can cause serious side effects. Therefore, many countries and regions have announced strict maximum residue limits (MRLs) for KAN residues. For example, the MRL of kanamycin sulfate in milk is 150 μg / kg. Therefore, the sensitive, selective, and rapid detection of KAN is of crucial significance for ensuring environmental and food safety.

[0003] Liquid chromatography (LC), as a standard method for kanamycin (KAN) detection, has advantages such as excellent separation ability, selectivity, and sensitivity. However, this method has limitations such as expensive instrument equipment, complex operation procedures, and the need for professional personnel to operate. To overcome these deficiencies, researchers have developed various optical analysis methods for KAN detection, including fluorescence analysis, colorimetry, surface-enhanced Raman spectroscopy (SERS), and chemiluminescence (CL) detection, etc. Among these methods, fluorescence analysis has received extensive attention due to its characteristics such as rapid response, simple operation, high sensitivity, and non-destructive detection. However, most reported fluorescent probes are monochromatic emission, which is easily affected by the external environment, resulting in low detection accuracy. In contrast, ratio fluorescence detection utilizes the self-calibration mechanism of dual emission centers, which can effectively eliminate systematic errors and improve the signal-to-noise ratio. By utilizing the multi-signal output of ratio fluorescence sensing of MOFs, it provides the possibility for constructing a highly sensitive and selective KAN fluorescence sensing platform.

[0004] Sensing based on enzyme inhibition provides another option, which realizes detection by monitoring the process of the analyte inhibiting the substrate conversion reaction. The commonly used enzymes are biological enzymes, such as acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, and alkaline phosphatase, and various analytes have been detected. However, these inhibition reactions based on biological enzymes lack sufficient selectivity and are prone to inactivation, and there is currently no report on the inhibition reaction of biological enzymes to KAN. Summary of the Invention

[0005] The object of the present invention is to provide a kanamycin sulfate (KAN) recognition fluorescence sensor and a method for detecting kanamycin sulfate. The present invention is a novel method for detecting KAN, which is realized based on the inhibitory effect of kanamycin sulfate on the AChE activity of MOF-808-Al and the fluorescence ratio response of mixed metal nanoclusters to thiocholine, and has high sensitivity and selectivity in detecting KAN.

[0006] The present invention is realized as follows:

[0007] By synthesizing a system with the AChE activity of MOF-808-Al and the fluorescence ratio response of mixed metal nanoclusters to thiocholine for the detection of KAN, an analytical detection method is constructed.

[0008] Specifically, the preparation method of MOF-808-Al includes the following steps:

[0009] (a) First, zirconium oxychloride octahydrate is dissolved in formic acid, 1,3,5-benzenetricarboxylic acid is added to an N,N-dimethylformamide solution, the solutions of zirconium oxychloride octahydrate and 1,3,5-benzenetricarboxylic acid are mixed, ultrasonically treated, then reacted, centrifuged, washed to obtain a solid, and finally dried to obtain MOF-808;

[0010] (b) MOF-808 is added to a DMF solution of aluminum nitrate, heated and stirred, and a solid is obtained by centrifugation, washed and dried to obtain MOF-808-Al.

[0011] The preparation method of Cu NCs includes the following steps: Polyvinylpyrrolidone is added to ultrapure water and ultrasonically dissolved, and the pH value is adjusted; then, ascorbic acid aqueous solution and copper sulfate pentahydrate solution are added to the above polyvinylpyrrolidone solution, and the reaction is carried out under continuous stirring. The obtained product is dialyzed through a dialysis membrane to obtain copper nanoclusters. The obtained copper nanoclusters need to be stored at 4 °C for standby.

[0012] The preparation method of Au NCs includes the following steps:

[0013] (a) 6-Aza-2-thiothymine containing NaOH is added to a solution of chloroauric acid trihydrate, and continuously stirred at room temperature in the dark, and ATT-Au NCs are synthesized by ultrafiltration purification;

[0014] (b) Arginine is added to the ATT-Au NCs solution, and gold nanoclusters are obtained after the reaction. The obtained gold nanoclusters are also stored in the dark at 4 °C.

[0015] The material prepared by the present invention can be used for the quantitative detection of KAN.

[0016] The method for detecting KAN substance includes the following steps:

[0017] (1) Preparation of Al 3+ Material MOF-808-Al for modifying MOF-808;

[0018] (2) Preparation of copper nanoclusters and gold nanoclusters;

[0019] (3) Preparation of aqueous solutions of kanamycin sulfate at different concentrations;

[0020] (4) Respectively mix the aqueous solutions of kanamycin sulfate at different concentrations prepared in step (3) with MOF-808-Al, then add acetylthiocholine and buffer for incubation. After the incubation ends, take the supernatant. Sequentially add copper nanoclusters and gold nanoclusters to the supernatant, and finally measure the ratio of the fluorescence intensities at 525 nm and 418 nm, and plot a linear curve of the fluorescence ratio versus the concentration of kanamycin sulfate;

[0021] (5) Mix the sample to be tested with MOF-808-Al, then add acetylthiocholine and buffer for incubation. After the incubation ends, take the supernatant. Sequentially add copper nanoclusters and gold nanoclusters to the supernatant, and finally measure the ratio of the fluorescence intensities at 525 nm and 418 nm, and calculate the concentration of kanamycin sulfate contained in the sample to be tested according to the linear curve of the fluorescence ratio versus the concentration of kanamycin sulfate in step (4).

[0022] The equation corresponding to the linear curve of the fluorescence ratio versus the concentration of kanamycin sulfate in step (4) above is: F 525 / F 418 = 2.294log 10 X - 2.408, where X represents the concentration of kanamycin sulfate, F 525 represents the fluorescence value at 525 nm, and F 418 represents the fluorescence value at 418 nm; the value range of X is 12.9 - 27.7 μM.

[0023] In the present invention, the fluorescence ratio response of MOF-808-Al and mixed metal nanoclusters to thiocholine is optimized. In steps (4) and (5), the optimized parameters are as follows: the concentration of MOF-808-Al is 2 mg / mL, and the volume is 30 μL; after the aqueous solutions of kanamycin sulfate at different concentrations (or the sample to be tested) are respectively mixed with MOF-808-Al, react at 37°C for 1 h; the concentration of acetylthiocholine is 15 mM, and the volume is 50 μL; the buffer is HEPES buffer, and the pH value of the buffer is 8.5; the temperature during the incubation with acetylthiocholine and buffer is 37°C, and the incubation time is 20 min; sequentially add 20 μL of copper nanoclusters to the supernatant, incubate for 10 min, then add 40 μL of gold nanoclusters, and incubate for 10 min.

[0024] Prepare aqueous KAN solutions with different concentrations. Utilize the inhibitory effect of KAN on the AChE-like activity of MOF-808-Al, and use the fluorescence ratio response of MOF-808-Al and mixed metal nanoclusters to thiocholine as a detection platform. Mix it with aqueous KAN solutions of different concentrations, react at 37 °C, measure the fluorescence spectrum, and calculate the fluorescence ratio. Take the concentration of KAN as the abscissa and the green-blue fluorescence ratio as the ordinate to determine the linear equation between the fluorescence ratio and the KAN concentration.

[0025] In the embodiments of the present invention, under the same synthesis conditions, different antibiotics and amino acids are selected to replace KAN to measure their fluorescence intensities, and it is found that their fluorescence intensities are negligible compared to the fluorescence intensity ratio of the KAN to be detected, indicating that this mode has good selectivity.

[0026] Meanwhile, by conducting standard addition experiments in actual samples, the accuracy of this method was evaluated. The added concentrations of the standard substance KAN were 14.7, 18.5, and 27.7 μM respectively. Record their fluorescence intensity ratios, and the recoveries of 98.2% - 108.8% were obtained, further confirming the reliability of the method for determination in the present invention.

[0027] The present invention combines the MOF inhibition reaction with thiol-responsive fluorescent metal nanoclusters (NCs) to prepare a fluorescent sensor for the detection and recognition of KAN. With the assistance of Lewis acid and metal - OH sites, Al 3+ -modified MOF-808 (MOF-808-Al) exhibits AChE-like activity and catalyzes the decomposition of acetylthiocholine (ATCh) into thiocholine. The chemical groups -OH and -NH in the KAN molecular skeleton 2 have a stronger binding ability to the Lewis acid sites than ATCh, thereby inhibiting the acetylcholinesterase-like activity of MOF-808-Al. In addition, thiocholine attacks the metal core of Au NCs, resulting in a decrease in fluorescence intensity. While the blue fluorescence of polymer-template Cu NCs is tolerant to thiocholine. Combining the inhibitory effect of KAN on the AChE-like activity of MOF-808-Al and the fluorescence ratio response of mixed metal nanoclusters to thiocholine, analyze the blue and green fluorescence signals through pattern recognition, and this fluorescent sensor shows good detection ability for KAN.

[0028] The raw materials used in the preparation of the materials of the present invention have low costs, the preparation method is simple, easy to operate, and can be mass-produced in batches. The detection system of the present invention has the ability to recognize substances related to KAN and conduct effective signal transduction, achieving a low background signal, high sensitivity and selectivity for KAN, and having broad potential application value. Description of the Drawings

[0029] Figure 1TEM images of Cu NCs and Au NCs prepared in Example 1 of the present invention; among them, a is the TEM image of Cu NCs, and b is the TEM image of Au NCs.

[0030] Figure 2 High-magnification TEM image of MOF-808-Al prepared in Example 2 of the present invention.

[0031] Figure 3 Fluorescence spectra of Cu NCs and Au NCs measured in Example 3 of the present invention; among them, a is the excitation-emission fluorescence spectrum of Cu NCs and Au NCs; b is the fluorescence spectrum with the volume of Cu NCs fixed and the volume of different Au NCs adjusted.

[0032] Figure 4 Absorption spectra of MOF-808-Al with AChE-like activity and the chromogenic reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) measured in Example 4 of the present invention.

[0033] Figure 5 Fluorescence spectra of the materials in Example 5 of the present invention; among them, a is the fluorescence spectrum of Cu / Au NCs and Cu / Au NCs plus MOF-808-Al and acetylthiocholine; b is the fluorescence spectrum of Cu NCs, Au NCs, and the fluorescence spectra of each of them plus acetylthiocholine and each of them plus MOF-808-Al and acetylthiocholine; c is the fluorescence spectrum of Cu NCs, Au NCs, and the fluorescence spectra of each of them plus thiocholine after addition.

[0034] Figure 6 Fluorescence ratios at 525 nm for different volumes of Au NCs in the presence and absence of MOF-808-Al with AChE-like activity in Example 6 of the present invention.

[0035] Figure 7 Fluorescence ratios at 525 nm for different concentrations of ATCh in the presence and absence of MOF-808-Al with AChE-like activity in Example 7 of the present invention.

[0036] Figure 8 Fluorescence ratios at 525 nm for different pH values in the presence and absence of MOF-808-Al with AChE-like activity in Example 8 of the present invention.

[0037] Figure 9 Fluorescence ratios at 525 nm for different reaction times in the presence and absence of MOF-808-Al with AChE-like activity in Example 9 of the present invention; among them, a is the change in fluorescence ratio at different incubation times of Cu NCs; b is the change in fluorescence ratio at different incubation times of Au NCs.

[0038] Figure 10 For the fluorescence ratio at 525 nm at different temperatures in the presence or absence of MOF-808-Al with pseudo-AChE activity in Example 10 of the present invention.

[0039] Figure 11 Fluorescence spectral and linear correlation curves of the fluorescence ratio response of MOF-808-Al and mixed metal nanoclusters to thiocholine as a detection platform for detecting KAN in Example 11 of the present invention; where a is the fluorescence spectral, and b is the linear correlation curve.

[0040] Figure 12 Result diagram of the selectivity ability to recognize KAN in Example 12 of the present invention.

[0041] Figure 13 Result diagram of the salt tolerance ability to recognize KAN in Example 13 of the present invention. Detailed implementation mode

[0042] The following examples are used to further illustrate the present invention in detail, but do not limit the present invention in any form.

[0043] Example 1

[0044] Add 1 g of polyvinylpyrrolidone (PVP) to 20 mL of ultrapure water, sonicate for 15 min, and adjust the pH to 6.0 with 0.5 M sodium hydroxide (NaOH) solution. Then, add 2 mL of ascorbic acid (AA) aqueous solution (0.1 M) and 0.2 mL of copper sulfate pentahydrate (CuSO 4 ·5H 2 O) solution (0.1 M) to the above PVP solution, and react for 6 days under continuous stirring. The obtained product is dialyzed through a dialysis membrane with a molecular weight of 30,000 for 24 h to obtain a copper nanocluster (Cu NCs) solution, which is stored at 4 °C for later use.

[0045] Add 6-aza-2-thiothymine (ATT, 15 mL, 80 mM) containing 0.2 M NaOH to chloroauric acid trihydrate (HAuCl 4 ·3H 2 O) solution (15 mL, 10 mg / mL), continuously stir for 1 h at room temperature in the dark, and purify the synthesized ATT-Au NCs solution by ultrafiltration (Millipore, 50 kDa). The obtained ATT-Au NCs are stored at 4 °C in the dark before use. Add 2 mL, 40 mM of arginine (Arg) to 18 mL of the prepared ATT-Au NCs solution, adjust the mixed solution to pH = 10, and react at 37 °C for 24 h to obtain a gold nanocluster (Au NCs) solution, which is stored in the dark at 4 °C.

[0046] After ultrasonic dispersion treatment of the Cu NCs and Au NCs solutions, they were dropped onto copper grids and then air-dried naturally. TEM was used to characterize them respectively, and the obtained results are as Figure 1 shown. As can be seen from Figure 1 , in the examples of the present invention, nano quantum dot materials were successfully synthesized. The particle size of the prepared Cu NCs material was 3.3 nm, and the particle size of the Au NCs material was 2.3 nm.

[0047] Example 2

[0048] Zirconium oxychloride octahydrate (ZrOCl 2 ·8H 2 O, 970 mg, 3 mM) was dissolved in 30 mL of formic acid, and 1,3,5-benzenetricarboxylic acid (210 mg, 1.26 mM) was added to 30 mL of N,N-dimethylformamide (DMF) solution. The formic acid solution of zirconium oxychloride octahydrate and the DMF solution of 1,3,5-benzenetricarboxylic acid were mixed, and the mixed solution was ultrasonically treated for about 10 minutes and then transferred to a glass jar made of Teflon. After reacting at 110 °C for 24 h, centrifugation (9500 rpm, 3 min) was carried out, and it was washed 3 times with DMF and acetone to obtain a solid. These solids were vacuum-dried at 100 °C for 1 h to prepare MOF-808.

[0049] MOF-808 (100 mg) was added to 40 mL of aluminum nitrate (Al(NO 3 ) 3 ) solution (0.1 M aluminum nitrate dissolved in DMF). The mixture was heated to 85 °C and continuously stirred for 6 h. After centrifugation (9500 rpm, 3 min), a solid was obtained, which was washed 3 times with DMF and acetone and vacuum-dried at 100 °C for 1 h to obtain the MOF-808-Al material modified with Al 3+ .

[0050] After ultrasonic dispersion treatment of the MOF-808-Al aqueous solution, it was dropped onto copper grids and then air-dried naturally. HAADF-STEM was used to characterize it, and the obtained results are as Figure 2 shown. As can be seen from Figure 2 , in the examples of the present invention, the MOF-808-Al material was successfully synthesized, and the particle size of the prepared material was 560 nm.

[0051] Example 3

[0052] Fluorescence tests were carried out on the Cu NCs and Au NCs prepared in Example 1, and the results are as Figure 3 shown. As shown in Figure 3As shown in a, Cu NCs and Au NCs exhibit strong blue and green fluorescence respectively, showing that the peaks of Cu NCs and Au NCs appear at 410 nm and 535 nm respectively. The fluorescence excitation spectra of Cu and Au have a relatively wide overlap in the range of 300 - 400 nm, and the overlap between the emission peaks is extremely small. A well-separated hump fluorescence emission peak is observed.

[0053] As Figure 3 shown in b, with the volume of Cu NCs fixed at 20 μL, by controlling the addition amounts of different Au NCs (0, 10, 15, 20, 22, 25, 28, 30 μL), the ratio between the blue and green emission components of Cu / Au NCs can be well adjusted. The figure shows that with the increase in the addition amount of Au NCs, the green luminescence intensity of Au NCs increases significantly, and the blue luminescence intensity of Cu NCs decreases, indicating that they have great potential in developing chemical analysis as fluorescence ratio probes.

[0054] Example 4

[0055] To verify the acetylcholinesterase (AChE)-like activity of MOF-808-Al, acetylthiocholine (ATCh) (50 μL, 15 mM) and 500 μL of HEPES buffer (0.1 M, pH = 9.0) were added to MOF-808-Al (2 mg / mL) with different volumes (0, 10, 20, 30, 40, 50, 60 μL), incubated at 37 °C for 20 min, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 150 μL, 4 mM) was added to the supernatant (150 μL) for reaction for 3 min, and the absorbance at 400 - 550 nm was measured. MOF-808-Al decomposes the substrate (ATCh) into acetic acid and thiocholine. As Figure 4 shown, when MOF-808-Al is introduced into the mixture of DTNB and ATCh, the absorption intensity increases at 412 nm, and the absorption intensity depends on the concentration of MOF-808-Al. The decomposition product thiocholine reacts with DTNB to form a yellow complex, demonstrating that MOF-808-Al has AChE-like activity.

[0056] Example 5

[0057] MOF-808-Al and ATCh were introduced into the Cu / Au NCs system. ATCh (50 μL, 15 mM) and 500 μL of HEPES buffer (0.1 M, pH = 8.5) were added to 30 μL of MOF-808-Al (2 mg / mL), and incubated at 37 °C for 20 min. 20 μL of Cu NCs was added to the supernatant (150 μL), incubated for 10 min, and then 40 μL of Au NCs was added and incubated for 10 min. The fluorescence spectrum was measured at an excitation wavelength of 350 nm. As Figure 5 shown in a, since MOF-808-Al has AChE-like activity, it can catalyze the generation of thiocholine from ATCh, resulting in a decrease in the fluorescence intensity in the green region and a slight increase in the fluorescence intensity in the blue region.

[0058] To further understand the fluorescence response of Cu / Au NCs to thiocholine, the product of the decomposition of MOF-808-Al and ATCh, parallel experiments were conducted. As Figure 5 shown in b, Cu NCs and Au NCs were separately introduced into the MOF-808-Al and ATCh systems for reaction. For example, ATCh + Cu NCs, ATCh (50 μL, 15 mM) and 500 μL of H 2 O were added to 30 μL of H 2 O, incubated at 37 °C for 20 min. 20 μL of Cu NCs was added to the supernatant (150 μL), incubated for 10 min, and then 40 μL of H 2 O was added and incubated for 10 min. The fluorescence spectrum was measured at an excitation wavelength of 350 nm. The reaction conditions for ATCh + Au NCs were the same as above, but 20 μL of H 2 O was added to the supernatant (150 μL), incubated for 10 min, and then 40 μL of Au NCs was added and incubated for 10 min. The fluorescence spectrum was measured at an excitation wavelength of 350 nm. For example, MOF-808-Al + ATCh + Cu NCs, ATCh (50 μL, 15 mM) and 500 μL of HEPES buffer (0.1 M, pH = 8.5) were added to 30 μL of MOF-808-Al (2 mg / mL), incubated at 37 °C for 20 min. 20 μL of Cu NCs was added to the supernatant (150 μL), incubated for 10 min, and then 40 μL of H 2O, incubate for 10 min, then add 40 μL of Au NCs and incubate for 10 min. Measure the fluorescence spectrum at an excitation wavelength of 350 nm. The results show that although ATCh can also decrease the green-emitting gold nanomaterials, the coexistence of MOF-808-Al and ATCh is a necessary condition for the fluorescence quenching of the green-emitting gold nanomaterials. The coexistence of MOF-808-Al and ATCh is crucial for the fluorescence quenching of the green-emitting Au NCs. To further verify the fluorescence response of Cu / Au NCs to thiocholine, the product of the decomposition of MOF-808-Al and ATCh, substitute the standard substance thiocholine for MOF-808-Al + ATCh, such as Thiocholine + Cu NCs. Add 80 μL of the standard substance thiocholine to 500 μL of HEPES buffer (0.1 M, pH = 8.5), incubate at 37 °C for 20 min, add 20 μL of Cu NCs to the supernatant (150 μL), incubate for 10 min, and then add 40 μL of H 2 O, incubate for 10 min, and measure the fluorescence spectrum at an excitation wavelength of 350 nm. For example, Thiocholine + Au NCs reacts under the same conditions as above, but add 20 μL of H to the supernatant (150 μL) 2 O, incubate for 10 min, then add 40 μL of Au NCs and incubate for 10 min. Measure the fluorescence spectrum at an excitation wavelength of 350 nm. As Figure 5 shown in c, the fluorescence intensity in the blue region of the standard substance thiocholine by Cu NCs increases slightly, the green fluorescence intensity of the standard substance thiocholine by Au NCs decreases, and the direct fluorescence response of Au NCs to the standard substance thiocholine confirms that the reaction between Au NCs and thiol molecules is the reason for the fluorescence quenching. The fluorescence responses of Cu NCs and Au NCs to the standard substance thiocholine further confirm that MOF-808-Al has AChE-like activity and can catalyze ATCh to generate thiocholine.

[0059] Example 6

[0060] Optimize the experimental conditions and record the fluorescence intensity in the green region. Add ATCh (50 μL, 15 mM) and 500 μL HEPES buffer (0.1 M, pH = 9.0) to 30 μL of MOF-808-Al (2 mg / mL), incubate at 37 °C for 20 min. Add 20 μL of Cu NCs to the supernatant (150 μL), incubate for 5 min, then add different volumes (10, 20, 25, 30, 35, 40, 45, 50 μL) of Au NCs, incubate for 5 min, and measure the fluorescence spectrum at an excitation wavelength of 350 nm. Meanwhile, measure the fluorescence spectrum without MOF-808-Al under the above conditions. The results are as Figure 6 shown. As the volume of Au NCs increases, the quenching ratio (F / F 0 ) at 525 nm between without MOF-808-Al (F) and with MOF-808-Al (F 0 ) reaches the maximum quenching efficiency when the volume of Au NCs is 40 μL.

[0061] Example 7

[0062] Optimize the experimental conditions and record the fluorescence intensity in the green region. Add 50 μL of ATCh with different concentrations (0, 2, 4, 6, 8, 10, 12, 15, 18, 20, 22, 24, 26, 28, 30 mM) and 500 μL of HEPES buffer (0.1 M, pH = 9.0) to 30 μL of MOF-808-Al (2 mg / mL), incubate at 37 °C for 20 min. Add 20 μL of Cu NCs to the supernatant (150 μL), incubate for 5 min, then add 40 μL of Au NCs, incubate for 5 min, and measure the fluorescence spectrum at an excitation wavelength of 350 nm. Meanwhile, measure the fluorescence spectrum without MOF-808-Al under the above conditions. The results are as Figure 7 shown. As the concentration of ATCh increases, the quenching ratio (F / F 0 ) at 525 nm between without MOF-808-Al (F) and with MOF-808-Al (F 0 ) reaches the maximum quenching efficiency when the concentration of ATCh is 15 mM.

[0063] Example 8

[0064] Optimize the experimental conditions and record the fluorescence intensity in the green region. Add ATCh (50 μL, 15 mM) and 500 μL HEPES buffer (0.1 M) to 30 μL of MOF-808-Al (2 mg / mL). Incubate at 37 °C for 20 min at different pH values (6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10). Add 20 μL of Cu NCs to the supernatant (150 μL), incubate for 5 min, then add 40 μL of Au NCs and incubate for 5 min. Measure the fluorescence spectrum at an excitation wavelength of 350 nm. Meanwhile, measure the fluorescence spectrum without MOF-808-Al under the above conditions. The results are as Figure 8 shown. As the pH value increases, the quenching ratio (F / F 0 ) at 525 nm of without MOF-808-Al (F) and with MOF-808-Al (F 0 ) is the largest when pH is 8.5.

[0065] Example 9

[0066] Optimize the experimental conditions and record the fluorescence intensity in the green region. Add ATCh (50 μL, 15 mM) and 500 μL HEPES buffer (0.1 M, pH = 8.5) to 30 μL of MOF-808-Al (2 mg / mL). Incubate at 37 °C for 20 min. Add 20 μL of Cu NCs to the supernatant (150 μL) and incubate for different times (5, 10, 20, 30, 40, 60, 80, 100 min), then add 40 μL of Au NCs and incubate for 10 min. Measure the fluorescence spectrum at an excitation wavelength of 350 nm. Meanwhile, perform parallel experiments. Add 20 μL of Cu NCs to the supernatant (150 μL) and incubate for 10 min, then add 40 μL of Au NCs and incubate for different times (5, 10, 20, 30, 40, 60, 80, 100 min), and measure the fluorescence spectrum at an excitation wavelength of 350 nm. Also, measure the fluorescence spectrum without MOF-808-Al under the above conditions. The results are as Figure 9 shown. As time increases, the quenching ratio (F / F 0 ) at 525 nm of without MOF-808-Al (F) and with MOF-808-Al (F 0 ) is the largest when adding Cu NCs and incubating for 10 min, then adding Au NCs and incubating for 10 min, with a total incubation time of 20 min.

[0067] Example 10

[0068] Optimize the experimental conditions and record the fluorescence intensity in the green region. Add ATCh (50 μL, 15 mM) and 500 μL HEPES buffer (0.1 M, pH = 8.5) to 30 μL of MOF-808-Al (2 mg / mL), react at different temperatures (0, 30, 37, 40, 50, 60, 70, 80, 90 °C) for 20 min, add 20 μL of Cu NCs to the supernatant (150 μL) and incubate for 10 min, then add 40 μL of AuNCs and incubate for 10 min, and measure the fluorescence spectrum at an excitation wavelength of 350 nm. Meanwhile, measure the fluorescence spectrum without MOF-808-Al under the above conditions. The results are as Figure 10 shown. As the temperature continuously increases, the quenching ratio (F / F 0 ) of without MOF-808-Al (F) and with MOF-808-Al (F 0 ) at 525 nm, when the temperature is 37 °C, the quenching efficiency is the largest.

[0069] Example 11

[0070] Use the ratio fluorescence method to detect KAN and record the fluorescence intensities in the blue and green regions. Mix 150 μL of aqueous solutions of different concentrations of KAN (0, 9.2, 11.1, 12.9, 14.7, 16.6, 18.5, 24.0, 27.7, 33.2, 36.9 μM) with 30 μL of MOF-808-Al (2 mg / mL), react at 37 °C for 1 h. Then add ATCh (50 μL, 15 mM) and 350 μL of HEPES buffer (0.1 M, pH = 8.5), incubate at 37 °C for 20 min, add 20 μL of Cu NCs to the supernatant (150 μL) and incubate for 10 min, then add 40 μL of AuNCs and incubate for 10 min, and measure the fluorescence spectrum at an excitation wavelength of 350 nm. Calculate the ratio of the fluorescence intensity at 525 nm to the fluorescence intensity at 418 nm, that is, calculate the F 525 / F 418 fluorescence ratio, make a linear correlation curve of the fluorescence ratio and the KAN concentration, and the obtained results are as Figure 11 shown. It can be seen from Figure 11 that the fluorescence ratio has a good linear relationship with the aqueous solution of KAN in the range of 12.9 μM - 27.7 μM, and the linear regression equation of F 525 / F 418 = 2.294log 10 X - 2.408, R 2 = 0.993 is obtained, and the detection limit (LOD) is as low as 4.5 nM.

[0071] Example 12

[0072] Separate solutions containing 1 mM of pefloxacin (PEF), amoxicillin (AMX), ciprofloxacin (CIP), ofloxacin (OFX), levofloxacin (LVX), erythromycin (ERY), rifampicin (RIF), lomefloxacin (LOM), ampicillin (AMP), sarafloxacin (SAR), gatifloxacin (GAT), azithromycin (AZM), flumequine (FQ), norfloxacin (OFX), enrofloxacin hydrochloride (EFX), crystal violet (CV), methyl orange (MO), azure II (AZ), alizarin red (AB), alizarin yellow (AY), brilliant green (SF), tartrazine (TZ), malachite green (MG), allura red (AR), victoria blue B (VB), pararosaniline (PRA), basic fuchsin (BF), ethyl violet (FV), and solutions containing 50 μg / mL of methyl parathion (MP), methyl parathion (PM), phoxim (BAY), demeton-S-methyl (DOM), trichlorfon (DIP), fenitrothion (FEN), imidacloprid (IMI), dinotefuran (DIN), acetamiprid (ACE), thiamethoxam (THI), nitenpyram (NIT), thiodicarb (Thi), aldicarb (Ald), isoprocarb (Phe), fenobucarb (Bas), carbaryl (Car), carbofuran (Cbf), fenoxycarb (Fen), methomyl (Mtm), glyphosate (Gly), carbosulfan (CS), indoxacarb (Ind) were mixed with 30 μL of MOF-808-Al (2 mg / mL) and reacted at 37 °C for 1 h. Then, ATCh (50 μL, 15 mM) and 350 μL of HEPES buffer (0.1 M, pH = 8.5) were added, and the mixture was incubated at 37 °C for 20 min. 20 μL of Cu NCs was added to the supernatant (150 μL) and incubated for 10 min, and then 40 μL of Au NCs was added and incubated for 10 min. The fluorescence spectrum was measured at an excitation wavelength of 350 nm. Calculate the fluorescence ratio (F 525 / F 418 ), and simultaneously measure the blank group without KAN (Blank) and the group with KAN as a control. The obtained selectivity ability results are as shown in Figure 12 . As can be seen from Figure 12 , the effects of these substances on the detection platform and its recognition ability for KAN are almost negligible.

[0073] Example 13

[0074] Mix 150 μL of KAN (36.9 μM) with 30 μL of MOF-808-Al (2 mg / mL), and react at 37 °C for 1 h. Then add ATCh (50 μL, 15 mM) and 300 μL of HEPES buffer (0.1 M, pH = 8.5). Add 50 μL of NaCl with different concentrations (0 mM, 20 mM, 40 mM, 60 mM, 80 mM, and 100 mM) to the buffer for the salt tolerance test. Incubate at 37 °C for 20 min. Add 20 μL of Cu NCs to the supernatant (150 μL) and incubate for 10 min, then add 40 μL of Au NCs and incubate for 10 min. Measure the fluorescence spectrum at an excitation wavelength of 350 nm and calculate the fluorescence ratio (F 525 / F 418 ). At the same time, measure the fluorescence ratio (F 525 / F 418 ) without adding KAN. The obtained results are as shown in Figure 13 . Figure 13 In [Figure Figure 13 ], Figure a shows the change in the fluorescence ratio F 525 / F 418 at different concentrations of NaCl with and without adding KAN. It can be seen that after adding KAN, the fluorescence ratio F 525 / F 418 increases significantly, and the system is not interfered by salt. Figure 13 In [Figure Figure 13 ], the upper row of Figure b represents the sample without adding KAN, and the lower row represents the sample with added KAN. It can be seen that without adding KAN, it emits blue light, and after adding KAN, it shows green light emission.

[0075] Example 14

[0076] Purchase milk samples from a local supermarket. The milk is deproteinized with acetone and sodium chloride and then ultrasonically filtered. No kanamycin sulfate is found in the tested milk samples. Then mix 75 μL of the actual sample with 75 μL of the standard sample KAN, and then mix with 30 μL of MOF-808-Al (2 mg / mL), and react at 37 °C for 1 h. Then add ATCh (50 μL, 15 mM) and 350 μL of HEPES buffer (0.1 M, pH = 8.5). Incubate at 37 °C for 20 min. Add 20 μL of Cu NCs to the supernatant (150 μL) and incubate for 10 min, then add 40 μL of Au NCs and incubate for 10 min. Measure the fluorescence spectrum at an excitation wavelength of 350 nm. Calculate the fluorescence ratio (F 525 / F 418 ). Further, based on the concentration of the KAN solution and the fluorescence ratio (F 525 / F 418) Calculate the concentration of the KAN solution based on the linear relationship, compare the calculated concentration of the KAN solution with the spiked concentration of the standard sample KAN, and calculate the spiked recovery rate and RSD value.

[0077] In this embodiment, 75 μL of the standard sample KAN is mixed with 75 μL of the actual milk sample. Since the actual milk sample does not contain kanamycin sulfate, the spiked concentration in the 150 μL solution can be obtained. Three tests were conducted in this embodiment, and the spiked concentrations for each test were 14.7, 18.5, and 27.7 μM respectively, and the corresponding detected concentrations were 16.0, 19.0, and 27.2 μM respectively. The final spiked recovery rate was 98.2% - 108.8%. This indicates the accuracy of the method for detecting kanamycin sulfate in this application. Table 1 below shows the spiked recovery results of this embodiment.

[0078] Table 1 Spiked Recovery Test Results of Milk Samples in Example 14

[0079]

Claims

1. A kanamycin sulfate recognition fluorescence sensor, characterized in that: The fluorescent sensor identifies kanamycin sulfate in the following way: MOF-808-Al catalyzes the decomposition of acetylthiocholine into thiocholine, the fluorescence emitted by gold nanoclusters can be quenched by thiocholine, and the fluorescence emitted by copper nanoclusters is not affected by thiocholine. Kanamycin sulfate can inhibit the acetylcholinesterase activity of MOF-808-Al, thereby restoring the fluorescence emitted by gold nanoclusters. The concentration of kanamycin sulfate can be quantitatively identified by the fluorescence ratio of gold nanoclusters and copper nanoclusters.

2. The KAN recognition fluorescence sensor according to claim 1, characterized in that: The relationship between the fluorescence ratio of gold nanoclusters and copper nanoclusters and the concentration of kanamycin sulfate is: 525 / F 418 =2.294log 10 X-2.408, X represents the concentration of kanamycin sulfate, F 525 Indicates the fluorescence value at 525 nm, F 418 Indicates the fluorescence value at 418 nm; X ranges from 12.9 to 27.7 μM.

3. A method for detecting kanamycin sulfate, characterized in that: The steps include: (1) Preparation of Al 3+ MOF-808-Al, a material for modifying MOF-808; (2) preparing copper nanoclusters and gold nanoclusters; (3) preparing kanamycin sulfate aqueous solutions of different concentrations; (4) mixing the kanamycin sulfate aqueous solutions of different concentrations prepared in step (3) with MOF-808-Al respectively, then adding acetylthiocholine and buffer for incubation, taking the supernatant after the incubation, adding copper nanoclusters and gold nanoclusters to the supernatant in sequence, and finally measuring the ratio of the fluorescence intensity at 525 nm and 418 nm, and drawing a linear curve of the fluorescence ratio and the kanamycin sulfate concentration; (5) The sample to be tested is mixed with MOF-808-Al, and then acetylthiocholine and buffer are added for incubation. After the incubation, the supernatant is taken, and copper nanoclusters and gold nanoclusters are added to the supernatant in sequence. Finally, the ratio of the fluorescence intensity at 525 nm and 418 nm is measured, and the concentration of kanamycin sulfate contained in the sample to be tested is calculated according to the linear curve of the fluorescence ratio and the kanamycin sulfate concentration in step (4).

4. The method for detecting kanamycin sulfate according to claim 3, wherein: The equation corresponding to the linear curve of fluorescence ratio and kanamycin sulfate concentration in step (4) is: 525 / F 418 =2.294log 10 X-2.408, X represents the concentration of kanamycin sulfate, F 525 Indicates the fluorescence value at 525 nm, F 418 Indicates the fluorescence value at 418 nm; X ranges from 12.9 to 27.7 μM.

5. The method for detecting kanamycin sulfate according to claim 3, wherein: Step (1) is as follows: First, zirconium oxychloride octahydrate is dissolved in formic acid, 1,3,5-benzenetricarboxylic acid is added to N,N-dimethylformamide solution, zirconium oxychloride octahydrate and 1,3,5-benzenetricarboxylic acid solution are mixed, ultrasonically treated, reacted, centrifuged, washed to obtain a solid, and finally dried to obtain MOF-808; MOF-808 is added to a DMF solution of aluminum nitrate, heated, stirred, and centrifuged to obtain a solid, which is then washed and dried to obtain MOF-808-Al.

6. The method for detecting kanamycin sulfate according to claim 3, wherein: The preparation method of copper nanoclusters in step (2) is as follows: polyvinyl pyrrolidone is added to ultrapure water and dissolved by ultrasonication; then, ascorbic acid aqueous solution and copper sulfate pentahydrate solution are added to the polyvinyl pyrrolidone solution, and reacted under continuous stirring, and the obtained product is dialyzed through a dialysis membrane to obtain copper nanoclusters.

7. The method for detecting kanamycin sulfate according to claim 3, wherein: The preparation method of the gold nanoclusters in step (2) is as follows: 6-aza-2-thiothymine containing NaOH was added to tetrachloroauric acid trihydrate solution, stirred continuously in the dark at room temperature, and ATT-Au NCs were synthesized by ultrafiltration purification; Arginine is added to the ATT-Au NCs solution, and gold nanoclusters are obtained after the reaction.

8. The method for detecting kanamycin sulfate according to claim 3, wherein: In steps (4) and (5), the buffer is HEPES buffer, and the pH value of the buffer is 8.

5.

9. The method for detecting kanamycin sulfate according to claim 3, wherein: In steps (4) and (5), copper nanoclusters and gold nanoclusters are added to the supernatant in sequence. Specifically, 20 μL of copper nanoclusters are added to the supernatant in sequence, incubated for 10 min, and then 40 μL of gold nanoclusters are added to the supernatant and incubated for 10 min.

10. The method for detecting kanamycin sulfate according to claim 3, wherein: In steps (4) and (5), the temperature when acetylthiocholine and buffer are added for incubation is 37° C. and the incubation time is 20 min.

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