Preparation method and application of fluorescence enhancement probe for rapid detection of moxifloxacin and norfloxacin

Abstract

The application discloses a preparation method and application of a fluorescence-enhanced probe for rapidly detecting moxifloxacin and norfloxacin, and belongs to the technical field of rapid antibiotic detection. The fluorescence-enhanced probe is prepared from p-aminosalicylic acid and L-cysteine by a hydrothermal method, and the optimal preparation process is that the p-aminosalicylic acid and the L-cysteine with a mass ratio of 4:1 are dissolved in anhydrous ethanol, and then heated at 200 DEG C for 14 hours. Moxifloxacin and norfloxacin can significantly enhance the fluorescence intensity of the fluorescence probe at 520 nm and 460 nm, respectively, and have a good linear relationship with the concentration. The application has the advantages of simple operation, low cost, rapid detection, high sensitivity, high selectivity, wide detection range and low detection limit, and can realize on-site rapid detection of moxifloxacin and norfloxacin in water and milk.

Description

Technical Field

[0001] The invention discloses a preparation method and application of a fluorescence enhancement probe for rapid detection of moxifloxacin and norfloxacin, belonging to the technical field of rapid antibiotic detection. Background Art

[0002] The quinolone antibiotics moxifloxacin and norfloxacin are primarily used to treat bacterial infections and are widely used in the medical and animal husbandry industries. However, the overuse and misuse of moxifloxacin and norfloxacin can lead to antibiotic residues in the environment and food, posing a variety of potential health risks to humans, including allergic reactions, neurotoxicity, and drug resistance. Therefore, detecting moxifloxacin and norfloxacin residues in the environment and food is crucial for protecting human health.

[0003] Traditional detection methods often require the use of large instruments in the laboratory, which is not only costly but also requires certain operating skills. However, the analysis and testing of environmental and food samples are usually time-sensitive, making traditional detection technologies difficult to achieve rapid on-site monitoring. Rapid detection methods are simple to operate and can produce test results in a very short time, making them suitable for rapid on-site screening of environmental and food safety. Therefore, the development of rapid detection technologies with high sensitivity and good specificity is of great significance to protecting human health.

[0004] Fluorescence analysis utilizes changes in the fluorescence signal emitted by fluorescent substances after being excited by light of a specific wavelength to perform qualitative and quantitative analysis of target substances. Fluorescence analysis offers advantages such as simple operation, rapid response, high sensitivity, and good specificity, and holds broad promise in the field of rapid detection. Fluorescent probes are the core of fluorescence detection technology. Fluorescent probes can specifically bind to non-fluorescent substances, causing changes in their fluorescence wavelength or intensity, thereby enabling the detection and analysis of non-fluorescent substances. Although some fluorescence-quenching probes have been used for antibiotic detection, their high background signal affects their sensitivity and accuracy. In contrast, fluorescence-enhanced probes offer high sensitivity and are particularly suitable for detecting low-concentration pollutants. Therefore, the development of fluorescence-enhanced probes for the rapid detection of moxifloxacin and norfloxacin in the environment and food is of great significance. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a preparation method and application of a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin, which can achieve rapid, highly sensitive and highly specific detection of moxifloxacin and norfloxacin in water and milk after simple pretreatment.

[0006] In order to achieve the above object, the present invention adopts a method for preparing a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin, comprising the following steps:

[0007] Step (1), dissolving p-aminosalicylic acid and L-cysteine ​​in anhydrous ethanol at a mass ratio of 4:1-8:1, stirring to fully dissolve them, to obtain a mixed solution;

[0008] Step (2), placing the mixed solution into a high-pressure reactor and heating it at 120° C.-200° C. for 10 h-18 h to obtain a reaction product;

[0009] Step (3), cooling the reaction product to room temperature, centrifuging, and obtaining a crude carbon quantum dot solution supernatant;

[0010] Step (4), after the supernatant of the crude carbon quantum dot solution is filtered through a filter membrane, the filtrate is transferred to a dialysis bag and dialyzed in ultrapure water to obtain a carbon quantum dot CQDs solution;

[0011] Step (5): dilute the carbon quantum dot CQDs solution with ultrapure water to obtain a fluorescent probe solution.

[0012] Preferably, the mass ratio of p-aminosalicylic acid to L-cysteine ​​in step (1) is 4:1; and the volume of anhydrous ethanol and the mass ratio of p-aminosalicylic acid are 500 mL:4 g.

[0013] Preferably, the high-pressure reactor in step (2) is provided with a Teflon lining, and the high-pressure reactor is heated at 200° C. for 14 h.

[0014] Preferably, the centrifugal speed in step (3) is 10,000 r / min, and the centrifugal time is 10 min.

[0015] Preferably, the filter membrane in step (4) is a 0.22 μm microporous filter membrane, the molecular weight cutoff of the dialysis bag is 1000 Da, and the dialysis time is 48 h-72 h.

[0016] Preferably, in step (5), the carbon quantum dot CQDs solution is diluted 50 times with ultrapure water.

[0017] The second object of the present invention is to provide a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin, which is prepared by the preparation method.

[0018] The fluorescent probe described in the present invention, when excited under 355 nm ultraviolet light, exhibits a strong fluorescence signal at 520 nm. Adding moxifloxacin to the fluorescent probe solution significantly increases the fluorescence signal at 520 nm. Adding norfloxacin to the fluorescent probe solution significantly increases the fluorescence signal at 460 nm. The detection mechanism is that hydrogen bonds or π-π bonds are formed between the fluorescent probe and moxifloxacin, increasing the conjugated surface of the fluorescent probe and making its rigid structure more stable, thereby enhancing the fluorescence of the fluorescent probe at 520 nm. Norfloxacin can bind to the fluorescent probe through electrostatic interactions, promoting electron transfer from the fluorescent probe to norfloxacin, resulting in a blue shift in the fluorescence emission peak of the fluorescent probe from 520 nm to 460 nm. Furthermore, hydrogen bonds or π-π bonds are formed between the fluorescent probe and norfloxacin, increasing the conjugated surface of the fluorescent probe and making its rigid structure more stable, thereby enhancing the fluorescence of the fluorescent probe at 460 nm. The concentrations of moxifloxacin and norfloxacin can be detected by recording the increase in fluorescence intensity after adding different concentrations of moxifloxacin or norfloxacin to the system. The fluorescence intensity of the fluorescent probe exhibits a good linear relationship with the concentrations of moxifloxacin and norfloxacin. The linear ranges for quantitative detection of moxifloxacin and norfloxacin concentrations are 0-249 μM and 0-313 μM, respectively, with minimum detection limits of 2.5 nM and 4.0 nM, respectively. The probe exhibits high sensitivity for moxifloxacin and norfloxacin and excellent selectivity, making it suitable for the rapid detection of moxifloxacin and norfloxacin in water and milk.

[0019] The third object of the present invention is to provide an application of the fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin, which is used to detect the content of moxifloxacin and norfloxacin in water and milk products.

[0020] Preferably, the steps of detecting the content of moxifloxacin in water using the fluorescent probe are as follows:

[0021] Step (1), the fluorescent probe solution and the moxifloxacin spiked solution (0-100 mg / L) were mixed evenly, and the fluorescence intensity of the mixed solution at 520 nm under an excitation wavelength of 355 nm was recorded; the increase factor F of the fluorescence intensity after adding moxifloxacin was used as the 520 / F0 was the ordinate and moxifloxacin concentration was the abscissa, and a linear regression model was established;

[0022] Step (2): centrifuge the water sample to be tested at a speed of 10,000 r / min for 3 minutes, and filter the supernatant with a 0.22 μm microporous filter membrane; mix the filtered test solution with the fluorescent probe solution, and measure the fluorescence intensity of the mixed solution at 520 nm under an excitation wavelength of 355 nm; substitute the measured fluorescence intensity into the obtained linear regression equation to calculate the concentration of moxifloxacin in the water.

[0023] Preferably, the volume ratio of the fluorescent probe solution to the spiked solution in step (1) is 50:1.

[0024] Preferably, the volume ratio of the fluorescent probe solution to the test solution in step (2) is 50:1.

[0025] Preferably, the steps of detecting the norfloxacin content in water are as follows:

[0026] Step (1), the fluorescent probe solution and norfloxacin spiked solution (0-100 mg / L) were mixed evenly, and the fluorescence intensity of the mixed solution at 460 nm under 355 nm excitation wavelength was recorded; the increase factor F of the fluorescence intensity after adding norfloxacin was used. 460 / F0 was the ordinate and norfloxacin concentration was the abscissa, and a linear regression model was established;

[0027] Step (2): centrifuge the water sample to be tested at a speed of 10,000 r / min for 3 minutes, and filter the supernatant with a 0.22 μm microporous filter membrane; mix the filtered test solution with the fluorescent probe solution, and measure the fluorescence intensity of the mixed solution at 460 nm under an excitation wavelength of 355 nm; substitute the measured fluorescence intensity into the obtained linear regression equation to calculate the concentration of norfloxacin in the water.

[0028] Preferably, the volume ratio of the fluorescent probe solution to the spiked solution in step (1) is 50:1.

[0029] Preferably, the volume ratio of the fluorescent probe solution to the test solution in step (2) is 50:1.

[0030] Preferably, the steps of detecting the moxifloxacin content in milk are as follows:

[0031] Step (1), the control milk was mixed with acetonitrile, sodium chloride was added, vortexed for 30 seconds to fully mix, centrifuged at 10000 r / min for 3 minutes, and the supernatant was filtered through a 0.22 μm microporous membrane to collect the milk extract;

[0032] Step (2): Evenly mix the fluorescent probe solution and the milk extract spiked with moxifloxacin (0-100 mg / L), and record the fluorescence intensity of the mixed solution at 520 nm under an excitation wavelength of 355 nm; the increase factor F of the fluorescence intensity after adding moxifloxacin is used as the value. 520 / F0 was the ordinate and moxifloxacin concentration was the abscissa, and a linear regression model was established;

[0033] Step (3), the milk to be tested was mixed with acetonitrile, sodium chloride was added, vortexed for 30 seconds to fully mix, centrifuged at 10000 r / min for 3 minutes, and the supernatant was filtered through a 0.22 μm microporous membrane;

[0034] Step (4): Evenly mix the filtered test solution and the fluorescent probe solution, and measure the fluorescence intensity of the mixed solution at 520 nm under an excitation wavelength of 355 nm; substitute the measured fluorescence intensity into the obtained linear regression equation to calculate the concentration of moxifloxacin in milk.

[0035] Preferably, the volume ratio of milk to acetonitrile in step (1) is 1:1, and the volume ratio of acetonitrile to sodium chloride is 5 mL:2 g.

[0036] Preferably, the volume ratio of the fluorescent probe solution to the spiked solution in step (2) is 50:1.

[0037] Preferably, the volume ratio of milk to acetonitrile in step (3) is 1:1, and the volume ratio of acetonitrile to sodium chloride is 5 mL:2 g.

[0038] Preferably, the volume ratio of the fluorescent probe solution to the sample solution in step (4) is 50:1.

[0039] Preferably, the steps of detecting the norfloxacin content in milk are as follows:

[0040] Step (1), milk and acetonitrile were mixed, sodium chloride was added, vortexed for 30 seconds to fully mix, and then centrifuged at 10000 r / min for 3 minutes. The supernatant was filtered through a 0.22 μm microporous filter membrane to collect the milk extract;

[0041] Step (2): Evenly mix the fluorescent probe solution and the milk extract spiked with norfloxacin (0-100 mg / L), and record the fluorescence intensity of the mixed solution at 520 nm under an excitation wavelength of 355 nm; the increase factor F of the fluorescence intensity after adding norfloxacin is used. 460 / F0 was the ordinate and norfloxacin concentration was the abscissa, and a linear regression model was established;

[0042] Step (3), the milk was mixed with acetonitrile, sodium chloride was added, and the mixture was vortexed for 30 seconds to fully mix, and then centrifuged at 10000 rpm for 3 minutes, and the supernatant was filtered through a 0.22 μm microporous filter membrane;

[0043] Step (4): Evenly mix the filtered test solution and the fluorescent probe solution, and measure the fluorescence intensity of the mixed solution at 460 nm under an excitation wavelength of 355 nm; substitute the measured fluorescence intensity into the obtained linear regression equation to calculate the concentration of norfloxacin in the milk.

[0044] Preferably, the volume ratio of milk to acetonitrile in step (1) is 1:1, and the volume ratio of acetonitrile to sodium chloride is 5 mL:2 g.

[0045] Preferably, the volume ratio of the fluorescent probe solution to the spiked solution in step (2) is 50:1.

[0046] Preferably, the volume ratio of milk to acetonitrile in step (3) is 1:1, and the volume ratio of acetonitrile to sodium chloride is 5 mL:2 g.

[0047] Preferably, the volume ratio of the fluorescent probe solution to the sample solution in step (4) is 50:1.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The fluorescence-enhanced probe prepared by the present invention significantly increases the fluorescence emission intensity at 520 nm after binding to moxifloxacin; and significantly increases the fluorescence emission intensity at 460 nm after binding to norfloxacin, with the fluorescence increase factor showing a good linear relationship with the concentration of moxifloxacin or norfloxacin. The detection limits of this probe for moxifloxacin and norfloxacin are 2.5 nM and 4.0 nM, respectively, making it suitable for the rapid detection of moxifloxacin and norfloxacin in water and milk products. The fluorescence-enhanced probe prepared by the present invention has the advantages of a simple preparation process, low cost, fast detection speed, high sensitivity, and good selectivity. It can simultaneously detect moxifloxacin and norfloxacin, providing a simple and rapid strategy for the on-site quantitative detection of moxifloxacin and norfloxacin. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of the preparation and detection of the fluorescence-enhanced probe for detecting moxifloxacin and norfloxacin involved in the present invention;

[0051] Figure 2 This is a diagram of the optimization of the synthesis time of carbon quantum dots CQDs involved in the present invention;

[0052] Figure 3 (A) UV absorption, fluorescence excitation, and emission spectra of the carbon quantum dots (CQDs) involved in the present invention, with the inset showing the color comparison of the solution under sunlight and 365 nm UV light; (B) infrared spectrum; (C) transmission electron microscopy image, with the inset showing the particle size distribution; (D) X-ray diffraction pattern; and (E) X-ray photoelectron spectroscopy.

[0053] Figure 4 High-resolution X-ray photoelectron spectra of (A) C1s, (B) O1s, (C) N1s and (D) S2p of carbon quantum dots CQDs involved in the present invention;

[0054] Figure 5 The fluorescence intensity stability of the fluorescent probe of the present invention is (A) stored at 25°C and 4°C for 14 days and (B) in different pH ranges;

[0055] Figure 6 The fluorescence emission spectra of the fluorescent probe of the present invention after mixing with different concentrations of (A) moxifloxacin and (B) norfloxacin;

[0056] Figure 7 The linear relationship between the increase in fluorescence intensity of the probe involved in the present invention and the concentrations of (A) moxifloxacin and (B) norfloxacin;

[0057] Figure 8 The fluorescence intensity increase factor after the fluorescent probe of the present invention is mixed with moxifloxacin or norfloxacin and stored at (A) 25°C for 14 days; (B) 4°C for 14 days and (C) different pH ranges;

[0058] Figure 9 This is a comparison chart of the fluorescence intensity increase after the fluorescent probe of the present invention is mixed with 5 mg / L moxifloxacin, norfloxacin and 100 mg / L different interfering substances (antibiotics, pesticides and metal ions);

[0059] Figure 10 The present invention provides fluorescent probes for identifying moxifloxacin and norfloxacin in (A) aqueous solution and (B) milk samples. DETAILED DESCRIPTION

[0060] Example 1: A method for preparing a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin, comprising the following steps:

[0061] Step (1), weighing 0.4 g of p-aminosalicylic acid and 0.1 g of L-cysteine, dissolving them in 50 mL of anhydrous ethanol, and stirring to fully dissolve them to obtain a mixed solution;

[0062] Step (2), placing the mixed solution in a polytetrafluoroethylene liner high-pressure reactor, heating at 200° C. for 14 h to obtain a reaction product;

[0063] Step (3), the reaction product was cooled to room temperature, transferred to a 50 mL centrifuge tube, and centrifuged at a speed of 10000 r / min for 10 min to remove some large solid impurities to obtain a crude carbon quantum dot solution supernatant;

[0064] Step (4), filtering the supernatant of the crude carbon quantum dot solution with a 0.22 μm filter membrane to remove small particulate impurities, and transferring the resulting filtrate into a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzing it in ultrapure water for 48 h-72 h to remove small molecular impurities to obtain a carbon quantum dot CQDs solution;

[0065] Step (5): dilute the carbon quantum dot CQDs solution 50 times with ultrapure water to obtain a fluorescent probe solution, and store it at 4°C in the dark.

[0066] Synthesis conditions of carbon quantum dots CQDs:

[0067] Figure 2 The effect of synthesis time on the fluorescence intensity of the prepared carbon quantum dots (CQDs) is shown. Within the synthesis time range of 10-18 hours, the fluorescence intensity reaches the highest at a synthesis time of 14 hours.

[0068] Structural characterization of carbon quantum dots CQDs:

[0069] like Figure 3 As shown in (A), the carbon quantum dot CQDs solution is colorless and transparent under fluorescent light, and exhibits green fluorescence under 365 nm ultraviolet light. The maximum excitation wavelength of carbon quantum dot CQDs is 355 nm, and the maximum emission wavelength is 520 nm. The fluorescence quantum yield of the carbon quantum dot CQDs solution is 10.2%. Figure 3 (B) shows that the wave number in the infrared spectrum of carbon quantum dots CQDs is 3348 cm -1 The broad peak at 1650 cm corresponds to the stretching vibration peak of OH or NH bond. -1 The peak at corresponds to the stretching vibration peak of the C=O bond. Therefore, the surface of carbon quantum dots CQDs has hydrophilic groups such as hydroxyl or amino groups, which makes carbon quantum dots CQDs have good water solubility; Figure 3 As shown in (C), the transmission electron microscopy image of carbon quantum dots CQDs shows that they are uniformly distributed spherical particles with an average particle size of 7.3 nm and no obvious agglomeration. Figure 3 As shown in (D), the strong diffraction peak at 2θ = 28.91° in the X-ray diffraction pattern of carbon quantum dots CQDs corresponds to the (002) diffraction plane of graphite carbon, and the diffraction peak at 2θ = 40.15° corresponds to the C (100) plane, indicating that carbon quantum dots CQDs have a standard amorphous carbon configuration and low crystallinity. Figure 3As shown in (E), the four characteristic peaks centered at 163.6 eV, 288.4 eV, 399.0 eV, and 532.2 eV in the X-ray photoelectron spectrum of carbon quantum dots CQDs can be attributed to the binding energies of S2p, C1s, N1s, and O1s, respectively, indicating that carbon quantum dots CQDs contain S, C, N, and O elements;

[0070] like Figure 4 As shown in (A), in the high-resolution XPS spectrum of C1s, three fitting peaks are shown at 284.8 eV, 286.2 eV, and 289.2 eV, which are attributed to CC / C=C, CO / CN, and C=O bonds, respectively; Figure 4 As shown in (B), in the high-resolution XPS spectrum of Ols, two fitting peaks are present at 531.4 eV and 532.7 eV, which are attributed to the C=O and CO bonds, respectively; Figure 4 As shown in (C), in the high-resolution XPS spectrum of N1s, two fitting peaks are shown at 399.4 eV and 400.8 eV, which are attributed to the CN and NH peaks, respectively; Figure 4 As shown in (D), the high-resolution XPS spectrum of S2p shows two fitting peaks at 163.7 eV and 164.8 eV, which are attributed to CSC S 2P3 / 2 and SO4 2- S 2P3 / 2 The above results are in good agreement with those of the infrared spectrum.

[0071] Stability of fluorescent probes:

[0072] like Figure 5 As shown in (A), the fluorescence intensity of the probe did not change significantly whether stored at 25°C or 4°C for 14 days; Figure 5 As shown in (B), the fluorescence intensity of the probe remains stable in the pH range of 7 to 14. These results verify the stability of the fluorescent probe and lay the foundation for its practical application.

[0073] Fluorescent probe detection of moxifloxacin and norfloxacin:

[0074] like Figure 6 As shown in (A), after adding moxifloxacin, the maximum emission wavelength of the fluorescent probe remained unchanged, and the fluorescence intensity increased; as the concentration of moxifloxacin increased, the fluorescence of the fluorescent probe at 520 nm gradually increased. Figure 7 (A) shows the fluorescence intensity increase factor F 520 / F0 showed a good linear relationship with moxifloxacin concentration in the range of 0-100 mg / L. The calculated LODs and LOQs of the fluorescent probe for moxifloxacin were 0.66 μg / L and 2.18 μg / L.

[0075] like Figure 6 As shown in (B), after the addition of norfloxacin, the maximum emission wavelength of the fluorescent probe shifted to 460 nm, and the fluorescence intensity increased; with the increase of norfloxacin concentration, the fluorescence of the fluorescent probe at 460 nm gradually increased. Figure 7 (B) shows the fluorescence intensity increase factor F 460 / F0 showed a good linear relationship with norfloxacin concentration in the range of 0-100 mg / L. The calculated LODs and LOQs of the fluorescent probe for norfloxacin were 0.42 μg / L and 1.39 μg / L, respectively.

[0076] like Figure 8 As shown in (A), at 25 °C, the fluorescence intensity of the fluorescent probe did not change significantly within 14 days after binding to moxifloxacin or norfloxacin; Figure 8 As shown in (B), at 4 °C, the fluorescence intensity of the fluorescent probe did not change significantly within 14 days after binding to moxifloxacin or norfloxacin. Figure 8 As shown in (C), the fluorescent probe binds to moxifloxacin or norfloxacin, and the fluorescence intensity remains stable in the pH range of 5-10.

[0077] Selectivity of fluorescent probes:

[0078] 100 mg / L of antibiotics (ampicillin, oxytetracycline, tetracycline hydrochloride, sulfamethoxazole, and sulfamethoxazole), pesticides (bifenthrin, boscalid, chlormequat, cyhalothrin, cypermethrin, dicamba, flonicamid, fluazifop, and propargite), and metal ions (Ag) were added to the fluorescent probes. + , Al 3+ , Ba 2+ , Ca 2+ , Cd 2+ ,Co 2+ , Cr 3+ , Cu 2+ , Fe 2 + , Fe 3+ , Hg 2+ , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Na + , Ni 2+ , Pb 2+ and Zn 2+ ) and other common pollutants, and the selectivity of the probe for moxifloxacin and norfloxacin was evaluated. Figure 9As shown in (A), compared with the control group, the fluorescence intensity of the probe increased by a factor of F after adding 5 mg / L moxifloxacin. 520 / F0 is significantly higher than other common pollutants; such as Figure 9 As shown in (B), compared with the control group, the fluorescence intensity of the probe increased by a factor of F after adding 5 mg / L norfloxacin. 460 / F0 was significantly higher than that of other common pollutants. These results indicate that the fluorescent probe has good selectivity for moxifloxacin and norfloxacin.

[0079] Example 2: Rapid detection of moxifloxacin and norfloxacin in water using a fluorescence-enhanced probe, comprising the following steps:

[0080] To 4 mL of the fluorescent probe solution from Example 1, add 80 μL of moxifloxacin-spiked aqueous solutions at varying concentrations (0-100 mg / L). After mixing, measure and record the fluorescence intensity at 520 nm under an excitation wavelength of 355 nm before and after the addition of the moxifloxacin solution. A linear regression model was constructed, plotting the increase in fluorescence intensity after the addition of moxifloxacin as the ordinate and the moxifloxacin concentration as the abscissa.

[0081] To 4 mL of the fluorescent probe solution from Example 1, add 80 μL of a spiked norfloxacin solution at varying concentrations (0-100 mg / L). After mixing, measure and record the fluorescence intensity at 460 nm under a 355 nm excitation wavelength before and after the addition of the norfloxacin solution. A linear regression model was constructed, plotting the increase in fluorescence intensity after the addition of norfloxacin as the y-axis and the norfloxacin concentration as the x-axis.

[0082] After the water sample to be tested was filtered through a 0.22 μm filter membrane, 80 μL of the filtrate was mixed with 4 mL of the fluorescent probe solution, and its fluorescence intensity was tested. The value was substituted into the linear regression equation obtained above to calculate the concentration of moxifloxacin and norfloxacin in the water to be tested.

[0083] Linear range and sensitivity of fluorescent probe for detection of moxifloxacin and norfloxacin in water:

[0084] like Figure 6 As shown in (A), as the concentration of moxifloxacin increases, the fluorescence intensity of the fluorescent probe at 520 nm gradually increases. Figure 7 (A) shows the fluorescence intensity increase factor F 520 There was a good linear relationship between / F0 and the concentration of moxifloxacin in the range of 0-100 mg / L, with the regression equation being y = 0.0657 x + 0.9495 and the correlation coefficient R 2=0.9918 (where x is the moxifloxacin concentration and y is the multiple increase in fluorescence intensity after the addition of moxifloxacin). According to the formula LOD = 3σ / k (where σ is the standard deviation of the fluorescence intensity ratio and k is the slope of the standard curve), the detection limit of this fluorescent probe for moxifloxacin in water was calculated to be 0.66 μg / L, indicating that it can achieve highly sensitive detection of moxifloxacin.

[0085] like Figure 6 As shown in (B), as the concentration of norfloxacin increases, the fluorescence intensity of the fluorescent probe at 460 nm gradually increases. Figure 7 (B) shows the fluorescence intensity increase factor F 460 There was a good linear relationship between / F0 and norfloxacin concentration in the range of 0-100 mg / L, with the regression equation being y = 0.7164 x + 2.1677 and the correlation coefficient R 2 =0.9887 (where x is the norfloxacin concentration and y is the multiple increase in fluorescence intensity after the addition of norfloxacin). According to the formula LOD = 3σ / k (where σ is the standard deviation of the fluorescence intensity ratio and k is the slope of the standard curve), the detection limit of this fluorescent probe for norfloxacin in water is calculated to be 0.42 μg / L, indicating that it can achieve highly sensitive detection of norfloxacin.

[0086] The recognition ability of the fluorescent probe for moxifloxacin, norfloxacin and their mixture in water:

[0087] The linear discriminant analysis (LDA) model was constructed using the fluorescence intensity of different groups of samples at 460 nm and 520 nm. Figure 10 As shown in (A), the LDA model can be used to separate moxifloxacin, norfloxacin, and their mixture in aqueous solution, with classification accuracy rates greater than 0.992, demonstrating the potential of this fluorescent probe in analyzing complex mixed samples.

[0088] Example 3: Rapid detection of moxifloxacin and norfloxacin in milk using a fluorescence-enhanced probe, comprising the following steps:

[0089] Milk was pretreated to remove protein and fat. Five milliliters of blank milk was mixed with 5 milliliters of acetonitrile and 2 g of sodium chloride was added. The mixture was vortexed for 30 seconds to thoroughly mix, and then centrifuged at 10,000 rpm for 3 minutes. The supernatant was filtered through a 0.22 μm microporous membrane to collect the milk extract.

[0090] To 4 mL of the fluorescent probe solution from Example 1, add 80 μL of a milk extract containing moxifloxacin at varying concentrations (0-100 mg / L). After mixing thoroughly, measure and record the fluorescence intensity at 520 nm under an excitation wavelength of 355 nm before and after the addition of the moxifloxacin solution. A linear regression model was constructed, plotting the increase in fluorescence intensity after the addition of moxifloxacin as the y-axis and the moxifloxacin concentration as the x-axis.

[0091] 4 mL of the fluorescent probe solution from Example 1 was added to 80 μL of milk extract containing varying concentrations of norfloxacin (0-100 mg / L). After mixing, the fluorescence intensity at 460 nm under an excitation wavelength of 355 nm was measured and recorded before and after the addition of the norfloxacin solution. A linear regression model was constructed, with the increase in fluorescence intensity after the addition of norfloxacin as the ordinate and the norfloxacin concentration as the abscissa.

[0092] Mix 5 mL of the milk sample to be tested with 5 mL of acetonitrile and add 2 g of sodium chloride. Vortex thoroughly for 30 seconds and then centrifuge at 10,000 rpm for 3 minutes. Filter the supernatant through a 0.22 μm microporous filter to collect the test solution. Mix 80 μL of the filtered sample with 4 mL of the fluorescent probe solution and measure the fluorescence intensity. Substituting this value into the linear regression equation obtained above, the concentrations of moxifloxacin and norfloxacin in milk can be calculated.

[0093] Fluorescent probe detection of moxifloxacin and norfloxacin spiked recovery in milk:

[0094] As shown in Table 2, after adding 0.1, 1, and 10 mg / L of moxifloxacin or norfloxacin to milk samples, the recoveries ranged from 85% to 109%, with relative standard deviations (RSDs) less than 3.9%. This demonstrates that the fluorescent probe of the present invention has good accuracy and reliability in detecting moxifloxacin and norfloxacin in milk.

[0095] Linear range and sensitivity of fluorescent probe for detection of moxifloxacin and norfloxacin in milk:

[0096] As the concentration of moxifloxacin increases, the fluorescence intensity of the fluorescent probe at 520 nm gradually increases. 520 There was a good linear relationship between / F0 and the concentration of moxifloxacin in the range of 0-100 mg / L, with the regression equation being y = 0.0710x + 0.9834 and the correlation coefficient R 2=0.9955 (where x is the moxifloxacin concentration and y is the increase in fluorescence intensity after the addition of moxifloxacin). According to the formula LOD = 3σ / k (where σ is the standard deviation of the fluorescence intensity ratio and k is the slope of the standard curve), the detection limit of this fluorescent probe for moxifloxacin in milk was calculated to be 1.01 μg / L, indicating that it can achieve highly sensitive detection of moxifloxacin in milk.

[0097] As the concentration of norfloxacin increases, the fluorescence intensity of the fluorescent probe at 460 nm gradually increases. 460 / F0 has a good linear relationship with the concentration of norfloxacin in the range of 0-100 mg / L, and the regression equation is y = 0.6092x + 1.0375, with a correlation coefficient R 2 =0.9993 (where x is the norfloxacin concentration and y is the increase in fluorescence intensity after the addition of norfloxacin). According to the formula LOD = 3σ / k (where σ is the standard deviation of the fluorescence intensity ratio and k is the slope of the standard curve), the detection limit of this fluorescent probe for moxifloxacin in milk was calculated to be 1.29 μg / L, indicating that it can achieve highly sensitive detection of norfloxacin in milk.

[0098] The recognition ability of the fluorescent probe for moxifloxacin, norfloxacin and their mixture in milk:

[0099] The linear discriminant analysis (LDA) model was constructed using the fluorescence intensity of different groups of samples at 460 nm and 520 nm. Figure 10 As shown in (B), the LDA model can be used to separate moxifloxacin, norfloxacin, and their mixture in milk samples, with classification accuracy rates greater than 0.989, demonstrating that the fluorescent probe has the potential to analyze moxifloxacin and norfloxacin in complex mixed real samples.

[0100] Table 1. Linear relationships and detection limits of moxifloxacin and norfloxacin in water and milk samples.

[0101]

[0102] Performance testing:

[0103] Recovery tests were conducted on spiked samples to evaluate the accuracy and precision of the fluorescent probe prepared in Example 1 of the present invention for detecting the contents of moxifloxacin and norfloxacin in milk. The results are shown in Table 2.

[0104] Table 2. Spiked recoveries of moxifloxacin and norfloxacin in milk samples.

[0105]

[0106] Data Analysis:

[0107] As shown in Table 2, when milk samples were spiked with 0.1, 1, and 10 mg / L of moxifloxacin or norfloxacin, the recoveries ranged from 85% to 109%, with relative standard deviations (RSDs) less than 3.9%. This demonstrates that the fluorescent probe of the present invention has good accuracy and reliability and is suitable for the rapid detection of moxifloxacin and norfloxacin in actual milk samples.

[0108] The above is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

Claims

1. A method for preparing a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin, characterized in that: The steps include: Step (1), dissolving p-aminosalicylic acid and L-cysteine ​​in anhydrous ethanol, stirring to fully dissolve them, to obtain a mixed solution; Step (2), placing the mixed solution in a high-pressure reactor, heating and reacting to obtain a reaction product; Step (3), cooling the reaction product to room temperature, centrifuging, filtering the supernatant, transferring the filtrate into a dialysis bag, and dialyzing in ultrapure water to obtain a carbon quantum dot CQDs solution; Step (4): dilute the carbon quantum dot CQDs solution with ultrapure water to obtain a fluorescent probe solution.

2. The method for preparing a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin according to claim 1, wherein: The mass ratio of p-aminosalicylic acid to L-cysteine ​​in step (1) is 4:

1.

3. The method for preparing a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin according to claim 1, wherein: The mass ratio of the anhydrous ethanol volume to the p-aminosalicylic acid in step (1) is 500 mL:4 g.

4. The method for preparing a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin according to claim 1, wherein: The autoclave in step (2) is provided with a Teflon lining, and the autoclave is heated at 200° C. for 14 hours.

5. The method for preparing a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin according to claim 1, wherein The molecular weight cut-off of the dialysis bag in step (3) is 1000 Da.

6. The method for preparing a fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin according to claim 1, wherein: In step (4), the carbon quantum dot CQDs solution is diluted 50 times with ultrapure water to obtain a fluorescent probe solution.

7. A fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin, characterized in that: The method is prepared according to any one of claims 1 to 5.

8. The use of the fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin according to claim 7, characterized in that: Used to detect the content of moxifloxacin and norfloxacin in water or milk products.

9. The use of the fluorescence-enhanced probe for rapid detection of moxifloxacin and norfloxacin according to claim 7, characterized in that: Testing for moxifloxacin and norfloxacin in water or milk products involves the following steps: Step (1), mixing the fluorescent probe solution and the moxifloxacin or norfloxacin spiked solution evenly, recording the fluorescence intensity of the mixed solution; establishing a linear regression model between the fluorescence intensity and the moxifloxacin or norfloxacin concentration; Step (2): uniformly mix the pretreated aqueous solution or milk extract to be tested with the fluorescent probe solution, and measure the fluorescence intensity of the mixed solution; substitute the measured fluorescence intensity into the obtained linear regression equation to calculate the concentration of moxifloxacin or norfloxacin.

10. The use of the fluorescence-enhanced probe for detecting moxifloxacin and norfloxacin in water and milk products according to claim 9, characterized in that: The volume ratio of the fluorescent probe solution to the spiked solution in step (1) is 50:

1.

11. The use of the fluorescence-enhanced probe for detecting moxifloxacin and norfloxacin in water and milk products according to claim 9, characterized in that: The volume ratio of the fluorescent probe solution to the test solution in step (2) is 50:1.

Images