Preparation method and application of fluorescence enhanced probe for rapidly detecting moxifloxacin and norfloxacin

Abstract

The invention 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 detection of antibiotics. The fluorescence enhanced probe is prepared by taking p-aminosalicylic acid and L-cysteine as raw materials through a hydrothermal method, and the optimal preparation process comprises the following steps: dissolving aminosalicylic acid and L-cysteine in a mass ratio of 4: 1 in absolute ethyl alcohol, and heating and reacting at 200 DEG C for 14 hours; the moxifloxacin and the norfloxacin can significantly enhance the fluorescence intensity of the fluorescent probe at 520 nm and 460 nm respectively, and have a good linear relationship with the concentration. The method has the advantages of simplicity in operation, low cost, rapidness in detection, high sensitivity, high selectivity, wide detection range, low detection limit and the like, 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] Quinolone antibiotics moxifloxacin and norfloxacin are mainly used to treat infections caused by bacteria and are widely used in the medical and animal husbandry industries. However, the abuse and misuse of moxifloxacin and norfloxacin may lead to antibiotic residues in the environment and food, and cause a variety of potential hazards to human health, including allergic reactions, neurotoxicity and drug resistance. Therefore, the detection of moxifloxacin and norfloxacin residues in the environment and food is of great significance to protect 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 techniques. However, the analysis and testing of environmental and food samples are usually time-sensitive, and traditional detection technologies are 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 for protecting human health.

[0004] Fluorescence method is to use the change of fluorescence signal emitted by fluorescent substances after being excited by light of specific wavelength to conduct qualitative and quantitative analysis of the target. Fluorescence method has the advantages of simple operation, fast response speed, high sensitivity and good specificity, and has broad prospects 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 realizing the detection and analysis of non-fluorescent substances. Although some fluorescence quenching probes can be used for the detection of antibiotics, the background signal of fluorescence quenching probes is high, which affects the sensitivity and accuracy of their detection. In contrast, fluorescence enhanced probes have high sensitivity and are particularly suitable for the detection of low-concentration pollutants. Therefore, it is of great significance to develop a fluorescence enhanced probe for the rapid detection of moxifloxacin and norfloxacin in the environment and food. 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, high-sensitivity and high-specificity rapid 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: Step (1), dissolving p-aminosalicylic acid and L-cysteine ​​in a mass ratio of 4:1-8:1 in anhydrous ethanol, stirring to fully dissolve them, and obtaining a mixed solution; Step (2), placing the mixed solution in a high pressure reactor and heating it at 120° C.-200° C. for 10 h-18 h to obtain a reaction product; Step (3), cooling the reaction product to room temperature, centrifuging, and obtaining a crude carbon quantum dot solution supernatant; Step (4), after the supernatant of the crude carbon quantum dot solution is filtered through a filter membrane, the filtrate is transferred into a dialysis bag and dialyzed in ultrapure water to obtain a carbon quantum dot CQDs solution; Step (5), diluting the carbon quantum dots CQDs solution with ultrapure water to obtain a fluorescent probe solution.

[0007] Preferably, in step (1), the mass ratio of p-aminosalicylic acid to L-cysteine ​​is 4:1; and the volume ratio of anhydrous ethanol to p-aminosalicylic acid is 500 mL:4 g.

[0008] Preferably, the autoclave in step (2) is provided with a Teflon lining, and the autoclave is heated at 200° C. for 14 h.

[0009] Preferably, the centrifugal speed in step (3) is 10000 r / min, and the centrifugal time is 10 min.

[0010] 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.

[0011] Preferably, in step (5), the carbon quantum dots CQDs solution is diluted 50 times with ultrapure water.

[0012] 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.

[0013] The fluorescent probe of the present invention is excited under 355 nm ultraviolet light and has a strong fluorescent signal at 520 nm; after adding moxifloxacin to the fluorescent probe solution, the fluorescent signal at 520 nm increases significantly; after adding norfloxacin to the fluorescent probe solution, the fluorescent signal at 460 nm increases significantly. The detection mechanism is that the fluorescent probe and moxifloxacin form hydrogen bonds or π-π bonds, so that the conjugated surface of the fluorescent probe is larger and the rigid structure is more stable, thereby enhancing the fluorescence of the fluorescent probe at 520 nm; norfloxacin can combine with the fluorescent probe through electrostatic interaction, promote the transfer of electrons from the fluorescent probe to norfloxacin, resulting in the blue shift of the fluorescent emission peak of the fluorescent probe from 520 nm to 460 nm; and the fluorescent probe and norfloxacin form hydrogen bonds or π-π bonds, so that the conjugated surface of the fluorescent probe is larger and the rigid structure is more stable, thereby enhancing the fluorescence of the fluorescent probe at 460 nm. The concentration of moxifloxacin and norfloxacin can be detected by recording the change in fluorescence intensity enhancement after adding different concentrations of moxifloxacin or norfloxacin into the system. The fluorescence intensity of the fluorescent probe has a good linear relationship with the concentration of moxifloxacin and norfloxacin, and the linear ranges of quantitative detection of moxifloxacin and norfloxacin concentrations are 0-249 μM and 0-313 μM, respectively, and the minimum detection limits are 2.5 nM and 4.0 nM, respectively. It has a high detection sensitivity for moxifloxacin and norfloxacin, and has good selectivity, and can be used for rapid detection of moxifloxacin and norfloxacin in water and milk.

[0014] 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.

[0015] Preferably, the steps of using the fluorescent probe to detect the content of moxifloxacin in water are as follows: Step (1), the fluorescent probe solution and the moxifloxacin spiked solution (0-100 mg / L) are mixed evenly, and the fluorescence intensity of the mixed solution at 520 nm under an excitation wavelength of 355 nm is recorded; the increase factor of the fluorescence intensity after adding moxifloxacin is taken as F 520 / F0 was taken as the ordinate and moxifloxacin concentration as the abscissa, and a linear regression model was established; Step (2), centrifuging the water sample at a speed of 10000 r / min for 3 min, filtering the supernatant with a 0.22 μm microporous filter membrane; mixing the filtered solution to be tested with the fluorescent probe solution, and measuring the fluorescence intensity of the mixed solution at 520 nm under an excitation wavelength of 355 nm; substituting the measured fluorescence intensity into the obtained linear regression equation to calculate the concentration of moxifloxacin in the water.

[0016] Preferably, the volume ratio of the fluorescent probe solution to the spiked solution in step (1) is 50:1.

[0017] Preferably, the volume ratio of the fluorescent probe solution to the test solution in step (2) is 50:1.

[0018] Preferably, the steps of detecting the norfloxacin content in water are as follows: Step (1), the fluorescent probe solution and the norfloxacin spiked solution (0-100 mg / L) are mixed evenly, and the fluorescence intensity of the mixed solution at 460 nm under an excitation wavelength of 355 nm is recorded; the increase factor of the fluorescence intensity after adding norfloxacin is F 460 / F0 was taken as the ordinate and norfloxacin concentration as the abscissa, and a linear regression model was established; Step (2), centrifuging the water sample at a speed of 10000 r / min for 3 min, filtering the supernatant with a 0.22 μm microporous filter membrane; mixing the filtered solution to be tested with the fluorescent probe solution, and measuring the fluorescence intensity of the mixed solution at 460 nm under an excitation wavelength of 355 nm; substituting the measured fluorescence intensity into the obtained linear regression equation to calculate the concentration of norfloxacin in the water.

[0019] Preferably, the volume ratio of the fluorescent probe solution to the spiked solution in step (1) is 50:1.

[0020] Preferably, the volume ratio of the fluorescent probe solution to the test solution in step (2) is 50:1.

[0021] Preferably, the steps of detecting the content of moxifloxacin in milk are as follows: Step (1), mixing the control milk with acetonitrile, adding sodium chloride, vortexing for 30 seconds to fully mix, centrifuging at 10000 r / min for 3 minutes, filtering the supernatant with a 0.22 μm microporous filter membrane, and collecting the milk extract; Step (2), the fluorescent probe solution and the milk extract spiked with moxifloxacin (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 of the fluorescence intensity after adding moxifloxacin was taken as F 520 / F0 was taken as the ordinate and moxifloxacin concentration as the abscissa, and a linear regression model was established; Step (3), mixing the milk to be tested with acetonitrile, adding sodium chloride, vortexing for 30 seconds to fully mix, centrifuging at 10000 r / min for 3 minutes, and filtering the supernatant with a 0.22 μm microporous filter membrane; Step (4), the filtered test solution and the fluorescent probe solution are mixed evenly, and the fluorescence intensity of the mixed solution at 520 nm under an excitation wavelength of 355 nm is measured; the measured fluorescence intensity is substituted into the obtained linear regression equation to calculate the concentration of moxifloxacin in the milk.

[0022] 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.

[0023] Preferably, the volume ratio of the fluorescent probe solution to the spiked solution in step (2) is 50:1.

[0024] 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.

[0025] Preferably, the volume ratio of the fluorescent probe solution to the sample solution in step (4) is 50:1.

[0026] Preferably, the steps of detecting the norfloxacin content in milk are as follows: Step (1), mixing milk and acetonitrile, adding sodium chloride, vortexing for 30 seconds to fully mix, and then centrifuging at 10000r / min for 3 minutes, filtering the supernatant with a 0.22 μm microporous filter membrane, and collecting the milk extract; Step (2), the fluorescent probe solution and the milk extract spiked with norfloxacin (0-100 mg / L) are mixed evenly, and the fluorescence intensity of the mixed solution at 520 nm under an excitation wavelength of 355 nm is recorded; the increase factor of the fluorescence intensity after adding norfloxacin is F 460 / F0 was taken as the ordinate and norfloxacin concentration as the abscissa, and a linear regression model was established; Step (3), mixing the milk with acetonitrile, adding sodium chloride, vortexing for 30 seconds to fully mix, and then centrifuging at 10000 r / min for 3 minutes, and filtering the supernatant with a 0.22 μm microporous filter membrane; Step (4), the filtered test solution and the fluorescent probe solution are mixed evenly, and the fluorescence intensity of the mixed solution at 460 nm under an excitation wavelength of 355 nm is measured; the measured fluorescence intensity is substituted into the obtained linear regression equation to calculate the concentration of norfloxacin in the milk.

[0027] 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.

[0028] Preferably, the volume ratio of the fluorescent probe solution to the spiked solution in step (2) is 50:1.

[0029] 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.

[0030] Preferably, the volume ratio of the fluorescent probe solution to the sample solution in step (4) is 50:1.

[0031] Compared with the prior art, the present invention has the following beneficial effects: After a fluorescence-enhanced probe prepared by the present invention is combined with moxifloxacin, the fluorescence emission intensity at 520 nm is significantly increased; after the probe is combined with norfloxacin, the fluorescence emission intensity at 460 nm is significantly increased, and the fluorescence increase factor is in a good linear relationship with the concentration of moxifloxacin or norfloxacin. The detection limits of the probe for moxifloxacin and norfloxacin are 2.5 nM and 4.0 nM, respectively, and the probe is suitable for rapid detection of moxifloxacin and norfloxacin in water and milk products. A fluorescence-enhanced probe prepared by the present invention has the advantages of simple preparation process, low cost, fast detection speed, high sensitivity and good selectivity, and can detect moxifloxacin and norfloxacin at the same time, providing a simple and rapid strategy for on-site quantitative detection of moxifloxacin and norfloxacin. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The figure is a schematic diagram of the preparation and detection of the fluorescence enhanced probe for detecting moxifloxacin and norfloxacin involved in the present invention; Figure 2 This is a synthesis time optimization diagram of carbon quantum dots CQDs involved in the present invention; Figure 3 (A) UV absorption, fluorescence excitation and emission spectra of the carbon quantum dots CQDs involved in the present invention, the inset is a color comparison of the solution under sunlight and 365 nm UV light; (B) infrared spectrum; (C) transmission electron microscope image, the inset is a particle size distribution diagram; (D) X-ray diffraction diagram and (E) X-ray photoelectron energy spectrum diagram; Figure 4 High-resolution X-ray photoelectron spectra of (A) C1s, (B) O1s, (C) N1s and (D) S2p of the carbon quantum dots CQDs involved in the present invention; 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; Figure 6 The fluorescence emission spectra of the fluorescent probe of the present invention after being mixed with (A) moxifloxacin and (B) norfloxacin of different concentrations; Figure 7The linear relationship between the increase factor of the fluorescence intensity of the probe involved in the present invention and the concentration of (A) moxifloxacin and (B) norfloxacin; Figure 8 The fluorescence intensity increase factor of the fluorescent probe of the present invention after being mixed with moxifloxacin or norfloxacin under (A) 25°C storage for 14 days; (B) 4°C storage for 14 days and (C) different pH ranges; Fig. 9 It is a comparison diagram of the fluorescence intensity increase factor after the fluorescent probe involved in the present invention is mixed with 5 mg / L moxifloxacin, norfloxacin and 100 mg / L different interfering substances (antibiotics, pesticides and metal ions); Fig.10 The present invention provides the fluorescent probe for the identification of moxifloxacin and norfloxacin in (A) aqueous solution and (B) milk samples. DETAILED DESCRIPTION

[0033] Example 1: A method for preparing a fluorescence enhanced probe for rapid detection of moxifloxacin and norfloxacin, comprising the following steps: Step (1), weigh 0.4 g of p-aminosalicylic acid and 0.1 g of L-cysteine, dissolve them in 50 mL of anhydrous ethanol, and stir to fully dissolve them to obtain a mixed solution; Step (2), placing the mixed solution in a polytetrafluoroethylene-lined high-pressure reactor, heating at 200° C. for 14 h to obtain a reaction product; Step (3), cooling the reaction product to room temperature, transferring it to a 50 mL centrifuge tube, and centrifuging it at 10000 r / min for 10 min to remove some large solid impurities to obtain a crude carbon quantum dot solution supernatant; Step (4), filtering the supernatant of the crude carbon quantum dot solution with a 0.22 μm filter membrane to filter out small particulate impurities, and transferring the resulting filtrate into a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzing in ultrapure water for 48 h-72 h to remove small molecular impurities, thereby obtaining a carbon quantum dot CQDs solution; 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 away from light.

[0034] Synthesis conditions of carbon quantum dots CQDs: Figure 2 The effect of synthesis time on the fluorescence intensity of the prepared carbon quantum dots CQDs is shown. When the synthesis time is in the range of 10-18h, the fluorescence intensity reaches the highest when the synthesis time is 14h.

[0035] Structural characterization of carbon quantum dots CQDs: like Figure 3 As shown in (A), the carbon quantum dot CQDs solution is a colorless and transparent solution 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-1 corresponds to the stretching vibration peak of OH or NH bonds. -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 phenomenon. 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 3 As 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; 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 shown 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), in the high-resolution XPS spectrum of S2p, there are two fitting peaks at 163.7 eV and 164.8 eV, which are attributed to CSC S 2P3 / 2 and SO4 2- S 2P3 / 2The above results are in good agreement with those of the infrared spectrum.

[0036] Stability of fluorescent probes: 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 7-14. These results verify the stability of the fluorescent probe and lay the foundation for its practical application.

[0037] Fluorescent probe detection of moxifloxacin and norfloxacin: 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 has a good linear relationship with the concentration of moxifloxacin in the range of 0-100 mg / L. The calculated LODs and LOQs of the fluorescent probe for moxifloxacin are 0.66 μg / L and 2.18 μg / L.

[0038] like Figure 6 As shown in (B), after the addition of norfloxacin, the maximum emission wavelength of the fluorescent probe blue-shifted to 460 nm, and the fluorescence intensity increased; as the concentration of norfloxacin increased, the fluorescence of the fluorescent probe at 460 nm gradually increased. Figure 7 (B) shows the fluorescence intensity increase factor F 460 / F0 has a good linear relationship with the concentration of norfloxacin in the range of 0-100 mg / L. The LODs and LOQs of the fluorescent probe for norfloxacin were calculated to be 0.42 μg / L and 1.39 μg / L.

[0039] 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 with 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 with 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.

[0040] Selectivity of fluorescent probes: 100 mg / L of antibiotics (ampicillin, oxytetracycline, tetracycline hydrochloride, sulfamethoxazole, and sulfamethoxazole), pesticides (bifenthrin, boscalid, chlormequat, cyhalothrin, cypermethrin, dicamba, flonicamid, fluazifop, and chlorfenapyr), and metal ions (Ag + , 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 evaluate the selectivity of the probe for moxifloxacin and norfloxacin. Fig. 9 As 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 Fig. 9 (B) shows that 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 is significantly higher than other common pollutants. These results indicate that the fluorescent probe has good selectivity for moxifloxacin and norfloxacin.

[0041] Example 2: Rapid detection of moxifloxacin and norfloxacin in water using a fluorescence-enhanced probe, comprising the following steps: Take 4 mL of the fluorescent probe solution in Example 1, add 80 μL of moxifloxacin spiked aqueous solution of different concentrations (0-100 mg / L) thereto, mix well, and measure and record the fluorescence intensity at 520 nm under 355 nm excitation wavelength before and after adding the moxifloxacin solution. A linear regression model is established with the increase multiple of the fluorescence intensity after adding moxifloxacin as the ordinate and the moxifloxacin concentration as the abscissa.

[0042] Take 4 mL of the fluorescent probe solution in Example 1, add 80 μL of different concentrations (0-100 mg / L) of norfloxacin spiked aqueous solution, mix well, and measure and record the fluorescence intensity at 460 nm under 355 nm excitation wavelength before and after adding the norfloxacin solution. A linear regression model is established with the increase in fluorescence intensity after adding norfloxacin as the ordinate and the norfloxacin concentration as the abscissa.

[0043] 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.

[0044] Linear range and sensitivity of fluorescent probe for detecting moxifloxacin and norfloxacin in water: 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 / F0 has a good linear relationship with the concentration of moxifloxacin in the range of 0-100 mg / L, and the regression equation is y = 0.0657 x + 0.9495, with a correlation coefficient R 2 =0.9918 (where x is the concentration of moxifloxacin, and y is the multiple of the fluorescence intensity increase 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 the fluorescent probe for moxifloxacin in water is calculated to be 0.66 μg / L, which means that high-sensitivity detection of moxifloxacin can be achieved.

[0045] 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 / 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 = 0.7164 x + 2.1677, with a correlation coefficient R 2 =0.9887 (where x is the concentration of norfloxacin, and y is the multiple of the fluorescence intensity increase 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 the fluorescent probe for norfloxacin in water is calculated to be 0.42 μg / L, which means that high-sensitivity detection of norfloxacin can be achieved.

[0046] The recognition ability of fluorescent probes for moxifloxacin, norfloxacin and their mixture in water: The linear discriminant analysis (LDA) model was constructed using the fluorescence intensity of different groups of samples at 460 nm and 520 nm. Fig.10 As shown in (A), the LDA model can be used to separate moxifloxacin, norfloxacin and their mixture in aqueous solution, and the classification accuracy is greater than 0.992, demonstrating the potential of this fluorescent probe in analyzing complex mixed samples.

[0047] Example 3: Rapid detection of moxifloxacin and norfloxacin in milk using a fluorescence-enhanced probe, comprising the following steps: The milk was pretreated to remove the protein and fat contained therein. 5 mL of blank milk was mixed with 5 mL of acetonitrile, and 2 g of sodium chloride was added. The mixture was vortexed for 30 seconds to mix thoroughly, and then centrifuged at 10,000 r / min for 3 minutes. The supernatant was filtered with a 0.22 μm microporous membrane to collect the milk extract.

[0048] Take 4 mL of the fluorescent probe solution in Example 1, add 80 μL of milk extract containing different concentrations (0-100 mg / L) of moxifloxacin, mix well, and measure and record the fluorescence intensity at 520 nm under an excitation wavelength of 355 nm before and after adding the moxifloxacin solution. A linear regression model is established with the increase in fluorescence intensity after adding moxifloxacin as the ordinate and the moxifloxacin concentration as the abscissa.

[0049] Take 4 mL of the fluorescent probe solution in Example 1, add 80 μL of milk extract containing different concentrations (0-100 mg / L) of norfloxacin, mix well, and measure and record the fluorescence intensity at 460 nm under an excitation wavelength of 355 nm before and after adding the norfloxacin solution. A linear regression model is established with the increase in fluorescence intensity after adding norfloxacin as the ordinate and the norfloxacin concentration as the abscissa.

[0050] 5 mL of the milk sample to be tested was mixed with 5 mL of acetonitrile, and 2 g of sodium chloride was added. The mixture was vortexed for 30 seconds to mix thoroughly, and then centrifuged at 10,000 r / min for 3 minutes. The supernatant was filtered with a 0.22 μm microporous filter membrane to collect the solution to be tested. 80 μL of the filtered sample to be tested 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 milk.

[0051] Fluorescence probe detection of moxifloxacin and norfloxacin spike recovery in milk: As shown in Table 2, after adding 0.1, 1 and 10 mg / L moxifloxacin or norfloxacin to the milk samples, the recovery range was 85%-109%, and the relative standard deviation (RSD) was less than 3.9%. This shows that the fluorescent probe of the present invention has good accuracy and reliability in detecting moxifloxacin and norfloxacin in milk.

[0052] Linear range and sensitivity of fluorescent probe for detecting moxifloxacin and norfloxacin in milk: As the concentration of moxifloxacin increases, the fluorescence intensity of the fluorescent probe at 520 nm gradually increases. 520 / F0 has a good linear relationship with the concentration of moxifloxacin in the range of 0-100 mg / L, and the regression equation is y = 0.0710x + 0.9834, with a correlation coefficient R 2 =0.9955 (where x is the concentration of moxifloxacin, and y is the multiple of the fluorescence intensity increase 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 the fluorescent probe for moxifloxacin in milk is calculated to be 1.01 μg / L, which means that high-sensitivity detection of moxifloxacin in milk can be achieved.

[0053] 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 multiple of the fluorescence intensity increase 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 the fluorescent probe for moxifloxacin in milk is calculated to be 1.29 μg / L, which means that high-sensitivity detection of norfloxacin in milk can be achieved.

[0054] The recognition ability of fluorescent probes for moxifloxacin, norfloxacin and their mixture in milk: The linear discriminant analysis (LDA) model was constructed using the fluorescence intensity of different groups of samples at 460 nm and 520 nm. Fig.10 As shown in (B), based on the LDA model, moxifloxacin, norfloxacin and their mixture in milk samples can be separated from each other, and the classification accuracy is greater than 0.989, which proves that the fluorescent probe has the potential to analyze moxifloxacin and norfloxacin in complex mixed actual samples.

[0055] Table 1. Linear relationships and detection limits of moxifloxacin and norfloxacin in water and milk samples.

[0056] Performance Testing: The recovery test of the spiked samples was carried out to evaluate the accuracy and precision of the fluorescent probe prepared in Example 1 of the present invention for detecting the content of moxifloxacin and norfloxacin in milk. The results are shown in Table 2.

[0057] Table 2. Spiked recoveries of moxifloxacin and norfloxacin in milk samples.

[0058] Data Analysis: As shown in Table 2, when 0.1, 1 and 10 mg / L moxifloxacin or norfloxacin were added to the milk samples, the recovery rate of the method ranged from 85% to 109%, and the relative standard deviation (RSD) was less than 3.9%. This shows 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.

[0059] 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 belong to 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), diluting the carbon quantum dots 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, characterized in that: 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, characterized in that: The mass ratio of the volume of anhydrous ethanol in step (1) to the mass ratio of p-aminosalicylic acid 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, characterized in that: 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, characterized in that: 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, characterized in that: 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 by the preparation method 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, and 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), mixing the pretreated aqueous solution or milk extract to be tested with the fluorescent probe solution, and measuring the fluorescence intensity of the mixed solution; substituting 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.

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