A method for on-site fluorescent visualization detection of antibiotic residues

Abstract

The present application belongs to the field of environment and food detection technology, and specifically relates to a kind of on-site fluorescent visualization detection method of antibiotic residue.The present application constructs a convenient, fast and sensitive sensing detection platform by designing fluorescent probe molecule Eu@CDs with target recognition function and combining with smart phone portable sensor.The fluorescent probe molecule Eu@CDs of the present application has blue fluorescence emission at 450 nm.When NFZ is introduced, blue light is quantitatively quenched.After adding TC, the beta-diketone group of TC is combined with Eu 3+ Chelate, absorb energy and sensitize Eu through "antenna effect" 3+ Emit 617 nm characteristic red light.Thereby, the present application realizes on-site single-probe synchronous sampling detection of NFZ and TC, and the detection limit is 0.0034 mg / L and 0.00870 mg / L respectively, and the linear range is 0.011-32.0 mg / L and 0.029-40.0 mg / L respectively, which provides an efficient and accurate solution for rapid screening of environmental and food pollutants.

Description

Technical Field

[0001] This invention belongs to the field of environmental and food testing technology. Specifically, this invention relates to a method for on-site fluorescence visualization detection of antibiotic residues. Background Technology

[0002] Nitrofurazone (NFZ) and tetracycline (TC) are widely used veterinary antibiotics in aquaculture. Their residues are continuously introduced into the aquatic environment through aquaculture wastewater, medical discharges, and agricultural runoff, forming highly toxic compound pollutants. Tetracycline has a half-life of up to 180 days in water and induces the horizontal spread of antibiotic resistance genes (ARGs); the nitrofurazone metabolite saminoglycan (SEM) can accumulate in sediment for up to 5 years, and the coexistence of the two produces a synergistic toxic effect. These pollutants not only disrupt the balance of aquatic microbial communities but also threaten human health through drinking water and the food chain. Tetracycline accumulation can cause liver damage and drug-resistant bacterial infections, while nitrofurazone is classified as a Group 2B carcinogen (causing gene mutations) by the World Health Organization (WHO). Against this backdrop, there is a need to develop highly sensitive and interference-resistant on-site water pollution monitoring technologies that can simultaneously detect NFZ / TC to support ecological safety early warning and public health governance.

[0003] Currently, three main analytical methods are used for the detection of NF-Z and TC residues: chromatography-mass spectrometry (GC-MS) is accurate but susceptible to matrix interference; immunoassay relies on large instruments and has complex pretreatment, making it difficult to meet the needs of on-site screening; and emerging biosensor technologies, while offering high throughput, suffer from insufficient specificity and poor stability when co-detecting NF-Z and TC due to cross-reactivity. In summary, existing technologies struggle to simultaneously address the requirements of sensitivity, specificity, on-site detection, and simultaneous multi-component detection, necessitating the development of a novel, universal platform to overcome these bottlenecks. Summary of the Invention

[0004] To address the need for rapid and accurate detection of nitrofurazone (NFZ) and tetracycline (TC), this invention, through systematic research, develops a method for on-site fluorescence visualization detection of antibiotic residues. Specifically, the technical solution of this invention is as follows:

[0005] In one aspect, this invention discloses a method for preparing Eu@CDs, a fluorescence sensor for on-site fluorescence visualization detection of antibiotic (NFZ, TC) residues, the method comprising:

[0006] (1) Preparation of Eu@CDs by one-pot hydrothermal method: Urea, citric acid monohydrate, and europium(III) nitrate hexahydrate were dissolved in N,N-dimethylformamide and sonicated until transparent. The solution was transferred to a 50.0 mL polytetrafluoroethylene reactor and reacted at 180 °C for 8.0 h. After cooling to room temperature, the solution was filtered through a 0.22 μm filter membrane. 0.2 mg / mL sodium hydroxide solution was added, and the reaction was continued for 30.0 min. After the reaction was completed, the solution was centrifuged at 10000.0 rpm for 10.0 min, the precipitate was collected, washed three times with deionized water, and finally freeze-dried to obtain the product Eu@CDs.

[0007] In one embodiment, the mass ratio of urea:citric acid monohydrate:europium(III) nitrate hexahydrate is 1.5:1.05:0.45, the time for ultrasonication to the transparent state is 30.0 min, and the centrifugation parameters are 10000.0 rpm and 10.0 min.

[0008] In one embodiment, the present invention discloses a method for preparing Eu@CDs, a fluorescence sensor for on-site fluorescence visualization detection of antibiotic residues, wherein the antibiotic residues are furacilin and tetracycline, and the method includes:

[0009] (1) Dissolve 1.05 g of citric acid monohydrate (molecular weight 210.14 g / mol, 250.0 mM), 1.5 g of urea (molecular weight 60.06 g / mol, 1250.0 mM), and 0.45 g of europium(III) nitrate hexahydrate (molecular weight 446.0 g / mol, 50.0 mM) in 20.0 mL of N,N-dimethylformamide. Sonicate for 30.0 min until transparent. Transfer to a 50.0 mL polytetrafluoroethylene reactor and react at 180 ℃ for 8.0 h. Cool to room temperature and filter through a 0.22 μm filter membrane. Add 0.2 mg / mL sodium hydroxide solution and react for 30.0 min. After the reaction is complete, centrifuge the solution at 10000.0 rpm for 10.0 min, collect the precipitate, wash the precipitate three times with deionized water, and finally freeze-dry to obtain the product Eu@CDs.

[0010] In one aspect, the present invention discloses a method for on-site fluorescence visualization detection of antibiotic residues, the method comprising the following steps:

[0011] S1: The Eu@CDs fluorescent probe was mixed with the test sample of furazolidone (NFZ). After reacting for a period of time, the mixture was transferred to a fluorescent dish. The fluorescence intensity value Fn of the reactant at 450 nm under the excitation wavelength of 370 nm was measured using a fluorescence spectrophotometer. Then, the fluorescence signal was captured using a smartphone, and the image RGB analysis was performed using ImageJ software to obtain the R / B value of NFZ.

[0012] S2: Substitute the fluorescence intensity value and R / B value obtained in step S1 into the linear equation constructed based on NFZ standards with different concentrations, and calculate the NFZ concentration in the sample respectively.

[0013] S3: Mix the Eu@CDs fluorescent probe with the tetracycline (TC) sample, react for a period of time, then transfer to a fluorescence dish. Measure the fluorescence intensity F of the reactants at 450 nm and 617 nm using a fluorescence spectrophotometer at an excitation wavelength of 370 nm. 450 F 617 Calculate the fluorescence intensity ratio F 617 nm / F 450 nm， Then, the fluorescence signal was captured using a smartphone, and the RGB values ​​of the image were analyzed using ImageJ software to obtain the R / B value of TC.

[0014] S4: The fluorescence intensity ratio F obtained in step S3 is... 617 nm / F 450 nm Substituting R / B into the linear equations constructed based on TC standards with different concentrations, the TC concentration in the samples was calculated.

[0015] In one embodiment, the preparation method of the furacilin standard curve is as follows: Under room temperature conditions, the Eu@CDs fluorescence sensor and furacilin (NFZ) at different concentration gradients are added to Tris-HCl buffer, thoroughly stirred and mixed, and after reacting for a period of time, transferred to a fluorescence dish. The fluorescence intensity of the reactants at 450 nm under an excitation wavelength of 370 nm is measured using a fluorescence spectrophotometer and denoted as F. q After irradiation with a 365 nm UV lamp, fluorescence signals were captured using a smartphone, and the R / B values ​​were obtained through RGB image analysis using ImageJ software. Fluorescence intensity values ​​F were measured using NFZ standards at different concentration gradients. q The fluorescence linear regression equation F = XC was constructed using the R / B value and the concentration of the standard. NFZ + y、R / B= XC NFZ + y, where C NFZ This refers to the concentration of the standard.

[0016] In one embodiment, the tetracycline standard curve is prepared as follows: At room temperature, the Eu@CDs fluorescence sensor and tetracycline at different concentration gradients are added to a Tris-HCl buffer solution, thoroughly stirred and mixed. After reacting for a period of time, the mixture is transferred to a fluorescence dish, and the fluorescence intensity of the reactants at 450 nm and 617 nm under a 370 nm excitation wavelength is measured using a fluorescence spectrophotometer. This intensity is denoted as F1. 450 F 617， Calculate F 617 nm / F 450 nm After irradiation with a 365 nm UV lamp, fluorescence signals were captured using a smartphone, and the R / B values ​​of tetracycline were obtained through RGB image analysis using ImageJ software. Fluorescence intensities (F) measured using TC standards at different concentration gradients were also analyzed. 617 nm / F 450 nm A fluorescence linear regression equation F was constructed using the R / B ratio and the concentration of the standard. 617 nm / F 450 nm = XC TC + y, R / B = XC TC + y, where C TC This refers to the concentration of the standard.

[0017] In one aspect, this invention discloses a method for on-site fluorescence visualization detection of antibiotic residues, comprising the following steps:

[0018] S1: Determination of fluorescence intensity at 450 nm for NFZ standard test solutions with different concentration gradients. F: Under room temperature conditions, the Eu@CDs fluorescence sensor and NFZ with different concentration gradients were added to Tris-HCl buffer solution for reaction. The mixture was thoroughly stirred and reacted at 35 °C for 1.5 min, then transferred to a fluorescence dish. The fluorescence intensity at 450 nm (excitation wavelength 370 nm) was then measured using a fluorescence spectrophotometer and recorded as F. q The sample was dropped onto a filter paper strip, excited by 365 nm ultraviolet light, and the fluorescence signal was captured using a smartphone. ImageJ software was used for RGB analysis of the image to obtain the R / B value of NFZ.

[0019] S2: Constructing a linear regression equation for the standards: using the fluorescence intensity values ​​F measured from NFZ standards at different concentration gradients. q The fluorescence linear regression equation F = XC was constructed using the R / B value and the concentration of the standard. NFZ + y、R / B= XC NFZ + y, where C NFZ For standard concentration;

[0020] S3: Determine the ratio F of fluorescence intensity at 450 nm and 617 nm for the test solution of TC standard at different concentration gradients.617 nm / F 450 nm At room temperature, the Eu@CDs fluorescence sensor and TC at different concentration gradients were added to Tris-HCl buffer for reaction. The mixture was thoroughly stirred and reacted at 35 °C for 1.5 min, then transferred to a fluorescence dish. The fluorescence intensity at 450 nm and 617 nm (excitation wavelength 370 nm) was then measured using a fluorescence spectrophotometer and denoted as F1. 450 and F 617 Calculate their ratio F 617 nm / F 450 nm The sample was dropped onto a filter paper strip, excited by 365 nm ultraviolet light, and the fluorescence signal was captured using a smartphone. ImageJ software was used for RGB analysis of the image to obtain the R / B value of TC.

[0021] S4: Constructing a linear regression equation for the standards: using the ratio F of fluorescence intensity measured from TC standards at different concentration gradients. 617 nm / F 450 nm A fluorescence linear regression equation F was constructed using the R / B ratio and the concentration of the standard. 617 nm / F 450 nm = XC TC + y, R / B = XC TC +y, where C TC For standard concentration;

[0022] S5: Take the sample to be tested, repeat S2 and S4 to obtain the fluorescence intensity value or ratio and R / B value of each sample, and substitute them into the corresponding linear equation to obtain the NFZ and TC concentrations in the sample to be tested.

[0023] In one embodiment: in steps S1 and S3, the pH of the Tris-HCl buffer is 8.3-8.7; the concentration of the Eu@CDs fluorescence sensor is 100.0 mg / L; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 35 ℃; and the reaction time is 1.5 min.

[0024] In one embodiment: In S2, the linear regression equation for the fluorescence spectrophotometer is F = -47.0608 C. NFZ +2748.1595, R / B = 0.01921 C NFZ + 0.10289;

[0025] In one embodiment: In S4, the linear regression equation for the fluorescence spectrophotometer is F. 617nm / F 450nm = 0.01402C TC +0.03211、R / B = 0.02926 C TC+ 0.19981;

[0026] In one aspect, the present invention discloses the application of the fluorescence sensor Eu@CDs in the detection of NFZ and TC.

[0027] In one aspect, the present invention discloses a reagent for detecting NFZ and TC, said reagent comprising a fluorescence sensor Eu@CDs.

[0028] In one aspect, the present invention discloses a kit for detecting NFZ and TC, the kit comprising a fluorescence sensor Eu@CDs, nitrofurazone standard, tetracycline standard, and buffer solution. Beneficial effects

[0029] This invention utilizes a dual-channel detection system with a single fluorescence sensor to simultaneously detect two antibiotics based on changes in fluorescence signals. The detection principle is simple and easy to use.

[0030] This invention does not require complex instruments, is low in cost, and can collect fluorescence signals using only a smartphone. It is simple to operate and can be used quickly and easily.

[0031] Compared with existing chromatographic and fluorescence detection technologies, this invention has a shorter detection time, requiring only 1.5 minutes to observe changes in fluorescence. Attached Figure Description

[0032] Figure 1 Design of an on-site fluorescence visualization detection platform for antibiotic residues.

[0033] Figure 2 (A) is the TEM image of Eu@CDs and (B) is the infrared spectrum of Eu@CDs and CDs.

[0034] Figure 3 , where (A) is the hydrated particle size of Eu@CDs and CDs and (B) is the Zeta potential of Eu@CDs and CDs.

[0035] Figure 4 Optimization of conditions for Eu@CDs detection of furazolidone, including pH and temperature.

[0036] Figure 5 Optimization of conditions for Eu@CDs detection of tetracycline, including pH and temperature.

[0037] Figure 6 To verify the feasibility of detecting furazolidone and tetracycline with Eu@CDs, different groups were used: (1) CDs, (2) Eu@CDs, (3) Eu@CDs + TC, (4) Eu@CDs + NFZ.

[0038] Figure 7 The fluorescence spectra and standard curves of standards containing different concentrations of furazolidone are shown.

[0039] Figure 8 The fluorescence spectra and standard curves of tetracycline standards containing different concentrations are shown.

[0040] Figure 9 Selectivity tests for the Eu@CDs detection system. (A) Fluorescence intensity values ​​at 450 nm. Values ​​1-15 represent blank, kanamycin, amoxicillin, chloramphenicol, gentamicin, ampicillin, streptomycin, histidine, alanine, glutamic acid, tyrosine, methionine, serine, TC, and NFZ, respectively. (B) Fluorescence intensity values ​​at 617 nm. Values ​​1-15 represent blank, kanamycin, amoxicillin, chloramphenicol, gentamicin, ampicillin, streptomycin, histidine, alanine, glutamic acid, tyrosine, methionine, serine, NFZ, and TC, respectively.

[0041] Figure 10 The standard curves are RGB signals (R / B) containing different concentrations of furazolidone and tetracycline standards. Detailed Implementation

[0042] Please see Figure 1-10 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and to provide a clear understanding of the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0043] Reagents used in the embodiments of this invention:

[0044] The raw materials used in this invention are: urea, citric acid monohydrate, europium(III) nitrate hexahydrate, tetracycline, and furazolidone, which were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Tris-HCl hydrochloride buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and deionized water was used in the experimental process.

[0045] Unless otherwise specified, all reagents or materials described in the following examples are commercially available.

[0046] The technical solution of the present invention will be further described in detail below with reference to examples.

[0047] Example 1: Synthesis of Eu@CDs Fluorescent Sensor

[0048] A method for fabricating a fluorescence sensor Eu@CDs, the method comprising:

[0049] (1) Preparation of Eu@CDs by one-pot hydrothermal method: Urea, citric acid monohydrate, and europium(III) nitrate hexahydrate were dissolved in N,N-dimethylformamide and sonicated until transparent. The solution was transferred to a 50.0 mL polytetrafluoroethylene reactor and reacted at 180 °C for 8.0 h. After cooling to room temperature, the solution was filtered through a 0.22 μm filter membrane. 0.2 mg / mL sodium hydroxide solution was added, and the reaction was continued for 30.0 min. After the reaction was completed, the solution was centrifuged at 10000.0 rpm for 10.0 min, the precipitate was collected, washed three times with deionized water, and finally freeze-dried to obtain the product Eu@CDs.

[0050] Preferably, the mass ratio of urea:citric acid monohydrate:europium(III) nitrate hexahydrate is 1.5:1.05:0.45, the time for ultrasonication to achieve transparency is 30.0 min, and the centrifugation parameters are 10000.0 rpm and 10.0 min.

[0051] More preferably, this embodiment discloses a method for preparing a fluorescence sensor Eu@CDs, the method comprising:

[0052] (2) 1.05 g of citric acid monohydrate (molecular weight 210.14 g / mol, 250.0 mM), 1.5 g of urea (molecular weight 60.06 g / mol, 1250.0 mM), and 0.45 g of europium(III) nitrate hexahydrate (molecular weight 446.0 g / mol, 50.0 mM) were dissolved in 20.0 mL of N,N-dimethylformamide and sonicated for 30.0 min until transparent. The solution was transferred to a 50.0 mL polytetrafluoroethylene reactor and reacted at 180 ℃ for 8.0 h. After cooling to room temperature, the solution was filtered through a 0.22 μm filter membrane. 0.2 mg / mL sodium hydroxide solution was added, and the reaction was continued for 30.0 min. After the reaction was completed, the solution was centrifuged at 10000.0 rpm for 10.0 min, the precipitate was collected, and the precipitate was washed three times with deionized water. Finally, it was freeze-dried to obtain the product Eu@CDs. The characterization of the synthesized Eu@CDs is as follows: Figure 2 , Figure 3 As shown.

[0053] Example 2: Optimal conditions for Eu@CDs detection of furazolidone and tetracycline

[0054] To optimize the analytical performance of Eu@CDs, detailed tests were conducted on the pH and temperature parameters in the furacilin (NFZ) and tetracycline (TC) detection systems.

[0055] (1) The properties of the system were investigated in the temperature range of 25-45 ℃. The results are as follows: Figure 4 As shown in Figure A, when the temperature range is 25 ℃ to 35 ℃, the fluorescence intensity difference of the reaction system at 450 nm increases with increasing reaction temperature. When the temperature exceeds 35 ℃ and increases to 50 ℃, the fluorescence intensity difference decreases with increasing temperature. Therefore, 35 ℃ was chosen as the optimal temperature for detecting the NFZ system.

[0056] (2) The performance of the system in the pH range of 8.0-10.0 was investigated, and Tris-HCl was selected as the reaction buffer. The results are as follows: Figure 4 As shown in Figure B, when the pH value is in the range of 8.0 to 9.0, the fluorescence intensity difference of the reaction system at 450 nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 9.0, the fluorescence intensity difference decreases with the increase of pH value. Therefore, 9.0 was selected as the optimal pH value for detecting the NFZ system.

[0057] (3) The performance of the system in the temperature range of 25-45 ℃ was investigated. The results are as follows: Figure 5 As shown in Figure A, when the temperature range is 25 ℃ to 35 ℃, the fluorescence intensity ratio F of the reaction system at 450 nm and 617 nm is... 617 nm / F 450 nm It increases with increasing reaction temperature. When the temperature exceeds 35 ℃, it increases to 50 ℃, F 617 nm / F 450 nm The ratio decreases as it increases, therefore 35 ℃ was chosen as the optimal temperature for detecting the TC system.

[0058] (4) The performance of the system in the pH range of 8.0-10.0 was investigated, and Tris-HCl was selected as the reaction buffer. The results are as follows: Figure 5 As shown in Figure B, when the pH value is in the range of 8.0 to 8.5, the fluorescence intensity ratio F of the reaction system at 450 nm and 617 nm is... 617 nm / F 450 nm The value increases with increasing pH of the buffer system. When the pH exceeds 8.5, F... 617nm / F 450nm The ratio decreases as it increases, so 8.5 was chosen as the optimal pH value for the system.

[0059] Based on the detection of NFZ and TC, temperature and pH optimization were performed, and the optimal conditions for the system were selected as 35 ℃ and pH 8.5.

[0060] Example 3: Eu@CDs Method System and Feasibility Verification for Detection of Furazolidone and Tetracycline

[0061] The following four reaction systems were designed: (1) CDs, (2) Eu@CDs, (3) Eu@CDs + TC, and (4) Eu@CDs + NFZ. Tris-HCl was used as the buffer solution. After the reaction was complete, the mixture was transferred to a fluorescence dish, and the fluorescence intensity at 450 nm and 617 nm was measured using a fluorescence spectrophotometer. The results are shown below. Figure 6 The results show that NFZ can significantly quench the fluorescence of Eu@CDs at 450 nm, while TC can enhance the fluorescence of Eu@CDs at 617 nm and cause a significant change in fluorescence intensity. This demonstrates the feasibility of the detection system proposed in this invention.

[0062] In the above verification steps, the concentration of Eu@CDs fluorescent probe was 100.0 mg / L; the concentration of NFZ was 32.0 mg / L; the concentration of TC was 40.0 mg / L; the pH of the added Tris-HCl buffer was 8.3-8.7; the reaction temperature was 35 ℃; and the mixing and reaction time was 1.5 min.

[0063] Example 4: Detection of different concentrations of furazolidone and tetracycline standards in Eu@CDs fluorescence mode

[0064] Different concentration gradients of NFZ solutions (including 0.0, 2.0, 5.0, 8.0, 11.0, 14.0, 17.0, 20.0, 23.0, 26.0, 29.0, 32.0, and 36.0 mg / L) were reacted with 100.0 mg / L Eu@CDs fluorescent probes in Tris-HCl buffer at 35°C for 1.5 min. After the reaction, the reaction solution was transferred to a fluorescence dish, and the fluorescence intensity F at 450 nm was measured using a fluorescence spectrophotometer. q The sample was dropped onto a filter paper strip and irradiated with a 365 nm UV lamp. Fluorescence signals were captured using a smartphone, and the R / B values ​​were obtained through RGB analysis using ImageJ software. The fluorescence intensity at 450 nm decreased with increasing NF2 concentration.

[0065] Therefore, the fluorescence intensity F measured using NFZ standards with different concentration gradients q The fluorescence linear regression equation F = XC was constructed using the fluorescence value, R / B value, and concentration of the standard. NFZ + y、 R / B= XC NFZ + y, where C NFZ This refers to the concentration of the standard. For example... Figure 7As shown, the concentration of NFZ and the fluorescence intensity F are obtained. q The fluorescence intensity was linear in the range of 0.011–32.0 mg / L, with a detection limit of 0.0034 mg / L. The regression equation for fluorescence intensity was F = -47.0608 C. NFZ + 2748.1595, R / B = 0.01921 C NFZ + 0.10289 ( Figure 10 The correlation coefficients were 0.99904 and 0.99029, respectively. Furthermore, as the concentration of NFZ increased, the solution fluorescence exhibited a color gradient (from dark blue to light blue), enabling linear detection of NFZ concentration.

[0066] Different concentration gradients of TC solutions (including 0.0, 1.0, 2.0, 5.0, 8.0, 10.0, 13.0, 16.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 mg / L) were reacted with 100.0 mg / L Eu@CDs fluorescent probes in Tris-HCl buffer at 35 °C for 1.5 min. After the reaction, the reaction solution was transferred to a fluorescence dish, and the fluorescence intensity ratio F at 450 nm and 617 nm (excitation wavelength 370 nm) was measured using a fluorescence spectrophotometer. 617 nm / F 450 nm The sample was dropped onto a filter paper strip, irradiated with a 365 nm UV lamp, and the fluorescence signal was captured using a smartphone. Image RGB analysis was performed using ImageJ software. The fluorescence ratio F increased with increasing TC concentration. 617 nm / F 450 nm Gradually increase.

[0067] A linear regression equation F was constructed using the fluorescence intensity ratio, R / B value, and standard concentrations obtained from TC standards at different concentration gradients. 617 nm / F 450 nm = XC TC + y, R / B = XC TC + y, where C TC For standard concentration;

[0068] F 617 nm / F 450 nm = 0.01402 C TC + 0.03211、R / B = 0.02926 C TC + 0.19981

[0069] Therefore, a fluorescence linear regression equation F was constructed using the fluorescence intensity ratio, R / B value, and standard concentrations obtained from TC standards at different concentration gradients. 617 nm / F450 nm = XC TC + y, R / B = XC TC + y, where C TC This refers to the concentration of the standard. For example... Figure 8 As shown, the ratio of TC concentration to fluorescence intensity exhibits a linear relationship between 0.029 and 40.0 mg / L, with a detection limit of 0.00870 mg / L. The regression equation for the fluorescence intensity ratio is F... 617 nm / F 450 nm = 0.01402 C TC + 0.03211、R / B =0.02926 C TC + 0.19981 ( Figure 10 (B) The correlation coefficients were 0.9959 and 0.98944, respectively. Furthermore, as the concentration of TC increases, the solution fluorescence exhibits a color gradient (from dark blue to red), enabling linear detection of TC concentration.

[0070] Example 5: Selectivity test of Eu@CDs for detecting furacilin and tetracycline

[0071] To evaluate the selectivity of Eu@CDs for NFZ, several representative substances were used as proof-of-concept, including blank, kanamycin, amoxicillin, chloramphenicol, gentamicin, ampicillin, streptomycin, histidine, alanine, glutamic acid, tyrosine, methionine, serine, TC, and NFZ. At room temperature, the Eu@CDs fluorescent probes were reacted with each of these substances in Tris-HCl buffer; after thorough mixing and reaction for 1.5 min, the mixture was transferred to a fluorescence dish. The fluorescence intensity at 450 nm was measured. The results are as follows: Figure 9 As shown in Figure A, only the fluorescence intensity value of NFZ shows a significant decrease, indicating that the detection system of the present invention has good selectivity for NFZ.

[0072] To evaluate the selectivity of Eu@CDs for TC, several representative substances were used as proof-of-concept, including blank, kanamycin, amoxicillin, chloramphenicol, gentamicin, ampicillin, streptomycin, histidine, alanine, glutamic acid, tyrosine, methionine, serine, NF2, and TC. At room temperature, the Eu@CDs fluorescent probes were reacted with each of these substances in Tris-HCl buffer; after thorough mixing and reaction for 1.5 min, the mixture was transferred to a fluorescent dish. The fluorescence intensity at 617 nm was measured. The results are as follows: Figure 9 As shown in Figure B, only the fluorescence intensity of TC shows a significant increase, indicating that the detection system of the present invention has good selectivity for TC.

[0073] In the above verification steps, the concentration of Eu@CDs fluorescent probe was 100.0 mg / L; the concentration of selective verification substance was 32.0 mg / L; the pH of the added Tris-HCl buffer was 8.3-8.7; the reaction temperature was 35 ℃; and the mixing and reaction time was 1.5 min.

[0074] Example 6: Detection and verification of NFZ and TC in actual samples using Eu@CDs.

[0075] To verify the sensing performance of Eu@CDs in detecting NFZ and TC in real-world sample applications, tap water was selected as a representative sample. The tap water used in this test was randomly sampled from Hefei, China, without any chemical pretreatment. The solution was filtered through a 0.22 μm filter membrane to reduce matrix effects, and the pH was adjusted to 8.5. Then, 0.0, 10.0, 20.0, and 30.0 mg / L of NFZ and TC were added, respectively. After thorough mixing, the mixture was reacted for 1.5 min and then transferred to a fluorescence dish. The fluorescence intensity value Fn at 450 nm and the fluorescence intensity ratio F at 450 nm and 617 nm were measured. 617 nm / F 450 nm The obtained fluorescence intensity Fn and fluorescence intensity ratio F are... 617 nm / F 450 nm Substituting the corresponding linear equations, the NFZ and TC results measured under fluorescence detection mode are shown in Table 1.

[0076] To verify the sensing performance of Eu@CDs in detecting NFZ and TC in real-world samples, lake water was selected as a representative sample. The lake water used in this test was randomly sampled from Hefei, China, without any chemical pretreatment. The solution was filtered through a 0.22 μm filter membrane to reduce matrix effects, and the pH was adjusted to 8.5. Then, 0.0, 10.0, 20.0, and 30.0 mg / L of NFZ and TC were added, respectively. After thorough mixing and reaction for 1.5 min, the mixture was transferred to a fluorescence dish. The fluorescence intensity value Fn at 450 nm and the fluorescence intensity ratio F at 450 nm and 617 nm were measured. 617 nm / F 450 nm The obtained fluorescence intensity Fn and fluorescence intensity ratio F are... 617 nm / F 450 nm Substituting the corresponding linear equations, the NFZ and TC results measured under fluorescence detection mode are shown in Table 1.

[0077] To verify the sensing performance of Eu@CDs in detecting NFZ and TC in real-world samples, urban wastewater was selected as a representative sample. The urban wastewater in this test was randomly sampled from Hefei, China, without any chemical pretreatment. The solution was filtered through a 0.22 μm filter membrane to reduce matrix effects, and the pH was adjusted to 8.5. Then, 0.0, 10.0, 20.0, and 30.0 mg / L of NFZ and TC were added, respectively. After thorough mixing, the mixture was reacted for 1.5 min and then transferred to a fluorescence dish. The fluorescence intensity value Fn at 450 nm and the fluorescence intensity ratio F at 450 nm and 617 nm were measured. 617 nm / F 450 nm The obtained fluorescence intensity Fn and fluorescence intensity ratio F are... 617 nm / F 450 nm Substituting the corresponding linear equations, the NFZ and TC results measured under fluorescence detection mode are shown in Table 1.

[0078] Table 1 shows the detection of NFZ and TC in actual samples by Eu@CDs. The actual samples were tap water, lake water, and urban sewage.

[0079]

[0080] The results show that the reaction system can be applied to the detection of actual samples, with a recovery rate between 97.54% and 106.81% and a relative standard deviation (RSD) of less than 2.10%.

[0081] In summary, this invention discloses a method for on-site fluorescence visualization detection of antibiotic residues. By designing fluorescent probe molecules with targeted recognition capabilities and combining them with portable sensors in smartphones, a convenient, rapid, and sensitive sensing and detection platform is constructed. This invention constructs an Eu@CDs fluorescence sensor, which emits blue fluorescence at 450 nm under 370 nm excitation. When NFZ is introduced, its nitro group (-NO2) acts as an electron acceptor, inducing photoelectron transfer (PET) from excited-state carbon dots to the nitro group, leading to blue light quenching. Furthermore, NFZ absorbs both 370 nm excitation light and 450 nm emission light, producing a synergistic internal filtering effect (IFE), jointly achieving quantitative blue light quenching. After adding TC, the β-diketone group of TC reacts with Eu... 3+ Chelation absorbs energy and sensitizes Eu through the "antenna effect". 3+It emits a characteristic 617 nm red light. This dual-signal orthogonal response mechanism, combined with a smartphone optical analysis platform, enables on-site single-probe simultaneous sampling and detection of NFZ and TC, with detection limits of 0.0034 mg / L and 0.00870 mg / L, respectively, and linear ranges of 0.011 - 32.0 mg / L and 0.029 - 40.0 mg / L, respectively. This provides an efficient and accurate solution for rapid screening of environmental and food contaminants.

[0082] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

[0083] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A method for preparing a fluorescent sensor Eu@CDs, characterized in that, The preparation of the Eu@CDs includes the following steps: dissolving urea, citric acid monohydrate, and europium(III) nitrate hexahydrate in... N,N In dimethylformamide, the mixture was sonicated until transparent, transferred to a polytetrafluoroethylene reactor, and reacted at 180 °C for 8.0 h. After cooling to room temperature, the mixture was filtered through a filter membrane, and 0.2 mg / mL sodium hydroxide solution was added. The reaction was continued for 30.0 min. After the reaction was completed, the solution was centrifuged at 10000.0 rpm for 10 min, the precipitate was collected, washed with deionized water, and finally freeze-dried to obtain the product Eu@CDs. The mass ratio of urea:citric acid monohydrate:europium(III) nitrate hexahydrate was 1.5:1.05:0.45, and the sonication time to the transparent state was 30.0 min.

2. A method for the in situ fluorescent visualisation of antibiotic residues, characterised in that, The antibiotic residues are furanocillin and tetracycline, and the method comprises the following steps, S1: The Eu@CDs fluorescent probe prepared in claim 1 is mixed with the sample to be tested of furanocillin (NFZ) and then reacted, the reaction temperature is 35 ℃, the reaction time is 1.5 min, is transferred to a fluorescence dish, the fluorescence intensity value Fn of the reactant at 370 nm excitation wavelength is measured by a fluorescence spectrophotometer, then the fluorescence signal is captured by a smart phone, and the image RGB analysis is carried out by an ImageJ platform to obtain the R / B value of NFZ; S2: The fluorescence intensity value and the R / B value obtained in step S1 are substituted into the NFZ linear equation constructed based on the NFZ standard with different concentrations to respectively calculate and obtain the concentration of NFZ in the sample. S3: After mixing the Eu@CDs fluorescent probe with the sample to be tested of tetracycline (TC), the reaction was carried out at 35 ℃; the reaction time was 1.5 min, and then the reaction was transferred to a fluorescence dish, and the fluorescence intensity value F of the reactant at 450 nm and 617 nm under the excitation wavelength of 370 nm was measured by a fluorescence spectrophotometer 450 , F 617 , the fluorescence intensity ratio F 617 nm / F 450 nm was calculated, then the fluorescence signal was captured by using a smart phone, and the image RGB analysis was carried out by using an ImageJ platform, so as to obtain the R / B value of TC; S4: The fluorescence intensity ratio F of step S3 is substituted into the linear equation of TC based on the TC standard containing different concentrations to calculate the TC concentration in the sample, respectively. 617 nm / F 450 nm , R / B value into the linear equation of TC based on the TC standard containing different concentrations to calculate the TC concentration in the sample, respectively.

3. The method for on-site fluorescent visualization of antibiotic residues according to claim 2, characterized in that, The preparation method of the NFZ linear equation is: under normal temperature conditions, the Eu@CDs fluorescent sensor and different concentration gradients of furacilin (NFZ) are added into Tris-HCl buffer solution, fully stirred and mixed, transferred to a fluorescence dish after reaction for a period of time, and the fluorescence intensity of the reactants at 450 nm under 370 nm excitation wavelength is measured by a fluorescence spectrophotometer, which is recorded as F q ; after irradiation by a 365 nm ultraviolet lamp, the fluorescence signal is captured by a smart phone, and the image RGB analysis is performed by using an ImageJ platform to obtain R / B value; the fluorescence intensity values F q , R / B values and the concentrations of the standard samples are used to construct a fluorescence linear regression equation F=XC NFZ + y, R / B=XC NFZ +y, wherein C NFZ is the concentration of the standard sample.

4. The method for on-site fluorescent visualization of antibiotic residues according to claim 2, characterized in that, The preparation method of the TC linear equation is: under normal temperature conditions, the Eu@CDs fluorescent sensor and different concentration gradients of tetracycline are added into Tris-HCl buffer solution, fully stirred and mixed, transferred to a fluorescence dish after reaction for a period of time, and the fluorescence intensity of the reactants at 450 nm and 617 nm under the excitation wavelength of 370 nm is measured by a fluorescence spectrophotometer, which is recorded as F 450 , F 617， The F 617 nm / F 450 nm is calculated, after irradiation by a 365 nm ultraviolet lamp, the fluorescence signal is captured by using a smart phone, and the image RGB analysis is carried out by using an ImageJ platform, the R / B value of tetracycline is obtained, the fluorescence intensity F 617 nm / F 450 nm of the standard sample of different concentration gradients of TC is measured, and the fluorescence linear regression equation F 617 nm / F 450 nm = XC TC + y, R / B = XC TC +y is constructed, wherein C TC is the concentration of the standard sample.

5. The method of claim 3 or claim 4, wherein, The pH value of the Tris-HCl buffer is 8.3-8.7; the concentration of the Eu@CDs fluorescent sensor is 100.0 mg / L; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 35 ℃; and the reaction time is 1.5 min.

6. The method of claim 2, wherein Linear equation for detection of furacilin is F = -47.0608 C NFZ +2748.1595, R / B = 0.01921 C NFZ +0.10289; linear equation for detection of tetracycline is F 617nm 450nm = 0.01402 C TC +0.03211, R / B = 0.02926 C TC +0.19981.​ 7. Use of the fluorescent sensor Eu@CDs in the detection of furacilin and tetracycline, characterized in that, The preparation method of the fluorescent sensor Eu@CDs is as claimed in claim 1.

8. A reagent for detecting furacilin and tetracycline, characterized in that, The reagent comprises the fluorescent sensor Eu@CDs, and the preparation method of the fluorescent sensor Eu@CDs is as claimed in claim 1.

9. A kit of reagents for the detection of furazolidone and tetracyclines, characterized in that, The kit comprises the fluorescent sensor Eu@CDs, furanocillin standard, tetracycline standard and buffer; and the preparation method of the fluorescent sensor Eu@CDs is as claimed in claim 1.

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