Fluorescent carbon dots and hydrogels thereof and applications, and enrofloxacin detection method

Abstract

The present application belongs to the technical field of fluorescent carbon nanomaterial preparation and antibiotic detection, and discloses fluorescent carbon dots, hydrogel thereof, and application, and an enrofloxacin detection method. The fluorescent carbon dot hydrogel is constructed based on the fluorescent carbon dots, and the enrofloxacin detection method is constructed based on the fluorescent carbon dots or the fluorescent carbon dot hydrogel. The detection method is to mix the fluorescent carbon dots with a sample to be detected, to realize quantitative analysis of enrofloxacin according to the fitting relationship between the I 411 / I 508 value and the enrofloxacin concentration; or to immerse the fluorescent carbon dot hydrogel in the sample to be detected for sufficient incubation, to obtain a color development photo under irradiation of 365 nm ultraviolet light, to obtain the G / B value of the fluorescent carbon dot hydrogel region in the color development photo, and to realize quantitative analysis of enrofloxacin according to the fitting relationship between the G / B value and the enrofloxacin concentration. The present application can improve the accuracy of enrofloxacin detection, and further, can realize rapid and high-throughput detection of enrofloxacin.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent carbon nanomaterial preparation and antibiotic detection technology, and relates to fluorescent carbon dots and their hydrogels and applications, as well as enrofloxacin detection methods. Background Technology

[0002] Enrofloxacin, a third-generation fluoroquinolone antibiotic, is widely used in livestock and aquaculture for the prevention and treatment of various bacterial infections due to its excellent antibacterial activity. However, its excessive and unregulated use has led to increasingly prominent residue problems in animal-derived foods (such as poultry, eggs, milk, and aquatic products) and aquaculture environments (water bodies and soil). Enrofloxacin residues can enter the human body through the food chain. Long-term intake of enrofloxacin, even at low doses, may induce drug resistance in pathogens, seriously threatening public health and safety. It may also cause health problems such as joint pain, liver and kidney damage, and allergic reactions. Therefore, developing rapid, sensitive, and reliable enrofloxacin detection technologies is of paramount importance for ensuring food safety, protecting the ecological environment, and promoting the healthy development of livestock and aquaculture industries.

[0003] Currently, routine methods for detecting enrofloxacin residues mainly include high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and enzyme-linked immunosorbent assay (ELISA). While HPLC and CE offer advantages such as high sensitivity and accuracy, they suffer from low detection efficiency and insufficient throughput. They rely on expensive, large-scale instruments, require complex sample pretreatment, involve cumbersome and time-consuming procedures, and must be performed by professionals in a laboratory environment, making them difficult to apply in the field for rapid detection. Furthermore, the cost per test is high. These inherent limitations severely restrict their application in rapid on-site screening, large-scale sample initial screening, and resource-constrained environments (such as farms and market supervision sites). Although ELISA offers some convenience, it typically suffers from high antibody preparation costs, poor stability, susceptibility to matrix interference, limited sensitivity, and is mostly semi-quantitative or qualitative. Therefore, developing an enrofloxacin detection technology that balances high sensitivity, high accuracy, low cost, and field application has become an urgent need in this field.

[0004] Fluorescence analysis has become a research hotspot for antibiotic detection due to its advantages such as high sensitivity, fast response speed, and convenient operation. Carbon dots (CDs), as carbon-based fluorescent nanomaterials, possess low toxicity, high water solubility, excellent photostability, and biocompatibility, showing significant potential in the field of fluorescence detection. Although significant progress has been made in the research of single-signal fluorescent probes ("on-off" or "off-on") for the detection of enrofloxacin, they still face serious challenges in the detection of complex samples in practice. Food and environmental samples have complex compositions, and factors such as the excitation light source, probe concentration, and slight fluctuations in the detection environment (temperature, pH) can all cause changes in the fluorescence signal, resulting in poor repeatability and large signal fluctuations in the detection results. This significantly reduces the reliability of detection results in complex real-world samples such as meat extracts and environmental water bodies.

[0005] Current fluorescence sensing research is mostly limited to sequential detection of single samples. It generally lacks high-throughput detection capabilities. Whether based on cuvettes or single test strips, both methods can only perform sequential detection of individual samples, resulting in low efficiency and lengthy processing times. This fails to meet the needs of rapid, simultaneous screening of large batches of samples, making its detection efficiency extremely low in practical applications requiring simultaneous processing of large numbers of samples (such as import / export port quarantine, routine monitoring of large-scale farms, and market supervision sampling). Furthermore, existing fluorescence analysis methods lack on-site application and visualization capabilities, exhibiting significant shortcomings in applications in food processing sites, farms, and the field: firstly, signal reading heavily relies on large fluorescence spectrometers, lacking portable dedicated detection equipment; secondly, the methods themselves typically cannot provide visually perceptible color changes, lacking a solution to convert fluorescence signals into intuitive and easily readable output results, severely limiting real-time applications in the field and workshops.

[0006] In summary, there is currently no enrofloxacin detection solution that integrates high sensitivity, high accuracy, high throughput, intelligence, and on-site detection capabilities. Summary of the Invention

[0007] To address the shortcomings of existing enrofloxacin detection technologies, such as poor anti-interference capabilities, low throughput, and difficulty in field application, this invention provides fluorescent carbon dots and their hydrogels, along with their applications and an enrofloxacin detection method. This aims to improve the accuracy of enrofloxacin detection, further enabling high-throughput detection of enrofloxacin and facilitating the field application of the detection method.

[0008] This invention provides fluorescent carbon dots, fluorescent carbon dot hydrogels containing fluorescent carbon dots, the application of fluorescent carbon dots or fluorescent carbon dot hydrogels in enrofloxacin detection, and enrofloxacin detection methods based on fluorescent carbon dots or fluorescent carbon dot hydrogels. They all contain the common technical feature of fluorescent carbon dots and belong to a general inventive concept.

[0009] The fluorescent carbon dots provided by this invention are synthesized using polyethyleneimine, coumarin, and sodium hydroxide as raw materials via a one-step hydrothermal method, and are prepared by the following method:

[0010] (1) Polyethyleneimine and coumarin were dissolved in ethanol at a mass ratio of 1:1 and ultrasonicated until completely dissolved to obtain a homogeneous precursor solution.

[0011] (2) Add sodium hydroxide solution to the precursor solution to adjust the pH to 9-11, then transfer it to a high-pressure reactor and react at 90-120 °C for 9-12 h. After natural cooling to room temperature, a crude carbon dot solution is obtained.

[0012] (3) After centrifuging the crude solution, take the supernatant, filter it through a 0.22 μm organic phase filter membrane, place the filtrate in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze for 24~72 h, freeze dry to obtain fluorescent carbon dots.

[0013] The fluorescent carbon dot hydrogel provided by the present invention is composed of a polyvinyl alcohol hydrogel matrix and the above-mentioned fluorescent carbon dots. The fluorescent carbon dots are fixed in the polyvinyl alcohol hydrogel matrix network by physical embedding, wherein the mass ratio of the fluorescent carbon dots to the polyvinyl alcohol hydrogel matrix is ​​(5~30):2000.

[0014] Furthermore, the above-mentioned fluorescent carbon dot hydrogel was prepared by the following method:

[0015] (1) Add polyvinyl alcohol to water, heat and stir until completely dissolved to obtain a polyvinyl alcohol solution with a concentration of 0.1~0.15 g / mL;

[0016] (2) Cool the polyvinyl alcohol solution to 40~60 ℃, add fluorescent carbon dots, stir evenly to obtain a fluorescent carbon dot-polyvinyl alcohol composite solution, wherein the mass ratio of fluorescent carbon dots to polyvinyl alcohol is (5~30):2000;

[0017] (3) The fluorescent carbon dot-polyvinyl alcohol composite solution was frozen at -20~-80 ℃ for 1~4 h, and then thawed at room temperature for 1~2 h. The freeze-thaw cycle was repeated 3 times to achieve physical cross-linking and obtain fluorescent carbon dot hydrogel.

[0018] The present invention also provides the application of the above-mentioned fluorescent carbon dots or fluorescent carbon dot hydrogels in the detection of enrofloxacin.

[0019] The fluorescent carbon dots described in this invention have uniform morphology, small size, and can be dispersed in water to form a colorless and transparent solution. The aqueous solution or hydrogel of the fluorescent carbon dots emits green fluorescence under ultraviolet light excitation at a wavelength of 365 nm, and the luminescence is stable. Using the fluorescent carbon dots described in this invention as a fluorescent carbon dot ratio sensing material, it exhibits excellent enrofloxacin detection performance. The fluorescent carbon dots can be detected by I...411 / I 508 Self-calibration eliminates environmental interference, and the detection limit for enrofloxacin is as low as 0.05 μM.

[0020] This invention also provides a method for detecting enrofloxacin, which employs either method one or method two as follows;

[0021] Method 1: Mix the above-mentioned fluorescent carbon dots with the sample to be tested, and then perform fluorescence spectroscopy on the resulting mixture. Calculate the fluorescence intensity I at a wavelength of 411 nm. 411 The fluorescence intensity I at a wavelength of 508 nm 508 The ratio I 411 / I 508 Then, the enrofloxacin concentration is calculated based on the fitted equation Y = 0.1990X + 0.0351, where X is the enrofloxacin concentration in µmol / L, and Y is the enrofloxacin concentration in µmol / L. 411 / I 508 value;

[0022] Method 2: Immerse the above-mentioned fluorescent carbon dot hydrogel in the sample to be tested and incubate it for a long time. Record the color development image under 365 nm ultraviolet light. Select the fluorescent carbon dot hydrogel area in the color development image, extract its RGB feature values, and calculate the ratio of the mean green component G to the mean blue component B, G / B. Calculate the enrofloxacin concentration according to the fitted equation y = -0.0268x + 1.1820, where x is the enrofloxacin concentration in µmol / L and y is the G / B value.

[0023] In the above-mentioned enrofloxacin detection method, the sample to be tested is obtained by pre-treating a solid or liquid sample to avoid interference from interfering substances (such as proteins and particulate matter) in the sample to the detection of enrofloxacin. The pre-treatment method for solid samples is as follows: homogenize the solid sample, take the sample after homogenization to precipitate the protein, centrifuge, take the supernatant and filter, and dilute the obtained filtrate. The pre-treatment method for liquid samples is as follows: precipitate the protein in the liquid sample containing protein, centrifuge, take the supernatant and filter, or filter the liquid sample without protein.

[0024] Typically, the procedure for precipitating proteins in homogenized samples is as follows: acetonitrile and ethanol are added to the homogenized sample, and the proteins are precipitated by sonication. The procedure for precipitating proteins in liquid samples containing protein is as follows: the liquid sample containing protein is mixed with an equal volume of acetonitrile, and the proteins are precipitated by vortexing or sonication. Solid samples typically include meat and egg samples; liquid samples containing protein typically include milk, biological fluids, and high-protein fermentation broth samples; liquid samples without protein typically include water and non-dairy beverage samples.

[0025] When using Method 2 for detection, the fluorescent carbon dot hydrogel is immersed in the sample to be tested and incubated for a sufficient period. The liquid on the surface of each fluorescent carbon dot hydrogel is then blotted dry, and a colorimetric photograph is recorded under 365 nm ultraviolet light. The incubation time for the fluorescent carbon dot hydrogel in the sample to be tested is determined by ensuring that the fluorescence intensity of the hydrogel reaches a stable state after incubation.

[0026] Furthermore, when using Method 2 for detection, a detection device and the aforementioned fluorescent carbon dot hydrogel are used for detection; the detection device includes a box body, a box cover, a power supply, and an ultraviolet light source. The box body is a cuboid with an internal cavity. The box cover matches the top opening of the box body and is hinged to the box body. The box cover has an observation hole, which is sealed with a filter lens. The ultraviolet light source emits ultraviolet light with a wavelength of 365 nm. The power supply is electrically connected to the ultraviolet light source. A switch is located outside the box body to control the on / off state of the ultraviolet light source. The method includes the following steps:

[0027] (1) Preprocessing and image acquisition: The batch of test samples are added to the fluorescent carbon dot hydrogels distributed in the well plate and the hydrogels are immersed and fully incubated. The well plate is placed in the box of the detection device, the box cover is closed, the ultraviolet light source is turned on, and the well plate is photographed through the observation hole to obtain the color development photos of the batch of test samples.

[0028] (2) Image analysis: Use image processing software to extract the RGB feature values ​​of the corresponding regions of each sample to be tested in the color photo obtained in step (1), and calculate the ratio G / B of the mean value of the green component G and the mean value of the blue component B of the corresponding regions of each sample to be tested.

[0029] (3) Concentration calculation: The enrofloxacin concentration x of each sample to be tested is calculated based on the fitted equation y = -0.0268x + 1.1820, in µmol / L. y is the G / B value obtained in step (2).

[0030] The housing and lid of the detection device can be manufactured using 3D printing. The 3D printed model is designed using SolidWorks software. During the printing process, the design drawings are imported into the 3D printer, which then slices and layers the model according to its structure, dividing it into a material printing area and a support printing area. Polylactic acid (PLA) black filament is typically used as the printing material.

[0031] Furthermore, when using Method 2 for detection, the image processing software is ImageJ, a mobile app, or a mobile mini-program; the mobile app or mini-program is equipped with an enrofloxacin high-throughput detection system. This system uses the fitting equation y = -0.0268x + 1.1820 as its basis, obtains color photos of a batch of test samples by taking pictures with a mobile phone, calculates the G / B value of the corresponding region of each test sample in the color photo, and substitutes it as the y value into the fitting equation to obtain the enrofloxacin concentration of each test sample in the batch of test samples in one go.

[0032] Current fluorescent probe detection methods largely rely on laboratory fluorescence spectrometers, making it impossible to achieve on-site visual detection without large instruments. With the development of mobile internet technology, smartphones, thanks to their widespread availability, powerful built-in sensors (high-definition cameras), high-performance central processing units, and flexible application development environments, can be used as portable detection terminals. Therefore, this invention further designs mobile software or a mobile app that integrates a high-throughput enrofloxacin detection system. This system analyzes chromogenic photographs to obtain enrofloxacin concentration, combining smartphones with fluorescence sensing technology. By capturing chromogenic photographs with the smartphone's camera and then using built-in algorithms to extract RGB feature values ​​and process data, quantitative analysis of enrofloxacin can be performed directly on the mobile phone. This completely eliminates the dependence on large instruments, making truly real-time on-site detection and home self-testing possible.

[0033] Furthermore, when using Method 2 for detection, the enrofloxacin high-throughput detection system includes an image preprocessing module, a grid segmentation module, an RGB sampling module, a concentration calculation module, and a result display module;

[0034] The image preprocessing module is used to acquire and preprocess images, and to complete the digital loading and caching of images through the canvas drawing component; the images are color photos of the batch of samples to be tested obtained in step (1);

[0035] The grid segmentation module uses an array-style equal division algorithm to divide the image into regions. The region division process is as follows: First, the pixel size parameters of the loaded image are obtained, the size of a single grid cell is calculated, and the image is evenly divided into array-style equal-area grid regions. Each grid region corresponds to the image of a single test sample in the color development photos of a batch of test samples. Then, the row and column numbers are traversed through a double loop to calculate the coordinates of the upper left corner of each grid region, and each grid region is independently drawn into a preset hidden canvas drawing component to achieve the separation and extraction of each test sample image in the color development photos of a batch of test samples.

[0036] The RGB sampling module is used to obtain the RGB feature values ​​of each grid region after separation and extraction. The acquisition process is as follows: determine the width and height of the image in each grid region after separation and extraction, and calculate the center coordinates of the grid region; set the sampling window parameters; read the pixel data in the sampling window through the canvas drawing component, and extract the average value of the RGB color components of all pixels in the sampling window as the RGB feature value of the corresponding grid region.

[0037] The concentration calculation module performs concentration conversion based on the fitted equation: according to the RGB characteristic values ​​of each grid region, the ratio G / B of the average green component G to the average blue component B of each grid region is calculated, and this ratio is used as the y value and substituted into the fitted equation x = (1.1820 - y) / 0.0268 to calculate the enrofloxacin concentration x of each sample to be tested, in µmol / L.

[0038] The results display module displays the image corresponding to the grid area, the extracted RGB feature values, and the calculated enrofloxacin concentration on the display interface.

[0039] The above-mentioned high-throughput enrofloxacin detection system mainly uses the following algorithms:

[0040] Grid segmentation algorithm: By calculating the image size equally and locating the coordinates, the algorithm separates the image regions of each test sample in the color development photograph of a batch of test samples, ensuring that each grid region contains only the complete image of a single test sample, thus providing a basis for the detection of batch test samples.

[0041] RGB sampling algorithm: A sampling window of 20×20 pixels in the central area is used. Multi-point pixel mean extraction is used to avoid interference from factors such as edge shadows and uneven ultraviolet illumination in the image area of ​​the sample to be tested, thereby improving the stability of RGB feature values.

[0042] Concentration calibration algorithm: A calculation model is built based on the linear relationship between G / B value and concentration to realize the conversion from color features to concentration, simplifying the detection process.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The fluorescent carbon dots provided by this invention have excellent enrofloxacin detection performance and can be detected by I... 411 / I 508 Self-calibration eliminates environmental interference, resulting in a detection limit for enrofloxacin as low as 0.05 μM. The method for preparing the fluorescent carbon dots is simple to operate, yields stable fluorescent carbon dots, uses readily available raw materials, does not contain toxic metal elements, and is environmentally friendly.

[0045] 2. Based on the fluorescent carbon dots, this invention also provides a fluorescent carbon dot hydrogel. The color change of this fluorescent carbon dot hydrogel is visually perceptible. By acquiring a colorimetric image of the sample after interaction with the hydrogel under 365 nm ultraviolet light irradiation, the RGB characteristic values ​​of the colorimetric image are obtained, and the ratio G / B (mean of the green component G to the mean of the blue component B) is calculated. The G / B value shows a good linear fit with the enrofloxacin concentration. Based on the fitting equation, enrofloxacin can be detected, with a detection limit of 0.76 μM. This hydrogel is formed by freeze-thaw molding, eliminating the need for chemical cross-linking agents, thus avoiding damage to the performance of the fluorescent carbon dots. Furthermore, the preparation cycle is short, making it suitable for mass production.

[0046] 3. The enrofloxacin detection method based on fluorescent carbon dot hydrogel provided by this invention combines fluorescent carbon dot hydrogel, ultraviolet lamp, camera, and image processing software capable of extracting RGB feature values. It enables quantitative detection of enrofloxacin without relying on specialized large instruments or professional testing and analysis personnel. It boasts advantages such as low detection cost and simple, convenient operation. The quantitative detection of enrofloxacin can be achieved in just three steps: taking a colorimetric photograph of the hydrogel after interaction with the sample, extracting RGB feature values ​​from the photograph to obtain the G / B value, and calculating the concentration. Even non-professionals can complete the detection in a short time, which is conducive to promoting the widespread application of the enrofloxacin detection method described in this invention in on-site testing scenarios.

[0047] 4. The enrofloxacin detection method based on fluorescent carbon dot hydrogel provided by this invention can realize the synchronous signal acquisition and reading of batches of test samples, fundamentally overcoming the efficiency bottleneck of traditional single-signal sensors that require sequential detection, and can realize the simultaneous parallel processing of batches of test samples, greatly improving the detection throughput per unit time, which can meet the urgent need for rapid screening of large batches of test samples.

[0048] 5. The enrofloxacin detection method provided by this invention has strong adaptability to complex matrices. After sample pretreatment to remove protein and particulate interference, the recovery rate in matrices such as beef and mutton, milk, fish and shrimp, eggs, and environmental water is stable at 90.8%~109.1%, with a relative standard deviation (RSD) of <6.8%. This solves the problem of large deviation in existing enrofloxacin detection methods in complex samples and can meet the needs of food safety and environmental monitoring. Attached Figure Description

[0049] Figure 1 The image is a transmission electron microscope image of the fluorescent carbon dots prepared in Example 1.

[0050] Figure 2The images show the XPS and FT-IR spectra of the fluorescent carbon dots prepared in Example 1 and Comparative Example 1. Images A to D are XPS spectra of the fluorescent carbon dots prepared in Comparative Example 1, images E to H are XPS spectra of the fluorescent carbon dots prepared in Example 1, and image I is the FT-IR spectra of the fluorescent carbon dots prepared in Example 1 and Comparative Example 1.

[0051] Figure 3 The graph shows the results of selective analysis of enrofloxacin detection based on fluorescent carbon dots, where the horizontal axis represents different substances and the vertical axis represents the ratio of fluorescence intensity.

[0052] Figure 4 The graph shows the test results of the anti-interference performance of enrofloxacin detection based on fluorescent carbon dots. The horizontal axis represents the mixture of different interfering substances and enrofloxacin, and the vertical axis represents the fluorescence intensity ratio.

[0053] Figure 5 For I 411 / I 508 The graph shows the linear relationship between the value and the concentration of enrofloxacin, where the concentration range of enrofloxacin is 0~13μM; the fitted equation is Y = 0.1990X + 0.0351, R0 2 = 0.9954.

[0054] Figure 6 The attached diagram is a schematic diagram of the detection device. The following labels are used to explain the structure: 1 is the power supply, which provides power to the ultraviolet light source; 2 is the ultraviolet light source, which emits ultraviolet light with a wavelength of 365 nm; 3 is the observation port, which is used for observation and to acquire colorimetric photographs; 4 is the switch; and 5 is the housing.

[0055] Figure 7 The fluorescence color changes of the fluorescent carbon dot hydrogel at different enrofloxacin concentrations (0~24 μM) are shown. The enrofloxacin concentrations from left to right are 0, 4, 8, 12, 16, 20, and 24 μM.

[0056] Figure 8 The graph shows the linear relationship between the G / B ratio of the fluorescent carbon dot hydrogel chromogenic image and the enrofloxacin concentration, where the enrofloxacin concentration ranges from 0 to 24 μM. The fitted equation is y = –0.0268x + 1.1820, RB. 2 = 0.9901.

[0057] Figure 9 The images shown are screenshots of the color development photos and enrofloxacin concentration calculation results obtained by a WeChat mini-program on a mobile phone equipped with an enrofloxacin high-throughput detection system. Image A shows the color development photos and some of the concentration calculation results, while Image B shows some of the concentration calculation results. Detailed Implementation

[0058] The technical solutions of various embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0059] Example 1

[0060] Fluorescent carbon dots were synthesized via a one-step hydrothermal method using coumarin, polyethyleneimine, and sodium hydroxide as raw materials. The specific steps are as follows:

[0061] (1) Polyethyleneimine and coumarin were dissolved in a small amount of ethanol at a mass ratio of 1:1 and ultrasonically treated until completely dissolved to obtain a homogeneous precursor solution.

[0062] (2) Adjust the pH of the precursor solution to 11 with sodium hydroxide solution, then transfer it to a high-pressure reactor lined with polytetrafluoroethylene, place the reactor in a 100 ℃ oven for 10 h, and allow it to cool naturally to room temperature to obtain a yellow carbon dot crude solution.

[0063] (3) Centrifuge the crude carbon dot solution, take the supernatant, filter it through a 0.22 μm organic phase filter membrane to remove large particulate impurities, place the filtrate in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze for 72 h, freeze dry to obtain fluorescent carbon dots (abbreviated as CDs-OH).

[0064] The morphology of the fluorescent carbon dots was characterized using transmission electron microscopy (TEM). Figure 1 As shown, the fluorescent carbon dots are spherical, well-dispersed, and approximately 2 nm in diameter. X-ray photoelectron spectroscopy (XPS) was used to analyze the chemical composition and state of the fluorescent carbon dot surface, and its full spectrum (…) Figure 2 The E-plot shows that the sample mainly contains three elements: carbon (C 1s, ~285 eV), nitrogen (N 1s, ~400 eV), and oxygen (O 1s, ~532 eV); the C 1s high-resolution spectrum ( Figure 2 The F-plot can be fitted with characteristic peaks of C=C / CC (284.6 eV), CN (285.2 eV), CO (288.0 eV), and C=O (288.9 eV), indicating the presence of multiple carbon-containing functional groups on the surface of the fluorescent carbon dots; the N1s high-resolution spectrum ( Figure 2 The G-plot can be fitted to nitrogen species such as NH (399.8 eV) and NC (401.2 eV), and the O 1s high-resolution spectrum ( Figure 2 The H-plot can be fitted to peaks corresponding to oxygen-containing functional groups such as OC (531.5 eV) and O=C (533.0 eV). FT-IR analysis of the fluorescent carbon dots yielded the following results: Figure 2 As shown in Figure I. 3200~3500 cm -1 The broad and strong absorption band is attributed to the superposition of OH and NH stretching vibrations, indicating that the surface is rich in hydroxyl and amino groups; 2800~3000 cm -1 The weak absorption corresponds to the saturated CH stretching vibration, indicating that the alkyl chain portion of polyethyleneimine is retained; 1600~1700 cm⁻¹ -1 The strong absorption peak is a superposition of C=O stretching, C=C skeletal vibration, and NH bending vibration of the amide bond, proving that the coumarin lactone ring opens under alkaline conditions and undergoes an amidation reaction with polyethyleneimine, forming a covalently grafted amide bond; 1000~1400 cm⁻¹ -1 The multiple absorption peaks correspond to CN and CO stretching vibrations, indicating the presence of oxygen- and nitrogen-containing functional groups. XPS and FT-IR tests confirm the successful synthesis of fluorescent carbon dots with surface-rich carboxyl, carbonyl, and amino functional groups in this embodiment.

[0065] Comparative Example 1

[0066] Fluorescent carbon dots were prepared according to the method of Example 1, but sodium hydroxide solution was not used to adjust the pH of the precursor solution during the preparation process. The obtained fluorescent carbon dots (CDs) were used as control samples and compared with the fluorescent carbon dots (CDs-OH) prepared in Example 1.

[0067] The XPS spectra of the fluorescent carbon dots prepared in Comparative Example 1 were tested, and the results are as follows: Figure 2 As shown in Figures A-D, the FT-IR spectra of the fluorescent carbon dots prepared in Comparative Example 1 were tested, and the results are as follows. Figure 2 As shown in Figure I. Based on the XPS spectra of the fluorescent carbon dots prepared in Example 1 and Comparative Example 1, the elemental composition of the fluorescent carbon dots prepared in Example 1 and Comparative Example 1 was calculated, and the results are as follows:

[0068]

[0069] In Example 1, the sodium hydroxide added during the preparation of fluorescent carbon dots did not merely provide an alkaline environment; rather, it participated in the reaction process and regulated the surface functional groups and molecular structure of the reaction products. XPS full-resolution spectra showed that the fluorescent carbon dots prepared in both Comparative Example 1 and Example 1 were mainly composed of C, N, and O elements. After sodium hydroxide treatment, the main framework of the fluorescent carbon dots and the nitrogen-containing structures introduced by polyethyleneimine were still retained. C 1s high-resolution, N 1s high-resolution, and O 1s high-resolution spectra showed that the fluorescent carbon dots prepared in both Comparative Example 1 and Example 1 possessed CC / C=C, CN, CO, and C=O structures. However, in Example 1, after the introduction of sodium hydroxide, the peak shapes and relative contributions of the CC / C=C, CN, CO, and C=O structures changed. The CO, OC, NH, NC, and O=C structures were more prominent in the fluorescent carbon dots prepared in Example 1, indicating that the alkaline environment promoted the formation or exposure of oxygen- and nitrogen-containing functional groups. Especially the O 1s high-resolution spectrum (… Figure 2 In the D and H diagrams, the oxygen environment associated with the OC and O=C structures changed significantly, indicating that sodium hydroxide mainly modulated the chemical state of oxygen on the surface of the fluorescent carbon dots. The fluorescent carbon dots prepared in Example 1 contained more hydroxyl, ether, carbonyl, or carboxylate-related structures on their surface, which is consistent with the FT-IR spectra ( Figure 2 The results from the FT-IR spectrum (Figure 1) corroborate each other. In the FT-IR spectrum, the CDs-OH prepared in Example 1 shows a wavelength range of 3200–3500 cm⁻¹. -1 The broad peaks in the region are more pronounced and can be attributed to the stretching vibrations of -OH and -NH; at 1600 cm⁻¹ -1 Absorptions associated with C=O, C=C, or NH bending vibrations appeared on both sides; in the range of 1000–1400 cm⁻¹ -1 Vibrational signals such as CO and CN can be observed in the fingerprint region. Compared with the CDs prepared in Comparative Example 1, these characteristic peaks of CDs-OH are more prominent, indicating that the polar functional groups on the surface of the fluorescent carbon dots become more abundant after sodium hydroxide is introduced during the preparation process.

[0070] The aforementioned differences enable the fluorescent carbon dots of the present invention to specifically detect enrofloxacin. The enrofloxacin molecule contains carboxyl groups, ketone carbonyl groups, protonable piperazine nitrogen, and a conjugated quinolone skeleton, which can respectively interact with the abundant hydroxyl / amine groups, carboxylate sites, and conjugated carbon domains on the surface of the fluorescent carbon dots prepared in Example 1 through hydrogen bonding, pH-dependent electrostatic interactions, and π-π interactions. This multi-site synergistic binding promotes the enrichment of enrofloxacin on the luminescent surface of the fluorescent carbon dots prepared in Example 1 and perturbs its surface electronic states and fluorescence processes, thereby endowing the fluorescent carbon dots of the present invention with a strong selective fluorescence response.

[0071] Example 2 This embodiment provides the application of fluorescent carbon dots in the detection of enrofloxacin.

[0072] (1) Preparation of blank control group

[0073] The fluorescent carbon dots prepared in Example 1 were used as a solvent to prepare a fluorescent carbon dot stock solution with a concentration of 1 mg / mL. 200 μL of the fluorescent carbon dot stock solution was placed in a quartz cuvette, and water was added to bring the volume to 3 mL. The fluorescence spectrum was measured at an excitation wavelength of 280 nm, and the fluorescence intensities at 411 nm and 508 nm were recorded. 411 with I 508 Calculate I 411 / I 508 value.

[0074] (2) Selective testing

[0075] Add 200 μL of solutions of different substances [1 mM, including enrofloxacin (ENR), azithromycin (AZM), erythromycin (EM), sulfamethoxazole (SMX), chloramphenicol (CAP), mitoxantrone (MX), tetracycline (TC), and vancomycin (VAN)] to 200 μL of fluorescent carbon dot stock solution, as well as Ag + Ca 2+ Fe 3+ NO3 - [19 common ions] were added to a final volume of 3 mL with water, and the fluorescence spectrum was measured at an excitation wavelength of 280 nm to calculate I. 411 / I 508 Value, result as Figure 3 As shown. Only enrofloxacin can cause I. 411 / I 508 The value increased significantly, and other substances had a greater impact on I. 411 / I 508 The value has a negligible effect, indicating that the fluorescent carbon dots have excellent selectivity for enrofloxacin.

[0076] (3) Anti-interference performance test

[0077] Add 200 μL of enrofloxacin solution (1 mM) and 200 μL of interferon solution (1 mM) to 200 μL of fluorescent carbon dot stock solution simultaneously. The interferon solution includes seven antibiotics: azithromycin (AZM), erythromycin (EM), sulfamethoxazole (SMX), chloramphenicol (CAP), mitoxantrone (MX), tetracycline (TC), and vancomycin (VAN), as well as Ag. + Ca 2+ Fe 3+ NO3 - [19 common ions] were added, and after adjusting the volume to 3 mL, the fluorescence spectrum was measured at an excitation wavelength of 280 nm, and I was calculated. 411 / I 508Value, result as Figure 4 As shown. I of the hybrid interference system and the single enrofloxacin system. 411 / I 508 The small deviation in the value indicates that the fluorescent carbon dots have good anti-interference ability.

[0078] (4) Sensitivity test

[0079] Different volumes of enrofloxacin standard solution (50 μM) were added to 200 μL of fluorescent carbon dot stock solution, and the volume was adjusted to 3 mL to prepare test solutions with a concentration range of 0–13 μM. The fluorescence spectra were measured at an excitation wavelength of 280 nm, and Ig was calculated. 411 / I 508 Value, result as Figure 5 As shown. With increasing enrofloxacin concentration, I 508 Gradually decrease, I 411 Gradually increase, I 411 / I 508 The value showed a good linear relationship with the enrofloxacin concentration, and the fitted equation was Y = 0.1990X + 0.0351 (X is the enrofloxacin concentration, μM), with a correlation coefficient R. 2 = 0.9954; the limit of detection (LOD) for enrofloxacin was calculated to be 0.05 μM based on LOD = 3σ / k (σ: blank standard deviation; k: slope).

[0080] (5) Validation of the ability to detect complex samples

[0081] Actual samples including beef, mutton, fish, shrimp, eggs, milk, lake water, highland pasture water, and tap water were selected for pretreatment.

[0082] Solid samples (meat, eggs, shrimp): Homogenize the solid sample, take 0.5 g of homogenized sample, add 5 mL of acetonitrile and 5 mL of ethanol, sonicate for 10 minutes (25 ℃) to precipitate protein, centrifuge at 10000 rpm for 10 minutes, take the supernatant and filter it through a 0.22 μm organic filter membrane, and dilute it.

[0083] Liquid samples (milk, water): Mix milk sample with an equal volume of acetonitrile, vortex for 5 minutes, centrifuge, and filter the supernatant through a 0.22 μm organic filter membrane; filter water sample directly through a 0.22 μm aqueous filter membrane.

[0084] Pretreated samples were mixed with 4, 8, and 12 μM enrofloxacin standard solutions to form test samples. Fluorescence spectra were measured at an excitation wavelength of 280 nm, and Ig was calculated. 411 / I 508The concentration of enrofloxacin and the spiked recovery rate were calculated. The results are shown in Table 1. In this embodiment, the spiked recovery rate of enrofloxacin was between 96.4% and 109.1%, and the relative standard deviation (RSD) was less than 6.8%, indicating that the method is suitable for the accurate detection of enrofloxacin in complex matrices.

[0085] Pretreated samples were mixed with 4, 8, and 12 μM enrofloxacin standard solutions to form test samples. Fluorescence spectra were measured at an excitation wavelength of 280 nm, and Ig was calculated. 411 / I 508 The concentration of enrofloxacin and the spiked recovery rate were calculated. The results are shown in Table 1. In this embodiment, the spiked recovery rate of enrofloxacin was between 96.4% and 109.1%, and the relative standard deviation (RSD) was less than 6.8%, indicating that the method is suitable for the accurate detection of enrofloxacin in complex matrices.

[0086] Table 1 shows the spiked recoveries and relative standard deviations (RSD) of enrofloxacin in actual samples.

[0087]

[0088] As can be seen from this embodiment, when using the fluorescent carbon dot-based ratio fluorescence detection strategy of the present invention to detect enrofloxacin, it has a wide linear range and a low detection limit. It can be directly used for the quantitative analysis of enrofloxacin in complex matrix samples such as environmental water, milk and animal meat homogenates, and can be applied in the detection of food and environmental pollutant residues.

[0089] Example 3

[0090] In this embodiment, a method for preparing fluorescent carbon dot hydrogels is provided.

[0091] (1) Add polyvinyl alcohol to water, stir at 150 rpm for 30 minutes, then heat to 90 ℃ and stir until completely dissolved to obtain a transparent homogeneous polyvinyl alcohol solution with a concentration of 0.1 g / mL.

[0092] (2) Cool the polyvinyl alcohol solution to 40 °C, add fluorescent carbon dots, the mass ratio of fluorescent carbon dots to polyvinyl alcohol is 5:2000, stir evenly to obtain a light yellow fluorescent carbon dots-polyvinyl alcohol composite solution.

[0093] (3) Inject 100 μL of fluorescent carbon dot-polyvinyl alcohol composite solution into each well of a 96-well plate. Freeze at -80 °C for 4 h and then thaw at room temperature for 2 h. Repeat the above operation of freezing at -80 °C for 4 h and thawing at room temperature for 2 h three times to achieve physical cross-linking and obtain fluorescent carbon dot hydrogel. The hydrogel emits yellow-green fluorescence under 365 nm ultraviolet light.

[0094] Example 4

[0095] This embodiment provides the application of the fluorescent carbon dot hydrogel prepared in Example 3 in the detection of enrofloxacin.

[0096] 0–24 μM enrofloxacin solution was injected into the fluorescent carbon dot hydrogels in the 96-well plate to completely immerse each hydrogel. After incubation for 30 minutes, the enrofloxacin solution was poured out and the surface liquid of the hydrogel was dried. Then, the 96-well plate was placed in the detection device box, the box was closed, and the ultraviolet light source was turned on. The fluorescence changes of the hydrogel were observed through the observation well under ultraviolet light at a wavelength of 365 nm.

[0097] The detection device includes a box body, a box cover, a power supply, and an ultraviolet light source. The box body is a cuboid with an internal cavity. The box cover matches the top opening of the box body and is hinged to the box body. The box cover has an observation hole, which is sealed with a filter lens. The ultraviolet light source emits ultraviolet light with a wavelength of 365 nm. The power supply is electrically connected to the ultraviolet light source to supply power to the ultraviolet light source. A switch is located outside the box body to control the on / off state of the ultraviolet light source.

[0098] A schematic diagram of the detection device is shown below. Figure 6 As shown, the box body and lid were prepared by 3D printing. The 3D printed model was designed using SolidWorks software, with external dimensions of 25 cm (length) × 25 cm (width) × 10 cm (height). The 3D model was divided into upper and lower parts, each 5 cm high. During the 3D printing process, the design drawings were imported into the Snapmaker 3D printer. The 3D printer performed slicing and layering according to the model structure, dividing it into a model material printing area and a support printing area. Polylactic acid (PLA) black filament was used as the printing material. After the box body and lid were printed, a filter lens was installed on the lid to seal the observation hole. The box body and lid were connected by hinges, and then assembled with an ultraviolet light source, power supply, and switch to form the detection device, which is used in conjunction with a smartphone.

[0099] With the ultraviolet light source of the detection device turned on, a colorimetric photograph of the hydrogel was taken using a smartphone through the observation port. Figure 7 This describes the fluorescence color change of fluorescent carbon dot hydrogels at different enrofloxacin concentrations (0–24 μM). With increasing enrofloxacin concentration, the fluorescence color emitted by the fluorescent carbon dot hydrogels under 365 nm ultraviolet light changes significantly, which can be used for semi-quantitative discrimination. The RGB feature values ​​of the fluorescent hydrogel regions in the colorimetric photographs were extracted using ImageJ image processing software, including the mean red component (R), the mean green component (G), and the mean blue component (B). The ratio of the mean green component (G) to the mean blue component (B), G / B, was calculated. The obtained G / B value showed a linear relationship with the enrofloxacin concentration in the range of 0–24 μM. Figure 8 As shown, the fitted equation is G / B = -0.0268x + 1.1820, R0 2 = 0.9901, and the detection limit for enrofloxacin is 0.76 μM, indicating that the fluorescent carbon dot hydrogel described in this invention is suitable for the quantitative detection of enrofloxacin.

[0100] Example 5

[0101] In this embodiment, a high-throughput detection method for enrofloxacin is provided.

[0102] The detection device described in Example 4 and the fluorescent carbon dot hydrogel prepared in Example 3 were used in conjunction with a smartphone to achieve high-throughput, rapid, and quantitative on-site detection of enrofloxacin via a WeChat mini-program equipped with an enrofloxacin high-throughput detection system.

[0103] The enrofloxacin high-throughput detection system uses the fitting equation y = -0.0268x + 1.1820 as its basis. It obtains color photos of a batch of test samples by taking pictures with a mobile phone, calculates the G / B value of the corresponding region of each test sample in the color photos of the batch of test samples, and substitutes it into the fitting equation as the y value to obtain the enrofloxacin concentration of each test sample in the batch in one go.

[0104] The enrofloxacin high-throughput detection system includes an image preprocessing module, a grid segmentation module, an RGB sampling module, a concentration calculation module, and a result display module.

[0105] The image preprocessing module is used to acquire and preprocess images. It uses a canvas drawing component to digitize and cache the images, providing standardized image data for subsequent grid segmentation. The images are color photos of a batch of samples to be tested, with each sample in the color photo being evenly distributed and uniformly colored.

[0106] The grid segmentation module uses an array-style equal division algorithm to divide the image into regions (e.g., 4×4 equal division). The region division process is as follows: First, obtain the pixel size parameters of the loaded image, including the image width imgWidth and the image height imgHeight; calculate the size of a single grid cell, where the cell width cellWidth = Math.floor (imgWidth / 4) and the cell height cellHeight = Math.floor (imgHeight / 4), and divide the image evenly into 16 array-style equal-area grid regions, with each grid region corresponding one-to-one with the image of a single sample in the color development photograph of the batch of samples; then, through a double loop traversal of row (0-3) and column number col (0-3), calculate the upper left corner coordinates of each grid region (x = col×cellWidth, y = row×cellHeight), and draw each grid region independently into a preset hidden canvas drawing component, thereby realizing the separation and extraction of the images of each sample in the color development photograph of the batch of samples to be tested, and avoiding image interference from different regions.

[0107] The RGB sampling module is used to obtain the RGB feature values ​​of each separated and extracted grid region. The acquisition process is as follows: Determine the width canvasWidth and height canvasHeight of the image in each separated and extracted grid region, and calculate the center coordinates of the grid region (centerX = Math.floor (canvasWidth / 2), centerY = Math.floor (canvasHeight / 2)); Set the sampling window parameters: the sampling window size sampleSize = 20, and the sampling window shape is a square, that is, a 20×20 pixel square sampling area. This sampling window covers 400 pixels in the center area of ​​the grid, ensuring that the sampling results can reflect the true color characteristics of each sample image; Read the pixel data in the sampling window through the canvas drawing component, that is, read the pixel data in the area centered at (centerX, centerY) with sampleSize as the side length, and extract the average RGB color component of all pixels in the sampling window as the RGB feature value of the corresponding grid region.

[0108] The concentration calculation module performs concentration conversion based on the fitted equation: according to the RGB characteristic values ​​of each grid region, the ratio G / B of the average green component G to the average blue component B of each grid region is calculated, and this ratio is used as the y value and substituted into the fitted equation x = (1.1820 - y) / 0.0268 to calculate the enrofloxacin concentration x of each sample to be tested, in µmol / L.

[0109] The results display module synchronously displays the image corresponding to the grid area, the extracted RGB feature values, and the calculated enrofloxacin concentration on the display interface, realizing the visualization of the test results of a batch of samples to be tested.

[0110] The specific testing steps are as follows:

[0111] (1) Prepare the sample to be tested

[0112] Actual samples including beef, mutton, fish, shrimp, eggs, milk, lake water, highland pasture water, and tap water were selected for pretreatment.

[0113] Solid samples (meat, eggs, shrimp): Homogenize the solid sample, take 0.5 g of homogenized sample, add 5 mL of acetonitrile and 5 mL of ethanol, sonicate for 10 minutes (25 ℃) to precipitate protein, centrifuge at 10000 rpm for 10 minutes, take the supernatant and filter it through a 0.22 μm organic filter membrane, and dilute it.

[0114] Liquid samples (milk, water): Mix milk sample with an equal volume of acetonitrile, vortex for 5 minutes, centrifuge, and filter the supernatant through a 0.22 μm organic filter membrane; filter water sample directly through a 0.22 μm aqueous filter membrane.

[0115] Add 4, 8, and 12 μM enrofloxacin standard solutions to the pretreated samples to form the test samples.

[0116] (2) Preprocessing and image acquisition: The batch of test samples were added to the fluorescent carbon dot hydrogel (prepared in Example 3) array distributed in the 96-well plate and immersed in the hydrogel for 30 minutes. The 96-well plate was placed in the box of the detection device, the box cover was closed, the ultraviolet light source was turned on, and the well plate was photographed through the observation hole to obtain the color development images of the batch of test samples.

[0117] (3) Image analysis: Run the WeChat mini program “ENRTester” that integrates the enrofloxacin high-throughput detection system, select to capture or upload images, extract the RGB feature values ​​of the corresponding regions of each sample in the color development photo obtained in step (2), and calculate the ratio G / B of the mean value of the green component G and the mean value of the blue component B of the corresponding regions of each sample.

[0118] (4) Concentration calculation: Based on the fitted equation y = -0.0268x + 1.1820, the enrofloxacin concentration x of each sample to be tested in the batch is automatically calculated, with the unit being µmol / L. y is the G / B value calculated in step (3) and the result is displayed.

[0119] Concentration calculation results are as follows Figure 9As shown in Table 2, the spiked recoveries and relative standard deviations (RSDs) of the method in this embodiment are 90.8%–109.2% with an RSD of <5.5% and a detection limit of 0.76 μM for enrofloxacin. This indicates that the method can be used for rapid on-site quantitative detection of enrofloxacin in complex samples.

[0120] Table 2 shows the spiked recoveries and relative standard deviations (RSD) of enrofloxacin in different real samples.

[0121]

[0122] like Figure 9 As shown, the enrofloxacin concentration of multiple samples was obtained at once and displayed in the mini-program using the method described in this embodiment, realizing high-throughput detection of enrofloxacin. Figure 9 The top of Figure A shows colorimetric images of a batch of samples to be tested (4×4 arranged areas, with different colors corresponding to the fluorescence response of different concentrations of enrofloxacin). Figure 9 In the middle of image A, there is a "Region Division and Concentration Calculation" button. Clicking it will trigger the image segmentation and concentration calculation function. Figure 9 Below Figure A and Figure 9 Figure B and the "Results Display" area present the enrofloxacin concentration detection results of different test samples in the chromogenic photograph, further verifying the correspondence between "color" and "concentration". The "Position", "RGB", and "Concentration" in the "Results Display" area correspond to 16 independent circular areas in the chromogenic photograph. Specifically, "Position", such as "Row 1, Column 1", corresponds to the circular area in Row 1, Column 1 of the chromogenic photograph; "RGB", such as "(144, 228, 182)", represents the three primary color values ​​corresponding to the circular area in Row 1, Column 1; and "Concentration", such as "0.00", represents the enrofloxacin concentration (in μM) corresponding to the circular area in Row 1, Column 1. The different colored circles visually demonstrate the difference in fluorescence color under ultraviolet light after the fluorescent carbon dot hydrogel comes into contact with different concentrations of enrofloxacin. Different enrofloxacin concentrations result in different RGB characteristic values ​​and visual colors.

[0123] Those skilled in the art will recognize that the embodiments described herein are intended to help the reader understand the principles of the invention, and should be understood that the scope of protection of the invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific modifications and combinations based on the technical teachings disclosed in this invention without departing from the spirit of the invention, and these modifications and combinations are still within the scope of protection of this invention.

Claims

1. A fluorescent carbon dot, characterized in that, It is prepared by the following method: (1) Polyethyleneimine and coumarin were dissolved in ethanol at a mass ratio of 1:1 and ultrasonicated until completely dissolved to obtain a homogeneous precursor solution. (2) Add sodium hydroxide solution to the precursor solution to adjust the pH to 9-11, then transfer it to a high-pressure reactor and react at 90-120 °C for 9-12 h. After natural cooling to room temperature, a crude carbon dot solution is obtained. (3) After centrifuging the crude solution, take the supernatant, filter it through a 0.22 μm organic phase filter membrane, place the filtrate in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze for 24~72 h, freeze dry to obtain fluorescent carbon dots.

2. A fluorescent carbon dot hydrogel, characterized in that, It is composed of a polyvinyl alcohol hydrogel matrix and fluorescent carbon dots as described in claim 1, wherein the fluorescent carbon dots are fixed in the polyvinyl alcohol hydrogel matrix network by physical embedding, and the mass ratio of the fluorescent carbon dots to the polyvinyl alcohol hydrogel matrix is ​​(5~30):2000.

3. The fluorescent carbon dot hydrogel according to claim 2, characterized in that, It is prepared by the following method: (1) Add polyvinyl alcohol to water, heat and stir until completely dissolved to obtain a polyvinyl alcohol solution of 0.1~0.15 g / mL; (2) Cool the polyvinyl alcohol solution to 40~60 ℃, add fluorescent carbon dots, stir evenly to obtain a fluorescent carbon dot-polyvinyl alcohol composite solution, wherein the mass ratio of fluorescent carbon dots to polyvinyl alcohol is (5~30):2000; (3) The fluorescent carbon dot-polyvinyl alcohol composite solution was frozen at -20~-80 ℃ for 1~4 h, and then thawed at room temperature for 1~2 h. The freeze-thaw cycle was repeated 3 times to achieve physical cross-linking and obtain fluorescent carbon dot hydrogel.

4. The application of the fluorescent carbon dots of claim 1 or the fluorescent carbon dot hydrogel of claim 2 or 3 in the detection of enrofloxacin.

5. A method for detecting enrofloxacin, characterized in that, The following method one or method two is used for testing; Method 1: Mix the fluorescent carbon dots described in claim 1 with the sample to be tested, and then perform fluorescence spectroscopy on the resulting mixture to calculate the fluorescence intensity I at a wavelength of 411 nm. 411 The fluorescence intensity I at a wavelength of 508 nm 508 The ratio I 411 / I 508 Then, the enrofloxacin concentration is calculated based on the fitted equation Y = 0.1990X + 0.0351, where X is the enrofloxacin concentration in µmol / L, and Y is the enrofloxacin concentration in µmol / L. 411 / I 508 value; Method 2: Immerse the fluorescent carbon dot hydrogel described in claim 2 or 3 in the sample to be tested and incubate it for a sufficient period of time. Record a colorimetric image under 365 nm ultraviolet light irradiation. Select the region of the fluorescent carbon dot hydrogel in the colorimetric image, extract its RGB feature values, calculate the ratio G / B of the mean value of the green component G to the mean value of the blue component, and calculate the enrofloxacin concentration according to the fitted equation y = -0.0268x +1.1820, where x is the enrofloxacin concentration in µmol / L and y is the G / B value.

6. The method according to claim 5, characterized in that, The sample to be tested is obtained by pre-treating a solid sample or a liquid sample. The pre-treatment method for solid samples is as follows: homogenize the solid sample, take the sample after homogenization to precipitate the protein, centrifuge, take the supernatant and filter, and dilute the obtained filtrate. The pre-treatment method for liquid samples is as follows: precipitate the protein in the liquid sample containing protein, centrifuge, take the supernatant and filter, or filter the liquid sample without protein.

7. The method according to claim 5, characterized in that, In Method 2, the fluorescent carbon dot hydrogel is immersed in the sample to be tested and incubated for a sufficient period of time. Then, the liquid on the surface of each fluorescent carbon dot hydrogel is first dried, and then a colorimetric photograph is recorded under 365 nm ultraviolet light.

8. The method according to any one of claims 5 to 7, characterized in that, Method 2 employs a detection device and the fluorescent carbon dot hydrogel described in claim 2 or 3 for detection. The detection device includes a box body, a lid, a power supply, and an ultraviolet light source. The box body is a rectangular parallelepiped with an internal cavity. The lid matches the top opening of the box body and is hinged to the box body. The lid has an observation hole, which is sealed with a filter. The ultraviolet light source emits ultraviolet light with a wavelength of 365 nm. The power supply is electrically connected to the ultraviolet light source. A switch is located outside the box body to control the on / off state of the ultraviolet light source. The method includes the following steps: (1) Preprocessing and image acquisition: The batch of test samples are added to the fluorescent carbon dot hydrogels distributed in the well plate and the hydrogels are immersed and fully incubated. The well plate is placed in the box of the detection device, the box cover is closed, the ultraviolet light source is turned on, and the well plate is photographed through the observation hole to obtain the color development photos of the batch of test samples. (2) Image analysis: Use image processing software to extract the RGB feature values ​​of the corresponding regions of each sample to be tested in the color photo obtained in step (1), and calculate the ratio G / B of the mean value of the green component G and the mean value of the blue component B of the corresponding regions of each sample to be tested. (3) Concentration calculation: The enrofloxacin concentration x of each sample to be tested is calculated based on the fitted equation y = -0.0268x + 1.1820, in µmol / L. y is the G / B value obtained in step (2).

9. The method according to claim 8, characterized in that, The image processing software is ImageJ, a mobile app, or a mobile app. The mobile app or mobile app is equipped with an enrofloxacin high-throughput detection system. This system uses the fitting equation y = -0.0268x + 1.1820 as its basis. It obtains color photos of a batch of test samples by taking pictures with a mobile phone, calculates the G / B value of the corresponding region of each test sample in the color photo, and substitutes it into the fitting equation as the y value to obtain the enrofloxacin concentration of each test sample in the batch of test samples in one go.

10. The method according to claim 9, characterized in that, The enrofloxacin high-throughput detection system includes an image preprocessing module, a grid segmentation module, an RGB sampling module, a concentration calculation module, and a result display module; The image preprocessing module is used to acquire and preprocess images, and to complete the digital loading and caching of images through the canvas drawing component; the images are color photos of the batch of samples to be tested obtained in step (1); The grid segmentation module uses an array-type equal division segmentation algorithm to divide the image into regions. The region division process is as follows: first, obtain the pixel size parameters of the loaded image, calculate the size of a single grid cell, and divide the image into array-type equal area grid regions. Each grid region corresponds to the image of a single test sample in the color development photo of a batch of test samples. Then, by iterating through the row and column numbers in a double loop, the coordinates of the top left corner of each grid region are calculated, and each grid region is drawn independently into a preset hidden canvas drawing component, so as to separate and extract the images of each test sample in the color development photos of a batch of test samples. The RGB sampling module is used to obtain the RGB feature values ​​of each grid region after separation and extraction. The acquisition process is as follows: determine the width and height of the image in each grid region after separation and extraction, calculate the center coordinates of the grid region; set the sampling window parameters; read the pixel data in the sampling window through the canvas drawing component, and extract the average RGB color component of all pixels in the sampling window as the RGB feature value of the corresponding grid region. The concentration calculation module performs concentration conversion based on the fitted equation: according to the RGB characteristic values ​​of each grid region, the ratio G / B of the average green component G to the average blue component B of each grid region is calculated, and this ratio is used as the y value and substituted into the fitted equation x = (1.1820 - y) / 0.0268 to calculate the enrofloxacin concentration x of each sample to be tested, in µmol / L. The results display module displays the image corresponding to the grid area, the extracted RGB feature values, and the calculated enrofloxacin concentration on the display interface.

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