Nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium as well as preparation method and application of nitrogen-doped sulfonated carbon dots

Abstract

The invention provides nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium as well as a preparation method and application of the nitrogen-doped sulfonated carbon dots, and belongs to the technical field of synthesis of new nano materials and detection of antibiotics. The method comprises the following steps: stirring and dissolving sodium diphenylaminesulfonate and sodium hydroxide in deionized water, heating, then applying direct current to obtain a mixed solution, then carrying out centrifugation, suction filtration and dialysis treatment on the mixed solution, and finally obtaining the carbon dots through a freeze-drying method. The nitrogen-doped sulfonated carbon dots prepared by the method disclosed by the invention show strong blue fluorescence emission, have a fluorescence quenching phenomenon and obvious change of fluorescence color after the ceftriaxone sodium is added, and can be used for measuring a fluorescent nano sensor of the ceftriaxone sodium. The ceftriaxone sodium in an actual sample can be rapidly and sensitively detected on the basis of the nitrogen-doped sulfonated carbon dots, and a new method and a new material are provided for application in the fields of biology, food safety and environmental monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterial synthesis and antibiotic detection, and relates to a nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium, a preparation method and an application thereof, in particular to a method for preparing nitrogen-doped sulfonated fluorescent carbon dots, and an application of the carbon dots as a fluorescent detection probe for detecting and identifying ceftriaxone sodium in aqueous solution and milk. Background Art

[0002] Ceftriaxone sodium (CTR) is a broad-spectrum third-generation cephalosporin antibiotic and has been widely used in medical treatment and animal husbandry. However, due to the frequent occurrence of situations such as some livestock and poultry farms failing to strictly follow the regulations on drug use, abusing antibiotics, and illegally using prohibited drugs, the veterinary drug residues in animal-derived foods have been greatly exceeded, posing a threat to human health and even causing death in severe cases. Among them, milk, as an important source of animal protein in people's lives, the problem of veterinary drug residues in it needs to be highly emphasized. Therefore, it is of great significance to simply, accurately and reliably determine the content of ceftriaxone sodium in environmental and food samples.

[0003] Currently, the analytical methods for detecting antibiotic residues include high performance liquid chromatography, capillary electrophoresis, microbial absorbance determination, chemiluminescence, electrochemistry, etc. However, most of these methods are time-consuming, expensive, and even require complex instrument equipment, which poses a great challenge to achieving rapid and convenient determination. Fluorescence method has become an effective analytical method due to its advantages such as high sensitivity, low detection limit, and high cost-effectiveness. In the past decade, fluorescence probes have made great progress in the field of environmental analysis and have been designed to respond to specific targets, including various new pollutants and small molecules such as picric acid, sulfite, hypochlorite, and doxycycline. However, so far, there have been few studies on the determination of ceftriaxone sodium by fluorescence spectroscopy. The challenge in the determination of ceftriaxone sodium is to find materials or fluorescence systems with specificity and high sensitivity for identification. The development and design of fluorescent materials for analysis are very important. As a new type of fluorescent nanomaterial that has attracted much attention in recent years, carbon dots have shown significant advantages in the field of fluorescence sensing due to their unique optical properties. This material can not only be used as an efficient optical sensor for substance analysis, but also effectively solve the technical bottlenecks in the operation convenience of traditional detection methods due to its good solubility, high stability, and strong anti-photobleaching ability during the detection process. Summary of the Invention

[0004] In view of the above-mentioned problems, the present invention provides carbon dots for detecting ceftriaxone sodium, a preparation method thereof, and an application thereof. The nitrogen-doped sulfonated carbon dots prepared by the method of the present invention exhibit strong blue fluorescence emission, and fluorescence quenching and a significant change in fluorescence color occur after the addition of ceftriaxone sodium, and can be used for a fluorescence nanosensor for determining ceftriaxone sodium. And the present invention constructs a portable sensing device based on nitrogen-doped sulfonated carbon dots, which can be used together with a smart phone to quickly and sensitively detect ceftriaxone sodium in actual samples, providing a new method and new material for applications in the fields of biology, food safety, and environmental monitoring.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A preparation method of nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium. First, sodium diphenylamine sulfonate is used as a carbon source. In an alkaline aqueous solution environment, a direct current is applied to the solution, and both the anode and cathode are platinum sheets. The sodium diphenylamine sulfonate molecules are oxidized and polymerized on the anode surface to obtain carbon dots, which are then dispersed in the solution. Finally, the carbon dots in the mixed solution are separated. The preparation method includes the following steps:

[0007] Step 1: At room temperature, using sodium diphenylamine sulfonate as a carbon source, it is added to deionized water solution together with an alkali and magnetically stirred until completely dissolved, and preheated to obtain a clear solution. The molar concentration of sodium diphenylamine sulfonate in the clear solution is 0.05 - 0.2 M, and the concentration of the alkali solution is 0.5 - 3 M.

[0008] Step 2: Apply a direct current to the clear solution obtained in Step 1 for electrolysis. During the electrolysis process, keep the temperature constant and perform magnetic stirring. After the electrolyte solution turns dark brown and there is no obvious color change, remove the current and let it cool naturally to room temperature to obtain a mixed solution. During the electrolysis process, the sodium diphenylamine sulfonate molecules are oxidized and polymerized on the anode surface to obtain carbon dots, which are then dispersed in the mixed solution.

[0009] Step 3: Centrifuge, filter by suction, and dialyze the mixed solution obtained in Step 2 to separate the carbon dots, and then freeze-dry them through a freeze dryer to obtain nitrogen-doped sulfonated carbon dots, and their form is a solid sample.

[0010] Further, in Step 1: The type of the alkali is a strong base, including one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, calcium hydroxide, barium hydroxide, ammonia water, etc., and preferably sodium hydroxide and potassium hydroxide.

[0011] Further, in Step 1: The preheating treatment temperature is 60 - 80 °C, and the time is 10 - 30 min.

[0012] Further, in Step 2: The system for applying a direct current consists of two platinum sheet electrodes and an electrolytic cell. The volume of the electrolyte accounts for 1 / 3 - 2 / 3 of the volume of the electrolytic cell. The intensity of the direct current is 0.5 - 3 A, and the time for the electrochemical reaction is 1 - 4 h. The electrolyte is the clarified solution from Step 1.

[0013] Further, in Step 3: The mixed solution is centrifuged at a speed of 8000 - 10000 r / min for 15 - 30 min, filtered through a PTFE filter paper with a pore size of 0.22 μm to obtain a filtrate, and the filtrate is dialyzed to neutral using a dialysis bag with a molecular weight cut-off of 500 - 1000 Da.

[0014] Further, in Step 3: The temperature for freeze-drying is -40 to -80 °C, and the time is 36 - 48 h.

[0015] Further, in Step 3, the pre-fitted linear equation is obtained by the following method: Take centrifuge tubes and quantitatively inject phosphate buffer solutions of carbon dots into them in sequence. Subsequently, accurately pipette equal volumes of ceftriaxone sodium samples with concentrations of 0, 4, 6, 10, 20, 30, 40, 50, and 60 μmol / L respectively into the system. The reaction system is uniformly mixed by vortex oscillation for 3 - 10 seconds, incubated in the dark at room temperature for 30 - 60 minutes, and then transferred to a standard quartz cuvette for image taking with a portable detection device. The color of the image is converted into corresponding R, G, B, H, S, and V values, and the linear equation of the value of B×H×100 / G×S×V corresponding to the nitrogen-doped sulfonated carbon dots and c is fitted, where c is the concentration of ceftriaxone sodium in the sample to be measured, and the values of B, H, G, S, and V represent the corresponding blue value, hue value, green value, saturation, and brightness of the image color conversion.

[0016] The present invention also provides a kind of nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium, which are prepared by the above preparation method. The nitrogen-doped sulfonated carbon dots have a nitrogen-doped carbon core structure, and are surface-modified with amino and sulfonic acid groups, with the characteristic of strong water dispersibility and excellent fluorescence performance.

[0017] An application of a kind of nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium, the nitrogen-doped sulfonated carbon dots are applied to the detection of ceftriaxone sodium, and the detection method includes the following steps:

[0018] Step 1: Add the phosphate buffer solution of the nitrogen-doped sulfonated carbon dots to the sample to be measured to obtain a mixed solution, and incubate for a period of time. The sample to be measured includes an aqueous solution or a milk sample. The incubation time is greater than 30 min.

[0019] Step 2: Transfer the mixed solution after incubation in Step 1 to a standard quartz cuvette for fluorescence emission spectrum measurement or fluorescence photograph taking.

[0020] Step 3: Convert the fluorescence emission intensity measured in Step 2 or the color of the captured image into corresponding numerical values, and substitute them into the pre-fitted linear equation to obtain the content of ceftriaxone sodium in the sample to be measured.

[0021] Further, in Step 1: the concentration of the phosphate buffer solution is 0.05 - 0.5 M, and the pH value is 7 - 9; the concentration of the nitrogen-doped sulfonated carbon dots in the phosphate buffer solution is 0.01 - 0.1 mg / mL. The method for preparing the milk sample is to mix milk and acetonitrile in a ratio of 1:1, ultrasonicate for 10 - 30 min, centrifuge at a centrifugation rate of 12,000 - 15,000 rpm for 10 min, and then filter with a microporous membrane with a pore size of 0.22 μm to obtain a clear and transparent pretreated milk solution; the phosphate buffer solution of the nitrogen-doped sulfonated carbon dots is mixed with the milk sample or the aqueous solution in a volume ratio of 1:1.

[0022] The principle and innovation of the present invention lie in:

[0023] In a strongly alkaline environment, hydroxyl radicals (·OH) and superoxide anions (·O 2- ) generated by the electrolysis of water will destroy the -NH- of sodium diphenylamine sulfonate; the sulfonic acid group in sodium diphenylamine sulfonate can improve the conductivity and promote the process of electrochemical oxidation. In the anodic region, the aromatic fragments and nitrogen-containing organic small molecules after the bond breakage of sodium diphenylamine sulfonate undergo condensation carbonization to form a nitrogen-doped carbon core, in which -NH- will be converted into pyrrole nitrogen and pyridine nitrogen; the surface is modified by the sulfonic acid group of sodium diphenylamine sulfonate and the amino group generated during the reaction process, thus exhibiting excellent fluorescence properties. This method of using electrochemical oxidation of small molecules is different from the previously reported top-down electrochemical exfoliation method and has the characteristics of easy synthesis and easy regulation. At the same time, the pyrrole nitrogen in the nitrogen-doped sulfonated carbon dots can selectively recognize ceftriaxone sodium in the sample to be measured and perform recognition detection through the generated fluorescence intensity or color change. This method is a simple, convenient to carry, and real-time rapid method for detecting ceftriaxone sodium.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] (1) The carbon dots prepared by the present invention have a unique composition and surface functional groups, with a nitrogen-doped core and sulfonate and amino groups on the surface;

[0026] (2) The preparation method of the present invention is simple, reliable, green, environmentally friendly, low-cost, and suitable for large-scale production;

[0027] (3) The carbon dots provided by the present invention can be efficiently quenched by ceftriaxone sodium, with high sensitivity and excellent anti-interference performance.

[0028] (4) The fluorescence detection probe and the portable detection device provided by the present invention have high sensitivity and good selectivity.

[0029] (5) The operation of the present invention is simple and does not require large-scale instruments. Through the smartphone software, on-site in-situ rapid detection can be carried out, which can better overcome the problems of complex food matrices such as milk and susceptibility to interference in detection. Brief Description of the Drawings

[0030] Figure 1 (a) is the transmission electron microscope image of the nitrogen-doped sulfonated carbon dots prepared in Example 1 of the present invention, Figure 1 (b) is the high-magnification transmission electron microscope image and lattice fringe image of the nitrogen-doped sulfonated carbon dots prepared in Example 1 of the present invention, Figure 1 (c) is Figure 1 (a) The corresponding particle size distribution diagram.

[0031] Figure 2 is the infrared spectrum diagram of the nitrogen-doped sulfonated carbon dots prepared in Example 1 of the present invention.

[0032] Figure 3 is the ultraviolet-visible absorption spectrum and fluorescence excitation-emission spectrum of the nitrogen-doped sulfonated carbon dots prepared in Example 1 of the present invention.

[0033] Figure 4 is the spectral diagram of the fluorescence emission curve of the nitrogen-doped sulfonated carbon dots prepared in Example 1 of the present invention changing with the excitation wavelength.

[0034] Figure 5 is the selectivity diagram of the nitrogen-doped sulfonated carbon dots for the detection of ceftriaxone sodium. Figure 5 (a) is the change in fluorescence intensity of the nitrogen-doped sulfonated carbon dot solution after adding different metal ions (300 μM) at an excitation wavelength of 423 nm; Figure 5 (b) is the change in fluorescence intensity of the nitrogen-doped sulfonated carbon dot solution after adding different small molecules (300 μM) at an excitation wavelength of 423 nm;

[0035] Figure 6 is the schematic diagram of the nitrogen-doped sulfonated carbon dots detecting the concentration of ceftriaxone sodium based on the change in fluorescence intensity; wherein, Figure 6 (a) is the change diagram of the fluorescence emission spectrum of the nitrogen-doped sulfonated carbon dots when the concentration of ceftriaxone sodium is 0 - 300 μM; Figure 6 (b) is the change diagram of the fluorescence emission spectrum of the nitrogen-doped sulfonated carbon dots when the concentration of ceftriaxone sodium is 0 - 24 μM; Figure 6 (c) is the piecewise linear relationship between the fluorescence intensity ratio of the nitrogen-doped sulfonated carbon dots before and after adding ceftriaxone sodium and the concentration of ceftriaxone sodium at 0 - 300 μM; Figure 6(d) is the linear relationship between the fluorescence intensity ratio of nitrogen-doped sulfonated carbon dots before and after adding ceftriaxone sodium and the concentration of ceftriaxone sodium in the range of 0 - 24 μM.

[0036] Figure 7 It is a schematic diagram for detecting the concentration of ceftriaxone sodium based on the color change of nitrogen-doped sulfonated carbon dots; among them, Figure 7 (a) shows the fluorescence color change of nitrogen-doped sulfonated carbon dots at ceftriaxone sodium concentrations in the range of 0 - 30 μM; Figure 7 (b) is a graph showing the relationship between the value of B×H×100 / G×S×V and the concentration of added ceftriaxone sodium. Detailed implementation manners

[0037] The present invention will be further described below in conjunction with the detailed implementation manners, but the present invention is not limited to the following examples.

[0038] Example 1:

[0039] Step 1: Magnetically stir and dissolve 2.768 g of sodium diphenylamine sulfonate and 4.000 g of sodium hydroxide in 100 mL of deionized water, and preheat from room temperature to 70 °C for 10 min.

[0040] Step 2: Apply a direct current of 1 A to the clear solution obtained in Step 1 for electrolysis. During the process, maintain a constant temperature of 70 °C and magnetic stirring conditions. The electrolysis time is 2 h. After electrolysis, naturally cool to room temperature.

[0041] Step 3: Centrifuge the mixture obtained in Step 2 at 9000 r / min for 20 min, filter it with a 0.22 μm microporous filter membrane, dialyze it to neutral using a 1000 Da dialysis bag, and then obtain a solid powder of carbon dots by freeze-drying using a freeze dryer. Among them, the temperature of freeze-drying is -40 °C and the time is 48 h. Measure the absolute quantum efficiency of a 0.01 mg / mL carbon dot solution with an ultraviolet-near-infrared absolute quantum yield instrument, which is 23.34%.

[0042] Example 2:

[0043] Step 1: Magnetically stir and dissolve 1.384 g of sodium diphenylamine sulfonate and 5.611 g of potassium hydroxide in 100 mL of deionized water, and preheat from room temperature to 60 °C for 20 min.

[0044] Step 2: Apply a direct current of 0.5 A to the clear solution obtained in Step 1 for electrolysis. During the process, maintain a constant temperature of 60 °C and magnetic stirring conditions. The electrolysis time is 1 h. After electrolysis, naturally cool to room temperature.

[0045] Step 3: Centrifuge the mixture obtained in Step 2 at 8000 r / min for 15 min, filter it through a 0.22 μm microporous membrane, dialyze it with a 500 Da dialysis bag until neutral, and then obtain the carbon dot solid powder by freeze-drying using a freeze dryer. Among them, the temperature of freeze-drying is -80 °C and the time is 36 h. Measure the absolute quantum efficiency of the 0.01 mg / mL carbon dot solution with an ultraviolet-near-infrared absolute quantum yield instrument, which is 11.53%.

[0046] Example 3:

[0047] Step 1: Magnetically stir and dissolve 5.536 g of sodium diphenylamine sulfonate and 8.000 g of sodium hydroxide in 100 mL of deionized water, and preheat from room temperature to 80 °C for 30 min.

[0048] Step 2: Apply a 2 A direct current to electrolyze the clarified solution obtained in Step 1. During the process, keep the temperature at 80 °C constant and under magnetic stirring conditions. The electrolysis time is 3 h, and after electrolysis, cool it naturally to room temperature.

[0049] Step 3: Centrifuge the mixture obtained in Step 2 at 8000 r / min for 25 min, filter it through a 0.22 μm microporous membrane, dialyze it with a 1000 Da dialysis bag until neutral, and then obtain the carbon dot solid powder by freeze-drying using a freeze dryer. Among them, the temperature of freeze-drying is -60 °C and the time is 40 h. Measure the absolute quantum efficiency of the 0.01 mg / mL carbon dot solution with an ultraviolet-near-infrared absolute quantum yield instrument, which is 13.33%.

[0050] Example 4:

[0051] Step 1: Magnetically stir and dissolve 8.304 g of sodium diphenylamine sulfonate and 12.001 g of sodium hydroxide in 100 mL of deionized water, and preheat from room temperature to 75 °C for 15 min.

[0052] Step 2: Apply a 3 A direct current to electrolyze the clarified solution obtained in Step 1. During the process, keep the temperature at 75 °C constant and under magnetic stirring conditions. The electrolysis time is 4 h, and after electrolysis, cool it naturally to room temperature.

[0053] Step 3: Centrifuge the mixture obtained in Step 2 at 10000 r / min for 30 min, filter it through a 0.22 μm microporous membrane, dialyze it with a 500 Da dialysis bag until neutral, and then obtain the carbon dot solid powder by freeze-drying using a freeze dryer. Among them, the temperature of freeze-drying is -70 °C and the time is 38 h. Measure the absolute quantum efficiency of the 0.01 mg / mL carbon dot solution with an ultraviolet-near-infrared absolute quantum yield instrument, which is 14.66%.

[0054] Comparative Example 1 (compared with Example 1):

[0055] Step 1: Dissolve 1.692 g of diphenylamine and 4.000 g of sodium hydroxide by magnetic stirring in 100 mL of deionized water, and preheat from room temperature to 70 °C for 15 min.

[0056] Step 2: Apply a direct current of 1 A to the clear solution obtained in Step 1 for electrolysis. During the process, maintain a constant temperature of 70 °C and magnetic stirring conditions. The electrolysis time is 2 h. After electrolysis, naturally cool to room temperature.

[0057] Step 3: Centrifuge the mixture obtained in Step 2 at 9000 r / min for 20 min, filter it with a 0.22 μm microporous filter membrane, dialyze it to neutral using a 500 Da dialysis bag, and then obtain the carbon dot solid powder by freeze-drying method using a freeze dryer. Among them, the temperature of freeze-drying is -40 °C and the time is 48 h. Measure the absolute quantum efficiency of the 0.01 mg / mL carbon dot solution with an ultraviolet-near-infrared absolute quantum yield instrument, which is 2.31%.

[0058] This Comparative Example 1 is compared with Example 1. The difference is that the carbon source is changed, and sodium diphenylamine sulfonate is replaced with diphenylamine. The results show the importance of the sulfonic acid group in sodium diphenylamine sulfonate for promoting the electrolysis reaction process.

[0059] Comparative Example 2 (compared with Example 1):

[0060] Step 1: Dissolve 2.768 g of sodium diphenylamine sulfonate by magnetic stirring in 100 mL of 1 M hydrochloric acid solution, and preheat from room temperature to 70 °C for 15 min.

[0061] Step 2: Apply a direct current of 1 A to the clear solution obtained in Step 1 for electrolysis. During the process, maintain a constant temperature of 70 °C and magnetic stirring conditions. The electrolysis time is 2 h. After electrolysis, naturally cool to room temperature.

[0062] Step 3: Centrifuge the mixture obtained in Step 2 at 9000 r / min for 20 min, filter it with a 0.22 μm microporous filter membrane, dialyze it to neutral using a 1000 Da dialysis bag, and then obtain the carbon dot solid powder by freeze-drying method using a freeze dryer. Among them, the temperature of freeze-drying is -40 °C and the time is 48 h. Measure the absolute quantum efficiency of the 0.01 mg / mL carbon dot solution with an ultraviolet-near-infrared absolute quantum yield instrument, which is 1.86%.

[0063] This Comparative Example 1 is compared with Example 1. The difference is that the electrolyte environment is changed, and sodium hydroxide is replaced with hydrochloric acid. The results show that the alkaline environment is one of the important conditions for electrolytic production of carbon dots.

[0064] The morphological characterization of the nitrogen-doped sulfonated carbon dots prepared in Example 1 of the present invention is as Figure 1 shown.Figure 1 The transmission electron microscope image shows that the carbon dots are in a monodispersed state, Figure 1 b is the high-resolution transmission electron microscope spectrum. The lattice spacing of 0.343 nm corresponds to the diffraction plane of graphite (sp 2 ) carbon. Figure 1 c The statistical analysis of the particle size of the carbon dots clearly shows that their particle size distribution is between 6 - 10 nm, and the average particle size is about 8.2 nm.

[0065] The structural characterization of the nitrogen-doped sulfonated carbon dots prepared in Example 1 of the present invention is as Figure 2 shown. The infrared spectrum shows that there are obvious differences in the peak patterns between the nitrogen-doped sulfonated carbon dots and the precursor sodium diphenylamine sulfonate, proving the successful occurrence of the reaction. The asymmetric stretching vibration of the O=S=O group at 1034 cm -1 and 1205 cm -1 , as well as the stretching vibration of the C-S bond at 631 cm -1 , indicate that there are sulfonic acid groups retained from sodium diphenylamine sulfonate in the nitrogen-doped sulfonated carbon dots. The characteristic vibrations involving nitrogen atoms are of special significance in the 1650 - 1300 cm -1 region. The strong absorption peaks at 1633 cm -1 , 1590 cm -1 and 1490 cm -1 correspond to the stretching vibration coupling modes of the C=C of the pyrrole ring skeleton, the C-C and C-N bonds of the pyridine ring skeleton respectively, confirming that the nitrogen-doped sulfonated carbon dots have a different form of nitrogen existence from that in sodium diphenylamine sulfonate and have an aromatic conjugated structure. In addition, the broad and strong absorption bands in the range of 3500 - 3300 cm -1 and 3600 - 3200 cm -1 are characteristic of the electronegative -OH, -COOH and -NH2 groups, supporting the conclusion that nitrogen and oxygen atoms have been doped onto the surface of the nitrogen-doped sulfonated carbon dots. By comparison with the raw materials, it can be seen that the nitrogen-doped sulfonated carbon dots retain the sulfonic acid functional groups of sodium diphenylamine sulfonate, and at the same time show the characteristics of new nitrogen-containing groups.

[0066] The optical property spectra of the nitrogen-doped sulfonated carbon dots prepared in Example 1 of the present invention are as Figure 3 , 4 shown. Figure 3 The optical properties of the nitrogen-doped sulfonated carbon dots were analyzed by ultraviolet-visible absorption spectroscopy. The results show two absorption bands that are very close to each other. The first absorption peak appears at 270 nm, attributed to the nuclear state sp 2The π→π* transition in the domain. The second absorption band was observed at 326 nm, which is related to the n→π* transition of the C=O bond and the surface functional groups of nitrogen-doped sulfonated carbon dots. This can be attributed to the surface functional groups and the doping of nitrogen and sulfur atoms. And the optimal excitation wavelength of the nitrogen-doped sulfonated carbon dots is 310 nm, corresponding to the emission peak at 423 nm. Figure 4 It is the emission spectrum of the carbon dots at different excitation wavelengths. When using excitation wavelengths of 270 - 340 nm, we observed that the central emission wavelength is 423 nm. It is worth noting that although the excitation wavelength changes, the maximum fluorescence emission peak of the nitrogen-doped sulfonated carbon dots remains consistent. This consistency indicates that the carbon dots have excitation wavelength independence, and the nitrogen-doped sulfonated carbon dots have a uniform size, thus producing a relatively stable fluorescence emission peak.

[0067] Application verification:

[0068] To investigate the selectivity of the prepared nitrogen-doped sulfonated carbon dots for ceftriaxone sodium, taking the nitrogen-doped sulfonated carbon dots prepared in Example 1 as an example, prepare BaCl2, CaCl2, CdCl2, AgNO3, CoCl2, CuCl2, FeCl2, FeCl3, MgCl2, MnCl2, NiCl2, and ZnCl2 solutions with a concentration of 0.1 mol / L, and dilute them to 1 mmol / L respectively. Take 1 mL of the above salt solutions and put them into centrifuge tubes. Then take 1 mL of the phosphate buffer solution containing 0.02 mg of nitrogen-doped sulfonated carbon dots, and achieve uniform mixing of the reaction system by vortex oscillation for 3 seconds; prepare valine, threonine, proline, glutamic acid, alanine, riboflavin, oxytetracycline, tetracycline, penicillin, ampicillin, and amoxicillin solutions with a concentration of 0.01 mol / L, and dilute them to 500 μmol / L respectively. Take 1 mL of the above small molecule solutions and put them into centrifuge tubes. Then take 1 mL of the phosphate buffer solution containing 0.02 mg of nitrogen-doped sulfonated carbon dots, and achieve uniform mixing of the reaction system by vortex oscillation for 3 seconds; use a fluorescence spectrometer to test whether the fluorescence of the nitrogen-doped sulfonated carbon dots quenches when adding the above substances under 310 nm incident light. Figure 5 It shows that the nitrogen-doped sulfonated carbon dots have good selectivity for the detection of ceftriaxone sodium, where Figure 5 (a) shows that most metal ions have no fluorescence quenching effect on the nitrogen-doped sulfonated carbon dots; Figure 5 (b) shows that the fluorescence quenching effect of some common small molecules on the nitrogen-doped sulfonated carbon dots is also negligible.

[0069] The nitrogen-doped sulfonated carbon dots obtained in Example 1, Example 2, Example 3, Example 4, Comparative Example 1 and Comparative Example 2 were respectively used as fluorescence probes for the detection test of ceftriaxone sodium. The specific method is as follows: Take a 4 mL centrifuge tube, and sequentially inject 1 mL of phosphate buffer solution containing 0.02 mg of nitrogen-doped sulfonated carbon dots, and then accurately pipette 1 mL of ceftriaxone sodium samples with concentrations of 4, 8, 16, 20, 24, 28, 32, 36, 40, 44 and 48 μmol / L into the system. The reaction system was uniformly mixed by vortex oscillation for 3 seconds, incubated in the dark at room temperature for 30 minutes, and then transferred to a standard quartz cuvette for fluorescence emission spectrum measurement. Record the change in fluorescence intensity, and fit the F0 / F and c(CTR) equations corresponding to each example, where c(CTR) is the concentration of ceftriaxone sodium in the solution to be measured, F0 is the fluorescence intensity value at 423 nm of the phosphate buffer solution of nitrogen-doped sulfonated carbon dots, and F is the fluorescence intensity value at 423 nm of the mixed solution after mixing the phosphate buffer solution of nitrogen-doped sulfonated carbon dots and the ceftriaxone sodium sample in a 1:1 ratio. It was calculated that Comparative Example 1 and Comparative Example 2 had no detection effect on ceftriaxone sodium, and the detection limits of Example 1, Example 2, Example 3 and Example 4 were 0.328 μM, 0.767 μM, 0.723 μM and 0.785 μM respectively. For Example 1, in Figure 6 (a), when the concentration of ceftriaxone sodium was 0 - 300 μM, we observed that the fluorescence of the nitrogen-doped sulfonated carbon dots quenched with the increase of the concentration of ceftriaxone sodium. In Figure 6 (b), we can perform piecewise linear fitting on this change in fluorescence intensity. In Figure 6 (c), it can be seen that as the concentration of ceftriaxone sodium increased from 2 μM to 24 μM, the blue fluorescence emission intensity at 423 nm gradually decreased, indicating that fluorescence quenching can also be sensitively triggered in the presence of low-concentration ceftriaxone sodium. This results in a stepwise change in the fluorescence intensity ratio F0 / F, ranging from 1 to 1.3. In Figure 6 (d), it can be seen that the fluorescence intensity ratio F0 / F and the CTR concentration showed a strong linear correlation (R 2 = 0.9957) in the range of 0 - 30 μM, and the fitting equation was F0 / F = 0.0107×c(CTR) + 0.9994. The detection limit (LOD) was calculated to be 0.298 μM according to the formula LOD = 3σ / k. The method was used to detect the spiked aqueous solution of ceftriaxone sodium, and the spiked recovery rate was measured to be 98.7% (n = 3).

[0070] The nitrogen-doped sulfonated carbon dots prepared in Example 1 were used to detect ceftriaxone sodium by observing color changes. Take a centrifuge tube and sequentially inject a phosphate buffer solution of carbon dots quantitatively. Subsequently, accurately pipette ceftriaxone sodium samples with concentrations of 0, 10, 20, 30, 40, 50, and 60 μmol / L respectively into the system. The reaction system was uniformly mixed by vortex oscillation for 10 seconds. After incubating in the dark at room temperature for 30 minutes, it was transferred to a standard quartz cuvette for image capture using a portable detection device. The color of the image was converted into the corresponding R, G, B, H, S, and V values, and the linear equation of B×H×100 / G×S×V corresponding to the nitrogen-doped sulfonated carbon dots and c(CTR) was fitted, where c(CTR) is the concentration of ceftriaxone sodium in the sample, and the B, H, G, S, and V values represent the corresponding blue value, hue value, green value, saturation, and brightness of the image color conversion respectively. Substituting the B×H×100 / G×S×V value corresponding to the photo of the unknown sample into the equation can obtain the content of ceftriaxone sodium in the sample. Figure 7 (a) A series of fluorescence images were taken of milk samples containing 0 - 30 μM ceftriaxone sodium. Figure 7 (b) The concentration of ceftriaxone sodium was evaluated by calculating the B×H×100 / G×S×V value. The B×H×100 / G×S×V value changes with the concentration of ceftriaxone sodium and shows a strong linear relationship in the range of 0 - 30 μM (R 2 = 0.9805). The fitted linear equation is B×H×100 / G×S×V = 0.1748c(CTR) + 9.4379, and the calculated detection limit (LOD) is 0.38 μM. The spiked recovery rate of ceftriaxone sodium in milk samples was measured to be 103.6% (n = 3) using this method.

[0071] Finally, it should be noted that the above-described embodiments only represent the implementation manners of the present invention, but should not be construed as limiting the scope of the present invention patent. It should be pointed out that for those skilled in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A preparation method of nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium, characterized in that, The described preparation method comprises the following steps: Step 1: Using sodium diphenylamine sulfonate as the carbon source, adding it and an alkali into deionized aqueous solution and magnetically stirring until completely dissolved, and then performing preheating treatment to obtain a clear solution; the molar concentration of sodium diphenylamine sulfonate in the clear solution is 0.05 - 0.2 M, and the concentration of the alkali solution is 0.5 - 3 M; Step 2: Applying a direct current to the clear solution obtained in Step 1 for electrolysis. During the electrolysis process, keep it at a constant temperature and perform magnetic stirring. During the electrolysis process, the sodium diphenylamine sulfonate molecules are oxidized and polymerized on the anode surface to obtain carbon dots, which are then dispersed into the mixed solution; Step 3: Centrifuging, filtering by suction, and dialyzing the mixed solution obtained in Step 2 to separate the carbon dots, and then performing freeze-drying to obtain nitrogen-doped sulfonated carbon dots.

2. The preparation method of nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium according to claim 1, characterized in that, In Step 1: The type of the alkali is a strong base, including one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, calcium hydroxide, barium hydroxide, and ammonia water; the preheating treatment temperature is 60 - 80 °C, and the time is 10 - 30 min.

3. The preparation method of nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium according to claim 1, characterized in that, In Step 1: The type of the alkali is preferably sodium hydroxide and potassium hydroxide.

4. The preparation method of nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium according to claim 1, wherein, In Step 2: After the electrolyte solution turns dark brown and there is no obvious color change, remove the current and let it cool naturally to room temperature to obtain the mixed solution.

5. The preparation method of nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium according to claim 1, wherein, In Step 2: The system for applying the direct current consists of two platinum sheet electrodes and an electrolytic cell. The volume of the electrolyte solution accounts for 1 / 3 - 2 / 3 of the volume of the electrolytic cell, the direct current intensity is 0.5 - 3 A, and the time for the electrochemical reaction is 1 - 4 h.

6. The preparation method of nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium according to claim 1, characterized in that, In Step 3: Centrifuge the mixed solution at a speed of 8000 - 10000 r / min for 15 - 30 min, filter by suction using a PTFE filter paper with a pore size of 0.22 μm to obtain the filtrate, and dialyze the filtrate to neutral using a dialysis bag with a molecular weight cut-off of 500 - 1000 Da; the temperature for freeze-drying is -40 to -80 °C, and the time is 36 - 48 h.

7. A nitrogen-doped sulfonated carbon dot for detecting ceftriaxone sodium, characterized in that, The nitrogen-doped sulfonated carbon dots are prepared by the preparation method described in any one of Claims 1 - 6; the nitrogen-doped sulfonated carbon dots have a nitrogen-doped carbon core structure, with amino and sulfonic acid groups modified on the surface, and have the characteristic of strong water dispersibility and exhibit fluorescence properties.

8. Use of the nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium according to claim 7, characterized in that, The described nitrogen-doped sulfonated carbon dots are applied to the detection of ceftriaxone sodium.

9. Use of the nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium according to claim 8, characterized in that, The detection method comprises the following steps: Step 1: Adding the phosphate buffer solution of the nitrogen-doped sulfonated carbon dots into the sample to be tested to obtain a mixed solution, and performing incubation, where the incubation time is greater than 30 min; Step 2: Transferring the mixed solution in Step 1 to a standard quartz cuvette for fluorescence emission spectrum measurement or fluorescence photograph taking; Step 3: Converting the fluorescence emission intensity measured in Step 2 or the color of the photographed image into corresponding numerical values, and substituting them into a pre-fitted linear equation to obtain the content of ceftriaxone sodium in the sample to be tested.

10. Use of the nitrogen-doped sulfonated carbon dots for detecting ceftriaxone sodium according to claim 9, characterized in that, In the described detection method: In Step 1, the sample to be tested includes an aqueous solution or a milk sample; among them, the production method of the milk sample is to mix milk and acetonitrile in a ratio of 1:1, and then perform ultrasonic treatment, centrifugation, and filtration to obtain it; In Step 1, the concentration of the phosphate buffer solution is 0.05 - 0.5 M, and the pH value is 7 - 9; In the step 1, the concentration of the nitrogen-doped sulfonated carbon dots in the phosphate buffer solution is 0.01 - 0.1 mg / mL; In the step 1, the phosphate buffer solution of the nitrogen-doped sulfonated carbon dots is mixed with the sample to be tested at a volume ratio of 1:1; In the step 3, the pre-fitted linear equation is obtained by the following method: Sequentially inject a quantitative amount of the phosphate buffer solution of the carbon dots into a centrifuge tube, and then accurately pipette the same volume of ceftriaxone sodium samples with concentrations of 0, 4, 6, 10, 20, 30, 40, 50, and 60 μmol / L respectively into the system; The reaction system is uniformly mixed by vortex oscillation, and after incubation in the dark at room temperature, it is transferred to a standard quartz cuvette for image capture; The color of the image is converted into the corresponding R, G, B, H, S, and V values, and the linear equation of the value of B×H×100 / G×S×V corresponding to the nitrogen-doped sulfonated carbon dots and c is fitted, where c is the concentration of ceftriaxone sodium in the sample to be tested, and the B, H, G, S, and V values respectively represent the corresponding blue value, hue value, green value, saturation, and brightness of the color of the image.

Images