N-doped carbon nanodots and application thereof in tinidazole detection

Abstract

This invention discloses a type of N-doped carbon nanodots and their application in the detection of tinidazole, belonging to the field of analytical detection. The preparation method of the N-doped carbon nanodots in this invention is as follows: L-lysine, ethylenediamine, and water are mixed to form a mixture, which is then heated to react. After the reaction, the N-doped carbon nanodots are purified. This invention is the first to use L-lysine and ethylenediamine as precursors to prepare carbon nanodots and achieve quantitative detection of tinidazole. It is low-cost, highly sensitive, and suitable for rapid on-site detection, possessing high practical value and promising prospects for widespread application.

Description

Technical Field

[0001] This invention relates to an N-doped carbon nanodot and its application in the detection of tinidazole, belonging to the field of analytical detection. Background Technology

[0002] With the rapid development of modern agriculture and animal husbandry, food safety issues have received increasing attention. Tinidazole (TNZ), a widely used nitroimidazole antibiotic, is commonly used to treat anaerobic bacterial infections in dairy cows. However, the use of tinidazole may result in residues in dairy cows, which can then enter the human food chain through dairy products. This not only poses a threat to human health but may also lead to antibiotic resistance. Therefore, detecting tinidazole residues is of significant practical importance to human health.

[0003] Currently, common methods for detecting tinidazole include chromatography, mass spectrometry, and immunoassay. While chromatography and mass spectrometry offer high sensitivity and accuracy, the equipment is expensive and the operation is complex, making them unsuitable for on-site detection. Examples include high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Immunoassay, although relatively simple, suffers from insufficient sensitivity, particularly in detecting low concentrations of the analyte.

[0004] In the existing technology, there is a technique for detecting tinidazole using carbon dots, such as patent CN 113025322 A -- a high fluorescence performance carbon dot powder and its preparation method and its application in the detection of nitroimidazole drugs. In this technique, defatted rice bran, water, and a nitrogen source are used to prepare a carbon dot for the detection of nitroimidazole drugs, but the linear range of tinidazole concentration detection is not large. Summary of the Invention

[0005] Technical issues

[0006] Among existing tinidazole residue detection technologies, chromatography and mass spectrometry, while highly accurate, suffer from drawbacks such as expensive equipment, complex operation, and unsuitability for rapid on-site detection. Immunoassay, though simpler, lacks sufficient sensitivity, particularly in detecting low concentrations of tinidazole. Other carbon dot detection techniques also exhibit limited linearity. Therefore, a simple, efficient, sensitive method with a wide linear range suitable for on-site detection of tinidazole is needed.

[0007] Technical solution

[0008] With the development of nanotechnology, the application of nanomaterials in analytical chemistry has received widespread attention and research. Among them, carbon nanodots (CDs), as a novel nanomaterial, possess high photostability, good spectral characteristics, and good biocompatibility, and have been widely used in sensors, imaging, and biolabeling. Therefore, this invention synthesizes an N-doped carbon nanodot using L-lysine and ethylenediamine as precursors via a hydrothermal method, and applies it to the detection of tinidazole.

[0009] This invention provides a method for preparing N-doped carbon nanodots for tinidazole detection, the method comprising:

[0010] L-lysine, ethylenediamine, and water were mixed to form a mixture, which was then heated to react. After the reaction was completed, the mixture was purified to obtain N-doped carbon nanodots.

[0011] Furthermore, the mass ratio of L-lysine to ethylenediamine is 3:1 to 1.5; the mass ratio of L-lysine to water is 3:5 to 15.

[0012] Furthermore, the volume of the container for the heating reaction is 1.5 to 2.5 times the volume of the mixed liquid.

[0013] Furthermore, the temperature of the heating reaction is 150–200°C; the heating reaction time is 5–10 h.

[0014] Furthermore, purification includes one or more purification methods such as centrifugation, filtration, and dialysis.

[0015] Furthermore, when centrifugation is used for purification, the centrifugation conditions are 8000-15000 rpm and 10-30 min.

[0016] Furthermore, when filtration is used for purification, the filtration conditions are to use a 0.2–0.25 μM microporous membrane.

[0017] Furthermore, when dialysis is chosen for purification, the dialysis conditions are as follows: use a dialysis membrane with a molecular weight cutoff of 3000–4000 Da for dialysis purification for 15–24 hours.

[0018] Furthermore, the N-doped carbon nanodots exist in the form of carbon dot mother liquor.

[0019] The present invention relates to the application of N-doped carbon nanodots for tinidazole detection in the field of tinidazole detection.

[0020] Furthermore, the application is to detect tinidazole in water or beverages; the water includes river water, groundwater, lake water, or seawater; the beverage includes milk, carbonated drinks, or fruit and vegetable juices.

[0021] The present invention also provides a method for detecting tinidazole, the method comprising the following steps:

[0022] Mix the carbon dot solution and the liquid to be tested, add PBS buffer and incubate for a period of time, then perform fluorescence spectroscopy detection. Substitute the detected fluorescence intensity peak at 390 nm into the linear relationship for calculation to obtain the concentration of tinidazole.

[0023] Furthermore, the carbon dot solution is prepared by diluting the carbon dot mother liquor by 35 to 45 times.

[0024] Furthermore, the liquid to be tested includes water or beverages.

[0025] Furthermore, the water body includes river water, groundwater, lake water, or seawater.

[0026] Furthermore, the beverage includes milk, carbonated drinks, or fruit and vegetable juices.

[0027] Furthermore, the mixing ratio of the carbon dot solution and the liquid to be tested is 1:1 to 2.

[0028] Furthermore, the pH of the PBS buffer is 7.5–8.5.

[0029] Furthermore, the mixing ratio of carbon dot solution and PBS buffer is 1:8 to 10.

[0030] Furthermore, the incubation time is 0.5 to 2 hours.

[0031] Furthermore, the conditions for fluorescence spectroscopy detection are as follows: the excitation slit width of the spectrometer is 1–2 nm, the emission slit width is 8–10 nm, and the integration time is 0.1–0.2 s; the excitation wavelength of the fluorescence spectrometer is 280–320 nm, the emission wavelength range is 340–540 nm, and the step size is 1–2 nm.

[0032] Specifically, the optional conditions for fluorescence spectroscopy detection are: the excitation slit width of the spectrometer is 2 nm, the emission slit width is 10 nm, and the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 320 nm, the emission wavelength range is 390 nm, and the step size is 1 nm.

[0033] Furthermore, the linear relationship is obtained as follows:

[0034] (1) Preparation of sample solution: Take the liquid to be tested and add a system of tinidazole at different concentrations to prepare tinidazole standard solutions of different concentrations; the different concentrations include a blank control group with a concentration of 0.

[0035] (2) The carbon dot mother liquor was diluted 35 to 45 times to prepare a carbon dot solution and mixed with tinidazole standard solutions of different concentrations. Then, PBS buffer with pH = 7.5 to 8.5 was added, and the mixture was incubated for 0.5 to 2 hours to obtain a spiked carbon dot solution. The spiked carbon dot solution was then subjected to fluorescence spectroscopy detection.

[0036] (3) Record the fluorescence intensity peak at 390 nm without the addition of tinidazole as F0, and the fluorescence intensity peak at 390 nm with the addition of tinidazole as F. Calculate the fluorescence quenching degree F0 / F, and then plot the relationship curve between the fluorescence quenching degree of the sample solution and the concentration of tinidazole to obtain the linear relationship.

[0037] Furthermore, in establishing the linear relationship, the conditions for fluorescence spectroscopy detection are as follows: the excitation slit width of the spectrometer is 1–2 nm, the emission slit width is 8–10 nm, and the integration time is 0.1–0.2 s; the excitation wavelength of the fluorescence spectrometer is 280–320 nm, the emission wavelength range is 340–540 nm, and the step size is 1–2 nm.

[0038] Furthermore, when the substance to be detected is milk or water, the linear relationship in step (2) is: F0 / F=0.01073C+1.0224, where F0 and F represent the peak fluorescence intensity of the system at 390nm before and after the addition of tinidazole, respectively, and C represents the concentration of the added tinidazole.

[0039] Furthermore, when the substance to be tested is milk or water, the concentration of tinidazole in the linear relationship is 0–100 μM.

[0040] Beneficial effects

[0041] 1. This invention is the first to synthesize carbon dots using L-lysine and ethylenediamine as precursors and use them as a fluorescent sensor to achieve quantitative detection of tinidazole. This method is simple and rapid to operate, has a wide linear range, can detect tinidazole from 0 to 100 μM, has high detection sensitivity, and a detection limit as low as 0.36 μM, making it very suitable for routine analysis.

[0042] 2. In this invention, the fluorescence excitation spectrum of N-doped carbon nanodots highly overlaps with the UV-Vis absorption spectrum of tinidazole, resulting in an internal filtration effect that quenches the fluorescence of the carbon nanodots, thus exhibiting good selectivity for tinidazole. Furthermore, these N-doped carbon nanodots possess strong anti-interference capabilities and can also detect tinidazole in complex liquid environments such as milk, making them significant in the field of food testing. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the detection of tinidazole in milk based on N-doped carbon nanodots.

[0044] Figure 2 The fluorescence spectra of the system in Example 2 when different concentrations of tinidazole were added are shown.

[0045] Figure 3 The curve showing the relationship between the degree of fluorescence quenching and the concentration of tinidazole in Example 2 is shown.

[0046] Figure 4 The linear fitting curves for the fluorescence quenching degree and the concentration range of 1–100 μM tinidazole in Example 2 are shown.

[0047] Figure 5 The graph shows the test results for the selectivity of tinidazole in Example 3.

[0048] Figure 6 The figure shows the test results of the anti-interference ability of tinidazole in Example 3. Detailed Implementation

[0049] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0050] Example 1

[0051] Weigh 3g of L-lysine and 1.5mL of ethylenediamine into a beaker, add 10mL of deionized water, sonicate for 10min, and after complete dissolution, transfer to a 25mL polytetrafluoroethylene-lined container and react at 180℃ for 8h. Then, centrifuge the reaction product at 10000rpm for 30min, filter using a 0.22μM microporous membrane, and then dialyze to purify for 24h using a dialysis membrane with a molecular weight cutoff of 3500Da to obtain the carbon dot mother liquor.

[0052] Example 2

[0053] This example demonstrates the construction of a linear assay model for tinidazole:

[0054] (1) Preparation of sample solutions: carbon dot solution (concentration: 40 times diluted with stock solution), PBS buffer (pH=8), and tinidazole standard solutions with concentrations of 0 μM (blank control), 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 120 μM, 150 μM, 170 μM, 200 μM, 220 μM, 250 μM, 270 μM, and 300 μM;

[0055] (2) Mix 0.3 mL of carbon dot solution and 0.3 mL of tinidazole standard solution of different concentrations, then adjust the volume to 3 mL with PBS buffer, and then incubate at 20 °C for 1 h to obtain carbon dot solutions of different tinidazoles, and perform fluorescence spectroscopy detection.

[0056] (3) Determination of the fluorescence spectrum of the system: Scanning conditions: excitation wavelength 320 nm, emission wavelength scanning range 340–540 nm, scanning once every 1 nm, slit width set to 2 nm / 10 nm (excitation slit / emission slit), the obtained fluorescence emission spectrum ( Figure 2 );

[0057] (4) The fluorescence intensity peak at 390 nm obtained with 0 μM tinidazole was recorded as F0 (1029867.043 au), and the fluorescence intensity peak at 390 nm obtained with tinidazole concentrations of 1–300 μM was recorded as F. The fluorescence quenching degree F0 / F was recorded, and then a curve showing the relationship between the fluorescence quenching degree of the sample solution and the tinidazole concentration was plotted, as follows: Figure 3 As shown in the figure. The fitting curves of fluorescence quenching degree and tinidazole concentration when the tinidazole concentration is 1–100 μM are as follows. Figure 4 As shown, the fluorescence quenching degree of the solution is linearly related to the concentration of tinidazole, with the linear equation being F0 / F = 0.01073C + 1.0224 and the correlation coefficient being R. 2 =0.9902, detection limit is 0.362μM.

[0058] Example 3

[0059] This embodiment demonstrates the selectivity and anti-interference capability of N-doped carbon dots for tinidazole:

[0060] (1) Selectivity: Referring to Example 2, different types of antibiotics (tinidazole, metronidazole, ornidazole, secnidazole, erythromycin, sulfadiazine, thiamphenicol, streptomycin, ampicillin, and chloramphenicol), each with a concentration of 200 μM, were mixed with carbon dot solution (concentration: 40 times diluted with the stock solution), then diluted to volume with PBS buffer (pH=8), incubated, and then subjected to fluorescence spectroscopy detection. Figure 5 As shown, F and F0 represent the fluorescence intensity before and after the addition of antibiotics, respectively. All fluorescence detections were performed under the same conditions. The results indicate that the carbon dots exhibit relatively low fluorescence response to other types of antibiotics, showing specific selectivity only for nitroimidazole antibiotics such as tinidazole, metronidazole, ornidazole, and secnidazole.

[0061] (2) Anti-interference ability: Referring to Example 2, six common metal ions (Ca) found in milk were selected. 2+ Na + K + Cu 2 + Mg 2+ Mn 2+As interfering substances, all ion concentrations were 1 mM, and all fluorescence detections were performed under identical conditions. The response of carbon dots to tinidazole in the presence of these ions was measured, such as... Figure 6 As shown, F and F0 represent the fluorescence intensity of the system before and after the addition of tinidazole, respectively. The detection results indicate that these ions did not significantly interfere with the detection of tinidazole.

[0062] Example 4

[0063] This example demonstrates the detection of tinidazole in milk samples.

[0064] Milk samples were pretreated as follows: 8 mL of milk was added to 1.6 mL of methanol solution containing 1% acetic acid, vortexed for 2 min, and then centrifuged at 5000 rpm for 10 min. The supernatant was filtered through a 0.22 μM microporous membrane to obtain the milk pretreatment solution. The milk pretreatment solution was used as a solvent to prepare 40, 60, 80, and 100 μM tinidazole standard solutions. 0.3 mL of carbon dot solution (concentration: diluted 40 times with the stock solution) and 0.3 mL of milk pretreatment solution containing different concentrations of tinidazole were mixed, and then the volume was adjusted to 3 mL with PBS buffer (pH=8). The mixture was then incubated at 20 °C for 1 h to obtain tinidazole-spiked carbon dot solutions in the milk sample environment. Fluorescence spectroscopy was performed, and the fluorescence intensity peak at 390 nm was substituted into the linear relationship obtained in Example 2 for calculation. The detection results are shown in Table 1.

[0065] Table 1 Test results of Example 4

[0066]

[0067] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for detecting tinidazole, characterized in that, The method includes the following steps: Mix the carbon dot solution and the liquid to be tested, add PBS buffer and incubate for a period of time, then perform fluorescence spectroscopy detection. Then substitute the detected fluorescence intensity peak at 390 nm into the linear relationship for calculation to obtain the concentration of tinidazole. The carbon dot solution was prepared by diluting the N-doped carbon nanodot mother liquor by 35 to 45 times. The method for preparing the N-doped carbon nanodot mother liquor is as follows: L-lysine, ethylenediamine, and water are mixed to form a mixture, which is then heated and reacted. After the reaction is completed, the mixture is purified. The mass ratio of L-lysine to ethylenediamine is 3:1~1.5; the mass ratio of L-lysine to water is 3:5~15; the temperature of the heating reaction is 150~200℃; and the heating reaction time is 5~10h.

2. The method for detecting tinidazole according to claim 1, characterized in that, Purification includes one or more purification methods such as centrifugation, filtration, and dialysis.

3. The method for detecting tinidazole according to claim 1, characterized in that, The liquid to be tested includes water or beverages.

4. The method for detecting tinidazole according to claim 3, characterized in that, The water body includes river water, groundwater, lake water, or seawater; the beverage includes milk, carbonated drinks, fruit and vegetable juices, or coffee.

5. The method for detecting tinidazole according to claim 1, characterized in that, The mixing ratio of carbon dot solution and test liquid is 1:1~2; the pH of PBS buffer is 7.5~8.5; The mixing ratio of carbon dot solution and PBS buffer is 1:8~10.

6. The method for detecting tinidazole according to claim 1, characterized in that, The linear relationship is obtained as follows: (1) Preparation of sample solution: Take the liquid to be tested and add a system of tinidazole at different concentrations to prepare tinidazole standard solutions of different concentrations; the different concentrations include a blank control group with a concentration of 0. (2) The N-doped carbon nanodot mother liquor described in claim 1 is diluted 35 to 45 times to prepare a carbon dot solution and mixed with tinidazole standard solutions of different concentrations. Then, PBS buffer with pH=7.5 to 8.5 is added, and the mixture is incubated for 0.5 to 2 hours to obtain a spiked carbon dot solution. The spiked carbon dot solution is then subjected to fluorescence spectroscopy detection. The mixing ratio of carbon dot solution and tinidazole standard solution is 1:1~2; The mixing ratio of carbon dot solution and PBS buffer is 1:8~10; (3) Record the fluorescence intensity peak at 390 nm when the concentration of tinidazole is 0 as F0, and the fluorescence intensity peak at 390 nm when the concentration of tinidazole is not 0 as F. Calculate the fluorescence quenching degree F0 / F, and then draw the relationship curve between the fluorescence quenching degree of the sample solution and the concentration of tinidazole to obtain the linear relationship.

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