N-doped carbon nanodot and application thereof in tinidazole detection

Abstract

The invention discloses an N-doped carbon nanodot and application thereof in tinidazole detection, and belongs to the field of analysis and detection. The preparation method of the N-doped carbon nanodots comprises the following steps: mixing L-lysine, ethylenediamine and water to form a mixed solution, then carrying out a heating reaction, and purifying after the reaction is finished to obtain the N-doped carbon nanodots. According to the method, the carbon dots are prepared by taking L-lysine and ethidene diamine as precursors for the first time, quantitative detection of tinidazole is realized, and the method is low in cost, high in sensitivity and suitable for on-site rapid detection and has relatively high practical value and popularization prospect.

Description

Technical Field

[0001] The invention relates to N-doped carbon nanodots and application thereof in metronidazole detection, belonging to the field of analysis and detection. Background Art

[0002] With the rapid development of modern agriculture and animal husbandry, food safety issues have received more and more attention. Tinidazole (TNZ), a widely used nitroimidazole antibiotic, is often used to treat anaerobic infections in dairy cows. However, the use of TNZ may cause its residues in dairy cows, which then enter the human food chain through dairy products. This not only poses a threat to human health, but may also cause drug resistance problems. Therefore, the detection of TNZ residues has important practical significance for human health.

[0003] At present, the common methods for detecting metronidazole are mainly chromatography, mass spectrometry and immunoassay. Among them, although chromatography and mass spectrometry have high sensitivity and high accuracy, the equipment is expensive and the operation is complicated, which is not suitable for on-site detection, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), etc. Although immunoassay is relatively simple, it faces the problem of insufficient sensitivity, especially in the detection of low concentrations of the analyte.

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

[0005] Technical issues

[0006] Among the existing metronidazole residue detection technologies, chromatography and mass spectrometry have high accuracy, but have the problems of expensive equipment, complicated operation, and unsuitability for rapid on-site detection. Although immunoassay is relatively simple, it is not sensitive enough, especially in the detection of low-concentration metronidazole. Other carbon dot detection technologies also have the defect of a small linear range. Therefore, it is necessary to provide a simple, efficient, sensitive, large linear range and suitable for on-site detection metronidazole detection method.

[0007] Technical Solution

[0008] With the development of nanotechnology, the application of nanomaterials in the field of analytical chemistry has received extensive attention and research. Among them, carbon nanodots (CDs), as a new type of nanomaterial, have high photostability, good spectral characteristics and good biocompatibility, and have been widely used in the fields of sensors, imaging and biomarkers. Therefore, the present invention uses L-lysine and ethylenediamine as precursors to synthesize a N-doped carbon nanodot by hydrothermal method, and applies it to the field of tinidazole detection.

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

[0010] L-lysine, ethylenediamine and water are mixed to form a mixed solution, which is then heated for reaction. After the reaction is completed, N-doped carbon nanodots are purified.

[0011] Furthermore, 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.

[0012] Furthermore, the volume of the heating reaction container 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.; and the time of the heating reaction is 5-10 hours.

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

[0015] Furthermore, when centrifugation is used for purification, the centrifugation conditions are a rotation speed of 8000 to 15000 rpm and a time of 10 to 30 min.

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

[0017] Furthermore, when dialysis is used for purification, the dialysis condition is to use a dialysis membrane with a molecular weight cutoff of 3000 to 4000 Da for dialysis purification for 15 to 24 hours.

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

[0019] The application of the N-doped carbon nanodots for metronidazole detection provided by the present invention in the field of metronidazole detection.

[0020] Furthermore, the application is to detect metronidazole in water or beverages; the water includes river water, groundwater, lake water or sea water; the beverage includes milk, carbonated beverage or fruit and vegetable juice.

[0021] The present invention also provides a method for detecting metronidazole, which comprises the following steps:

[0022] The carbon dot solution and the liquid to be detected are mixed, PBS buffer is added and incubated for a period of time, and then the fluorescence spectrum is detected. Then, the peak value of the fluorescence intensity at 390 nm is substituted 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 solution by 35 to 45 times.

[0024] Furthermore, the liquid to be detected includes water or beverage.

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

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

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

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

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

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

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

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

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

[0034] (1) preparing a sample solution: taking a liquid to be tested, adding a system of different concentrations of metronidazole thereto, and preparing metronidazole standard solutions of different concentrations; the different concentrations include a blank control group with a concentration of 0;

[0035] (2) diluting the carbon dot mother solution by 35 to 45 times to obtain a carbon dot solution and mixing it with a standard solution of metronidazole of different concentrations, then adding a PBS buffer solution of pH=7.5 to 8.5, and then incubating for 0.5 to 2 hours to obtain a spiked carbon dot solution, and performing fluorescence spectrum detection on the spiked carbon dot solution;

[0036] (3) The peak fluorescence intensity at 390 nm obtained when no tinidazole was added was recorded as F 0 The peak fluorescence intensity at 390 nm obtained when tinidazole was added was recorded as F, and the degree of fluorescence quenching F was calculated. 0 / F, and then draw a curve of the relationship between the fluorescence quenching degree of the sample solution and the concentration of tinidazole to obtain a linear relationship.

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

[0038] Furthermore, when the substance to be detected is milk or water, the linear relationship in step (2) is: 0 / F=0.01073C+1.0224, where F 0 and F represent the peak fluorescence intensity of the system at 390 nm before and after the addition of tinidazole, respectively, and C represents the concentration of the added tinidazole.

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

[0040] Beneficial Effects

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

[0042] 2. In the present invention, the fluorescence excitation spectrum of the N-doped carbon nanodots highly overlaps with the ultraviolet-visible absorption spectrum of metronidazole, resulting in an inner filter effect that causes fluorescence quenching of the carbon dots, and has good selectivity for metronidazole. In addition, the N-doped carbon nanodots have strong anti-interference ability and can also detect metronidazole in a complex liquid environment such as milk, which is of great significance in the field of food testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1Schematic diagram of the detection of metronidazole in milk based on N-doped carbon nanodots.

[0044] Figure 2 This is the fluorescence spectrum when different concentrations of metronidazole are added to the system in Example 2.

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

[0046] Figure 4 It is a linear fitting curve of the fluorescence quenching degree in Example 2 and the concentration range of tinidazole in the range of 1 to 100 μM.

[0047] Figure 5 This is a graph showing the test results for detecting the selectivity of metronidazole in Example 3.

[0048] Figure 6 This is a test result diagram for detecting the anti-interference ability of metronidazole in Example 3. DETAILED DESCRIPTION

[0049] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0050] Example 1

[0051] Weigh 3 g of L-lysine and 1.5 mL of ethylenediamine in a beaker, add 10 mL of deionized water, and ultrasonicate for 10 min. After fully dissolving, transfer into a 25 mL polytetrafluoroethylene liner and react at 180 ° C for 8 h. Then, the reaction product was centrifuged at 10000 rpm for 30 min, filtered with a 0.22 μM microporous filter membrane, and then dialyzed and purified with a dialysis membrane with a molecular weight cutoff of 3500 Da for 24 h to obtain a carbon dot mother liquor.

[0052] Example 2

[0053] This embodiment is to construct a linear determination model for metronidazole:

[0054] (1) preparing sample solutions: carbon dot solution (concentration: 40-fold dilution of the mother 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, respectively;

[0055] (2) 0.3 mL of the carbon dot solution and 0.3 mL of different concentrations of metronidazole standard solution were mixed, and then the volume was adjusted to 3 mL with PBS buffer, and then incubated at 20°C for 1 h to obtain different metronidazole spiked carbon dot solutions, and fluorescence spectrum detection was performed;

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

[0057] (4) The peak fluorescence intensity at 390 nm obtained by adding tinidazole at a concentration of 0 μM is recorded as F 0 (1029867.043au), the peak fluorescence intensity at 390nm obtained by adding tinidazole at a concentration of 1 to 300 μM is recorded as F, and the degree of fluorescence quenching F is recorded 0 / F, and then draw a curve of the relationship between the fluorescence quenching degree of the sample solution and the concentration of tinidazole, such as Figure 3 When the concentration of metronidazole is 1-100 μM, the fitting curve of fluorescence quenching degree and metronidazole concentration is as shown in Figure 4 As shown, it can be seen that the degree of fluorescence quenching of the solution is linearly related to the concentration of metronidazole, and the linear equation is F 0 / F=0.01073C+1.0224, the correlation coefficient is R 2 =0.9902, and the detection limit is 0.362μM.

[0058] Example 3

[0059] This example shows the selectivity and anti-interference ability of N-doped carbon dots to metronidazole:

[0060] (1) Selectivity: Referring to Example 2, different types of antibiotics (tinidazole, metronidazole, ornidazole, secnidazole, erythromycin, sulfadiazine, thiamphenicol, streptomycin, ampicillin and chloramphenicol) with a concentration of 200 μM were mixed with the carbon dot solution (concentration: 40 times dilution of the mother solution), and then fixed to volume with PBS buffer (pH = 8), and fluorescence spectrum detection was performed after static incubation. Figure 5 As shown, F and F 0 Respectively represent the fluorescence intensity before and after the addition of antibiotics, all fluorescence detections are carried out under the same conditions. According to the test results, the fluorescence response intensity of carbon dots to other types of antibiotics is relatively low, and it only has specific selectivity for nitroimidazole antibiotics such as metronidazole, metronidazole, ornidazole, and secnidazole.

[0061] (2) Anti-interference ability: Referring to Example 2, six common metal ions in milk (Ca 2+ 、Na + , K + , Cu 2 + Mg 2+ , Mn 2+ ) as interfering substances, all ion concentrations were 1 mM, and all fluorescence measurements were performed under the same conditions. The responses of carbon dots to tinidazole in the presence of these ions were measured, such as Figure 6 As shown, F and F 0 They represent the fluorescence intensity of the system before and after the addition of metronidazole. According to the test results, these ions did not cause significant interference to the detection of metronidazole.

[0062] Example 4

[0063] This example is for detecting tinidazole in a milk sample environment:

[0064] The milk sample was pretreated: 8 mL of milk was taken, 1.6 mL of methanol solution containing 1% acetic acid was added, vortexed for 2 min, and then centrifuged at 5000 rpm for 10 min, and the supernatant was filtered using a 0.22 μM microporous filter membrane to obtain a milk pretreatment solution. The milk pretreatment solution was used as a solvent to prepare 40, 60, 80, and 100 μM metronidazole standard solutions. 0.3 mL of carbon dot solution (concentration: mother liquor diluted 40 times) and 0.3 mL of milk pretreatment solution containing different concentrations of metronidazole were mixed, and then the volume was fixed to 3 mL with PBS buffer (PH=8), and then incubated at 20°C for 1 h to obtain different metronidazole-spiked carbon dot solutions in the milk sample environment, and fluorescence spectrum detection was performed, and then the peak fluorescence intensity at 390 nm was substituted into the linear relationship obtained in Example 2 for calculation, and the obtained 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 present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing N-doped carbon nanodots for tinidazole detection, characterized in that: The method is: L-lysine, ethylenediamine and water are mixed to form a mixed solution, which is then heated for reaction. After the reaction is completed, N-doped carbon nanodots are purified.

2. The preparation method according to claim 1, characterized in that: 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.

3. The preparation method according to claim 1, characterized in that: The temperature of the heating reaction is 150-200°C; the time of the heating reaction is 5-10 hours.

4. The preparation method according to claim 1, characterized in that: Purification includes one or more purification methods including centrifugation, filtration, and dialysis.

5. An N-doped carbon nanodot for the detection of metronidazole, characterized in that: The N-doped carbon nanodots are prepared according to the preparation method according to any one of claims 1 to 4; the N-doped carbon nanodots exist in the form of N-doped carbon nanodot mother liquor.

6. A method for detecting metronidazole, characterized in that: The method comprises the following steps: The carbon dot solution and the liquid to be tested were mixed, PBS buffer was added and incubated for a period of time, and then the fluorescence spectrum was detected. Then, the peak value of the fluorescence intensity at 390 nm was substituted into the linear relationship for calculation to obtain the concentration of tinidazole. The carbon dot solution is prepared by diluting the N-doped carbon nanodot mother solution described in claim 5 by 35 to 45 times.

7. The method for detecting metronidazole according to claim 6, characterized in that: The liquid to be detected includes water or beverages.

8. The method for detecting metronidazole according to claim 7, characterized in that: The water body includes river water, groundwater, lake water or sea water; the beverage includes milk, carbonated beverage, fruit and vegetable juice or coffee.

9. The method for detecting metronidazole according to claim 6, characterized in that: The mixing ratio of the carbon dot solution and the liquid to be detected is 1:1-2; the pH of the PBS buffer is 7.5-8.5; and the mixing ratio of the carbon dot solution and the PBS buffer is 1:8-10.

10. The method for detecting metronidazole according to claim 6, characterized in that: The linear relationship is obtained as follows: (1) preparing a sample solution: taking a liquid to be tested, adding a system of different concentrations of metronidazole thereto, and preparing metronidazole standard solutions of different concentrations; the different concentrations include a blank control group with a concentration of 0; (2) diluting the N-doped carbon nanodot mother solution described in claim 5 by 35 to 45 times to obtain a carbon dot solution and mixing it with a standard solution of metronidazole of different concentrations, then adding a PBS buffer solution of pH = 7.5 to 8.5, and then incubating for 0.5 to 2 hours to obtain a spiked carbon dot solution, and performing fluorescence spectrum detection on the spiked carbon dot solution; The mixing ratio of the carbon dot solution and the metronidazole standard solution is 1:1-2; The mixing ratio of carbon dot solution and PBS buffer was 1:8-10; (3) The peak fluorescence intensity at 390 nm obtained when the concentration of metronidazole is 0 is recorded as F0, and the peak fluorescence intensity at 390 nm obtained when the concentration of metronidazole is not 0 is recorded as F. The fluorescence quenching degree F0 / F is calculated, and then a curve of the relationship between the fluorescence quenching degree of the sample solution and the metronidazole concentration is plotted to obtain a linear relationship.

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