Method for detecting nitrite content based on nitrogen-doped carbon quantum dot fluorescent probe

Abstract

The invention discloses a method for detecting nitrite content based on a nitrogen-doped carbon quantum dot (N-CDs) fluorescent probe, which comprises the following steps: adding 2, 3-diaminopyridine and ultrapure water into polytetrafluoroethylene, uniformly mixing, sealing in a reaction kettle, reacting at 180 DEG C for 8 hours, cooling at room temperature, centrifuging in a centrifugal machine, and separating non-fluorescent sediments to obtain light brown supernate; separating insoluble solid particles from the supernate by using a microporous filter membrane, dialyzing the filtrate by using a dialysis bag, replacing a dialysis medium once every 6 hours, drying to obtain the nitrogen-doped carbon quantum dots, and determining the nitrite concentration in the sample solution containing nitrite to be detected by using a standard curve drawing method based on the nitrogen-doped carbon quantum dots. The N-CDs prepared by the invention shows a good application prospect in the aspect of rapid, efficient, high-selectivity and high-sensitivity detection of nitrite.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence detection of nitrite, and particularly relates to a method for detecting the content of nitrite based on a fluorescence probe of nitrogen-doped carbon quantum dots. Background Art

[0002] Nitrite is an inorganic nitrogen compound widely existing in nature, and it mainly comes from the oxidation of ammonia and the reduction of nitrate. In daily life, nitrite can be used as a food additive and also as a preservative to enhance the flavor and color of food. However, excessive nitrite will seriously pollute the environment and pose a serious threat to human health. When the intake of nitrite reaches 0.3 - 0.5 g, it can cause poisoning, and the intake of 3 g can even be fatal. When excessive nitrite is absorbed by the human body, hemoglobin in the blood will be oxidized and lose its oxygen-carrying capacity, leading to ischemic anemia, dyspnea, and even death. In addition, whether in vivo or in vitro, under acidic conditions, nitrite can react with secondary amines to produce highly carcinogenic nitrosamines. Nitrosamines have strong teratogenic and mutagenic effects and can enter the fetus through the placental barrier, causing fetal malformations, premature birth, growth retardation, developmental defects, etc. Therefore, nitrite, as a precursor of nitrosamines, has received extensive attention, and strict standards have been set for its usage in food processing in various countries. China has formulated national health standards for nitrite and nitrate in some foods and domestic drinking water. The Hygienic Standard for the Use of Food Additives in China stipulates that the content of nitrite in meat products shall not exceed 0.15 g / kg. At present, there is no good method at home and abroad for reducing the content of nitrite in food, so it is of great significance to develop a simple, sensitive, and selective method for detecting the content of nitrite in food.

[0003] So far, spectrophotometry is the most widely adopted technique for the determination of nitrite. It is widely used due to its low cost and simple preparation. However, this technique has poor sensitivity and is prone to interference from other analytes. Recently, a variety of analytical techniques have been developed, such as capillary electrophoresis, chemiluminescence, chromatography, electrochemical methods, microfluidic devices, surface-enhanced Raman spectroscopy, etc., for sensitive nitrite detection. However, these techniques still have some drawbacks: low sensitivity, poor selectivity, long analysis time, and complex and costly instrumentation. Compared with traditional analytical techniques, fluorescence method, as an indispensable tool among various analytical detectors, has received increasing attention in recent years due to its high sensitivity, good selectivity, low cost, simple operation, etc. Among them, nitrogen-doped carbon quantum dots are a new type of carbon-based fluorescent material with many excellent properties, such as excellent optical properties, good water solubility, low toxicity, environmental friendliness, wide raw material sources, low cost, and good biocompatibility. The surface functional groups (amines, carboxyl groups, hydroxyl groups, etc.) of nitrogen-doped carbon quantum dots can mediate chemical reactions to achieve the detection purpose, and have great application potential in fluorescence analysis.

[0004] With the improvement of the quality of life of the people in our country, the issue of food safety has increasingly attracted people's high attention. The guarantee of food safety depends on reliable quality monitoring. Therefore, there is an urgent need to develop a detection technique with simple preparation and easy operation to detect nitrite in food. Therefore, the preparation of a fluorescent probe with excellent performance has become the mainstream technology for the rapid on-site detection of nitrite in food, and is the preferred method for the rapid detection of nitrite in food in the current and future periods, providing a strong technical guarantee for the self-monitoring of food manufacturers and market supervision. Summary of the Invention

[0005] The present invention provides a method for detecting the content of nitrite based on a nitrogen-doped carbon quantum dot fluorescent probe, which is fast, efficient, highly selective and highly sensitive, to overcome the deficiencies in the performance and structure of the tools for detecting nitrite in food in the prior art.

[0006] The method for detecting the content of nitrite based on a nitrogen-doped carbon quantum dot fluorescent probe adopted by the present invention to solve the above technical problems is characterized in that the specific steps are as follows:

[0007] Step S1: Disperse nitrogen-doped carbon quantum dots in deionized water, and adjust the pH of the mixed system to 3 with hydrochloric acid to obtain a nitrogen-doped carbon quantum dot dispersion with a concentration of 0.65 mg / mL. Take 0.5 mL of the nitrogen-doped carbon quantum dot dispersion and add standard nitrite solutions with known concentrations respectively, and make up the volume to 5 mL. After mixing evenly, react at 25 °C for 20 min. Measure the fluorescence intensity of the mixed system at an excitation wavelength of 530 nm or 565 nm and an emission wavelength of 590 nm. When the nitrite concentration is in the range of 0.2 - 0.5 μM, there is a good linear relationship between the fluorescence quenching efficiency F / F0, and the linear equation is F / F0 = -2.43×10 -3 X + 1.33226, and the correlation coefficient R 2 = 0.9909, where F0 and F are the fluorescence intensities of the mixed system before and after adding the standard nitrite solution respectively, and X is the concentration of nitrite;

[0008] Step S2: Take 0.5 mL of the nitrogen-doped carbon quantum dot dispersion obtained in Step S1 and add the sample solution containing nitrite to be measured, make up the volume to 5 mL. After mixing evenly, react at 25 °C for 20 min. Measure the fluorescence intensity of the mixed system at an excitation wavelength of 530 nm or 565 nm and an emission wavelength of 590 nm. Then, based on the measured fluorescence intensities of the mixed system before and after adding the sample solution containing nitrite to be measured, and combined with the linear equation obtained in Step S1, calculate the nitrite concentration in the sample solution containing nitrite to be measured;

[0009] The specific preparation process of the nitrogen-doped carbon quantum dots is as follows: Add 2,3-diaminopyridine and ultrapure water into polytetrafluoroethylene, mix evenly and seal in a reaction kettle, react at 180 °C for 8 h, cool to room temperature and then centrifuge in a centrifuge to separate non-fluorescent sediments, obtain a light brown supernatant, separate insoluble solid particles from the supernatant with a microporous filter membrane, dialyze the filtrate with a dialysis bag, change the dialysis medium every 6 h, and dry to obtain nitrogen-doped carbon quantum dots, namely N-CDs.

[0010] Further defined, the nitrogen-doped carbon quantum dots emit 590 nm fluorescence under the excitation of light at 530 nm or 565 nm, and the fluorescence of the nitrogen-doped carbon quantum dots can be specifically quenched by nitrite.

[0011] Further defined, the nitrogen-doped carbon quantum dots can be used for selective detection of nitrite in a system with interference from common amino acids or / and common ions, where the common amino acids are one or more of Lys, Glu, Ser, Val, Ile, Leu, Asp, Cys-Cys, Cys, Met, Tyr, Hey or GSH, and the common ions are Fe 3+ 、Cu 2+ 、K + 、Al3+ , Zn 2+ , Fe 2+ , Mg 2+ , Ca 2+ , Mn 2+ , Na + , Co 2+ , Ba 2+ , I - , Br - , Cl - , SO4 2- , NO3 - , CO3 2- , HCO3 - or CH3COO - or more than one of them.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects: The N-CDs prepared by the present invention emit 590 nm fluorescence when excited by light at 530 nm or 565 nm, and the fluorescence of the N-CDs can be specifically quenched by nitrite. The N-CDs prepared by the present invention show good application prospects in the rapid, efficient, highly selective and highly sensitive detection of nitrite. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 are the transmission electron microscope (a) and particle size distribution diagram (b) of N-CDs.

[0014] Figure 2 is the stability of N-CDs.

[0015] Figure 3 is the influence of pH on the fluorescence performance of N-CDs.

[0016] Figure 4 is the influence of ionic strength on the fluorescence performance of N-CDs.

[0017] Figure 5 are the excitation and emission spectra of N-CDs.

[0018] Figure 6 are the ultraviolet-visible absorption spectra of N-CDs and 2,3-diaminopyridine.

[0019] Figure 7 is the infrared spectrum of N-CDs.

[0020] Figure 8 is the X-ray photoelectron spectrum of N-CDs.

[0021] Figure 9 is the nuclear magnetic resonance hydrogen spectrum of N-CDs.

[0022] Figure 10 The linear equation for the determination of nitrite by N-CDs.

[0023] Figure 11 The influence of common amino acid interference systems on the selectivity and anti-interference performance of the determination method.

[0024] Figure 12 The influence of common ion interference systems on the selectivity and anti-interference performance of the determination method. Specific implementation manners

[0025] The above content of the present invention will be further described in detail below through examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. Any technology implemented based on the above content of the present invention belongs to the scope of the present invention.

[0026] Synthesis of nitrogen-doped carbon quantum dots

[0027] In polytetrafluoroethylene, 306.0 mg of 2,3-diaminopyridine and 3 mL of ultrapure water were added to prepare a suspension. After mixing evenly, it was sealed in a reaction kettle and reacted at 180 °C for 8 h. After cooling to room temperature, it was centrifuged at a rate of 12,000 revolutions per minute in a centrifuge for 20 min to separate non-fluorescent deposits, and a light brown supernatant was obtained. The supernatant was filtered through a 0.22 μm microporous filter membrane to separate insoluble solid particles. The filtrate was dialyzed with a 500 Da dialysis bag for 24 h, and the dialysis medium was changed every 6 h. After drying the dialysis solution, nitrogen-doped carbon quantum dots, namely N-CDs, were obtained.

[0028] Fluorescence properties and characterization of nitrogen-doped carbon quantum dots

[0029] Figure 1 It is the transmission electron microscope characterization and particle size distribution diagram of N-CDs. Figure 1 In a, it is the transmission electron microscope image of N-CDs. It can be seen from the figure that N-CDs are quasi-spherical. Figure 1 In b, it is the particle size distribution diagram of N-CDs. It can be seen from the figure that the particle size of N-CDs is mainly distributed in the range of 2.3 - 3.7 nm, and the average particle size is 2.94 nm.

[0030] Figure 2 It is the influence of the storage time of N-CDs on their fluorescence properties. After being stored at 25 °C indoors for 7 days, the fluorescence intensity of N-CDs still reaches 90.8% of its initial fluorescence intensity, which proves that N-CDs have good stability.

[0031] Figure 3Effect of pH on its fluorescence intensity. In the pH range of 3 - 9 investigated, the fluorescence properties of N-CDs did not change significantly. Under the condition of pH = 10, the fluorescence intensity of N-CDs still reached 84.1% of its initial fluorescence intensity, which proved that N-CDs had good stability.

[0032] Figure 4 Effect of ionic strength on the fluorescence properties of N-CDs. When the sodium chloride concentration increased to 800 mM, the fluorescence properties of N-CDs were still not significantly affected.

[0033] Figure 5 Excitation and emission spectra of N-CDs. N-CDs had two obvious excitation peaks, located at 530 nm and 565 nm respectively, and the emission peaks at these two excitation wavelengths were both at 590 nm.

[0034] Figure 6 UV-visible absorption spectra of N-CDs and 2,3-diaminopyridine. The absorption peaks of n→σ* transition of N-CDs at 241 nm and n→π* at 308 nm showed obvious red shifts compared with the absorption peaks of n→σ* transition of 2,3-diaminopyridine at 237 nm and n→π* at 301 nm. An absorption peak that 2,3-diaminopyridine did not have appeared at 413 nm, which was one of the evidences for the successful synthesis of N-CDs.

[0035] Figure 7 Infrared spectrum of N-CDs. The absorption peaks of N-CDs at 3356 cm -1 and 3329 cm -1 were caused by the asymmetric and symmetric stretching vibrations of N-H. The absorptions at 1598 cm -1 and 752 cm -1 were caused by the in-plane and out-of-plane bending vibrations of N-H. The peak at 1250 cm -1 was the absorption peak of C-N. The absorption peak at 3180 cm -1 was caused by the stretching vibration of the -OH bond; the absorption peak at 1761 cm -1 appeared due to the vibration absorption peak of the -C=O bond, and the -OH and -C=O groups were not present in the 2,3-diaminopyridine molecule. This proved that N-CDs not only retained part of the amino groups in 2,3-diaminopyridine, but also introduced -OH and -C=O groups during the reaction, indicating the successful synthesis of N-CDs.

[0036] Figure 8 X-ray photoelectron spectrum of N-CDs. Figure 8 In a, it shows that N-CDs mainly contain carbon, nitrogen, oxygen and hydrogen elements; Figure 8In b, it indicates that N-CDs contain C-C, C-N, C=N, C=O and C-O bonds; Figure 8 In c, it indicates that N-CDs contain C-C, N-H, C=N bonds; Figure 8 In d, it indicates that N-CDs contain C=O and -OH bonds; these results are consistent with those of infrared.

[0037] Figure 9 It is the 1H NMR spectrum of N-CDs. The peak with a chemical shift of 6.18 is the active hydrogen of the amino group of N-CDs, and the hydrogens with chemical shifts of 7.26, 6.82 and 6.51 are aromatic hydrogens, indicating the presence of aromatic hydrogen (Ar-H) and aromatic amine (Ar-NH2) on the surface of N-CDs. The results are consistent with those of infrared and X-ray photoelectron spectroscopy, indicating the successful synthesis of N-CDs.

[0038] Highly selective and sensitive determination of nitrite content

[0039] Add N-CDs to a container to prepare an aqueous dispersion with a certain concentration and adjust the pH of the mixed system to 3 with hydrochloric acid to prepare an N-CDs dispersion with a concentration of 0.65 mg / mL. Add 0.5 mL of the 0.65 mg / mL N-CDs dispersion to the reaction container, then add the sample solution containing nitrite to be measured, make up the volume to 5 mL, mix well, react at 25 °C for 20 min, and measure the fluorescence intensity of the mixed system at an excitation wavelength of 530 nm or 565 nm and an emission wavelength of 590 nm. There is a good linear relationship between the nitrite concentration in the range of 0.2 - 0.5 μM and the fluorescence quenching efficiency F / F0. Repeat the determination of 0.3 μM nitrite 11 times, and calculate the detection limit to be 1.6 nM according to the relative standard deviation.

[0040] Figure 10 It is the linear equation for the determination of nitrite by N-CDs. The linear equation between the quenching efficiency of N-CDs and the concentration of nitrite is F / F0 = -2.43×10 -3 X + 1.33226, the correlation coefficient R 2 = 0.9909, where F0 and F are the fluorescence intensities of the mixed system before and after adding the nitrite standard solution respectively, X is the concentration of nitrite, with the unit of μM, and the concentrations X of the nitrite standard solution selected for drawing the standard curve are 0.20 μM, 0.25 μM, 0.30 μM, 0.35 μM, 0.40 μM, 0.45 μM, 0.50 μM in turn.

[0041] Prepare N-CDs dispersions containing 50 μM different amino acids using a 6.5 mg / mL N-CDs (pH = 3) dispersion and ordinary amino acid solutions. After mixing well, measure the fluorescence intensity of N-CDs with a fluorometer. The results are shown in Figure 11。It has been experimentally proven that common amino acids do not interfere with the determination of nitrite in the system, thus confirming that the N-CDs synthesized in the present invention have high selectivity for nitrite.

[0042] Prepare N-CDs dispersions containing 50 μM of different ions using a 6.5 mg / mL N-CDs (pH = 3) solution and common ion solutions. After mixing evenly, measure their fluorescence spectra with a fluorometer. The results are shown in Figure 12 。It has been experimentally proven that common ions do not interfere with the determination of nitrite in the system, thus confirming that the N-CDs synthesized in the present invention have high selectivity for nitrite.

[0043] Example 1

[0044] Application of the determination method in the determination of nitrite in water samples

[0045] Table 1 Detection of nitrite in actual water samples by N-CDs fluorescence probe (n = 3)

[0046]

[0047] The collected water samples include tap water, river water, lake water, aquarium water and aquaculture water. The water samples are centrifuged, filtered and concentrated. The nitrite contents detected by the N-CDs-based fluorescence analysis method constructed in the present invention all meet the national standards. The results are shown in Table 1, and the relative standard deviation (RSD) of the measurement results is 0.39% - 1.62%. This indicates that the N-CDs fluorescence probe synthesized in this detection method is feasible and reliable for the detection of nitrite in actual water samples.

[0048] Example 2

[0049] Application of the determination method in the determination of nitrite in actual meat samples

[0050] The collected meat samples include cooked beef, cooked chicken, chicken ham and starch-free ham. First, grind the meat samples into meat paste. Take 3.0 g and place it in a beaker, add 6.3 mL of saturated borax solution, stir evenly, then add 10 mL of hot water at 70 °C, heat in a boiling water bath for 15 min, add 5 mL of potassium ferrocyanide solution while stirring, stir evenly, then add 5 mL of zinc acetate solution to precipitate proteins, let it stand for 30 min, remove the upper layer of fat, filter, remove the precipitate, centrifuge the filtrate at 12,000 revolutions per minute for 20 min, and then filter with a 0.22 μm filter membrane. The obtained filtrate is for detection. The preparation of potassium ferrocyanide solution: Dissolve 10.6 g of potassium ferrocyanide in 100 mL of water to obtain it; The preparation of zinc acetate solution: Dissolve 22 g of zinc acetate and 3.0 mL of glacial acetic acid in 100 mL of water to obtain it.

[0051] The filtrate obtained above was centrifuged, filtered and diluted. The nitrite contents detected by the fluorescence analysis method based on N-CDs constructed in the present invention all met the national standards. The results are shown in Table 2, and the relative standard deviation (RSD) of the measurement results was 0.43% - 1.06%. This indicates that the N-CDs fluorescence probe synthesized in this detection method is feasible and reliable for the detection of nitrite in actual meat samples.

[0052] Table 2 Detection of nitrite in actual meat samples by N-CDs fluorescence probe (n = 3)

[0053]

[0054] In summary, a kind of nitrogen-doped carbon quantum dots synthesized in the present invention can accurately quantitatively detect nitrite in water and meat products. And this fluorescence probe has high selectivity and high sensitivity, and the synthesis method is simple, which is suitable for market promotion and application. For the method disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the description in the method part.

[0055] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for detecting the content of nitrite based on a nitrogen-doped carbon quantum dot fluorescence probe, characterized in that The specific steps are as follows: Step S1: Disperse nitrogen-doped carbon quantum dots in deionized water, and adjust the pH of the mixed system to 3 with hydrochloric acid to obtain a nitrogen-doped carbon quantum dot dispersion with a concentration of 0.65 mg / mL. Take 0.5 mL of the nitrogen-doped carbon quantum dot dispersion and add standard nitrite solutions with known concentrations respectively, and make up the volume to 5 mL. After mixing evenly, react at 25 °C for 20 min. Measure the fluorescence intensity of the mixed system at an excitation wavelength of 530 nm or 565 nm and an emission wavelength of 590 nm. When the nitrite concentration is in the range of 0.2 - 0.5 μM, there is a good linear relationship between the fluorescence quenching efficiency F / F0, and the linear equation is F / F0 = –2.43×10 –3 X + 1.33226, and the correlation coefficient R 2 = 0.9909, where F0 and F are the fluorescence intensities of the mixed system before and after adding the standard nitrite solution respectively, and X is the concentration of the standard nitrite solution; Step S2: Take 0.5 mL of the nitrogen-doped carbon quantum dot dispersion obtained in Step S1, add the sample solution containing nitrite to be measured, make up the volume to 5 mL, mix evenly, react at 25 °C for 20 min, measure the fluorescence intensity of the mixed system at the excitation wavelength of 530 nm or 565 nm and the emission wavelength of 590 nm, and then calculate the nitrite concentration in the sample solution containing nitrite to be measured according to the fluorescence intensities of the mixed system before and after adding the sample solution containing nitrite to be measured and combined with the linear equation obtained in Step S1; The specific preparation process of the nitrogen-doped carbon quantum dots is as follows: Add 2,3-diaminopyridine and ultrapure water into polytetrafluoroethylene, mix evenly, seal in a reaction kettle, react at 180 °C for 8 h, cool to room temperature, centrifuge in a centrifuge, separate the non-fluorescent sediment, obtain a light brown supernatant, separate the insoluble solid particles from the supernatant with a microporous filter membrane, dialyze the filtrate with a dialysis bag, change the dialysis medium every 6 h, and dry to obtain the nitrogen-doped carbon quantum dots, namely N-CDs.

2. The method for detecting nitrite content based on a nitrogen-doped carbon quantum dot fluorescence probe according to claim 1, wherein: The nitrogen-doped carbon quantum dots emit 590 nm fluorescence under the excitation of light at 530 nm or 565 nm, and the fluorescence of the nitrogen-doped carbon quantum dots can be specifically quenched by nitrite.

3. The method for detecting nitrite content based on a nitrogen-doped carbon quantum dot fluorescence probe according to claim 1, wherein: The nitrogen-doped carbon quantum dots can be used for the selective detection of nitrite in a system containing common amino acids or / and common ion interferences, where the common amino acids are one or more of Lys, Glu, Ser, Val, Ile, Leu, Asp, Cys-Cys, Cys, Met, Tyr, Hey or GSH, and the common ions are Fe 3+ , Cu 2+ , K + , Al 3+ , Zn 2+ , Fe 2+ , Mg 2+ , Ca 2 + , Mn 2+ , Na + , Co 2+ , Ba 2+ , NO 2- , I - , Br - , Cl - , SO4 2- , NO3 - , CO3 2- , HCO3 - or CH3COO - one or more of.

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