Fluorescence detection method and detection kit for nitrate

Abstract

The present invention discloses a fluorescence detection method for nitrate. The present invention uses zinc to first reduce nitrate to nitrite, and then uses a method for detecting nitrite to indirectly detect nitrate. According to the Griess reaction principle, nitrite forms a diazotization process and develops color, thereby constructing a nitrate detection kit. The present invention can detect in real time, quickly and visually, and the material preparation process is simple and inexpensive; the detection process is also convenient and fast. The detection limit of the visualization kit for nitrate detection reaches 0.1ppm, which is lower than the national standard for nitrate detection in water quality or soil. It is convenient for different farmers to detect the nitrate content in the soil, and then determine the level of fertilizer efficiency in water and soil, and take corresponding measures to deal with water and soil.

Description

Technical Field

[0001] The invention relates to a fluorescence detection method and a detection kit for nitrate, and is used for detecting nitrate in an environment such as water and soil. Background Art

[0002] High concentrations of nitrate ions are a ubiquitous pollutant. Excessive nitrate from overuse of nitrogen fertilizers in soils leads to excessive algal growth and hypoxia in aquatic species. This nitrate ion can harm human health through the food chain, causing hemoglobin to convert to methemoglobin, leading to hypoxia in humans. It can also cause hemoglobinemia and blue baby syndrome in aquatic animals. In 2002, the Food and Agriculture Organization of the United Nations and the World Health Organization established an acceptable daily intake of nitrate as 3.7 mg per kilogram of body weight. The U.S. Environmental Protection Agency sets maximum contamination levels for nitrite and nitrate ions in drinking water at 71.4 μM and 714 μM, respectively. my country's Ministry of Environmental Protection limits the concentration of nitrate nitrogen in surface water sources to ≤10 mg / L and stipulates that the concentration of nitrate nitrogen in groundwater from centralized drinking water sources must be ≤20 mg / L.

[0003] Currently, a variety of methods for nitrate detection have been developed, including chromatography, electrochemistry, capillary electrophoresis, chemiluminescence, and colorimetry. Fluorescence determination of nitrate is widely used. Most methods rely on reducing nitrate to nitrite using various reducing agents, such as hydrazine hydrate, V(III) or enzyme systems, Cu-Cd reducing agent columns, Zn, and Zn-Cd reducing agents [1-5]. The Griess reaction, which acidifies nitrite to diazotize aromatic amines, determines the nitrite content, and thus the nitrate content. Most of these nitrate detection methods rely on flow analysis to reduce nitrate to nitrite, and then measure nitrate based on the color change of the Griess reaction. This has played a role in nitrate detection and analysis. However, the reduction process is complex and time-consuming, and the analysis requires sophisticated instrumentation. These methods limit the use of nitrate sensors for on-site, real-time analysis. Colorimetry is also commonly used for nitrite analysis. Nitrite reacts with sulfonamide to form a diazotization process, and the color change of the red azo dye is used to visualize the detection of nitrite [6]. However, the color formation process takes a long time, and it is not well suited for scenarios where timely and on-site analysis of nitrate is required. Summary of the Invention

[0004] In response to the deficiencies in the prior art, the present invention provides a fluorescence detection method and a fluorescence detection kit for rapid nitrate detection, which overcome the problem that traditional nitrate detection takes a long time and is not suitable for rapid on-site detection. The fluorescent material in the fluorescence detection kit dissolves in the aqueous phase, and the fluorescence intensity gradually decreases during the detection process, making it suitable for rapid detection of nitrate in water and soil. The technical problem to be solved by the present invention is to achieve rapid, on-site detection of nitrate in an environment, such as water or soil.

[0005] The present invention combines the response of naphthylethylenediamine hydrochloride to nitrite with the fluorescent characteristic of citric acid carbon dots, which emit a bright blue light under ultraviolet light, to prepare fluorescent carbon dots. Nitrite is detected by fluorescence quenching. Combined with the nitrite ions generated by the reduction of nitrate, fluorescent detection of nitrate is achieved.

[0006] The fluorescent detection method for nitrate ions of the present invention uses a hydrothermal method to synthesize fluorescent carbon dots using naphthylethylenediamine hydrochloride and citric acid. A nitrate-containing test solution is reduced with a reducing agent to obtain a nitrite-containing solution. The fluorescent carbon dots and the nitrite-containing solution are mixed under ultraviolet light, and the color change is observed. This method can achieve qualitative detection of nitrate ions.

[0007] In the detection method, preferably, the hydrothermal synthesis of fluorescent carbon dots involves continuous heating in a reactor at 170-190 degrees Celsius for 4-6 hours to form a fluorescent carbon dot solution. Phosphate is added during the synthesis of the fluorescent carbon dots, with a molar ratio of 1:1.8-2.2:0.4-0.8 for naphthalene ethylenediamine hydrochloride, citric acid, and phosphate, preferably 1:2:0.5. The reducing agent is zinc powder. Studies have shown that using only naphthalene ethylenediamine hydrochloride and citric acid is also acceptable, but the detection limit is higher and the sensitivity is lower. Too high a temperature for the hydrothermal synthesis of fluorescent carbon dots will result in carbonization, while too low a temperature will result in incomplete reaction. A heating time of 3 hours results in incomplete reaction, but this is too long and wastes energy. The phosphate can be a soluble salt, such as sodium phosphate or potassium phosphate.

[0008] In order to achieve quantitative detection of nitrate, the present invention adopts a fluorescence detection method for nitrate, which comprises the following steps: (1) synthesizing a fluorescent carbon dot solution by hydrothermal method by doping naphthylethylenediamine hydrochloride, citric acid and phosphate at 180 degrees Celsius and continuously heating for 5 hours in a reaction kettle; the molar ratio of naphthylethylenediamine hydrochloride, citric acid and phosphate is 1:2:0.5;

[0009] (2) using zinc powder to catalyze the reduction of a standard solution containing nitrate to obtain a solution containing nitrite, and then mixing the solution with the fluorescent carbon dot solution in step (1) under ultraviolet light to develop color;

[0010] (3) Repeating steps (1) and (2) multiple times to establish a linear relationship between the concentration of the nitrate standard solution and the fluorescence response intensity, obtain a standard curve, and prepare a standard colorimetric card;

[0011] (4) Taking the test solution containing nitrate, catalytically reducing the test solution with zinc powder to obtain a test solution containing nitrite, and then mixing it with the fluorescent carbon dot solution in step (1) under ultraviolet light, and comparing the color development result with the standard colorimetric card.

[0012] In the above-mentioned detection method, preferably, the fluorescent carbon point contact is based on the principle of diazotization reaction, and different fluorescent carbon dots are quenched by reduced nitrite. In step (2), an acidic solution with a pH of 1.6-2.0, preferably a pH of 1.8, is added. In the acidic solution, zinc powder can better catalyze the reaction of nitrate to obtain nitrite. The acidic solution is a mixed solution containing 10-15wt% ammonium chloride and 8-12wt% potassium chloride and / or sodium chloride, and the pH is adjusted to 1.6-2.0 with hydrochloric acid, preferably a mixed solution containing 13wt% ammonium chloride and 10wt% potassium chloride and / or sodium chloride, and the pH is adjusted to 1.8 with hydrochloric acid.

[0013] Based on the above detection method, a fluorescent kit for detecting nitrate can be obtained, which includes a fluorescent carbon dot solution, zinc powder, and a standard colorimetric card. The kit sensor designed by the present invention is based on fluorescence analysis, which combines the characteristics of flow analysis reduction and fluorescence instrumentation. It does not require expensive instruments or complicated reduction processes, and can be detected using only a 360-366nm ultraviolet lamp. This study developed NO3 - The detection method of the kit's fluorescence sensor can achieve on-site, rapid, and timely detection of nitrate ions in the environment. It only takes 1-2 minutes for the color change to detect nitrite.

[0014] The fluorescence detection method of nitrate according to the present invention comprises the following steps:

[0015] (1) Fluorescent carbon dot solution was synthesized by hydrothermal method by doping naphthylethylenediamine hydrochloride with citric acid and phosphoric acid;

[0016] (2) using zinc powder to catalyze the reduction of a standard solution containing nitrate to obtain a solution containing nitrite, which is then mixed with the fluorescent carbon dot solution in step (1) to develop color;

[0017] (3) Repeat steps (1) and (2) multiple times to establish a linear relationship between the concentration of the nitrate standard solution and the fluorescence response intensity, obtain a standard curve, and prepare a standard colorimetric card; and use a fluorescence meter to establish a standard curve and detect nitrate, as shown in Table 1. (4) Take the test solution, perform steps (1) and (2), and compare the color development results with the standard colorimetric card.

[0018] In the above-mentioned detection method, preferably, in step (1), the mixed solution of citric acid-naphthylethylenediamine hydrochloride-sodium phosphate is continuously heated at 180 degrees in a reactor for 5 hours to form a fluorescent carbon dot solution.

[0019] In the above-mentioned detection method, preferably, the volume ratio of the fluorescent carbon dot solution in step (1) to the standard solution containing nitrate in step (2) is 0.5-0.8:1, more preferably 0.7:1. The volume ratio of the fluorescent carbon dot solution in step (1) to the test solution containing nitrate in step (4) is 0.5-0.8:0.5-1, more preferably 0.7:0.5.

[0020] The present invention also provides a fluorescent sensor for detecting nitrate, which is obtained based on the above-mentioned detection method.

[0021] The present invention uses zinc to reduce nitrate to nitrite, then uses a method for detecting nitrite to indirectly detect nitrate. Based on the Griess reaction principle, nitrite undergoes a diazotization process, resulting in color development, thereby constructing a nitrate detection kit. Carbon dots are made of naphthylethylenediamine hydrochloride and citric acid, doped with phosphate to form a novel composite fluorescent carbon dot—which exhibits a pale blue to yellowish color under ultraviolet light. The composite carbon dots are then analyzed to achieve excellent color development and a low detection limit. Nitrite undergoes diazotization with naphthylethylenediamine hydrochloride, while citric acid and phosphoric acid enhance color recognition and reduce the detection limit.

[0022] Photoelectron spectroscopy of carbon dot materials, Figure 1 , showing that C, H, N, and O are all present; Figure 2 Transmission electron microscopy of carbon dot materials, the color range of the kit is clear, such as Figure 3 The novel carbon dot design of the present invention reduces the detection limit and has a good anti-interference effect. It can better detect nitrite and then effectively detect the content of nitrate in soil or water. When the instrument is used to detect standard nitrate solution, a good linear relationship is found in the range of 3-140ppm, as shown in Figure 4. Compared with the national standard method (Table 1), the two have a good equivalence relationship.

[0023] The designed kit is within the range of 0.1-150ppm of the standard solution. Nitrite can make the carbon dots appear in different colors, such as Figure 3 From left to right, as the nitrite concentration increases (from left to right, corresponding to 0.1, 1, 10, 20, 50, 100, 150 ppm nitrate standard solution), the solution color gradually changes from blue to yellowish to bright blue, until at high concentrations (above 100 ppm), the blue fades and disappears to dark yellow. Figure 3A standard colorimetric card can be made as shown. This effectively enables visual detection of nitrate in the environment. The fluorescent carbon dots in the present invention are carbon dot composite materials that exhibit a composite fluorescent color—blue with a yellowish tint—under ultraviolet light. Upon exposure to a certain concentration of nitrite, one color is quenched, while at higher concentrations, another color is quenched.

[0024] The present invention tests common ions in soil such as sodium ions, potassium ions, sulfate ions K + , Cl - , NH4 + , Na + , Cu 2 + , Cr 3+ , Pb 2+ , PO4 3- , CO3 2- , SO4 2- However, no changes in fluorescence peaks were found in the tests, and the fluorescence intensity was not affected, indicating that the present invention is not interfered with by these common ions in soil. The kit designed by the present invention can sensitively respond to the displayed color, which can be compared with the color displayed by the standard solution to determine the nitrate content in the water or soil of the environment.

[0025] The present invention enables real-time, rapid, and visual detection, and the material preparation process is simple and inexpensive. The detection process is also convenient and quick. The visual test kit can detect nitrate to 0.1 ppm, which is lower than the national standard for nitrate detection in water or soil. This allows farmers to conveniently test soil nitrate content, thereby determining water pollution and soil fertilizer efficiency, and taking appropriate measures to control environmental pollution and conserve resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Photoelectron spectroscopy of composite carbon dots.

[0027] Figure 2 Transmission electron microscopy of composite carbon dots.

[0028] Figure 3 The color changes of carbon dots after different concentrations of nitrate are reduced.

[0029] Figure 4 Fluorescence test graph (4a) and standard curve (4b). DETAILED DESCRIPTION

[0030] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0031] Materials: Zinc powder was purchased from McLean Pharmaceutical Reagent Company. Other experimental reagents, such as those used for preparing buffer solutions and interfering ion tests, such as sodium chloride, citric acid, sodium phosphate, ammonium chloride, naphthylethylenediamine hydrochloride, potassium chloride, hydrochloric acid, copper sulfate, lead nitrate, cadmium chloride, zinc sulfate, etc., were all from McLean Pharmaceutical Co., Ltd.

[0032] Example 1

[0033] Preparation of fluorescent carbon dot material: A mixed solution of citric acid, naphthalene ethylenediamine hydrochloride and sodium phosphate was heated in a reaction kettle at 180 degrees Celsius for 5 hours to form a fluorescent carbon dot solution for use. Figure 3 Transmission electron microscopy of the fluorescent carbon dots shown.

[0034] Example 2: (Determination of nitrate in water environment)

[0035] (1) Add a full spoonful (the spoon is the size of the one in the product, used for ear cleaning) of zinc powder to a 1-2 ml centrifuge tube.

[0036] (2) Add 100 ml of nitrate standard solution, then add 200 μl of acidic solution with a pH of about 1.8 (containing a mixed solution of 13 wt% ammonium chloride and 10 wt% potassium chloride, adjusted to pH 1.8 with hydrochloric acid), shake briefly, and after 1-2 minutes, take the clear solution for use.

[0037] (3) Take 70 ml of the carbon dot solution and add it to another centrifuge tube. Then take the clarified solution from (2) and drop it into this small test tube. Color will develop. Use the same method to test different concentrations of nitrate and establish a standard colorimetric card.

[0038] (4) Determination of unknown aqueous solution: Take a water sample (about 50 mL) from the environment, filter it through a 0.25 μm filter membrane, and set aside.

[0039] (5) Follow the steps (1)(2)(3) to replace the standard nitrate solution with the unknown aqueous solution, develop the color, and then compare it with the standard colorimetric card to determine the nitrate content of the unknown aqueous solution.

[0040] Example 3: (Determination of Nitrate in Soil)

[0041] Take a portion of dry soil, sieve it through a 20-mesh sieve, and weigh it. Extract nitrate solution according to the national standard method. Save it for later use.

[0042] Follow steps (1)(2)(3). After color development, compare with the standard color card to determine the nitrate content of the unknown soil.

[0043] Soil extraction: For soil, first extract nitrate using aqueous solution according to the national standard method, then reduce it to nitrite, detect nitrite, and then determine nitrate. For water in the environment, a 0.25μM filter can be used to remove insoluble matter, and then reduce nitrate in the water to nitrite for detection.

[0044] Determine the concentration of nitrate or nitrite in the environment, compare its color with that of the standard solution, and determine whether the nitrate in the environment exceeds the standard or its content.

[0045] Nitrate determination by the national standard method (UV-Vis spectrophotometry): A standard curve for nitrate was established, and nitrate was extracted using the national standard method. Ten soil samples were then tested using UV light to calculate the nitrate concentration in the soil. After establishing a standard curve for nitrate using the fluorescence method, the nitrate in the soil extract was reduced and compared with the standard curve to calculate the nitrate concentration in the soil. As shown in Table 1, the fluorescence and UV detection results were generally similar.

[0046] Table 1 Comparison of soil testing by national standard method and fluorescence method

[0047]

[0048] It should be noted that the technical contents of the present invention described above are only for the purpose of explaining and illustrating the technical essence of the present invention to enable those skilled in the art to understand the technical essence of the present invention. Therefore, the technical contents described above are not intended to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be based on the claims. Those skilled in the art should be aware that any modifications, equivalent substitutions, and improvements based on the substantial spirit of the present invention shall fall within the substantial protection scope of the present invention.

[0049] References

[0050] [1]An improved hydrazine reduction method for the automated determination of low nitrate levels in freshwater. Water Research 12(1978)673-675.

[0051] [2]A review on spectroscopic methods for determination of nitrite andnitrate in environmental samples.Priyanka Singh,Manish Kumar Singh,YounusRaza Beg,Gokul Ram Nishad.Talanta 191(2019)364–381.

[0052] [3]G.F.Wang,M.Satake,K.Horita,Spectrophotometric determination ofnitrate and nitrite in water and some fruit samples using columnpreconcentration.Talanta 46(1998)671–678.

[0053] [4]Determination of Nitrate in Natural Waters by Vanadium Reductionand the Griess Assay:Reassessment and Optimization,Su-Cheng Pai,Yu-Ting Su,Mei-Chen Lu,Yalan Chou,Tung-YuanHo.ACS EST Water 1(2021)1524–1532.

[0054] [5]MdE.E.Alahi,S.C.Mukhopadhyay,Detection methods of nitrate inwater:a review.Sens.Actuators Phys.280(2018)210–221.

[0055] [6]Fluorescent and colorimetric dual-readout sensor based on Griessassay for nitrite detection.Huixiang Wu,Xin Shen,Danqun Huo,Yi Ma,MinghongBian,CaihongShen,Changjun Hou.Spectrochimica Acta Part A:Molecular andBiomolecular Spectroscopy 225(2020)117470.

Claims

1. Fluorescence detection method for nitrate: Fluorescent carbon dots are synthesized by hydrothermal method using naphthylethylenediamine hydrochloride and citric acid. The nitrate-containing test solution is reduced with a reducing agent to obtain a nitrite-containing solution. The fluorescent carbon dots and the nitrite-containing solution are mixed under ultraviolet light and the color change is observed.

2. The detection method according to claim 1, wherein The hydrothermal method for synthesizing fluorescent carbon dots is to continuously heat the reaction vessel at 170-190 degrees Celsius for 4-6 hours to form a fluorescent carbon dot solution.

3. The detection method according to claim 1, wherein In the synthesis of fluorescent carbon dots, phosphate, naphthylethylenediamine hydrochloride, citric acid and phosphate are added in a molar ratio of 1:1.8-2.2:0.4-0.

8.

4. The detection method according to claim 3, wherein The molar ratio of naphthylethylenediamine hydrochloride, citric acid and sodium phosphate is 1:2:0.

5.

5. The detection method according to claim 1 or 2, wherein: The reducing agent is zinc powder.

6. A fluorescence detection method for nitrate, characterized in that: The steps include: (1) A fluorescent carbon dot solution was synthesized by hydrothermal method by doping naphthylethylenediamine hydrochloride, citric acid and phosphate in a reactor at 180 degrees Celsius for 5 hours; The molar ratio of naphthylethylenediamine hydrochloride, citric acid and phosphate is 1:2:0.5; (2) using zinc powder to catalyze the reduction of a standard solution containing nitrate to obtain a solution containing nitrite, and then mixing the solution with the fluorescent carbon dot solution in step (1) under ultraviolet light to develop color; (3) Repeating steps (1) and (2) multiple times to establish a linear relationship between the concentration of the nitrate standard solution and the fluorescence response intensity, obtain a standard curve, and prepare a standard colorimetric card; (4) Taking the test solution containing nitrate, catalytically reducing the test solution with zinc powder to obtain a test solution containing nitrite, and then mixing it with the fluorescent carbon dot solution in step (1) under ultraviolet light, and comparing the color development result with the standard colorimetric card.

7. The detection method according to claim 6, wherein: The volume ratio of the fluorescent carbon dot solution in step (1) to the standard solution containing nitrate in step (2) is 0.5-0.8:

1.

8. The detection method according to claim 6, wherein: The volume ratio of the fluorescent carbon dot solution in step (1) to the test solution containing nitrate in step (4) is 0.5-0.8:0.5-1.

9. The detection method according to claim 6, wherein: In step (2), an acidic solution with a pH of 1.6-2.0 is added.

10. A fluorescent kit for detecting nitrate obtained based on the detection method according to any one of claims 1 to 6, comprising a fluorescent carbon dot solution, zinc powder, and a standard colorimetric card.

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