Method for rapidly extracting and detecting nitrate nitrogen in soil in situ

Abstract

The invention relates to a novel method for in-situ rapid extraction and detection of nitrate nitrogen in soil, and the principle is that a gradient diffusion film (DGT) technology can enrich and extract nitrate nitrogen in the soil in situ, and after elution, a fluorescent paper-based sensor is matched for color development reading, so that in-situ rapid extraction and detection of the nitrate nitrogen content in the soil are realized. The method mainly comprises the following steps: (1) preparing DGT for adsorbing nitrate nitrogen by using a developed nitrate nitrogen obligate adsorption material, placing the prepared DGT in a soil environment for a plurality of hours, taking out a binding membrane in a DGT device, and placing the binding membrane in an eluent for elution; and (2) carrying out simple pretreatment on the obtained eluent, carrying out fluorescent color development reaction by using a fluorescent paper-based sensor, and finally reading the content of nitrate nitrogen through color change. The invention provides a rapid, simple and convenient prediction method for evaluating the soil fertility and the absorption of the plants to the nitrogen in the soil.

Description

Technical Field

[0001] The present invention belongs to the field of rapid detection of nitrate nitrogen, and particularly relates to a method for in-situ rapid enrichment and extraction of soil nitrate nitrogen and fluorescence colorimetric detection of nitrate nitrogen content in soil environmental media by using the diffusive gradients in thin films (DGT) technology. Background Art

[0002] Soil nitrate nitrogen refers to the nitrogen in the soil in the form of nitrate, which is one of the main nitrogen forms available to plants. Nitrate nitrogen can be directly absorbed and utilized by most plants and is crucial for plant growth. Excessive application of nitrogen fertilizers can lead to the accumulation of nitrate nitrogen in the soil, which may enter water bodies through runoff and leaching, causing environmental problems such as water eutrophication. Reasonable fertilization and soil management are very important for controlling the loss of nitrate nitrogen and improving its utilization efficiency. Therefore, the rapid detection of soil nitrate nitrogen is of great significance for evaluating soil fertility and guiding agricultural production.

[0003] At present, the commonly used method for detecting soil nitrate nitrogen is potassium chloride solution extraction - spectrophotometry. This is a method recommended by the national standard GB / T 32737 - 2016 for determining the content of nitrate nitrogen in soil. The principle of this method is to extract nitrate nitrogen in the soil sample with potassium chloride solution, measure the absorbance of nitrate nitrogen in the soil leachate by an ultraviolet spectrophotometer, and then calculate the content of nitrate nitrogen according to the standard curve. The detection limit of this method for soil nitrate nitrogen is 0.5 mg / kg, and the quantification limit is 1 mg / kg. This method may have certain limitations on the detection lower limit of nitrate nitrogen and may not be applicable to the determination of low-concentration nitrate nitrogen. At the same time, the process of preparing samples by this method is complex and not applicable to in-situ detection.

[0004] In view of the above problems, the present invention has developed a new method for rapid in-situ detection of soil nitrate nitrogen based on the diffusive gradients in thin films (DGT) and fluorescence colorimetric technology. The DGT technology is based on Fick's first law of diffusion, and the effective state concentration of an ion is obtained by quantitatively measuring and calculating a certain ion passing through a specific thickness of the diffusion membrane within a specific time. Therefore, the determination of low-concentration nitrate nitrogen content can be achieved by controlling the enrichment time of the adsorption material for the target ion. At the same time, the DGT technology is an ideal in-situ passive sampling technology and can be perfectly combined with the fluorescence colorimetric technology to achieve in-situ detection of soil nitrate nitrogen content. Summary of the Invention

[0005] The purpose of the present invention is to provide a new method for solving the problem of rapid in-situ detection of soil nitrate nitrogen. This method perfectly combines the DGT enrichment and extraction technology with the fluorescence paper-based detection technology, with high detection sensitivity and applicability to complex soil environments.

[0006] The technical solution for the present invention to achieve the objective is as follows:

[0007] A method for rapidly detecting the content of nitrate nitrogen in soil, characterized by comprising the following steps:

[0008] S1. In-situ sampling is carried out using an adsorption nitrate nitrogen DGT device, and the adsorption is maintained for 1 - 4 h. The depth of the DGT device in the soil sample ranges from 1 - 2 cm;

[0009] S2. After the adsorption time is reached, the DGT device is taken out. The binding membrane in the DGT device is placed in a 1 M sodium chloride solution and eluted for 1 h, then filtered, and the DGT binding membrane eluate is collected;

[0010] S3. Preparation of the iron-based material: Aqueous sodium borohydride solution is added dropwise and uniformly to the aqueous ferrous sulfate solution, and then placed in an ultrasonic cleaner and ultrasonicated for 10 - 20 minutes; the ultrasonicated solution is poured into a 5 ml centrifuge tube, centrifuged at 8000 revolutions for 2 - 3 minutes, the centrifuge tube is taken out, the supernatant is poured off, deionized water is added, and the above operation is repeated for two water washings to obtain the iron-based material;

[0011] S4. Nitrate nitrogen reduction: The DGT binding membrane eluate is added to the iron-based material, shaken and reduced for 5 - 15 minutes, and then filtered through a filter membrane to obtain the reduced solution;

[0012] S5. Test strip fluorescence color development: Take the reduced solution, add aqueous sodium sulfite solution, shake well, put in a test strip, react for 3 - 5 minutes, take out the test strip, irradiate the test strip with a 365 nm ultraviolet lamp, compare with the standard colorimetric card, and read the concentration C of nitrate nitrogen in the eluate through the fluorescence color, and calculate the available content C of nitrate nitrogen in the soil DGT .

[0013] Moreover, the calculation method for the adsorbed nitrate nitrogen content M of the binding membrane is:

[0014] M = C × 20 / 0.95

[0015] The available content C of nitrate nitrogen in the soil DGT The calculation method is:

[0016]

[0017] Wherein, C DGT is the available content of nitrate nitrogen in the soil (μg / mL), M is the accumulated nitrate nitrogen content (μg) on the binding membrane within the DGT development time t, A is the DGT window area (cm 2 ), the diffusion layer thickness Δg is the sum of the diffusion gel layer thickness and the cellulose filter membrane thickness (cm), and D is the diffusion coefficient of nitrate nitrogen in the diffusion gel (cm2 ( / s), where t is the device deployment time (s).

[0018] Moreover, the nitrate nitrogen adsorption DGT device includes a binding membrane, a diffusion membrane, and a filtration membrane, which are placed and fixed in the DGT housing in sequence to form an adsorption device.

[0019] Moreover, the preparation method of the binding membrane in the DGT device is as follows: after reacting chloromethylated polystyrene-divinylbenzene copolymer with an amination reagent, it is added to an agarose gel solution to form a binding membrane; the mass ratio of chloromethylated polystyrene-divinylbenzene to the amination reagent is copolymer:amination reagent = 1:1 - 2.

[0020] Moreover, the in-situ sampling method of the nitrate nitrogen adsorption DGT device is as follows:

[0021] Method 1: In-situ collect some surface soil samples of 0 - 20 cm and place them in a container, add ultrapure water to the saturated water holding capacity, stir the soil sample evenly and then incubate for 1 h;

[0022] Or Method 2: In-situ select a piece of soil to be measured, add water externally until the soil is moist, use a glass rod to stir thoroughly to make the soil surface smooth and flat, and then incubate for 1 h.

[0023] Moreover, the relationship between the diffusion coefficient of the nitrate nitrogen and temperature is as follows:

[0024]

[0025] Where Dt is the diffusion coefficient of nitrate nitrogen at t °C, and D25 is the diffusion coefficient of nitrate nitrogen at 25 °C.

[0026] 7. The method for rapidly detecting the nitrate nitrogen content in soil according to claim 1, wherein the concentration of the ferrous sulfate solution is 5 - 10 wt%, the concentration of sodium borohydride is 0.2 - 0.7 wt%, and the concentration of sodium sulfite is 0.1 - 0.5 wt%.

[0027] The advantages and positive effects of the present invention are:

[0028] (1) The DGT device for adsorbing nitrate nitrogen adopted in the present invention can be deployed and applied outdoors on-site, which well solves the problem of in-situ detection.

[0029] (2) The nitrate nitrogen adsorption material adopted in the present invention has a fast adsorption rate and can rapidly absorb nitrate ions in the soil environment, providing a basis for rapid detection.

[0030] (3) The materials used in the present invention have the characteristics of specific adsorption. Therefore, the components of the eluate after eluting the binding membrane adsorbed by DGT are simpler than those of the potassium chloride eluate and are easy to detect.

[0031] (4) The fluorescence test strip detection technology adopted by the present invention is simple to operate, has high sensitivity and fast detection speed, and can be directly detected on site.

[0032] (5) The present invention combines the DGT technology to enrich and extract nitrate nitrogen in the soil with the fluorescence test strip detection technology, providing a complete set of rapid detection methods for the in-situ extraction and detection of soil nitrate nitrogen. At the same time, it also enables the evaluation of soil fertility and the guidance of agricultural production to be carried out more accurately. Description of the Drawings

[0033] Figure 1 It is a graph showing the relationship between the adsorption time and adsorption amount of DGT for adsorbing nitrate nitrogen;

[0034] Figure 2 It is a graph showing the detection results of different concentrations of nitrate nitrogen;

[0035] Figure 3 It is a standard colorimetric card for the fluorescence color development of the test strip;

[0036] Figure 4 It is an effect diagram of the detection of actual soil samples. Detailed Embodiments

[0037] The method of the present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all well-known methods to those skilled in the art. In addition, the implementation examples should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims.

[0038] The method for in-situ rapid extraction and detection of soil nitrate nitrogen provided by the present invention uses the DGT technology to in-situ enrich and extract soil nitrate nitrogen, elutes it with sodium chloride solution to obtain an eluate, reduces the nitrate nitrogen in the eluate to ammonium nitrogen and then conducts a fluorescence color development reaction, and finally determines the content of nitrate nitrogen in the soil through color change, so as to evaluate the soil fertility.

[0039] A method for in-situ rapid extraction of soil nitrate nitrogen, the steps are as follows:

[0040] S1: The DGT device includes a binding membrane, a diffusion membrane, a filtration membrane and a housing. The binding membrane is the DGT binding membrane given by the DGT binding membrane for the material for specifically adsorbing nitrate nitrogen disclosed in CN118649666A. The diffusion membrane is made of an agarose gel solution with a concentration of 1.5 wt%. The DGT device for adsorbing nitrate nitrogen is prepared together with the DGT housing in the order of the binding membrane, the diffusion membrane, and the filtration membrane.

[0041] S2: Mode 1: In-situ collect some soil samples from the surface layer of 0 - 20 cm and place them in a container. Add ultrapure water to the saturated water holding capacity, stir the soil samples evenly, and then incubate for 1 h.

[0042] Mode 2: In-situ select a soil sample to be measured. After adding water externally to make the soil moist (no need to add water externally for soils with long-term flooding or sufficient humidity), use a glass rod to stir thoroughly to make the soil surface smooth and flat, and then incubate for 1 h.

[0043] S3: Use a small wooden chip to scrape a small amount of soil sample to fill the DGT adsorption window, then insert the DGT device into the treated soil sample, and maintain a certain adsorption time of 1 - 2 h. The depth of the DGT device in the soil sample ranges from 1 - 2 cm. At the same time, record the real-time temperature during the adsorption process.

[0044] S4: After reaching the adsorption time, take out the DGT device, rinse the residual soil on the DGT outer shell with ultrapure water, and take out the binding membrane fixing glue in the DGT device. Place the binding membrane fixing glue in a 20 ml, 1 M sodium chloride solution and elute for 1 h. Filter out the binding membrane fixing glue and collect the DGT eluate.

[0045] A method for rapidly detecting the nitrate nitrogen content in soil, comprising the following steps:

[0046] S1. Preparation of iron-based material: Gradually and uniformly drop the aqueous sodium borohydride solution into the aqueous ferrous sulfate solution, and place it in an ultrasonic cleaner for ultrasonic treatment for 10 - 20 minutes. Pour the ultrasonicated solution into a 5 ml centrifuge tube, and centrifuge at 8000 revolutions for 2 - 3 minutes. Take out the centrifuge tube, pour out the supernatant, add deionized water, and repeat the above operation for two water washes to obtain the iron-based material.

[0047] S2. Nitrate nitrogen reduction: Add the above-mentioned DGT eluate to the iron-based material, shake it by hand for reduction for 10 minutes, and then filter it through a 0.45 μm filter membrane to obtain the reduced solution. Add 0.01 - 0.02 g of iron-based material to 3 ml of eluate. The iron-based material is sufficient for the reduction amount and is slightly excessive.

[0048] S3. Fluorescent color development of test paper: Take 255 μl of the above-mentioned reduced solution, add 210 μl of aqueous sodium sulfite solution, shake well, put in a test paper strip, react for 3 - 5 minutes, take out the test paper strip, irradiate the test paper strip with a 365 nm ultraviolet lamp, compare with the standard colorimetric card, and read the concentration C of nitrate nitrogen in the eluate through the fluorescent color, and calculate the available content C of nitrate nitrogen in the soil DGT 。

[0049] In step S1, the concentration of the ferrous sulfate solution is 5 - 10 wt%, the concentration of sodium borohydride is 0.2 - 0.7 wt%, and the concentration of sodium sulfite is 0.1 - 0.5 wt%.

[0050] The calculation method for the available content of soil nitrate nitrogen is as follows:

[0051]

[0052] Among them, C DGT is the available content of nitrate nitrogen in the soil (μg / mL), M is the content of nitrate nitrogen accumulated on the binding membrane during the DGT deployment time t (μg), A is the DGT window area (cm 2 ), the diffusion layer thickness Δg is the sum of the diffusion gel layer thickness and the cellulose filter membrane thickness (cm), D is the diffusion coefficient of nitrate nitrogen in the diffusion gel (cm 2 / s), and t is the device deployment time (s).

[0053] The calculation method for the content M of nitrate nitrogen adsorbed by the binding membrane is:

[0054] M = C × 20 / 0.95

[0055] The relationship between the diffusion coefficient of nitrate nitrogen in the diffusion gel and temperature is:

[0056]

[0057] Among them, D t is the diffusion coefficient of nitrate nitrogen at t °C, D 25 is the diffusion coefficient of nitrate nitrogen at 25 °C (D 25 is calculated by the following formula for D).

[0058] During the experiment for measuring the diffusion coefficient of nitrate nitrogen, 3.5 L of a mixed solution containing 2 mg·L -1 NO3 - -N and 0.001 mol·L -1 NaCl was prepared in a large container; the assembled DGT device was placed in the solution and fully adsorbed at 25 °C and pH 5.5; at 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, and 8 h, 3 devices were taken out each time, the device was opened to take out the binding membrane, and after being rinsed clean, it was placed in a sodium chloride solution for elution; the linear regression slope relationship of the accumulated nitrate nitrogen in the binding gel with time was obtained as shown in Figure 1 ; the diffusion coefficient of NO3 - -N was calculated to be 8.92 х 10 -6 cm 2 / s through the following formula.

[0059]

[0060] In the formula: Slope is the linear regression slope of the cumulative amount of NO3 - in the binding membrane with time at each time point (slope isFigure 1 The slope represented), Δg is the diffusion layer thickness (cm), C sol is the NO3 in the prepared solution - concentration (mg / L), A is the sampling window area (cm 2 ).

[0061] During the experiment to test the accuracy of DGT for extracting nitrate nitrogen, solutions of 0.1, 0.2, 0.5, 1, 5, 10, 50, 100, and 200 mg·L -1 NO3 - -N were respectively prepared. Three DGTs were placed in each solution for adsorbing and extracting nitrate nitrogen. After adsorption for several hours, the DGT devices were taken out. Elution was carried out with 1M sodium chloride solution, and the concentration of the eluate was measured. Finally, C DGT / C soln (C soln is the concentration of the nitrate nitrogen solution with a known concentration prepared in the experiment) was calculated. When 0.9 ≤ C DGT / C soln ≤ 1.1, it is considered that accurate determination of nitrate nitrogen can be achieved using DGT. The experimental results are as Figure 2 shown.

[0062] According to the above method, the soils in Tibet and Sichuan were actually detected. The DGT device was used for enriching and extracting soil nitrate nitrogen, and then elution treatment was carried out. Finally, colorimetric detection was carried out using a fluorescent test paper. The results are as Figure 4 shown. The nitrate nitrogen content in the soil of Sichuan is much higher than that in the soil of Tibet, which is consistent with the actual situation.

[0063] The preparation method of the DGT binding membrane for the material for specifically adsorbing nitrate nitrogen disclosed in CN118649666A is as follows:

[0064] S1. Preparation of the material for specifically adsorbing nitrate nitrogen:

[0065] Take 10 g of 100-mesh chloromethylated polystyrene-divinylbenzene copolymer in a 500 ml flask, add 50 ml of deionized water, and swell at room temperature for 12 h. Among them, the chloromethylated polystyrene resin contains 1% DVB cross-linking, the concentration of the chloromethylated polystyrene resin is 1.0 - 1.24 mmol / g, the particle size is 100 - 200 mesh, the CAS number is 55844-94-5, and the molecular weight is 602.70038.

[0066] Add 15 ml of amination reagent (triethylamine: dimethyloctylamine = 1 - 2:1), and the mass ratio is W(copolymer): W(amination reagent) = 1: 1 - 2. Stir and react at 40 °C for 6 h;

[0067] Wash the material with a large amount of deionized water until the effluent is neutral. Then, add an equal volume of 1 M hydrochloric acid and soak for 2 h. Next, wash the material with deionized water until the effluent is neutral. Finally, wash the material with an appropriate amount of absolute ethanol three times to obtain a material for specifically adsorbing nitrate nitrogen, and store it under low-temperature vacuum conditions.

[0068] S2. Preparation of the DGT binding membrane:

[0069] Weigh 1.5 g of the adsorbent material prepared in step S1 and mix it with a melted agarose gel solution (2 wt%). Stir until the material is evenly dispersed in the gel solution.

[0070] Ultrasonic for 2 - 3 minutes to make the material and the gel solution evenly mixed and without bubbles in the mixed solution. Then, quickly inject the mixed solution into a glass trough with a certain thickness. During the injection process, the glass plate should be placed on a hot plate heated to 70 °C throughout to prevent the agarose gel solution from solidifying halfway. Transfer the glass plate filled with the gel solution to a low-temperature incubator and incubate for 1 hour, then take it out, soak it in ultrapure water for 30 minutes, and then peel off the gel membrane from the glass plate. Soak the obtained gel membrane in 0.1 M sodium chloride solution for 24 hours, and then change to pure water at least three times to obtain the DGT binding membrane.

[0071] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that various substitutions, changes, and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A method for quickly detecting the content of nitrate nitrogen in soil, characterized in that: It includes the following steps: S1. In-situ sampling is carried out with a nitrate-nitrogen adsorption DGT device, and the adsorption is maintained for 1 - 4 h. The depth of the DGT device in the soil sample ranges from 1 - 2 cm; S2. After the adsorption time is reached, the DGT device is taken out. The binding membrane in the DGT device is placed in a 1M sodium chloride solution and eluted for 1 h, then filtered to collect the DGT binding membrane eluate; S3. Preparation of iron-based materials: Sodium borohydride aqueous solution is slowly added dropwise to ferrous sulfate aqueous solution, and then placed in an ultrasonic cleaner for ultrasonic treatment for 10 - 20 minutes; the ultrasonicated solution is poured into a 5 ml centrifuge tube and centrifuged at 8000 revolutions for 2 - 3 minutes. The centrifuge tube is taken out, the supernatant is poured off, deionized water is added, and the above operation is repeated for two water washings to obtain iron-based materials; S4. Nitrate-nitrogen reduction: The DGT binding membrane eluate is added to the iron-based materials, shaken and reduced for 5 - 15 minutes, and then filtered through a filter membrane to obtain the reduced solution; S5. Fluorescent color development of the test strip: Take the reducing solution, add an aqueous solution of sodium sulfite, shake well, put in the test strip, react for 3 - 5 minutes, take out the test strip, irradiate the test strip with a UV lamp at 365 nm, compare with the standard colorimetric card, read the concentration C of nitrate nitrogen in the eluate through the fluorescence color, and calculate the available content C of nitrate nitrogen in the soil DGT .

2. The method for rapidly detecting the content of nitrate nitrogen in soil according to claim 1, wherein: The calculation method for the nitrate-nitrogen content M adsorbed by the binding membrane is: M = C×20 / 0.95 The available content C of nitrate nitrogen in soil DGT The calculation method is as follows: Among them, C DGT is the available content of nitrate nitrogen in the soil (μg / mL), M is the content of nitrate nitrogen accumulated on the binding membrane during the DGT deployment time t (μg), A is the DGT window area (cm 2 ), the diffusion layer thickness Δg is the sum of the diffusion gel layer thickness and the cellulose filter membrane thickness (cm), D is the diffusion coefficient of nitrate nitrogen in the diffusion gel (cm 2 / s), and t is the device deployment time (s).

3. The method for rapidly detecting the content of nitrate nitrogen in soil according to claim 1, wherein: The nitrate-nitrogen adsorption DGT device includes a binding membrane, a diffusion membrane, and a filter membrane placed and fixed in the DGT housing in sequence to form an adsorption device.

4. The method for rapidly detecting the nitrate nitrogen content in soil according to claim 1 or 3, characterized in that: The preparation method of the binding membrane in the DGT device is: After reacting chloromethylated polystyrene-divinylbenzene copolymer with an amination reagent, it is added to an agarose gel solution to form a binding membrane; the mass ratio of chloromethylated polystyrene-divinylbenzene to the amination reagent is copolymer: amination reagent = 1:1 - 2.

5. The method for rapidly detecting the content of nitrate nitrogen in soil according to claim 1, characterized in that: The in-situ sampling method of the nitrate-nitrogen adsorption DGT device is: Method 1: In-situ collect some surface soil samples of 0 - 20 cm and place them in a container, add ultrapure water to the saturated water holding capacity, stir the soil sample evenly and then incubate for 1 h; Or Method 2 can be adopted: In-situ select a piece of soil to be measured, add water externally until the soil is moist, and use a glass rod to fully stir to make the soil surface smooth and flat, and then incubate for 1 h.

6. The method for rapidly detecting the content of nitrate nitrogen in soil according to claim 1, characterized in that: The relationship between the diffusion coefficient of the nitrate-nitrogen and temperature is: where Dt is the diffusion coefficient of nitrate-nitrogen at t °C, and D25 is the diffusion coefficient of nitrate-nitrogen at 25 °C.

7. The method for rapidly detecting the content of nitrate nitrogen in soil according to claim 1, characterized in that: The concentration of the ferrous sulfate solution is 5 - 10 wt%, the concentration of sodium borohydride is 0.2 - 0.7 wt%, and the concentration of sodium sulfite is 0.1 - 0.5 wt%.

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