A paper-based ammonia-nitrogen detection chip and a preparation method and application thereof

Abstract

The application relates to the field of microfluidic paper chips, and discloses a paper-based ammonia nitrogen detection chip as well as a preparation method and application thereof. The chip comprises, from bottom to top, a background layer, a color developing layer and a sealing layer; the color developing layer is provided with a reaction area, a color developing area and a channel connecting the reaction area and the color developing area; a detection reagent A is prepositioned in the reaction area; a detection reagent B is prepositioned in the color developing area; the sealing layer is provided with a sample inlet and a color developing port corresponding to the reaction area and the color developing area respectively; the detection reagent A solution is added dropwise to the reaction area and then freeze-dried, so that the detection reagent A is prepositioned in the reaction area; the detection reagent B solution is added dropwise to the color developing area and then freeze-dried, so that the detection reagent B is prepositioned in the color developing area. The detection reagent is evenly spread on the filter paper through the freeze-drying mode, and the amount of the carried reagent is more than that of the immersion method; and the detection reagent is loose and porous inside, the reaction rate is faster when contacting the to-be-measured solution, and the color developing uniformity is better.

Description

Technical Field

[0001] This invention relates to the field of microfluidic paper chips, specifically to a paper-based ammonia nitrogen detection chip, its preparation method, and its application. Background Technology

[0002] Ammonia nitrogen refers to ammonia in its free state (NH3) or ammonium salt (NH4+). 4+ Ammonia nitrogen exists in water in the form of nitrogen oxides (NOx). Excessive ammonia nitrogen can lead to eutrophication and is toxic to fish and certain aquatic organisms. The main sources of ammonia nitrogen in water are the decomposition products of nitrogenous organic matter in domestic sewage by microorganisms, some industrial wastewater, and agricultural drainage. Ammonia nitrogen concentration is an important indicator of the degree of water pollution, and timely and accurate detection of ammonia nitrogen in water bodies is of great significance.

[0003] Paper-based microfluidic chips, or μPADs for short, are microfluidic chips that use porous paper materials as a substrate and analytical platform. By combining hydrophobic materials with hydrophilic paper fibers through various processing techniques to form hydrophilic-hydrophobic channels, the flow direction of fluids can be controlled. Compared with traditional microfluidic chips, μPADs do not require additional liquid driving devices, relying on capillary action to deliver liquids. They also have advantages such as low cost, small sample and reagent volume, simple result recognition, good biocompatibility, and biodegradability.

[0004] Currently, conventional paper-based microarrays fall into two categories: the dropwise method, where reagents and the test solution are added dropwise gradually, and colorimetric quantification is performed after color development; and the immersion method, where the chip is first immersed in a reagent solution, then dried (either by baking or air drying) to obtain the detection chip, which is then reacted with the test solution for colorimetric quantification. The first method requires on-site reagent preparation, is complex, and is significantly affected by environmental factors. The second method has slower solid-liquid color development, a lower effective reagent loading, and slightly weaker color development; furthermore, the coffee ring effect occurs when the paper-based material comes into contact with the liquid, resulting in uneven color distribution on the chip.

[0005] Therefore, how to quickly and uniformly detect ammonia nitrogen using paper chip technology is an urgent problem that needs to be solved. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of uneven color development and low color development efficiency of existing ammonia nitrogen detection chips, and to provide a paper-based ammonia nitrogen detection chip, its preparation method and application.

[0007] To achieve the above objectives, the present invention provides a paper-based ammonia nitrogen detection chip, which comprises, from bottom to top, a background layer, a color development layer and a sealing layer;

[0008] The colorimetric layer has a reaction zone, a colorimetric zone, and a channel connecting the reaction zone and the colorimetric zone; the reaction zone contains a pre-placed detection reagent A, and the colorimetric zone contains a pre-placed detection reagent B;

[0009] The sealing layer is provided with an inlet and a colorimetric port corresponding to the reaction zone and the colorimetric zone, respectively, and the size of the colorimetric port is smaller than the size of the colorimetric zone;

[0010] The color developing layer is made of filter paper, and the background layer and the sealing layer are both made of hydrophobic materials.

[0011] The detection reagent A comprises a first excipient, a chromogenic agent, a first masking agent, a stabilizer, and a catalyst. The detection reagent A is pre-positioned in the reaction zone by adding the solution of the detection reagent A dropwise to the reaction zone and then freeze-drying it.

[0012] The detection reagent B contains a second excipient, a buffer, an oxidant, and a second masking agent. The detection reagent B solution is added dropwise to the color development area and then freeze-dried to pre-place the detection reagent B in the color development area.

[0013] Preferably, the color-developing area is a circle with a diameter of 4-12 mm;

[0014] Preferably, the width of the channel is 1-5mm and the length is 8-20mm;

[0015] Preferably, the reaction zone is a circle with a diameter of 3-10 mm.

[0016] Preferably, the sealing layer is made of PTFE or PP;

[0017] Preferably, the sealing layer has a length of 20-50 mm and a width of 15-40 mm.

[0018] Preferably, the injection port is a circle with a diameter of 2 mm, and the colorimetric port is a circle with a diameter of 4 mm.

[0019] Preferably, the background layer is made of an optical film, PET, or PP.

[0020] Preferably, the background layer has a length of 20-50 mm and a width of 15-40 mm.

[0021] A second aspect of the present invention provides a method for preparing a paper-based ammonia nitrogen detection chip, the method comprising the following steps:

[0022] (1) Use a laser engraving machine to engrave the filter paper to obtain a color development layer; the color development layer includes a reaction zone, a color development zone and a channel connecting the reaction zone and the color development zone;

[0023] (2) Add the test reagent A solution and the test reagent B solution to the color development area and the reaction area respectively, and then freeze dry to obtain a color development layer with test reagent A pre-placed in the reaction area and test reagent B pre-placed in the color development area;

[0024] (3) A sealing layer is provided, wherein the sealing layer is provided with an injection port and a color development port corresponding to the reaction zone and the color development zone respectively, and the size of the color development port is smaller than the size of the color development zone;

[0025] (4) Provide a background layer, and stack the background layer, the color development layer and the sealing layer obtained in step (2) from bottom to top;

[0026] Both the background layer and the sealing layer are made of hydrophobic materials.

[0027] The detection reagent A comprises a first excipient, a chromogenic agent, a first masking agent, a stabilizer, and a catalyst; the detection reagent B comprises a second excipient, a buffer, an oxidant, and a second masking agent.

[0028] Preferably, the first excipient is selected from one or more of mannitol, trehalose, dextran, and polyethylene glycol;

[0029] Preferably, the color developer is salicylic acid and / or sodium salicylate;

[0030] Preferably, the first masking agent is one or more of sodium tartrate, sodium citrate, sodium hexametaphosphate, and sodium EDTA.

[0031] Preferably, the stabilizer is selected from one or more of sodium chloride, sodium sulfate, and potassium chloride;

[0032] Preferably, the catalyst is sodium nitrosoferricyanide or potassium nitrosoferricyanide.

[0033] Preferably, in the detection reagent A, the weight ratio of the first excipient, the colorimetric agent, the first masking agent, the stabilizer, and the catalyst is 5.6-7.8:12-14.5:1.8-4.2:0.3-0.9:1;

[0034] Preferably, the concentration of test reagent A in the test reagent A solution is 1-15 wt%.

[0035] Preferably, the second excipient is selected from one or more of mannitol, trehalose, dextran, and polyethylene glycol;

[0036] Preferably, the buffer is selected from one or more of lithium hydroxide, disodium hydrogen phosphate, sodium hydroxide, and sodium phosphate;

[0037] Preferably, the oxidant is sodium dichloroisocyanurate;

[0038] Preferably, the second masking agent is selected from one or more of sodium tartrate, sodium citrate, sodium hexametaphosphate, and sodium EDTA salts.

[0039] Preferably, in the display reagent, the weight ratio of the second excipient, buffer, oxidant, and second masking agent is 8.9-11.7:4.8-6.5:0.8-1.4:1;

[0040] Preferably, the concentration of detection reagent B in the detection reagent B solution is 1-15 wt%.

[0041] Preferably, in step (2), the freeze-drying conditions include: a vacuum degree of 1-100 Pa and a time of 2-6 h.

[0042] A third aspect of the present invention provides a paper-based ammonia nitrogen detection chip prepared according to the method described above.

[0043] The fourth aspect of this invention provides the application of the paper-based ammonia nitrogen detection chip described above in the detection of ammonia nitrogen in water.

[0044] The fifth aspect of this invention provides a method for detecting ammonia nitrogen concentration in water, the method comprising the following steps:

[0045] The initial RGB value in the colorimetric port of the paper-based ammonia nitrogen detection chip is tested. Then, the solution to be tested is added dropwise to the injection port of the paper-based ammonia nitrogen detection chip. After the reaction, the measured RGB value in the colorimetric port is tested. The chromaticity distance D is determined based on the initial RGB value and the measured RGB value. Then, the concentration of ammonia nitrogen in the solution to be tested is determined by the chromaticity distance D.

[0046] The paper-based ammonia nitrogen detection chip is the same as the paper-based ammonia nitrogen detection chip described above.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1. This invention uses freeze-drying to evenly spread the detection reagents (including detection reagent A and detection reagent B) on the filter paper, which can carry more reagents than the impregnation method; and the internal structure of the detection reagents is loose and porous, resulting in a faster reaction rate and better color uniformity when in contact with the test solution.

[0049] 2. The paper-based ammonia nitrogen detection chip of the present invention uses hydrophobic materials for both the background layer and the sealing layer, eliminating the need for other reagents and methods to treat the paper-based chip with hydrophilicity or hydrophobicity, making the operation simple.

[0050] 3. The paper-based ammonia nitrogen detection chip of the present invention has the advantages of slowing down the liquid evaporation rate and preventing interference from external factors by setting a sealing layer; limiting the size of the color development port to be smaller than the color development area can weaken the coffee ring effect, gather the color intensity of colored particles to the central area, and increase the color intensity.

[0051] 4. The preparation method of the paper-based ammonia nitrogen detection chip of the present invention is simple and can be mass-produced.

[0052] 5. The paper-based ammonia nitrogen detection chip of this invention only requires adding the test solution to the sample inlet. The solution reacts sequentially with reagents built into the reaction zone and the colorimetric zone, and then develops color in the colorimetric zone. Concentration is identified by colorimetric quantification. The colorimetric results within the colorimetric port can be obtained with the help of imaging equipment and image processing software. The concentration of the test solution can be quickly and accurately analyzed using the colorimetric-concentration analysis method, which has higher accuracy and avoids the influence of subjective judgment on the results. Attached Figure Description

[0053] Figure 1 This is an exploded schematic diagram of the paper-based ammonia nitrogen detection chip described in this invention;

[0054] Figure 2 This is a schematic diagram of the colorimetric layer in the paper-based ammonia nitrogen detection chip of the present invention;

[0055] Figure 3 These are photographs taken after the paper-based ammonia nitrogen detection chip prepared in Example 1 has developed color after the detection solution has been added;

[0056] Figure 4 This is a graph showing the fitting results of chromaticity distance D and concentration in test example 2.

[0057] Explanation of reference numerals in the attached figures

[0058] 1. Background layer 2. Developing layer

[0059] 3 sealing layer 21 reaction zone

[0060] 22 channels, 23 colorimetric zones

[0061] 31 Inlet port 32 Developing port Detailed Implementation

[0062] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0063] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0064] In the description of this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating relative importance or implying the number of technical features indicated. Therefore, unless otherwise stated, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature; "multiple" means two or more. The term "comprising" and any variations thereof mean non-exclusive inclusion, where one or more other features, integers, steps, operations, units, components, and / or combinations thereof may be present or added.

[0065] This invention provides a paper-based ammonia nitrogen detection chip, in conjunction with reference to... Figure 1-2 The chip consists of a background layer 1, a color development layer 2, and a sealing layer 3 from bottom to top.

[0066] The color development layer 2 is provided with a reaction zone 21, a color development zone 23, and a channel 22 connecting the reaction zone and the color development zone 23; a detection reagent A is pre-placed in the reaction zone 21, and a detection reagent B is pre-placed in the color development zone 23;

[0067] The sealing layer 3 is provided with an inlet 31 and a colorimetric port 32 corresponding to the reaction zone 21 and the colorimetric zone 23, respectively, and the size of the colorimetric port 32 is smaller than the size of the colorimetric zone;

[0068] The color developing layer 2 is made of filter paper, and the background layer 1 and the sealing layer 3 are both made of hydrophobic materials;

[0069] The detection reagent A contains a first excipient, a chromogenic agent, a first masking agent, a stabilizer, and a catalyst. The detection reagent A solution is added dropwise to the reaction zone 21 and then freeze-dried to pre-place the detection reagent A in the reaction zone.

[0070] The detection reagent B contains a second excipient, a buffer, an oxidant, and a second masking agent. The detection reagent B solution is added dropwise to the color development area 23 and then freeze-dried to pre-place the detection reagent B in the color development area.

[0071] In this invention, by using freeze-drying to embed test reagent A and test reagent B within the reaction zone 21 and the color development zone 23, the reagents can be made into a loose and porous state, resulting in faster reaction and more uniform color development when in contact with the solution. By setting a sealing layer 3, and having a color development port 32 with a size smaller than that of the color development zone 23, the evaporation rate of the solution can be slowed down, the coffee ring effect can be weakened, and the color particles can be better reflected in the color development zone, increasing the color intensity.

[0072] In a preferred embodiment, the color development area 23 is a circle with a diameter of 4-12 mm, more preferably a circle with a diameter of 5-8 mm.

[0073] In this invention, the reaction zone 21 and the color development zone 23 are connected by a channel 22, which can be elongated. Preferably, the width of the channel 22 is 1-5 mm, more preferably 2-4 mm; and the length is 8-20 mm, more preferably 12-16 mm.

[0074] In this invention, the color development layer 2 can be directly constructed using filter paper. The filter paper can be a medium-flow-rate filter paper with a particle retention diameter of 6-20 μm. For example, Whatman No. 1 filter paper can be used.

[0075] In a preferred embodiment, the reaction zone 21 is a circle with a diameter of 3-10 mm.

[0076] In this invention, the sealing layer 3 is preferably made of a transparent material with hydrophobic properties, such as PTFE (polytetrafluoroethylene) or PP (polypropylene). More preferably, the sealing layer 3 has a length of 20-50 mm and a width of 15-40 mm.

[0077] In a preferred embodiment, the injection port 31 is a circle with a diameter of 2-3 mm, and the colorimetric port 32 is a circle with a diameter of 3-5 mm.

[0078] In this invention, the background layer 1 is preferably made of a white material with hydrophobic and adhesive properties. Its hydrophobic properties allow for the construction of hydrophobic regions on the chip, while its adhesive properties stably fix the color development layer 2 and the sealing layer 3 onto the background layer 1. The white color provides a base color background for the developed chip. The material of the background layer 1 can be, for example, an optical film, PET (polyethylene terephthalate), or PP. Preferably, the background layer 1 has a length of 20-50 mm and a width of 15-40 mm.

[0079] Conventional paper-based chips typically require the construction of hydrophilic and hydrophobic channels on a paper base. However, in this invention, both the background layer 1 and the sealing layer 3 are made of hydrophobic materials and bonded together, naturally forming a hydrophobic region. Since the paper-based chip is hydrophilic, the detection solution flows only along the hydrophilic paper base and does not flow to other areas.

[0080] In this invention, ammonia nitrogen in water reacts with a detection reagent to generate a blue substance, and the ammonia nitrogen in the water can be detected by the color change.

[0081] In a preferred embodiment, the weight ratio of the first excipient, chromogenic agent, first masking agent, stabilizer and catalyst in the detection reagent A is 5.6-7.8:12-14.5:1.8-4.2:0.3-0.9:1.

[0082] According to some preferred embodiments of the present invention, the first excipient is selected from mannitol, trehalose, dextran, or polyethylene glycol; the color developer is salicylic acid or sodium salicylate; the first masking agent is one or more of sodium tartrate, sodium citrate, sodium hexametaphosphate, and sodium EDTA; the stabilizer is one or more of sodium chloride, sodium sulfate, and potassium chloride; and the catalyst is sodium nitrosoferricyanide or potassium nitrosoferricyanide.

[0083] In a preferred embodiment, the weight ratio of the second excipient, buffer, oxidant and second masking agent in the detection reagent B is 8.9-11.7:4.8-6.5:0.8-1.4:1.

[0084] According to some preferred embodiments of the present invention, the second excipient is selected from mannitol, trehalose, dextran, or polyethylene glycol; the buffer is selected from one or more of lithium hydroxide, disodium hydrogen phosphate, sodium hydroxide, and sodium phosphate; the oxidant is sodium dichloroisocyanurate; and the second masking agent is selected from one or more of sodium tartrate, sodium citrate, sodium hexametaphosphate, and sodium EDTA salts.

[0085] A second aspect of the present invention provides a method for preparing a paper-based ammonia nitrogen detection chip, the method comprising the following steps:

[0086] (1) Use a laser engraving machine to engrave the filter paper to obtain a color development layer; the color development layer includes a reaction zone, a color development zone and a channel connecting the reaction zone and the color development zone;

[0087] (2) Add the test reagent A solution and the test reagent B solution to the color development area and the reaction area respectively, and then freeze dry to obtain a color development layer with test reagent A pre-placed in the reaction area and test reagent B pre-placed in the color development area;

[0088] (3) A sealing layer is provided, wherein the sealing layer is provided with an injection port and a color development port corresponding to the reaction zone and the color development zone respectively, and the size of the color development port is smaller than the size of the color development zone;

[0089] (4) Provide a background layer, and stack the background layer, the color development layer and the sealing layer obtained in step (2) from bottom to top;

[0090] Both the background layer and the sealing layer are made of hydrophobic materials.

[0091] The detection reagent A comprises a first excipient, a chromogenic agent, a first masking agent, a stabilizer, and a catalyst; the detection reagent B comprises a second excipient, a buffer, an oxidant, and a second masking agent.

[0092] In the method described in this invention, a color-developing layer with a specific composition can be obtained by engraving the filter paper using a laser engraving machine; that is, the color-developing layer is directly constructed from the filter paper. There are no special requirements for the selection of the laser engraving machine; any conventional choice in the art can be used.

[0093] In the method described in this invention, the color-developing area and the reaction area in the color-developing layer are connected by channels. Preferably, the color-developing area is a circle with a diameter of 4-12 mm, more preferably a circle with a diameter of 5-8 mm; the channel can be elongated, with a channel width of 1-5 mm, more preferably 2-4 mm; and a channel length of 8-20 mm, more preferably 12-16 mm; the reaction area is a circle with a diameter of 3-10 mm.

[0094] In a preferred embodiment, in detection reagent A, the first excipient is selected from mannitol, trehalose, dextran, or polyethylene glycol; the colorimetric agent is salicylic acid or sodium salicylate; the first masking agent is one or more of sodium tartrate, sodium citrate, sodium hexametaphosphate, and sodium EDTA; the stabilizer is one or more of sodium chloride, sodium sulfate, and potassium chloride; and the catalyst is sodium nitrosoferricyanide or potassium nitrosoferricyanide.

[0095] In this invention, the amount of each component in the detection reagent A can be reasonably controlled, which is beneficial to increasing the colorimetric stability and accelerating the reaction rate. In a preferred embodiment, the weight ratio of the first excipient, the colorimetric agent, the first masking agent, the stabilizer, and the catalyst in the detection reagent A is 5.6-7.8:12-14.5:1.8-4.2:0.3-0.9:1.

[0096] In the method described in this invention, if the concentration of reagent A in the reagent A solution is too low, the internal crystals after lyophilization will be large, resulting in a lower color density after color development and reducing the accuracy of the detection results. If the concentration is too high, the internal density after lyophilization will be greater, the crystals will be smaller, and the color density after color development will be higher, leading to a decrease in the reaction rate between reagent A and the test solution. Preferably, the concentration of reagent A in the reagent A solution is 1-15 wt%, more preferably 5-12 wt%. The solvent in the reagent A solution can be water. The reagent A solution can be obtained by mixing water and reagent A.

[0097] In a preferred embodiment, in the detection reagent B, the second excipient is selected from mannitol, trehalose, dextran, or polyethylene glycol; the buffer is selected from one or more of lithium hydroxide, disodium hydrogen phosphate, sodium hydroxide, and sodium phosphate; the oxidant is sodium dichloroisocyanurate; and the second masking agent is selected from one or more of sodium tartrate, sodium citrate, sodium hexametaphosphate, and sodium EDTA.

[0098] In the method described in this invention, the amount of each component in the detection reagent B can be reasonably controlled, which is beneficial for uniform color development and accelerating the color development reaction rate. In a preferred embodiment, the weight ratio of the second excipient, buffer, oxidant and second masking agent in the detection reagent B is 8.9-11.7:4.8-6.5:0.8-1.4:1.

[0099] In the method described in this invention, if the concentration of reagent B in the reagent B solution is too low, the internal crystals after lyophilization will be large, resulting in a lower color density after color development and reducing the accuracy of the detection results; if the concentration is too high, the reaction rate between reagent B and the test solution will decrease. Therefore, in a preferred embodiment, the concentration of reagent B in the reagent B solution is 1-15 wt%, more preferably 5-12 wt%. In the method described in this invention, in step (2), the colorimetric layer obtained in step (1) can be pre-frozen in liquid nitrogen, and then reagent A solution and reagent B solution can be added dropwise to the reaction zone and colorimetric zone respectively, followed by freeze-drying.

[0100] In this invention, if the volume of the detection reagent A solution added to the reaction zone is too large, it will cause the paper substrate to bear too much liquid, forming arc-shaped droplets on the paper substrate surface. After freeze-drying, this forms a raised reagent layer, resulting in uneven reagent distribution. Therefore, the relationship between the added detection reagent A solution and the circular diameter of the reaction zone can be reasonably controlled. In a preferred embodiment, the ratio of the volume of the detection reagent A solution to the circular diameter of the reaction zone is 2-5 μL:1 mm.

[0101] In this invention, if the volume of the detection reagent B solution added to the display area is too large, the paper substrate will bear too much liquid, forming arc-shaped droplets on the surface. After freeze-drying, this forms a raised reagent layer, resulting in uneven reagent distribution. Therefore, the relationship between the added detection reagent B solution and the diameter of the display area can be reasonably controlled. In a preferred embodiment, the ratio of the volume of the display reagent solution to the circular diameter of the display area is 2-5 μL: 1 mm.

[0102] In the method described in this invention, the pre-positioning of reagent B and reagent A is achieved by dropwise addition followed by freeze-drying. This allows the reagents to be evenly distributed on the filter paper in a loose and porous state. This improves the reaction efficiency between the solution and the reagents, while also enhancing the uniformity of color development.

[0103] In a preferred embodiment, the freeze-drying conditions in step (2) include: a vacuum degree of 1-100 Pa and a time of 2-6 h.

[0104] In this invention, the sealing layer is preferably made of a transparent material with hydrophobic properties, such as PTFE or PP. More preferably, the sealing layer has a length of 20-50 mm and a width of 15-40 mm.

[0105] In a preferred embodiment, the injection port is a circle with a diameter of 2-3 mm, and the colorimetric port is a circle with a diameter of 3-5 mm.

[0106] In this invention, the background layer is preferably made of a white material with hydrophobic and adhesive properties. Its hydrophobic properties allow for the construction of hydrophobic channels, while its adhesive properties ensure stable fixation of the developing layer and the sealing layer to the background layer. The white color effectively contrasts with the color of the developed chip. The background layer can be made of materials such as optical film, PET, or PP. Preferably, the background layer has a length of 20-50 mm and a width of 15-40 mm.

[0107] In the method described in this invention, during chip assembly in step (4), the background layer, color development layer, and sealing layer can be stacked sequentially from bottom to top. The background layer is adhesive, allowing the three layers to adhere tightly. Preferably, by stacking, the injection port and reaction zone are coaxially aligned (i.e., their centers are on the same axis), and the color development port and color development zone are coaxially aligned. After assembly, a portion of the reaction zone is exposed through the injection port, and a portion of the color development zone is exposed through the color development port.

[0108] A third aspect of the present invention provides a paper-based ammonia nitrogen detection chip prepared according to the method described above.

[0109] The fourth aspect of this invention provides the application of the paper-based ammonia nitrogen detection chip described above in the detection of ammonia nitrogen in water.

[0110] The paper-based ammonia nitrogen detection chip described in this invention can be used in environmental and water quality monitoring for the detection of ammonia nitrogen in water bodies, enabling both qualitative and quantitative detection. It offers advantages such as fast detection speed, ease of operation, and high accuracy. It is suitable for detecting ammonia nitrogen in water from industrial wastewater, domestic sewage, aquaculture water, and natural water sources.

[0111] The fifth aspect of this invention provides a method for detecting ammonia nitrogen concentration in water, the method comprising the following steps:

[0112] The initial RGB value in the colorimetric port 32 of the paper-based ammonia nitrogen detection chip is tested. Then, the solution to be tested is added dropwise to the sample inlet 31 of the paper-based ammonia nitrogen detection chip. After the reaction, the measured RGB value in the colorimetric port 32 is tested. The chromaticity distance D is determined based on the initial RGB value and the measured RGB value. Then, the concentration of ammonia nitrogen in the solution to be tested is determined by the chromaticity distance D.

[0113] The paper-based ammonia nitrogen detection chip is the same as the paper-based ammonia nitrogen detection chip described above.

[0114] In a preferred embodiment, the volume of the test solution can be 15-30 μL. Using the paper-based ammonia nitrogen detection chip of the present invention to detect ammonia nitrogen in water requires only a small amount of solution to obtain accurate results quickly.

[0115] In this invention, in the paper-based ammonia nitrogen detection chip, a portion of the color development area 23 is exposed through the color development port 32. Therefore, during use, the RGB value within the color development port 32 refers to the RGB value of the area of ​​the color development area 23 exposed through the color development port 32.

[0116] In this invention, the detection reagent A pre-placed in the reaction zone 21 and the detection reagent B pre-placed in the color development zone 23 of the paper-based ammonia nitrogen detection chip can both react rapidly with the water sample; it has the advantages of fast detection speed and accurate detection results. In a preferred embodiment, the water sample is added to the inlet 31 of the paper-based ammonia nitrogen detection chip. After blue appears in the color development port 32, wait 2-4 minutes, and then test the RGB value in the color development port 32.

[0117] In this invention, the RGB values ​​include R, G, and B values, which refer to the brightness values ​​of the three basic colors: red (R), green (G), and blue (B), respectively. The initial and measured RGB values ​​of the color display port can be obtained by taking a picture of the color display port 32 using a camera (e.g., a mobile phone), and then using MATLAB to identify and analyze the valid image within the color display port 32.

[0118] In the method described in this invention, the formula for calculating the chromaticity distance D is:

[0119]

[0120] Where D is the chromaticity distance, R, G and B are the measured R, G and B values ​​respectively, and R0, G0 and B0 are the initial R, G and B values ​​respectively.

[0121] In a preferred embodiment, the external standard method can be used to quantitatively detect the concentration of ammonia nitrogen in a water sample. At least five ammonia nitrogen standard solutions of known concentrations are prepared. The chromaticity distance D of each standard solution is measured using a paper-based ammonia nitrogen detection chip, and the relationship between concentration and chromaticity distance D is calculated. Then, the chromaticity distance D of the water sample is measured using the same method, and the concentration of ammonia nitrogen in the test solution is calculated using the relationship between the chromaticity distance D of the water sample and the concentration and chromaticity distance D. The paper-based ammonia nitrogen detection chips used for the standard solutions and the water sample are from the same batch (i.e., prepared under the same conditions), and the injection volumes of the standard solution and the water sample are the same during detection. In a specific embodiment, the ammonia nitrogen standard substance (100 mg / L) can be purchased from the National Institute of Metrology of China and diluted a certain factor before use.

[0122] According to some preferred embodiments of the present invention, the method for detecting ammonia nitrogen concentration in water includes the following steps:

[0123] S1: Test the initial RGB value in the color development port 32 of the paper-based ammonia nitrogen detection chip, and then add the standard solution to the injection port 31 of the paper-based ammonia nitrogen detection chip. After the blue color appears in the color development port 32, wait for 2-4 minutes, and then test the measured RGB value in the color development port 32 after the reaction. Determine the color distance D of the standard solution based on the initial RGB value and the measured RGB value.

[0124] S2: Fit the equations with chromaticity distance D as the ordinate and ammonia nitrogen concentration as the abscissa to obtain the relationship between chromaticity distance D and ammonia nitrogen concentration;

[0125] S3: Test the initial RGB value in the colorimetric port 32 of the paper-based ammonia nitrogen detection chip, then add the water sample to the inlet 31 of the paper-based ammonia nitrogen detection chip. After blue appears in the colorimetric port 32, wait for 2-4 minutes, then test the measured RGB value in the colorimetric port after the reaction. Determine the chromaticity distance D of the water sample based on the initial RGB value and the measured RGB value. Substitute the chromaticity distance D of the water sample into the relationship obtained in step S2 for calculation to obtain the concentration of ammonia nitrogen in the water sample.

[0126] The formula for calculating the chromaticity distance D is as follows:

[0127]

[0128] Where D is the chromaticity distance, R, G and B are the measured R, G and B values ​​respectively, and R0, G0 and B0 are the initial R, G and B values ​​respectively.

[0129] In the preferred embodiment, the paper-based ammonia nitrogen detection chips used for the standard solution and water sample are produced in the same batch, meaning that the chip parameters and reagents contained in the chip are the same; and the volume of the standard solution and water sample used is the same.

[0130] The method for detecting ammonia nitrogen concentration in solution described in this invention utilizes the aforementioned paper-based ammonia nitrogen detection chip, which can quickly and accurately detect the ammonia nitrogen concentration in water samples. Since the detection chip described in this invention can be used with a mobile phone, it has advantages such as ease of operation and fast detection speed, providing a convenient and effective new approach for non-professionals to detect ammonia nitrogen concentration in water in real time on-site.

[0131] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0132] Example 1

[0133] Preparation of paper-based ammonia nitrogen detection chip S1:

[0134] (1) Use a laser engraving machine to engrave Whatman No. 1 filter paper to obtain a color development layer; the color development layer includes a reaction area, a color development area and a channel connecting the reaction area and the color development area; wherein, the reaction area is a circle with a diameter of 7mm, the channel is a rectangle with a width of 3mm and a length of 10mm, and the color development area is a circle with a diameter of 5mm.

[0135] (2) Prepare a solution of reagent A with a concentration of 8 wt% by mixing reagent A with deionized water. The reagent A is composed of a first excipient (mannitol), a colorimetric agent (sodium salicylate), a first masking agent (sodium tartrate), a stabilizer (sodium chloride), and a catalyst (sodium nitrosoferricyanide) in a weight ratio of 5.8:10.2:2.4:0.6:1.

[0136] The test reagent B is mixed with deionized water to prepare a test reagent B solution with a concentration of 8 wt%. The test reagent B is composed of a second excipient (mannitol), a buffer (lithium hydroxide), an oxidant (sodium dichloroisocyanurate), and a second masking agent (sodium tartrate) in a weight ratio of 8:6.5:1.2:1.

[0137] The colorimetric layer was pre-frozen in liquid nitrogen for 30 seconds. Then, 20 μL of reagent A solution and 15 μL of reagent B solution were added dropwise to the colorimetric area and the reaction area, respectively. The mixture was then freeze-dried to obtain a colorimetric layer with reagent A pre-placed in the reaction area and reagent B pre-placed in the colorimetric area. The freeze-drying was performed under a vacuum of 10 Pa for 3 hours.

[0138] (3) Provide a sealing layer (PTFE material, 35mm in length and 20mm in width), the sealing layer is provided with an injection port (a hollow circle with a diameter of 2mm) and a color development port (a hollow circle with a diameter of 4mm) corresponding to the reaction zone and the color development zone respectively;

[0139] (4) Provide a background layer (adhesive optical film, 35mm in length and 20mm in width), and stack the background layer, the color development layer obtained in step (2), and the sealing layer in sequence from bottom to top to obtain the paper-based ammonia nitrogen detection chip S1. The exploded schematic diagram of the detection chip is shown below. Figure 1 As shown, a schematic diagram of the color development layer in the detection chip is as follows: Figure 2 As shown.

[0140] Example 2

[0141] Preparation of paper-based ammonia nitrogen detection chip S2:

[0142] (1) Use a laser engraving machine to engrave Whatman No. 1 filter paper to obtain a color development layer; the color development layer includes a reaction area, a color development area and a channel connecting the reaction area and the color development area; wherein, the reaction area is a circle with a diameter of 7mm, the channel is a rectangle with a width of 3mm and a length of 10mm, and the color development area is a circle with a diameter of 5mm.

[0143] (2) Prepare a solution of test reagent A with a concentration of 8 wt% by mixing test reagent A with deionized water. The test reagent A is composed of a first excipient (mannitol), a colorimetric agent (sodium salicylate), a first masking agent (sodium tartrate), a stabilizer (sodium chloride), and a catalyst (sodium nitrosoferricyanide) in a weight ratio of 5.4:11:2:0.6:1.

[0144] The test reagent B is mixed with deionized water to prepare a test reagent B solution with a concentration of 8 wt%. The test reagent B is composed of a second excipient (mannitol), a buffer (lithium hydroxide), an oxidant (sodium dichloroisocyanurate), and a second masking agent (sodium tartrate) in a weight ratio of 7.5:6.7:1.4:1.

[0145] The colorimetric layer was pre-frozen in liquid nitrogen for 30 seconds. Then, 20 μL of reagent A solution and 15 μL of reagent B solution were added dropwise to the colorimetric area and the reaction area, respectively. The mixture was then freeze-dried to obtain a colorimetric layer with reagent A pre-placed in the reaction area and reagent B pre-placed in the colorimetric area. The freeze-drying was performed under a vacuum of 30 Pa for 4 hours.

[0146] (3) Provide a sealing layer (PTFE material, 35mm in length and 20mm in width), the sealing layer is provided with an injection port (a hollow circle with a diameter of 2mm) and a color development port (a hollow circle with a diameter of 4mm) corresponding to the reaction zone and the color development zone respectively;

[0147] (4) Provide a background layer (adhesive optical film, 35 mm in length and 20 mm in width), and stack the background layer, the color development layer and the sealing layer obtained in step (2) in sequence from bottom to top.

[0148] Example 3

[0149] Preparation of paper-based ammonia nitrogen detection chip S3:

[0150] (1) Use a laser engraving machine to engrave Whatman No. 1 filter paper to obtain a color development layer; the color development layer includes a reaction area, a color development area and a channel connecting the reaction area and the color development area; wherein, the reaction area is a circle with a diameter of 7mm, the channel is a rectangle with a width of 3mm and a length of 10mm, and the color development area is a circle with a diameter of 5mm.

[0151] (2) Prepare a solution of reagent A with a concentration of 8 wt% by mixing reagent A with deionized water. The reagent A is composed of a first excipient (mannitol), a colorimetric agent (sodium salicylate), a first masking agent (sodium tartrate), a stabilizer (sodium chloride), and a catalyst (sodium nitrosoferricyanide) in a weight ratio of 6.5:9.1:2.6:0.8:1.

[0152] The test reagent B is mixed with deionized water to prepare a test reagent B solution with a concentration of 8 wt%. The test reagent B is composed of a second excipient (mannitol), a buffer (lithium hydroxide), an oxidant (sodium dichloroisocyanurate), and a second masking agent (sodium tartrate) in a weight ratio of 7:7.1:1.6:1.

[0153] The colorimetric layer was pre-frozen in liquid nitrogen for 30 seconds. Then, 15 μL of reagent A solution and 12 μL of reagent B solution were added dropwise to the colorimetric area and the reaction area, respectively. The mixture was then freeze-dried to obtain a colorimetric layer with reagent A pre-placed in the reaction area and reagent B pre-placed in the colorimetric area. The freeze-drying was performed under a vacuum of 10 Pa for 3 hours.

[0154] (3) Provide a sealing layer (PTFE material, 35mm in length and 20mm in width), the sealing layer is provided with an injection port (a hollow circle with a diameter of 2mm) and a color development port (a hollow circle with a diameter of 4mm) corresponding to the reaction zone and the color development zone respectively;

[0155] (4) Provide a background layer (adhesive optical film, 35 mm in length and 20 mm in width), and stack the background layer, the color development layer and the sealing layer obtained in step (2) in sequence from bottom to top.

[0156] Example 4

[0157] Preparation of paper-based ammonia nitrogen detection chip S4:

[0158] (1) Use a laser engraving machine to engrave Whatman No. 1 filter paper to obtain a color development layer; the color development layer includes a reaction area, a color development area and a channel connecting the reaction area and the color development area; wherein, the reaction area is a circle with a diameter of 7mm, the channel is a rectangle with a width of 3mm and a length of 10mm, and the color development area is a circle with a diameter of 5mm.

[0159] (2) Prepare a 6wt% solution of test reagent A by mixing test reagent A with deionized water. The test reagent A is composed of a first excipient (mannitol), a colorimetric agent (sodium salicylate), a first masking agent (sodium tartrate), a stabilizer (sodium chloride), and a catalyst (sodium nitrosoferricyanide) in a weight ratio of 6.1:10:2.5:1:1.

[0160] The test reagent B is mixed with deionized water to prepare a 6 wt% test reagent B solution, wherein the test reagent B is composed of a second excipient (mannitol), a buffer (lithium hydroxide), an oxidant (sodium dichloroisocyanurate), and a second masking agent (sodium tartrate) in a weight ratio of 8.7:6:0.9:1.

[0161] The colorimetric layer was pre-frozen in liquid nitrogen for 30 seconds. Then, 20 μL of reagent A solution and 15 μL of reagent B solution were added dropwise to the colorimetric area and the reaction area, respectively. The mixture was then freeze-dried to obtain a colorimetric layer with reagent A pre-placed in the reaction area and reagent B pre-placed in the colorimetric area. The freeze-drying was performed under a vacuum of 10 Pa for 3 hours.

[0162] (3) Provide a sealing layer (PTFE material, 35mm in length and 20mm in width), the sealing layer is provided with an injection port (a hollow circle with a diameter of 2mm) and a color development port (a hollow circle with a diameter of 4mm) corresponding to the reaction zone and the color development zone respectively;

[0163] (4) Provide a background layer (adhesive optical film, 35 mm in length and 20 mm in width), and stack the background layer, the color development layer and the sealing layer obtained in step (2) in sequence from bottom to top.

[0164] Example 5

[0165] The method described in Example 1 is implemented, except that in step (2), the concentration of reagent A solution is 3 wt% and the concentration of reagent B solution is 3 wt%.

[0166] Example 6

[0167] The method described in Example 1 is implemented, except that in step (2), the concentration of reagent A solution is 15 wt% and the concentration of reagent B solution is 15 wt%.

[0168] Example 7

[0169] The method described in Example 1 was implemented, except that in step (2), 40 μL of reagent A solution and 30 μL of reagent B solution were added dropwise to the color development area and the reaction area, respectively.

[0170] Example 8

[0171] The method described in Example 1 is implemented, except that in step (2), the amount of the first excipient and the second excipient is reduced by 50%, that is, the weight ratio of the first excipient to the catalyst in the detection reagent A is adjusted to 2.9:1, and the weight ratio of the second excipient to the second masking agent in the detection reagent B is adjusted to 4:1.

[0172] Comparative Example 1

[0173] The method described in Example 1 is implemented, except that the detection reagent A does not contain the first excipient, and the detection reagent B does not contain the second excipient.

[0174] Comparative Example 2

[0175] The method described in Example 1 is implemented, except that in step (2), the test reagent A and test reagent B are not pre-prepared by freeze drying.

[0176] Specifically, step (2) is as follows:

[0177] The test reagent A is mixed with deionized water to prepare a test reagent A solution with a concentration of 8 wt%. The test reagent A is composed of a first excipient (mannitol), a colorimetric agent (sodium salicylate), a first masking agent (sodium tartrate), a stabilizer (sodium chloride), and a catalyst (sodium nitrosoferricyanide) in a weight ratio of 5.8:10.2:2.4:0.6:1.

[0178] The test reagent B is mixed with deionized water to prepare a test reagent B solution with a concentration of 8 wt%. The test reagent B is composed of a second excipient (mannitol), a buffer (lithium hydroxide), an oxidant (sodium dichloroisocyanurate), and a second masking agent (sodium tartrate) in a weight ratio of 8:6.5:1.2:1.

[0179] The reaction zone and the color development zone of the color development layer are immersed in the prepared test reagent A solution and test reagent B solution, respectively, for 10 minutes. After immersion, they are removed and dried in a drying oven at 30°C.

[0180] Test Example 1

[0181] The color development effect of the detection chips prepared in the examples and comparative examples was tested, and the test results are shown in Table 1.

[0182] The detection method is as follows: use a smartphone to take a picture of the color display port of the detection chip, then use MATLAB to identify and analyze the valid image in the color display port to obtain the initial RGB value in the color display port.

[0183] Take 20 μL of ammonia nitrogen standard solution with a concentration of 2.5 mg / L using a pipette and inject it into the detection chip through the injection port. After the color port appears blue, wait for a certain period of time and take a picture of the color port of the detection chip with a smartphone. Then, MATLAB identifies and analyzes the effective image in the color port to obtain the measured RGB value in the color port.

[0184] The chromaticity distance D is calculated based on the initial RGB values ​​and the measured RGB values. The calculation formula is as follows:

[0185]

[0186] Where D is the chromaticity distance, R, G and B are the measured R, G and B values ​​respectively, and R0, G0 and B0 are the initial R, G and B values ​​respectively.

[0187] The test results are shown in Table 1, where the chromatic distance D is the average value of four repeated tests. The photographs taken after the detection chip developed color during the testing of the product prepared in Example 1 are shown below. Figure 3 As shown.

[0188] Table 1

[0189] Test sample waiting time min Chromaticity distance D Example 1 2 75 Example 2 2 71 Example 3 2 69 Example 4 2 64 Example 5 2 60 Example 6 2 62 Example 7 2 61 Example 8 2 60 Example 1 0.5 40 Example 1 5 65 Comparative Example 1 2 53 Comparative Example 2 2 50

[0190] As shown in Table 1, the paper-based ammonia nitrogen detection chip described in the examples has the advantages of fast detection rate, accurate detection results, and uniform color development. However, the chip prepared in Comparative Example 1, due to the lack of excipients, suffered from lyophilized structure collapse, affecting reagent pre-conditioning and resulting in uneven color development and a small chromaticity distance. Comparative Example 2, which used a non-freeze-treatment method for chip preparation, produced a chip with no loose reagent structure, dense reagent particles, a slower reaction rate, and a significantly smaller chromaticity distance, thus failing to achieve rapid detection.

[0191] Test Example 2

[0192] Ammonia nitrogen standard solutions with concentrations of 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1 mg / L, 2.5 mg / L, 5 mg / L, and 8 mg / L were prepared. Six paper-based ammonia nitrogen detection chips S1 were prepared, and detection was performed according to the method described in Test Example 1 (waiting time was 2 min). The chromaticity distances obtained were 17, 25, 39, 70, 120, and 168, respectively. Concentration-chromaticity distance curves were plotted and fitted using the above data. The results are as follows. Figure 4 As shown, and R 2 =0.9912, and the fitted relationship is y = 16.238x + 21.086, indicating a good linear relationship. This shows that the ammonia nitrogen detection chip described in this invention can achieve quantitative detection of ammonia nitrogen in water samples.

[0193] Test Example 3

[0194] The method described in Test Example 1 (with a waiting time of 2 minutes) was used to test six ammonia nitrogen detection chips prepared according to the method described in Example 1. The measured chromaticity distances were 73, 75, 71, 70, 76, and 70, with an average value of 72.5 and a relative standard deviation of 3.57%.

[0195] The results above show that the paper-based ammonia nitrogen detection chip described in this invention has the advantages of high detection efficiency and good repeatability in detecting ammonia nitrogen in water.

[0196] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A paper-based ammonia nitrogen detection chip, characterized in that, The chip consists of a background layer (1), a color development layer (2), and a sealing layer (3) from bottom to top. The color development layer (2) is provided with a reaction zone (21), a color development zone (23), and a channel (22) connecting the reaction zone (21) and the color development zone (23); a detection reagent A is pre-placed in the reaction zone (21), and a detection reagent B is pre-placed in the color development zone (23); The sealing layer (3) is provided with an injection port (31) and a color development port (32) corresponding to the reaction zone (21) and the color development zone (23) respectively, and the size of the color development port (32) is smaller than the size of the color development zone (23); The color development layer (2) is made of filter paper, and the background layer (1) and the sealing layer (3) are both made of hydrophobic materials; The detection reagent A contains a first excipient, a colorimetric agent, a first masking agent, a stabilizer, and a catalyst. The detection reagent A solution is added dropwise to the reaction zone (21) and then freeze-dried to pre-place the detection reagent A in the reaction zone (21). The detection reagent B contains a second excipient, a buffer, an oxidant, and a second masking agent. The detection reagent B solution is added dropwise to the color development area (23) and then freeze-dried to pre-place the detection reagent B in the color development area (23).

2. The paper-based ammonia nitrogen detection chip according to claim 1, characterized in that, The color-developing area (23) is a circle with a diameter of 4-12 mm; Preferably, the width of the channel (22) is 1-5 mm and the length is 8-20 mm; Preferably, the reaction zone (21) is a circle with a diameter of 3-10 mm.

3. The paper-based ammonia nitrogen detection chip according to claim 1 or 2, characterized in that, The material of the sealing layer (3) is PTFE or PP; Preferably, the sealing layer (3) has a length of 20-50 mm and a width of 15-40 mm.

4. The paper-based ammonia nitrogen detection chip according to claim 1 or 2, characterized in that, The injection port (31) is a circle with a diameter of 2 mm, and the color development port (32) is a circle with a diameter of 4 mm.

5. The paper-based ammonia nitrogen detection chip according to claim 1, characterized in that, The background layer (1) is made of optical film, PET or PP; Preferably, the background layer (1) has a length of 20-50 mm and a width of 15-40 mm.

6. A method for preparing a paper-based ammonia nitrogen detection chip, characterized in that, The method includes the following steps: (1) Use a laser engraving machine to engrave the filter paper to obtain a color development layer; the color development layer includes a reaction zone, a color development zone, and a channel connecting the reaction zone and the color development zone; (2) Add the test reagent A solution and the test reagent B solution to the color development area and the reaction area respectively, and then freeze dry to obtain a color development layer with test reagent A pre-placed in the reaction area and test reagent B pre-placed in the color development area; (3) A sealing layer is provided, wherein the sealing layer is provided with an injection port and a color development port corresponding to the reaction zone and the color development zone respectively, and the size of the color development port is smaller than the size of the color development zone; (4) Provide a background layer, and stack the background layer, the color development layer and the sealing layer obtained in step (2) from bottom to top; Both the background layer and the sealing layer are made of hydrophobic materials. The detection reagent A comprises a first excipient, a chromogenic agent, a first masking agent, a stabilizer, and a catalyst; the detection reagent B comprises a second excipient, a buffer, an oxidant, and a second masking agent.

7. The method according to claim 6, characterized in that, The first excipient is selected from one or more of mannitol, trehalose, dextran, and polyethylene glycol; Preferably, the color developer is salicylic acid and / or sodium salicylate; Preferably, the first masking agent is one or more of sodium tartrate, sodium citrate, sodium hexametaphosphate, and sodium EDTA. Preferably, the stabilizer is selected from one or more of sodium chloride, sodium sulfate, and potassium chloride; Preferably, the catalyst is sodium nitrosoferricyanide or potassium nitrosoferricyanide.

8. The method according to claim 6 or 7, characterized in that, In the detection reagent A, the weight ratio of the first excipient, chromogenic agent, first masking agent, stabilizer, and catalyst is 5.6-7.8:12-14.5:1.8-4.2:0.3-0.9:

1. Preferably, the concentration of test reagent A in the test reagent A solution is 1-15 wt%.

9. The method according to claim 6, characterized in that, The second excipient is selected from one or more of mannitol, trehalose, dextran and polyethylene glycol; Preferably, the buffer is selected from one or more of lithium hydroxide, disodium hydrogen phosphate, sodium hydroxide, and sodium phosphate; Preferably, the oxidant is sodium dichloroisocyanurate; Preferably, the second masking agent is selected from one or more of sodium tartrate, sodium citrate, sodium hexametaphosphate, and sodium EDTA salts.

10. The method according to claim 9, characterized in that, In the detection reagent B, the weight ratio of the second excipient, buffer, oxidant, and second masking agent is 8.9-11.7:4.8-6.5:0.8-1.4:

1. Preferably, the concentration of the detection reagent B in the detection reagent B solution is 1-15 wt%.

11. The method according to any one of claims 6-10, characterized in that, In step (2), the freeze-drying conditions include: a vacuum degree of 1-100 Pa and a time of 2-6 h.

12. The paper-based ammonia nitrogen detection chip prepared by the method according to any one of claims 6-11.

13. The application of the paper-based ammonia nitrogen detection chip according to claims 1-5 and 12 in the detection of ammonia nitrogen in water.

14. A method for detecting ammonia nitrogen concentration in water, characterized in that, The method includes the following steps: Test the initial RGB value in the colorimetric port (32) of the paper-based ammonia nitrogen detection chip, then drop the solution to be tested into the injection port (31) of the paper-based ammonia nitrogen detection chip, and then test the measured RGB value in the colorimetric port (32) after the reaction. Determine the chromaticity distance D based on the initial RGB value and the measured RGB value, and then determine the concentration of ammonia nitrogen in the solution to be tested through the chromaticity distance D. The paper-based ammonia nitrogen detection chip is the paper-based ammonia nitrogen detection chip according to any one of claims 1-5 and 12.

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