A paper-based nitrate ion detection chip and a preparation method and application thereof

Abstract

The application relates to the field of water quality detection, in particular to a paper-based nitrate ion detection chip and a preparation method and application thereof. The paper-based nitrate ion detection chip comprises a bottom plate and a paper-based layer on the bottom plate; the paper-based layer comprises a color developing area, a reduction area and a sample inlet area which are sequentially communicated; a detection reagent is pre-fixed in the color developing area, the detection reagent comprises glucose, p-aminobenzenesulfonic acid, N-1-naphthyl ethylenediamine and ascorbic acid; a reduction reagent is pre-fixed in the reduction area, the reduction reagent comprises zinc powder and cadmium chloride; the detection reagent solution is added to the color developing area and then freeze-dried, so that the detection reagent is pre-fixed in the color developing area. The freeze-drying technology is used to uniformly pre-fix the detection reagent on the filter paper, a loose structure is formed, the color developing uniformity of the detection reagent after reacting with nitrate ions can be effectively improved, and the reaction time is reduced.

Description

Technical Field

[0001] This invention relates to the field of water quality testing, specifically to a paper-based nitrate ion detection chip, its preparation method, and its application. Background Technology

[0002] Nitrates, a crucial component of the nitrogen cycle, are widely present in nature and in daily life. Common nitrates include ammonium nitrate, sodium nitrate, potassium nitrate, and calcium nitrate. Currently, excessive nitrate pollution in aquatic environments primarily stems from factors such as fertilizer use, domestic sewage and excrement, industrial wastewater, atmospheric nitrogen oxide deposition (both dry and wet), and over-extraction of groundwater. Nitrates can interfere with the oxygen-carrying function of human red blood cells, causing hypoxia and, in severe cases, various cancers including esophageal cancer. Furthermore, excessive nitrates in water can lead to eutrophication, disrupting normal ecological cycles and causing serious environmental pollution. Therefore, the detection of nitrates in water bodies is of great significance for human health and environmental protection.

[0003] Microfluidic paper chips, or paper chips for short, are microfluidic analysis devices based on microfluidic channels formed from paper, and represent a cutting-edge area of ​​microfluidic technology research. Paper chips possess several advantages, including the wide availability and low cost of paper materials, ease of transportation, high portability, real-time operation, no need for an external power source (due to the capillary forces within the paper), and excellent biocompatibility. Therefore, paper chips are receiving increasing attention in environmental monitoring. How to achieve rapid and highly sensitive detection of nitrates using paper chip technology is a pressing technical challenge that needs to be addressed.

[0004] Currently, nitrate detection is attracting increasing attention. Patent application CN218872246U discloses a microfluidic paper chip for multi-target biological detection, including hydrophobic and hydrophilic regions. A sample application area is located in the center of the hydrophilic region, and the sample application area is radially and uniformly distributed outside the area. This invention achieves multi-target detection of a single organism by setting multiple detection units. Furthermore, the microfluidic paper chip provided by this invention has a simple structure, is easy to manufacture, and is portable. Patent application CN115290712A discloses a paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes, belonging to the field of disease detection sensor technology. This sensor includes a microfluidic pretreatment module and an electrochemical detection module. The microfluidic module uses cellulose chromatography filter paper as the substrate and constructs a three-dimensional fluid flow channel using a wax-spray printer; the electrochemical detection module uses a polyimide film as the substrate and fabricates double-sided graphene electrodes using a laser engraving machine. The two modules are assembled by pressing with a fixture. Patent application CN116879284A provides a kit for detecting nitrate in water. The kit includes a nitrate detection reagent comprising the following components in parts by weight: 0.1 to 10.0 parts of reducing agent, 0.5 to 50.0 parts of p-aminobenzenesulfonic acid, 0.1 to 20.0 parts of naphthylethylenediamine hydrochloride, 1.0 to 60.0 parts of masking agent, 10.0 to 90.0 parts of weighting agent, and 0 to 10.0 parts of accelerator.

[0005] The aforementioned patents all involve directly adding the detection reagent to filter paper, drying it to obtain a colored area, and then reacting it with nitrate. However, the uniformity of the color distribution needs improvement, and the color development speed is relatively slow. Therefore, how to quickly and uniformly detect nitrate using paper-based chip technology is a problem worthy of further research. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of slow color development, uneven color development, and incomplete nitric acid reduction in the existing technology, and to provide a paper-based nitrate ion detection chip, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention provides a paper-based nitrate ion detection chip, characterized in that the paper-based nitrate ion detection chip includes a base plate and a paper base layer located on the base plate;

[0008] The paper substrate includes a color development zone, a reduction zone, and a sample injection zone that are connected in sequence;

[0009] The colorimetric region is pre-filled with a detection reagent, which includes glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine, and ascorbic acid.

[0010] The reduction zone is pre-filled with a reducing agent, which includes zinc powder and cadmium chloride.

[0011] In this process, the test reagent solution is added dropwise to the color development area and then freeze-dried, so that the test reagent is pre-positioned in the color development area.

[0012] Preferably, the base plate is made of PP, PET, PDMS, silicon wafer, or glass;

[0013] Preferably, the base plate is a rectangle with a length of 20-60mm and a width of 10-30mm.

[0014] Preferably, the color development area is a circle with a diameter of 3-10 mm, the reduction area is a strip with a length of 3-7 mm and a width of 2-4 mm, and the sample injection area is a strip with a length of 6-10 mm and a width of 2-4 mm.

[0015] Preferably, the width of the reduction zone and the sample injection zone are the same.

[0016] Preferably, in the test, the weight ratio of glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid is 25-35:0.8-2.1:1-3:1.

[0017] Preferably, in the reducing agent, the weight ratio of zinc powder to cadmium chloride is 0.2-0.8:1.

[0018] A second aspect of the present invention provides a method for preparing a paper-based nitrate ion detection chip, the method comprising the following steps:

[0019] (1) The filter paper is cut using a laser engraving machine to obtain a paper base layer, which includes a color development zone, a reduction zone and a sample injection zone connected in sequence;

[0020] (2) The paper substrate is pre-frozen in liquid nitrogen, and then the detection reagent solution is dropped onto the colorimetric area and then freeze-dried;

[0021] (3) Add the mixed solution containing the reducing agent dropwise to the reduction zone of the paper base obtained in step (2), and then heat it;

[0022] (4) Provide a base plate and place the paper base obtained in step (3) on the base plate;

[0023] The detection reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid, and the reducing reagent contains zinc powder and cadmium chloride.

[0024] Preferably, in the test, the weight ratio of glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid is 25-35:0.8-2.1:1-3:1.

[0025] Preferably, in the reducing agent, the weight ratio of zinc powder to cadmium chloride is 0.2-0.8:1.

[0026] Preferably, the concentration of the detection reagent in the detection reagent solution is 0.3-1.3 wt%.

[0027] Preferably, the mixed solution containing the reducing agent is obtained by mixing the reducing agent and water;

[0028] Preferably, the content of the reducing agent in the mixed solution containing the reducing agent is 0.1-0.5 wt%.

[0029] Preferably, in step (2), the freeze-drying conditions include: a pressure of 3-15 Pa and a time of 12-30 h.

[0030] Preferably, in step (3), the heating conditions include: a temperature of 40-60°C and a time of 3-15 minutes.

[0031] A third aspect of the present invention provides a paper-based nitrate ion detection chip prepared according to the method described above.

[0032] The fourth aspect of this invention provides the application of the paper-based nitrate ion detection chip described above in the detection of nitrate ions in water.

[0033] The fifth aspect of this invention provides a method for detecting the concentration of nitrate ions in water, the method comprising the following steps:

[0034] The initial RGB value of the color development area of ​​the paper-based nitrate ion detection chip is tested. Then, the water sample to be tested is added to the paper-based nitrate ion detection chip. The measured RGB value of the color development area after the reaction is then tested. The chromaticity distance D is determined based on the initial RGB value and the measured RGB value. Then, the concentration of nitrate ions in the water sample to be tested is determined by the chromaticity distance D.

[0035] The paper-based nitrate ion detection chip is the paper-based nitrate ion detection chip according to any one of claims 1-4 and 10.

[0036] Preferably, the formula for calculating the chromaticity distance D is:

[0037]

[0038] 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.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The paper-based nitrate ion detection chip provided by this invention directly constructs a functional layer using filter paper. The filter paper effectively filters out large particles that affect color development, ensuring that the water sample is not interfered with by particulate matter when it comes into contact with the reducing and detection reagents. Furthermore, by combining color development with photographic extraction of colorimetric values, accurate test results can be obtained quickly.

[0041] 2. The present invention proposes to obtain a paper base layer by laser engraving and cutting filter paper, and to use freeze-drying technology to uniformly pre-place the detection reagent on the filter paper to form a loose structure, which can effectively improve the color uniformity of the detection reagent after reacting with nitrate ions and reduce the reaction time.

[0042] 3. The paper-based nitrate ion detection chip of the present invention has a simple preparation method, low economic loss, and is conducive to further promotion and application. It is suitable for detecting nitrate ions in various water environments such as domestic sewage, industrial water of petrochemical enterprises, and swimming pool water. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the structure of the paper-based nitrate ion detection chip described in this invention;

[0044] Figure 2 These are images of the paper-based nitrate ion detection chip prepared in Example 1;

[0045] Figure 3 This is the standard curve obtained from the fitting in Test Example 3.

[0046] Explanation of reference numerals in the attached figures

[0047] 1. Base plate 2. Color display area

[0048] 3 Reduction Zone 4 Sample Injection Zone Detailed Implementation

[0049] 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.

[0050] 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.

[0051] The first aspect of this invention provides a paper-based nitrate ion detection chip, which can be referred to in conjunction with the above. Figure 1(Top view) The paper-based nitrate ion detection chip includes a base plate 1 and a paper base layer located on the base plate 1;

[0052] The paper substrate includes a color development zone 2, a reduction zone 3, and a sample injection zone 4 connected in sequence;

[0053] The color development area 2 is pre-filled with a detection reagent, which includes glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid;

[0054] The reduction zone 3 is pre-filled with a reducing agent, which includes zinc powder and cadmium chloride.

[0055] In this process, the test reagent solution is added dropwise to the color development area 2 and then freeze-dried, so that the test reagent is pre-placed in the color development area 2.

[0056] In this invention, the paper-based nitrate ion detection chip uses a paper substrate directly constructed from filter paper. The substrate is divided into a sample introduction zone, a reduction zone, and a colorimetric zone, with a reducing reagent and a detection reagent pre-placed in the reduction and colorimetric zones, respectively. During use, the solution is added through the sample introduction zone. Since the sample introduction zone is made of filter paper, the solution is filtered to reduce the influence of particulate matter. The solution then enters the reduction zone and reacts with the reducing reagent, reducing nitrate ions in the solution to nitrate ions. Next, the solution enters the colorimetric zone and reacts with the detection reagent. After the reaction, an image is captured, and software is used to process the image to obtain the chromaticity distance of the colorimetric zone. The concentration of nitrate ions in the solution can then be determined using the chromaticity distance.

[0057] In this invention, the base plate is used to support the paper substrate. To reduce the influence of the base plate's color on color development, the base plate is preferably made of a transparent material. Preferably, the base plate 1 is made of PP (polypropylene), PET (polyethylene terephthalate), PDMS (polydimethylsiloxane), silicon wafer, or glass.

[0058] More preferably, the base plate 1 is a rectangle with a length of 20-60mm and a width of 10-30mm.

[0059] In a preferred embodiment, the colorimetric region 2 is a circle with a diameter (D) of 3-10 mm, the reduction region 3 is an elongated strip with a length (L2) of 3-7 mm and a width of 2-4 mm, and the injection region 4 is an elongated strip with a length (L1) of 6-10 mm and a width of 2-4 mm. Setting the colorimetric region 2 as a circle, as described above, facilitates the adhesion of the detection reagent solution to the colorimetric region and allows for lyophilization.

[0060] More preferably, the reduction zone 3 and the sample injection zone 4 have the same width.

[0061] In this invention, the paper base layer is directly constructed from filter paper. Preferably, the paper base layer is made of Waterman 4# filter paper with a pore size of 25 μm.

[0062] In a preferred embodiment, to further improve the colorimetric effect and accuracy of the detection reagent, the amount of each component in the detection reagent can be reasonably controlled. In the test, the weight ratio of glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine, and ascorbic acid is 25-35:0.8-2.1:1-3:1; specifically, the weight ratio of glucose to ascorbic acid can be 25:1, 26:1, 28:1, 30:1, 32:1, or 35:1, the weight ratio of p-aminobenzenesulfonic acid to ascorbic acid can be 0.8:1, 1:1, 1.2:1, 1.4:1, 1.5:1, 1.6:1, 1.8:1, or 2:1, and the weight ratio of N-1-naphthylethylenediamine to ascorbic acid can be 1:1, 1.5:1, 2:1, 2.5:1, or 3:1.

[0063] In a preferred embodiment, the weight ratio of zinc powder to cadmium chloride in the reducing agent is 0.2-0.8:1; specifically, it can be 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1 or 0.8:1.

[0064] A second aspect of the present invention provides a method for preparing a paper-based nitrate ion detection chip, the method comprising the following steps:

[0065] (1) The filter paper is cut using a laser engraving machine to obtain a paper base layer, which includes a color development zone, a reduction zone and a sample injection zone connected in sequence;

[0066] (2) The paper substrate is pre-frozen in liquid nitrogen, and then the detection reagent solution is dropped onto the colorimetric area and then freeze-dried;

[0067] (3) Add the mixed solution containing the reducing agent dropwise to the reduction zone of the paper base obtained in step (2), and then heat it;

[0068] (4) Place the paper base obtained in step (3) on the base plate;

[0069] The detection reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid, and the reducing reagent contains zinc powder and cadmium chloride.

[0070] In the conventional preparation of paper chips, filter paper is typically soaked in a solution or the solution is dropped onto the filter paper and then dried. These methods often result in uneven distribution of the reagents on the filter paper, leading to uneven color development and a slow reaction rate. In the method described in this invention, freeze-drying allows the colorimetric reagents to be evenly distributed on the filter paper in a loose state, effectively improving color uniformity and reducing reaction time.

[0071] In this invention, the base plate is used to support the paper substrate. To reduce the influence of the base plate's color on color development, the base plate is preferably made of a transparent material. Preferably, the base plate 1 is made of PP, PET, PDMS, silicon wafer, or glass.

[0072] More preferably, the base plate 1 is a rectangle with a length of 20-60mm and a width of 10-30mm.

[0073] In a preferred embodiment, the color development area 2 is a circle with a diameter of 3-10 mm, the reduction area 3 is a strip with a length of 3-7 mm and a width of 2-4 mm, and the sample injection area 4 is a strip with a length of 6-10 mm and a width of 2-4 mm. More preferably, the reduction area 3 and the sample injection area 4 have the same width. The filter paper (i.e., the paper base layer) with the above shapes can be obtained by laser engraving.

[0074] In a preferred embodiment, to further improve the colorimetric effect and accuracy of the detection reagent, the amount of each component in the detection reagent can be reasonably controlled. In the test, the weight ratio of glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine, and ascorbic acid is 25-35:0.8-2.1:1-3:1; specifically, the weight ratio of glucose to ascorbic acid can be 25:1, 26:1, 28:1, 30:1, 32:1, or 35:1, the weight ratio of p-aminobenzenesulfonic acid to ascorbic acid can be 0.8:1, 1:1, 1.2:1, 1.4:1, 1.5:1, 1.6:1, 1.8:1, or 2:1, and the weight ratio of N-1-naphthylethylenediamine to ascorbic acid can be 1:1, 1.5:1, 2:1, 2.5:1, or 3:1.

[0075] In a preferred embodiment, the weight ratio of zinc powder to cadmium chloride in the reducing agent is 0.2-0.8:1; specifically, it can be 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1 or 0.8:1.

[0076] In the method described in this invention, if the concentration of the detection reagent in the reagent solution is too low, the amount of reagent will be insufficient, and the colorimetric reaction will not proceed completely; if the concentration of the detection reagent is too high, the amount of reagent in the colorimetric area after lyophilization will be excessive, and some reagent will fall off the filter paper, thus affecting the uniformity of color development. Preferably, the concentration of the detection reagent in the reagent solution is 0.3-1.3 wt%. The solvent used in the detection reagent solution can be water.

[0077] According to some preferred embodiments of the present invention, the solution containing the detection reagent can be obtained by mixing the detection reagent and water.

[0078] In the method described in this invention, if the volume of the solution added to the paper substrate is too large, the solution cannot adhere to the paper substrate, and the droplets will fall off the paper substrate. If the volume is too small, the amount of reagent in the colorimetric area will be insufficient, and the colorimetric reaction cannot proceed completely. In a preferred embodiment, in step (2), the ratio of the volume of the detection reagent solution to the area of ​​the colorimetric area is 0.1-0.8 μL: 1 mm. 2 .

[0079] In the method described in this invention, the mixed solution (i.e., suspension) containing the reducing agent can be obtained by mixing the reducing agent and water. More preferably, the content of the reducing agent in the mixed solution containing the reducing agent is 0.1-0.5 wt%.

[0080] In a preferred embodiment, in step (3), the ratio of the area of ​​the reduction zone to the volume of the mixed solution containing the reducing reagent is 1 mm. 2 : 0.15-0.7μL.

[0081] In the method described in this invention, step (2) allows the detection reagent to be pre-placed within the colorimetric region of the paper substrate. In a preferred embodiment, step (2) involves freeze-drying under the following conditions: a pressure of 3-15 Pa and a time of 12-30 h.

[0082] In this invention, step (3) allows the reducing agent to be pre-placed within the reduction zone of the paper substrate. In a preferred embodiment, step (3) involves heating conditions of 40-60°C for 3-15 minutes. The heating can be performed on a heating plate.

[0083] In the method described in this invention, in step (4), conventional means in the art, such as adhesive, can be used to fix the paper base to the substrate. Transparent adhesive is preferred to reduce the impact on the color development effect.

[0084] A third aspect of this invention provides a paper-based nitrate ion detection chip prepared according to the method described above. This paper-based silver nitrate ion detection chip can quickly and accurately detect the concentration of nitrate ions in a solution, and the chip has a simple preparation process, small size, and is easy to promote and apply on a large scale.

[0085] The fourth aspect of this invention provides the application of the paper-based nitrate ion detection chip described above in the detection of nitrate ions in water.

[0086] In this invention, the paper-based silver nitrate detection chip can be used to detect nitrate ions in various types of water bodies. For example, the water body can be domestic sewage, industrial water from petrochemical plants, swimming pool water, and many other types.

[0087] The fifth aspect of this invention provides a method for detecting the concentration of nitrate ions in water, the method comprising the following steps:

[0088] The initial RGB value of the color development area of ​​the paper-based nitrate ion detection chip is tested. Then, the water sample to be tested is added to the paper-based nitrate ion detection chip. The measured RGB value of the color development area after the reaction is then tested. The chromaticity distance D is determined based on the initial RGB value and the measured RGB value. Then, the concentration of nitrate ions in the water sample to be tested is determined by the chromaticity distance D.

[0089] The paper-based nitrate ion detection chip is the same as the paper-based nitrate ion detection chip described above.

[0090] In this invention, the detection reagent pre-placed in the colorimetric region of the paper-based nitrate ion detection chip can complete the reaction with nitrite ions (obtained by the reaction of nitrate ions and a reducing agent) in the solution in only 2 minutes; it has the advantages of fast detection speed and accurate detection results. In a preferred embodiment, the solution to be tested is added dropwise to the sample introduction area of ​​the paper-based nitrate ion detection chip. After the solution passes through the reduction zone and reaches the colorimetric region, wait 2-4 minutes, and then test the RGB value of the colorimetric region after the reaction.

[0091] In this invention, the detection of nitrate ion concentration in water using the above method requires only 10-20 μL of solution.

[0092] 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 area can be obtained by taking a picture using a camera (e.g., a mobile phone), and then processing the picture using software (Photoshop or ImageJ) to obtain the RGB values ​​of the color display area.

[0093] In the method described in this invention, the formula for calculating the chromaticity distance is:

[0094]

[0095] 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.

[0096] In a preferred embodiment, the concentration of nitrate ions in the solution can be quantitatively detected using the external standard method. At least five standard solutions with known concentrations of nitrate ions are prepared. The chromaticity distance D of each standard solution is measured using a paper-based nitrate ion detection chip. The relationship between concentration and chromaticity distance D is calculated. Then, the same method is used to measure the chromaticity distance D of the water sample. The concentration of nitrate ions 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 nitrate ion 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. The standard solutions can be prepared by mixing nitrate and water.

[0097] According to some specific embodiments of the present invention, the method for detecting the concentration of nitrate ions in a solution includes the following steps:

[0098] S1: Test the initial RGB value of the color development area of ​​the paper-based nitrate ion detection chip, and then add the standard solution to the sample injection area of ​​the paper-based nitrate ion detection chip. After the standard solution reaches the color development area, wait for 2-4 minutes, and then test the measured RGB value of the color development area after the reaction. Determine the chromaticity distance D of the standard solution based on the initial RGB value and the measured RGB value.

[0099] S2: Fit the equation with chromatic distance D as the ordinate and nitrate ion concentration as the abscissa to obtain the relationship between chromatic distance D and nitrate ion concentration.

[0100] S3: Test the initial RGB value of the color development area of ​​the paper-based nitrate ion detection chip, then add the water sample to the paper-based nitrate ion detection chip. After the test solution reaches the color development area, wait for 2-4 minutes, and then test the measured RGB value of the color development area after the reaction. Determine the color distance D of the water sample based on the initial RGB value and the measured RGB value. Substitute the color distance D of the water sample into the relationship obtained in step S2 for calculation to obtain the concentration of nitrate ions in the water sample.

[0101] The formula for calculating the chromaticity distance D is as follows:

[0102]

[0103] 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.

[0104] In the method described in this invention, in a preferred embodiment, when detecting the concentration of nitrate ions in a solution, the paper-based nitrate ion detection chips used for the standard solution and the test solution are from the same batch (i.e., the preparation conditions are the same), and the volume of the standard solution and the test solution used is the same.

[0105] The method for detecting nitrate ion concentration in solution described in this invention utilizes the aforementioned paper-based nitrate ion detection chip, which can quickly and accurately detect the concentration of nitrate ions in solution. 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 conduct real-time on-site detection of nitrate ion concentration in water.

[0106] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0107] Example 1

[0108] Preparation of paper-based nitrate ion detection chip S1:

[0109] (1) Waterman 4# filter paper is processed by laser engraving machine to obtain paper base layer, which includes a color development area, a reduction area and a sample injection area connected in sequence; wherein, the sample injection area is a strip with a length of 8mm and a width of 2.6mm, the reduction area is a strip with a length of 5mm and a width of 2.6mm, and the color development area is a circle with a diameter of 6mm.

[0110] (2) Mix the test reagent with water to prepare a test reagent solution with a concentration of 1 wt%, wherein the test reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid in a weight ratio of 30:1.5:2:1;

[0111] The paper substrate obtained in step (1) was placed in liquid nitrogen for pre-freezing. 8 μL of the test reagent solution was dropped onto the color development area and then placed in a freeze dryer and treated at a pressure of 7 Pa for 22 hours to pre-place the test reagent in the color development area.

[0112] (3) Mix the reducing agent with water to prepare a 0.3wt% suspension. The reducing agent is composed of zinc powder and cadmium chloride in a weight ratio of 0.6:1. Add 5uL of the suspension to the reduction zone of the paper base layer in step (2), and then place it on a hot plate at 50°C for 6 minutes to pre-place the reducing agent in the reduction zone.

[0113] (4) The PP material is processed using a laser engraving machine to obtain a base plate with a length of 45mm and a width of 22mm. The paper base layer obtained in step (3) is placed on top of the base plate and fixed with adhesive. The resulting paper-based nitrate ion detection chip is shown in the image below. Figure 2 As shown.

[0114] Example 2

[0115] Preparation of paper-based nitrate ion detection chip S2:

[0116] (1) Waterman 4# filter paper is processed by laser engraving machine to obtain paper base layer, which includes a color development area, a reduction area and a sample injection area connected in sequence; wherein, the sample injection area is a strip with a length of 8mm and a width of 2.6mm, the reduction area is a strip with a length of 5mm and a width of 2.6mm, and the color development area is a circle with a diameter of 6mm.

[0117] (2) The test reagent is mixed with water to prepare a test reagent solution with a concentration of 1 wt%, wherein the test reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid in a weight ratio of 30:1.8:1.8:1;

[0118] The paper substrate obtained in step (1) was placed in liquid nitrogen for pre-freezing. 8 μL of the test reagent solution was dropped onto the color development area and then placed in a freeze dryer and treated at a pressure of 7 Pa for 22 hours to pre-place the test reagent in the color development area.

[0119] (3) Mix the reducing agent with water to prepare a 0.3wt% suspension. The reducing agent is composed of zinc powder and cadmium chloride in a weight ratio of 0.6:1. Add 5uL of the suspension to the reduction zone of the paper base layer in step (2), and then place it on a hot plate at 50°C for 6 minutes to pre-place the reducing agent in the reduction zone.

[0120] (4) Use a laser engraving machine to process the PP material to obtain a base plate with a length of 45mm and a width of 22mm. Place the paper base layer obtained in step (3) on the base plate and fix it with adhesive.

[0121] Example 3

[0122] Preparation of paper-based nitrate ion detection chip S3:

[0123] (1) Waterman 4# filter paper is processed by laser engraving machine to obtain paper base layer, which includes a color development area, a reduction area and a sample injection area connected in sequence; wherein, the sample injection area is a strip with a length of 8mm and a width of 2.6mm, the reduction area is a strip with a length of 5mm and a width of 2.6mm, and the color development area is a circle with a diameter of 6mm.

[0124] (2) Mix the test reagent with water to prepare a test reagent solution with a concentration of 1 wt%, wherein the test reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid in a weight ratio of 30:1.5:2:1;

[0125] The paper substrate obtained in step (1) was placed in liquid nitrogen for pre-freezing. 10 μL of the test reagent solution was dropped onto the color development area and then placed in a freeze dryer and treated at a pressure of 7 Pa for 22 h to pre-place the test reagent in the color development area.

[0126] (3) Mix the reducing agent with water to prepare a 0.3wt% suspension. The reducing agent is composed of zinc powder and cadmium chloride in a weight ratio of 0.6:1. Add 5uL of the suspension to the reduction zone of the paper base layer in step (2), and then place it on a hot plate at 50°C for 6 minutes to pre-place the reducing agent in the reduction zone.

[0127] (4) Use a laser engraving machine to process the PP material to obtain a base plate with a length of 45mm and a width of 22mm. Place the paper base layer obtained in step (3) on the base plate and fix it with adhesive.

[0128] Example 4

[0129] Preparation of paper-based nitrate ion detection chip S4:

[0130] (1) Waterman 4# filter paper is processed by laser engraving machine to obtain paper base layer, which includes a color development area, a reduction area and a sample injection area connected in sequence; wherein, the sample injection area is a strip with a length of 8mm and a width of 2.6mm, the reduction area is a strip with a length of 5mm and a width of 2.6mm, and the color development area is a circle with a diameter of 6mm.

[0131] (2) Mix the test reagent with water to prepare a test reagent solution with a concentration of 1 wt%, wherein the test reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid in a weight ratio of 30:1.5:2:1;

[0132] The paper substrate obtained in step (1) was placed in liquid nitrogen for pre-freezing. 6 μL of the test reagent solution was dropped onto the color development area and then placed in a freeze dryer and treated at a pressure of 7 Pa for 22 h to pre-place the test reagent in the color development area.

[0133] (3) Mix the reducing agent with water to prepare a 0.3wt% suspension. The reducing agent is composed of zinc powder and cadmium chloride in a weight ratio of 0.6:1. Add 5uL of the suspension to the reduction zone of the paper base layer in step (2), and then place it on a hot plate at 50°C for 6 minutes to pre-place the reducing agent in the reduction zone.

[0134] (4) Use a laser engraving machine to process the PP material to obtain a base plate with a length of 45mm and a width of 22mm. Place the paper base layer obtained in step (3) on the base plate and fix it with adhesive.

[0135] Example 5

[0136] Preparation of paper-based nitrate ion detection chip S5:

[0137] (1) Waterman 4# filter paper is processed by laser engraving machine to obtain paper base layer, which includes a color development area, a reduction area and a sample injection area connected in sequence; wherein, the sample injection area is a strip with a length of 8mm and a width of 2.6mm, the reduction area is a strip with a length of 5mm and a width of 2.6mm, and the color development area is a circle with a diameter of 6mm.

[0138] (2) Mix the test reagent with water to prepare a test reagent solution with a concentration of 1 wt%, wherein the test reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid in a weight ratio of 30:1.5:2:1;

[0139] The paper substrate obtained in step (1) was placed in liquid nitrogen for pre-freezing. 8 μL of the test reagent solution was dropped onto the color development area and then placed in a freeze dryer and treated at a pressure of 7 Pa for 22 hours to pre-place the test reagent in the color development area.

[0140] (3) Mix the reducing agent with water to prepare a 0.4wt% suspension. The reducing agent is composed of zinc powder and cadmium chloride in a weight ratio of 0.6:1. Add 6uL of the suspension to the reduction zone of the paper base layer in step (2), and then place it on a hot plate at 50°C for 6 minutes to pre-place the reducing agent in the reduction zone.

[0141] (4) Use a laser engraving machine to process the PP material to obtain a base plate with a length of 45mm and a width of 22mm. Place the paper base layer obtained in step (3) on the base plate and fix it with adhesive.

[0142] Example 6

[0143] Preparation of paper-based nitrate ion detection chip S6:

[0144] (1) Waterman 4# filter paper is processed by laser engraving machine to obtain paper base layer, which includes a color development area, a reduction area and a sample injection area connected in sequence; wherein, the sample injection area is a strip with a length of 8mm and a width of 2.6mm, the reduction area is a strip with a length of 5mm and a width of 2.6mm, and the color development area is a circle with a diameter of 6mm.

[0145] (2) Mix the test reagent with water to prepare a test reagent solution with a concentration of 1 wt%, wherein the test reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid in a weight ratio of 30:1.5:2:1;

[0146] The paper substrate obtained in step (1) was placed in liquid nitrogen for pre-freezing. 8 μL of the test reagent solution was dropped onto the color development area and then placed in a freeze dryer and treated at a pressure of 7 Pa for 22 hours to pre-place the test reagent in the color development area.

[0147] (3) Mix the reducing agent with water to prepare a 0.25wt% suspension. The reducing agent is composed of zinc powder and cadmium chloride in a weight ratio of 0.6:1. Add 6uL of the suspension to the reduction zone of the paper base layer in step (2), and then place it on a hot plate at 50°C for 6 minutes to pre-place the reducing agent in the reduction zone.

[0148] (4) Use a laser engraving machine to process the PP material to obtain a base plate with a length of 45mm and a width of 22mm. Place the paper base layer obtained in step (3) on the base plate and fix it with adhesive.

[0149] Example 7

[0150] Preparation of paper-based nitrate ion detection chip S7:

[0151] (1) Waterman 4# filter paper is processed by laser engraving machine to obtain paper base layer, which includes a color development area, a reduction area and a sample injection area connected in sequence; wherein, the sample injection area is a strip with a length of 8mm and a width of 2.6mm, the reduction area is a strip with a length of 5mm and a width of 2.6mm, and the color development area is a circle with a diameter of 6mm.

[0152] (2) Mix the test reagent with water to prepare a test reagent solution with a concentration of 1 wt%, wherein the test reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid in a weight ratio of 30:1.5:2:1;

[0153] The paper substrate obtained in step (1) was placed in liquid nitrogen for pre-freezing. 12 μL of the test reagent solution was dropped onto the color development area and then placed in a freeze dryer and treated at a pressure of 7 Pa for 22 h to pre-place the test reagent in the color development area.

[0154] (3) Mix the reducing agent with water to prepare a 0.3wt% suspension. The reducing agent is composed of zinc powder and cadmium chloride in a weight ratio of 0.5:1. Add 4.5uL of the suspension to the reduction zone of the paper base layer in step (2), and then place it on a hot plate at 50°C for 6 minutes to pre-place the reducing agent in the reduction zone.

[0155] (4) The PP material is processed using a laser engraving machine to obtain a base plate with a length of 45mm and a width of 22mm. The paper base layer obtained in step (3) is placed on top of the base plate and fixed with adhesive. The resulting paper-based nitrate ion detection chip is shown in the image below. Figure 2 As shown.

[0156] Example 8

[0157] Preparation of paper-based nitrate ion detection chip S8:

[0158] (1) Waterman 4# filter paper is processed by laser engraving machine to obtain paper base layer, which includes a color development area, a reduction area and a sample injection area connected in sequence; wherein, the sample injection area is a strip with a length of 8mm and a width of 2.6mm, the reduction area is a strip with a length of 5mm and a width of 2.6mm, and the color development area is a circle with a diameter of 6mm.

[0159] (2) Mix the test reagent with water to prepare a test reagent solution with a concentration of 1.1 wt%, wherein the test reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid in a weight ratio of 33:1.5:2.2:1;

[0160] The paper substrate obtained in step (1) was placed in liquid nitrogen for pre-freezing. 8 μL of the test reagent solution was dropped onto the color development area and then placed in a freeze dryer and treated at a pressure of 7 Pa for 22 hours to pre-place the test reagent in the color development area.

[0161] (3) Mix the reducing agent with water to prepare a 0.35wt% suspension. The reducing agent is composed of zinc powder and cadmium chloride in a weight ratio of 0.6:1. Add 5uL of the suspension to the reduction zone of the paper base layer in step (2), and then place it on a hot plate at 50°C for 6 minutes to pre-place the reducing agent in the reduction zone.

[0162] (4) The PP material is processed using a laser engraving machine to obtain a base plate with a length of 45mm and a width of 22mm. The paper base layer obtained in step (3) is placed on top of the base plate and fixed with adhesive. The resulting paper-based nitrate ion detection chip is shown in the image below. Figure 2 As shown.

[0163] Comparative Example 1

[0164] The method described in Example 1 is implemented, except that in step (2), 8 μL of the detection reagent solution is dropped onto the color development area and then placed on a hot plate for heating so that the detection reagent is pre-placed in the color development area; wherein, the heating temperature is 60°C and the heating time is 5 min.

[0165] Test Example 1

[0166] 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.

[0167] The detection method is as follows:

[0168] The image is taken by taking a picture of the color display area of ​​the detection chip with a smartphone, and then the image is processed with Photoshop software to obtain the initial RGB values ​​of the color display area.

[0169] Take 12 μL of nitrate solution (nitrate ion concentration of 30 mg / L) with a pipette and inject it into the detection chip from the end of the injection area away from the reduction area. After the solution reaches the color development layer, wait for 2 minutes. Take a picture of the color development area of ​​the detection chip with a smartphone and then process the picture with Photoshop software to obtain the measured RGB value of the color development area.

[0170] The chromaticity distance D is calculated based on the initial RGB values ​​and the measured RGB values. The calculation formula is as follows:

[0171]

[0172] 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.

[0173] Table 1

[0174] Chromaticity distance D Example 1 81 Example 2 78 Example 3 83 Example 4 79 Example 5 84 Example 6 80 Example 7 79 Example 8 84 Comparative Example 1 71

[0175] As shown in Table 1, the detection chip prepared in the examples has the characteristics of fast color development rate and good color development effect, which can shorten the detection time of nitrate ions. However, the color distance of the detection chip prepared in Comparative Example 1 is significantly smaller than that of the examples. This is because the detection reagent in the preparation process of the detection chip in Comparative Example 1 is directly dropped onto the filter paper, which makes the detection reagent particles compact and without a loose structure, resulting in a slower reaction rate.

[0176] Test Example 2

[0177] The color uniformity of the detection chips prepared in Example 1 and Comparative Example 1 was tested.

[0178] Detection method: Take a picture of the color display area of ​​the detection chip with a smartphone to obtain the picture. Randomly select 8 areas of the same size from the color display area, and then process the picture with Photoshop software to obtain the initial RGB values ​​of the above 8 areas.

[0179] Use a pipette to take 12 μL of nitrate solution (nitrate ion concentration of 30 mg / L) and inject it into the detection chip from the end of the injection area away from the reduction area. After the solution reaches the color development layer, wait for a certain period of time, take a picture of the color development area of ​​the detection chip with a smartphone, and then use Photoshop software to process the picture to obtain the measured RGB values ​​of the above 8 areas.

[0180] The chromaticity distance D is calculated based on the initial RGB values ​​and the measured RGB values. The calculation formula is the same as that in Test Example 1.

[0181] When using the detection chip prepared in Example 1, after the solution reached the chromogenic layer, the process was allowed to proceed for 2 minutes before imaging. The chromatic distances of the eight detected regions were 81, 84, 76, 83, 79, 78, 84, and 78, with a relative standard deviation of 3.82%.

[0182] In the case where the detection chip prepared in Comparative Example 1 was used, after the solution reached the chromogenic layer, the process was allowed to proceed for 5 minutes before imaging. The chromatic distances of the eight detected regions were 89, 81, 71, 76, 85, 87, 73, and 84, with a relative standard deviation of 8.23%.

[0183] The above test results show that the paper-based nitrate ion detection chip of the present invention can quickly react with nitrate ions to produce a colorimetric reaction, and the color development is uniform. In contrast, the detection chip prepared in Comparative Example 1, even with sufficient reaction between the detection reagent and the solution, exhibits significantly lower color uniformity than that of Example 1.

[0184] Test Example 3

[0185] Silver nitrate solutions with nitrate ion concentrations of 5 mg / L, 10 mg / L, 15 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L were prepared as standard solutions. Six paper-based nitrate detection chips prepared in Example 1 were used, and detection was performed according to the method described in Test Example 1. The resulting chromaticity distances were 38, 50, 58, 81, 97, and 115, respectively. A standard curve was constructed with nitrate ion concentration as the ordinate and chromaticity distance as the abscissa. The results are shown below. Figure 3 As shown, the nitrate ion concentration and the chromaticity distance exhibit a good functional relationship, with the fitting result being: y = 0.603x - 19.16, where x is the chromaticity distance, y is the nitrate ion concentration, and the correlation coefficient R0 is 0.603x - 19.16. 2 =0.998. Based on this mathematical relationship, after measuring the color value, the concentration of nitrate ions in the solution can be calculated, thus achieving quantitative detection of nitrate ions.

[0186] Nitrate solutions with nitrate ion concentrations of 8 mg / L, 20 mg / L, and 45 mg / L were prepared as test solutions. Three detection chips prepared according to the method described in Example 1 were prepared, and the colorimetric distance was measured according to the method described in Test Example 1. The concentration was calculated by substituting the values ​​into the above formula. The measured results were compared with the theoretical values ​​of the test solutions, and the results are shown in Table 1.

[0187] Table 1

[0188] Theoretical value Chromaticity distance Detection value error 8mg / L 45.3 8.14 mg / L 1.75％ 20mg / L 65.4 20.25 mg / L 1.25％ 45mg / L 104.3 43.71 mg / L 2.87％

[0189] The results above show that the nitrate ion concentration and the chromaticity distance exhibit a good linear relationship, indicating that the paper-based nitrite ion detection chip described in this invention can accurately detect the nitrate ion concentration in the solution.

[0190] 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 nitrate ion detection chip, characterized in that, The paper-based nitrate ion detection chip includes a base plate (1) and a paper base layer located on the base plate (1); The paper substrate includes a color development zone (2), a reduction zone (3), and a sample injection zone (4) connected in sequence; The color development area (2) is pre-filled with a detection reagent, which includes glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid; The reduction zone (3) is pre-filled with a reducing agent, which includes zinc powder and cadmium chloride; In this process, the test reagent solution is added dropwise to the color development area (2) and then freeze-dried, so that the test reagent is pre-placed in the color development area (2).

2. The paper-based nitrate ion detection chip according to claim 1, characterized in that, The base plate (1) is made of PP, PET, PDMS, silicon wafer or glass; Preferably, the base plate (1) is a rectangle with a length of 20-60mm and a width of 10-30mm.

3. The paper-based nitrate ion detection chip according to claim 1 or 2, characterized in that, The color development area (2) is a circle with a diameter of 3-10 mm, the reduction area (3) is a strip with a length of 3-7 mm and a width of 2-4 mm, and the sample injection area (4) is a strip with a length of 6-10 mm and a width of 2-4 mm. Preferably, the width of the reduction zone (3) and the sample injection zone (4) are the same.

4. The paper-based nitrate ion detection chip according to claim 1, characterized in that, In the aforementioned test, the weight ratio of glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine, and ascorbic acid was 25-35: 0.8-2.1:1-3:1。 5. The paper-based nitrate ion detection chip according to claim 1 or 4, characterized in that, In the reducing agent, the weight ratio of zinc powder to cadmium chloride is 0.2-0.8:

1.

6. A method for preparing a paper-based nitrate ion detection chip, characterized in that, The method includes the following steps: (1) The filter paper is cut using a laser engraving machine to obtain a paper base layer, which includes a color development zone, a reduction zone and a sample injection zone connected in sequence; (2) The paper substrate is pre-frozen in liquid nitrogen, and then the detection reagent solution is dropped onto the colorimetric area and then freeze-dried; (3) Add the mixed solution containing the reducing agent dropwise to the reduction zone of the paper base obtained in step (2), and then heat it; (4) Provide a base plate and place the paper base obtained in step (3) on the base plate; The detection reagent contains glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine and ascorbic acid, and the reducing reagent contains zinc powder and cadmium chloride.

7. The method according to claim 6, characterized in that, In the aforementioned test, the weight ratio of glucose, p-aminobenzenesulfonic acid, N-1-naphthylethylenediamine, and ascorbic acid was 25-35: 0.8-2.1:1-3:1。 8. The method according to claim 6 or 7, characterized in that, In the reducing agent, the weight ratio of zinc powder to cadmium chloride is 0.2-0.8:

1.

9. The method according to claim 6 or 7, characterized in that, The concentration of the detection reagent in the detection reagent solution is 0.3-1.3 wt%.

10. The method according to claim 6 or 8, characterized in that, The mixed solution containing the reducing agent is obtained by mixing the reducing agent and water; Preferably, the content of the reducing agent in the mixed solution containing the reducing agent is 0.1-0.5 wt%.

11. The method according to claim 6, characterized in that, In step (2), the freeze-drying conditions include: a pressure of 3-15 Pa and a time of 12-30 h.

12. The method according to any one of claims 6-11, characterized in that, In step (3), the heating conditions include: a temperature of 40-60℃ and a time of 3-15min.

13. The paper-based nitrate ion detection chip prepared by the method according to any one of claims 6-12.

14. The application of the paper-based nitrate ion detection chip according to any one of claims 1-5 and 13 in the detection of nitrate ions in water.

15. A method for detecting the concentration of nitrate ions in water, characterized in that, The method includes the following steps: The initial RGB value of the color development area of ​​the paper-based nitrate ion detection chip is tested. Then, the water sample to be tested is added to the paper-based nitrate ion detection chip. The measured RGB value of the color development area after the reaction is then tested. The chromaticity distance D is determined based on the initial RGB value and the measured RGB value. Then, the concentration of nitrate ions in the water sample to be tested is determined by the chromaticity distance D. The paper-based nitrate ion detection chip is the paper-based nitrate ion detection chip according to any one of claims 1-4 and 10.

16. The method according to claim 15, characterized in that, The formula for calculating the chromaticity distance D is: 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.

Images