Preparation method and application of nitrate ion detection sensor based on screen-printed electrode

Abstract

The invention relates to a preparation method of a nitrate ion detection sensor based on a screen-printed electrode, which comprises the following steps: preparation of conductive substrates: respectively printing first slurry and second slurry on a polyester substrate based on a screen printing technology, and respectively forming conductive substrates of a working electrode and a reference electrode after air drying and cutting; insulating treatment: pasting insulating layers on non-working areas of the conductive substrates of the working electrode and the reference electrode; preparing a nitrate ion-selective membrane: preparing an ion-selective membrane precursor solution, stirring the ion-selective membrane precursor solution, then coating the ion-selective membrane precursor solution on a conductive substrate of the working electrode by adopting a slit coating technology to form a functional membrane layer, and drying the functional membrane layer to obtain a nitrate ion-selective electrode; and preparation of the reference electrode: preparing the reference membrane precursor solution, stirring, coating a conductive substrate of the reference electrode by adopting a slit coating technology to form a reference membrane layer, and drying to obtain the reference electrode. The invention further relates to a detection sensor for nitrate ions and application of the detection sensor.

Description

Technical Field

[0001] The present invention relates to the technical field of sensors, and particularly to a preparation method and application of a nitrate ion detection sensor based on a screen-printed electrode. Background Art

[0002] Nitrate nitrogen, as a commonly existing nitrogen compound, plays a crucial role in multiple fields such as food, agriculture, marine ecology, and human physiology. However, with the in-depth research, the potential health risks of nitrate nitrogen in multiple environments have gradually emerged. In the agricultural field, nitrate nitrogen, as the main component of nitrogen fertilizer, has significantly promoted the increase in crop yields. However, the excessive use of nitrogen fertilizer has led to the accumulation of nitrate nitrogen in soil and water bodies, and triggered a series of environmental problems such as groundwater pollution, aggravated greenhouse effect, and nitrogen deposition. Therefore, accurately monitoring the nitrate concentration in soil or water medium has become the key, which not only concerns the effectiveness of nitrate management but also is an important prerequisite for ensuring human health and ecological balance. In nitrate management, rapid and high-throughput on-site detection technologies play an indispensable role.

[0003] There are various types of nitrate detection technologies, covering different detection requirements such as temperature, sample volume, interfering ion concentration, etc. These technologies can be mainly divided into two categories: direct method and indirect method. The indirect method reduces nitrate to nitrite or nitric oxide, and then measures the nitrate concentration. Spectral technologies such as colorimetric spectrophotometry, atomic absorption spectrometry, and chemiluminescence method are used to measure the nitrite concentration. However, such methods require discrete soil sampling and are equipped with complex instruments, so they are limited in terms of time-consuming, labor-intensive, and extensive laboratory analysis. The direct method for measuring nitrate concentration has the characteristics of rapid response and directness. The chromatograph can accurately identify the nitrate concentration. First, nitrate is extracted from the sample, and then its concentration is measured by different technologies (such as ultraviolet, fluorescence, or mass spectrometry). However, the chromatograph is large in volume and difficult to miniaturize, which is not conducive to on-site application. The diffuse reflectance spectroscopy technology can perform on-site nitrate measurement by detecting the light energy level reflected by soil particles and nutrient ions, but this technology highly depends on the specific calibration of the soil and is not suitable for large-scale and high-resolution on-site deployment. In comparison, electrochemical sensors, especially ion-selective electrodes (ISEs), can quickly obtain the logarithmic relationship between the concentration of the target ion and the potential of the indicator electrode through the ion-selective membrane (ISM), and have the characteristics of high selectivity, rapid response, simple operation, wide measurement range, long service life, and no dependence on expensive reagents. This method shows great potential in in-situ rapid measurement of nitrate concentration and is applicable to various application fields.

[0004] However, in practical applications, traditional ion-selective electrodes (LC-ISEs) often suffer from unstable potential due to the leakage of the internal reference solution, failing to meet the requirements of real-time monitoring. To solve this problem, researchers have developed all-solid-state ion-selective electrodes (SC-ISEs), which abandon the easily leaky internal filling solution and have become an emerging trend in this field. The solid-state ion-selective electrodes are divided into two categories: (1) Direct coating type: The ion-sensitive membrane (ISM) is directly coated on the substrate electrode. The technology is simple but has problems such as high resistance, low capacitance, and water layer, and the stability of the internal potential is relatively poor. (2) Solid contact type: A solid contact layer (such as a conductive polymer, carbon-based material, etc.) is added between the ISM and the substrate electrode to replace the traditional internal solution, significantly improving the potential stability. Although SC-ISEs have advantages in potential stability, they still face some challenges: They cannot completely block water and oxygen, which may affect the measurement accuracy; the solid contact layer / intermediate layer may cause electrochemical side reactions, interfering with the electrode performance; the introduction of the intermediate layer increases the preparation time and cost, which is not conducive to large-scale production. Therefore, how to develop a simple, economical, and scalable sensor for nitrate solid-state ion-selective electrodes to promote its application in environmental monitoring and agricultural management is an urgent problem to be solved at present. Summary of the Invention

[0005] Aiming at the technical problems existing in the prior art, the first object of the present invention is to provide a preparation method for a nitrate ion detection sensor based on a screen-printed electrode, and to prepare a sensor for nitrate solid-state ion-selective electrode that is simple, economical, has high measurement accuracy, and good anti-interference performance.

[0006] The second object of the present invention is to provide a nitrate ion detection sensor based on a screen-printed electrode obtained by the above preparation method. Based on the combination of a working electrode and a reference electrode, a conductive substrate is prepared by using screen-printing technology and insulated with a silicone patch. The functional solution is deposited by slit coating technology to generate a nitrate ion-selective electrode and a reference electrode respectively, thereby preparing a nitrate ion detection sensor.

[0007] The third object of the present invention is to provide the application of the above-mentioned nitrate ion detection sensor based on a screen-printed electrode in the detection of nitrate concentration in environmental water bodies or soil. The working electrode and the reference electrode of the sensor are connected to an electrochemical workstation, and the concentration of nitrate ions is determined by measuring the change in the potential value.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A preparation method for a nitrate ion detection sensor based on a screen-printed electrode, comprising the following steps:

[0010] (1) Preparation of the conductive substrate: Based on screen printing technology, the first paste and the second paste are respectively printed on a polyester substrate, and after drying and cutting, the conductive substrates of the working electrode and the reference electrode are respectively formed;

[0011] (2) Insulation treatment: An insulating layer is pasted on the non-working areas of the conductive substrates of the working electrode and the reference electrode;

[0012] (3) Preparation of the nitrate ion-selective electrode: A quaternary ammonium salt, a polymer, a plasticizer, and an organic solvent are mixed in proportion to obtain a precursor solution of the ion-selective membrane. After stirring, the precursor solution is coated on the conductive substrate of the working electrode by slit coating technology to form a functional membrane layer (ISM), and after drying, a nitrate ion-selective electrode is obtained

[0013] (4) Preparation of the reference electrode: A potassium salt, a polymer, and an organic solvent are mixed in proportion to obtain a precursor solution of the reference membrane. After stirring, the precursor solution is coated on the conductive substrate of the reference electrode by slit coating technology to form a reference membrane layer, and after drying, a reference electrode (Ag / AgCl-REs) is obtained, where the functional membrane layer is defined as a nitrate ion-selective membrane.

[0014] Further, in step (1), the polyester substrate is a polybutylene terephthalate (PBT), polycarbonate (PC), polypropylene (PP), or polyethylene terephthalate (PET) substrate, and the preferred polyester substrate is a polyethylene terephthalate substrate; the first paste is a silver paste or a carbon paste, and the preferred first paste is a carbon paste, and the second paste is a silver / silver chloride paste.

[0015] Further, the working electrode and the reference electrode in step (2) are both composed of a plurality of dumbbell-shaped structures. The two ends of the dumbbell-shaped structure form square regions, which are respectively defined as the active sensing region and the external connection region, and the active sensing region and the external connection region are separated by the insulating layer. The length of each dumbbell-shaped structure is 28 mm, and the square regions at its two ends are defined as 2×2 mm 2 、3×3 mm 2 、4×4 mm 2 or 5×5 mm 2 of square regions, and the preferred area of the square region is 4×4 mm 2 or 5×5 mm 2 .

[0016] Further, in step (3), the polymer and the quaternary ammonium salt are added in a mass ratio of 10-20:1, the polymer and the plasticizer are added in a mass ratio of 1:1-3, the organic solvent and the polymer are added in a mass ratio of 3-6:1, and the stirring time is at least 24 h. Among them, the quaternary ammonium salt is tetra-n-octylammonium bromide (TOA-bromide), the polymer is polyvinyl chloride (PVC), the plasticizer is dibutyl phthalate, and the organic solvent is tetrahydrofuran (THF). Preferably, polyvinyl chloride and tetra-n-octylammonium bromide are added in a mass ratio of 15:1, and polyvinyl chloride and dibutyl phthalate are added in a mass ratio of 1:2. According to the polyvinyl chloride with different molecular weights, the ratio of tetrahydrofuran to polyvinyl chloride needs to be adjusted. For low-molecular-weight polyvinyl chloride, the mass ratio of tetrahydrofuran to polyvinyl chloride is 3.33:1, while for high-molecular-weight polyvinyl chloride, the mass ratio of tetrahydrofuran to polyvinyl chloride is 5:1 or 5.4:1. After mixing, the material is magnetically stirred. The thickness of the functional film layer is 70-210 μm, and the preferred thickness is 75 μm.

[0017] Further, in step (3), the ion-selective membrane precursor solution is coated on the active sensing area of the conductive substrate; in step (4), the reference membrane precursor solution is coated on the active sensing area of the conductive substrate.

[0018] Further, in step (3), the mass ratio of the potassium salt, the polymer and the organic solvent is 1:1:9-11; among them, the potassium salt is potassium chloride, the polymer is polyvinyl chloride, and the organic solvent is tetrahydrofuran.

[0019] Further, the insulating layer is a silica gel patch with a thickness of 0.1-1.5 mm, and the preferred thickness is 0.6 mm. The silica gel patch can be cut into strips or a square window array with specific intervals. The array windows are slightly larger than the square areas at both ends of the dumbbell-shaped electrodes to improve the adhesion performance of the film layer.

[0020] Further, the coating parameters of the slot coating technique in step (3) are: the shear speed is 10±2 mm / s, the pumping speed is 0.05±0.01 m / s, the distance between the coater and the insulating layer is 10±2 μm, and the drying time after coating is at least 48 h. Preferably, the shear speed is 10 mm / s, the pumping speed is 0.05 m / s, and the distance between the coater and the surface of the insulating layer is 10 μm.

[0021] A nitrate ion detection sensor based on a screen-printed electrode, obtained by the above preparation method, includes: a dumbbell-shaped working electrode and a reference electrode formed on a polyethylene terephthalate substrate based on the screen-printed electrode. The surface of the working electrode is covered with a nitrate ion-selective membrane made of tetraoctylammonium bromide, polyvinyl chloride, dibutyl phthalate, and tetrahydrofuran. The surface of the reference electrode is covered with a reference membrane made of potassium chloride, polyvinyl chloride, and tetrahydrofuran. A silicone insulating layer is pasted on the non-working areas of the working electrode and the reference electrode.

[0022] The application of the above nitrate ion detection sensor based on a screen-printed electrode in the detection of nitrate concentration in environmental water bodies or soil. The working electrode and the reference electrode of the sensor are connected to an electrochemical workstation, and the nitrate ion concentration is determined by measuring the change in the potential value.

[0023] The present invention has the following advantages:

[0024] 1. The present invention uses screen printing technology to prepare a conductive substrate and a sensing window, and uses a silicone patch for insulation treatment. Subsequently, through slot coating technology, the functional solution is uniformly deposited to generate nitrate ion-selective electrodes (NO 3 - -ISEs) or reference electrodes (REs), and combines them into a potential measurement system. This system has simplicity, economy, and scalability, providing a practical solution for constructing an efficient and economical nitrate ion detection sensor. This sensor not only supports large-scale production but also can accurately monitor the nitrate concentration in soil or water bodies, with the ability of rapid analysis and in-situ measurement. By providing nitrate concentration data in real time and accurately, this sensor improves the accuracy and efficiency of environmental monitoring and agricultural management. In addition, the large-scale production and application of the nitrate ion detection sensor will further promote its development and application in multiple fields such as environmental monitoring and agricultural management.

[0025] 2. The nitrate ion-selective electrode of the present invention adopts a double-layer structure design and is composed of a functional membrane layer and a carbon electrode conductive substrate. By screen-printing high-quality nano-conductive paste (especially carbon materials), a planar electrode is prepared as the conductive substrate, and the ion-selective membrane (ISM) is directly coated by combining the slot-die coating technology. Without the need for an additional intermediate layer (SC layer), the large capacitance and large surface area characteristics of the printed electrode itself are utilized to achieve the direct conversion of electron-ion signals, replacing the function of the intermediate layer in the traditional three-layer structure. This design simplifies the sensor structure, avoids the introduction of complex intermediate layer materials, improves the manufacturing efficiency, and effectively solves the problem of high potential drift caused by the absence of the intermediate layer. By eliminating the introduction of the intermediate layer, the present invention also avoids problems such as interface instability, material degradation, and signal noise caused by it. Further, geometric parameters such as the thickness (75μm) and active area (4×4mm 2 or 5×5mm 2 ) of the functional membrane layer are adjusted to make the performance of the double-layer structure reach or exceed the performance level of the traditional three-layer electrode. The present invention is applicable to high-performance nitrate sensors for low-cost and large-scale production.

[0026] 3. The nitrate ion detection sensor of the present invention combines Bluetooth communication technology and has the ability of rapid analysis and in-situ measurement, and can provide nitrate concentration data in real time and accurately. Description of the Drawings

[0027] Figure 1 is the process flow chart for the preparation of the nitrate ion detection sensor of the present invention.

[0028] Figure 2 is the microscopic morphology diagram of the working electrode and reference electrode of the present invention. Among them, Figure 2 a shows the SEM image of the carbon-based working electrode conductive substrate, Figure 2 b shows the SEM image of the silver-based reference electrode conductive substrate, Figure 2 c shows the SEM image of the Ag / AgCl reference electrode conductive substrate, Figure 2 d shows the SEM image of the nitrate ion-selective membrane / functional membrane layer, Figure 2 e shows the SEM image of the reference membrane.

[0029] Figure 3 is the analysis diagram of the selection of the conductive substrate and the influence of the nitrate ion-selective electrode on the potential performance.

[0030] Among them, Figure 3 a- Figure 3 c are the data diagrams of the silver-based working electrodes (Ag-ISEs) with three different thicknesses of ion-selective membranes / functional membrane layers (ISM) prepared in Comparative Examples 1-3, Figure 3a shows the trend of the time-dependent potential behavior of 50 μm, 75 μm, and 150 μm Ag-ISEs in 0.1 M KNO 3 solution over 90 hours, Figure 3 b and Figure 3 c are the trend graphs of the steady-state Nernst response of ISEs after being immersed in 0.1 M KNO 3 solution for 24 hours.

[0031] Among them, Figure 3 d- Figure 3 f are the data graphs of the carbon-based working electrodes (C-ISEs) with four different thicknesses of ion-selective membrane / functional membrane layers (ISM) prepared in Examples 1-4, Figure 3 d shows the potential changes of C-ISEs with functional membrane layer thicknesses of 75 μm, 100 μm, 150 μm, and 200 μm within 60 hours, Figure 3 e and Figure 3 f are the corresponding Nernst responses under the same conditions.

[0032] Among them, Figure 3 g- Figure 3 i are the data graphs of the carbon-based working electrodes (C-ISEs) with four different areas of ion-selective membrane / functional membrane layers (ISM) prepared in Examples 1, 5-7, Figure 3 g is the trend graph of the time-dependent potential behavior change of C-ISEs with different surface areas, Figure 3 h is the transient response of C-ISEs with different areas, Figure 3 i is the steady-state Nernst response.

[0033] Figure 4 is the electrochemical characterization analysis graph of C-ISEs based on cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Among them, Figure 4 a and Figure 4 d are C-ISEs with different ISM thicknesses in Examples 1-4, Figure 4 b and Figure 4 e are C-ISEs with different surface areas in Examples 1, 5-7, Figure 4 c and Figure 4 f are C-ISEs with different time frames in Examples 1-4, Figure 4 c and Figure 4 f are the equivalent circuit models of Examples 1-4, including solution resistance (Rs), ISM bulk resistance (Rm), geometric capacitance (Cm), charge transfer resistance (Rct), and double-layer capacitance (Cdl) at the C / ISM interface.

[0034] Figure 5 is the difference graph analyzing the carbon and silver conductive electrodes through CV and EIS, among which,Figure 5 a is the cyclic voltammogram (CV) of the Ag electrode and the C electrode in a 0.1 M potassium nitrate solution, Figure 5 b is an enlarged view of the cyclic voltammogram of the C electrode, Figure 5 c is the electrochemical impedance spectrum (EIS) of the Ag electrode and the C electrode.

[0035] Figure 6 is a data graph of the potential response analysis of the Ag / AgCl reference electrode, where, Figure 6 a is the long-term stability test of the Ag / AgCl reference electrode prepared in Example 1 of the present invention in 0.1 M potassium nitrate, 0.1 M potassium chloride, 0.5 M potassium chloride, and river water samples, measured by comparing with the electromotive force (EMF) of a commercial Ag / AgCl reference probe, Figure 6 b is the analytical response reproducibility of the Ag / AgCl reference electrode prepared in the present invention in different solutions.

[0036] Figure 7 is a graph of the performance characterization of the ISE in Example 1 of the present invention in soil, where, Figure 7 a is the anti-interference ability of the ISE under common ion interferences in soil, Figure 7 b is the steady-state Nernst performance of the ISE in the nitrate concentration range from 10^-9 M to 2.7 M (saturated KNO 3 solution), Figure 7 c is the electrode reproducibility characterization, Figure 7 d is the transient Nernst response and its calibration curve of the C-ISEs and the SSRE in a nitrate solution from 0.0001 M to 0.1 M.

[0037] Figure 8 is the in-situ and on-site characterization graph of Example 1 and Example 7 of the present invention, where, Figure 8 a is the in-situ characterization of the nitrate ISEs in Example 1 in soil in an indoor environment, Figure 8 b and Figure 8 c are the schematic diagram and photo of four pairs of nitrate ISEs and reference electrodes in Example 7, which integrate Bluetooth modules for testing the sampled solution and the coarse filtered solution from the nitrifying bacteria reaction tank, showing the continuous monitoring of nitrate concentration on the developed smartphone APP, Figure 8 d and Figure 8 e are the schematic diagrams of the experimental setups of two tests showing the gradual diffusion of water from two bottles to a stable 30% humidity level in the soil mixed with deionized water in Example 1, Figure 8 f is the result of the ISEs detecting nitrate in the nitrifying bacteria reaction tank at different pH values in Example 7. Figure 8 g is the sensor in Example 7 being used to measure three different weights of water samples after being treated by nitrifying bacteria. Detailed implementation mode

[0038] The present invention will be further described in detail below in conjunction with embodiments, but the implementation modes of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. The test methods without specific experimental conditions in the following embodiments usually follow the conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially. Among them, tetraoctylammonium bromide (TOA-bromide), polyvinyl chloride (PVC), dibutyl phthalate (plasticizer), tetrahydrofuran (THF), monopotassium phosphate (KH 2 PO 4 ), dipotassium hydrogen phosphate (K 2 HPO 4 ), sodium sulfate (Na 2 SO 4 ), potassium chloride (KCl) and potassium nitrate (KNO 3 ) were all purchased from Macklin. The carbon paste (JC81 of Julong Huina) was purchased from Julong, and the silver / silver chloride paste (DuPont 5880) was purchased from DuPont.

[0039] Example 1

[0040] As Figure 1 shown, this example provides a preparation method for a nitrate ion detection sensor based on a screen-printed electrode. The specific preparation steps are as follows:

[0041] (1) Preparation of the conductive substrate: Use AI software (Autodesk, San Rafael) to design a mesh through-hole frame template with dimensions of 400 mm × 300 mm to customize the electrode pattern. Through a fully automatic screen printer in a semi-continuous manner, carbon paste and silver / silver chloride paste are respectively printed on a polyethylene terephthalate (PET) substrate. After drying and cutting, the conductive substrates of the working electrode and the reference electrode are respectively formed.

[0042] Among them, both the working electrode and the reference electrode are composed of multiple dumbbell-shaped structures. The multiple dumbbell-shaped structures are arranged in sequence at a certain interval along their length direction. The two ends of the dumbbell-shaped structure form a 5×5 mm 2 square area, which are respectively defined as the active sensing area and the external connection area. The active sensing area is covered with a nitrate-selective membrane or a reference membrane, and the external connection area serves as the electrical connection window of the sensor.

[0043] (2) Insulation treatment: Paste a silicone patch on the non-working area of the conductive substrates of the working electrode and the reference electrode. The thickness of the silicone patch is 0.6 mm. After natural drying at room temperature, use a Cameo4 precision cutting machine to precisely cut the silicone patch. The silicone patch is strip-shaped as a whole and is pasted on the non-working area between the active sensing area and the external connection area to achieve insulation treatment of the non-working area. Since the silicone patch has a certain thickness, a groove is formed in the active sensing area for filling the nitrate ion-selective membrane and the reference membrane.

[0044] (3) Preparation of nitrate ion-selective electrode: Polyvinyl chloride and tetraoctylammonium bromide are added in a mass ratio of 15:1, polyvinyl chloride and dibutyl phthalate are added in a mass ratio of 1:2, and high-molecular-weight polyvinyl chloride is used. The mass ratio of tetrahydrofuran to polyvinyl chloride is 5.4:1. Mix tetraoctylammonium bromide, dibutyl phthalate, polyvinyl chloride, and tetrahydrofuran, and magnetically stir the mixed material for at least 24 h to achieve uniform distribution of the mixture, obtaining an ion-selective membrane precursor solution.

[0045] Use a heating type slot coater and integrate a 3D-printed guiding device. Set the width of the coating area to be wider than the expected sensor window by a certain value to ensure that the functional membrane can completely fill the active sensing area and prevent situations that may damage the sensor performance, such as the functional membrane peeling off from the substrate, external solution penetrating into the sensor, and the electrode oxidizing in the environment. To meet the requirements of functional membrane coatings with different areas, the opening width of the 3D-printed mold is set to 15 mm.

[0046] Place the conductive substrate of the working electrode at an appropriate position on the heating type coating machine platform. Use an injection pump to deliver the ion-selective membrane precursor solution to the substrate at a speed of 0.05 mL / s to fully cover the active sensing area of the dumbbell-shaped electrode. The applicator moves back and forth at a constant speed of 10 mm / s for coating. Since the conductive substrate is evenly adsorbed on the flat plate and maintains a constant shear rate and applied force during the coating process, this greatly improves the reproducibility of the preparation. The height of the applicator relative to the silicone surface of the substrate is set to 10 μm. After the functional membrane layer is deposited, air-dry it naturally at room temperature for more than 48 h to fully dry, obtaining nitrate ion-selective electrodes (NO 3 - -ISEs), where the thickness of the functional membrane layer (ISM) is 75 ± 2.35 μm.

[0047] (4) Preparation of reference electrode: The solid reference electrode uses a printed silver / silver chloride electrode and is combined with a salt-saturated reference membrane. During the preparation of the salt-saturated reference membrane, first, potassium chloride is ground to a size of 635 mesh (20 μm opening). Then, the ground potassium chloride, high molecular weight polyvinyl chloride, and tetrahydrofuran are mixed in a mass ratio of 1:1:10 to form a reference membrane precursor solution. To ensure the uniformity of the solution, the stirring time is at least 24 h. The reference membrane precursor solution is coated on the active sensing area of the conductive substrate using the slit coating technique in step (3) to form a reference membrane layer, forming a continuous strip of coating area, and the reference electrode (Ag / AgCl-REs) is obtained after drying.

[0048] Finally, the nitrate ion-selective electrode and the reference electrode are combined to form a nitrate ion detection sensor.

[0049] Example 2

[0050] The difference between this example and Example 1 is that the thickness of the functional membrane layer of the nitrate ion-selective electrode is 100 ± 2.7 μm, and the parts not mentioned are the same as those in Example 1.

[0051] Example 3

[0052] The difference between this example and Example 1 is that the thickness of the functional membrane layer of the nitrate ion-selective electrode is 150 ± 3.09 μm, and the parts not mentioned are the same as those in Example 1.

[0053] Example 4

[0054] The difference between this example and Example 1 is that the thickness of the functional membrane layer of the nitrate ion-selective electrode is 200 ± 2.58 μm, and the parts not mentioned are the same as those in Example 1.

[0055] Example 5

[0056] The difference between this example and Example 1 is that square regions of 2 × 2 mm are formed at both ends of the dumbbell-shaped structure, and the parts not mentioned are the same as those in Example 1. 2

[0057] Example 6

[0058] The difference between this example and Example 1 is that square regions of 3 × 3 mm are formed at both ends of the dumbbell-shaped structure, and the parts not mentioned are the same as those in Example 1. 2

[0059] Example 7

[0060] The difference between this example and Example 1 is that square regions of 4 × 4 mm are formed at both ends of the dumbbell-shaped structure, and the parts not mentioned are the same as those in Example 1. 2

[0061] Comparative Example 1

[0062] The difference between this comparative example and Example 1 is that the conductive substrate of the working electrode is silver paste (Ag-ISEs) printed on polyethylene terephthalate, the thickness of the functional film layer of the nitrate ion-selective electrode is 50 ± 4.9 μm, and the parts not mentioned are the same as those in Example 1.

[0063] Comparative Example 2

[0064] The difference between this comparative example and Example 1 is that the conductive substrate of the working electrode is silver paste printed on polyethylene terephthalate, the thickness of the functional film layer of the nitrate ion-selective electrode is 75 ± 5.9 μm, and the parts not mentioned are the same as those in Example 1.

[0065] Comparative Example 3

[0066] The difference between this comparative example and Example 1 is that the conductive substrate of the working electrode is silver paste printed on polyethylene terephthalate, the thickness of the functional film layer of the nitrate ion-selective electrode is 150 ± 9.7 μm. Among them, low-molecular-weight polyvinyl chloride is used, and the mass ratio of tetrahydrofuran to polyvinyl chloride is 3.33:1. The parts not mentioned are the same as those in Example 1.

[0067] Characterization of the nitrate ion detection sensor

[0068] Next, the nitrate ion detection sensors of Examples 1-7 and Comparative Examples 1-3 were characterized and analyzed.

[0069] (1) Composition of the electrode material

[0070] Figure 2 a- Figure 2 e shows the microscopic morphologies of the electrodes prepared in the above examples and comparative examples. Among them, Figure 2 a shows the scanning electron microscope (SEM) image of the carbon-based electrode, Figure 2 b shows the SEM image of the silver-based electrode. They both show a combination of interconnected conductive particles and an inert polymer binder on the substrate. Figure 2 c shows that the Ag / AgCl reference electrode is patterned using the same printing process as the Ag electrode, which is a thin silver conductor mixed with AgCl particles. The Ag particles provide the required electrical connection for the reference electrode and participate in a reversible redox reaction with AgCl to maintain a constant interfacial potential at the reference electrode. Figure 2 d shows the scanning electron microscope (SEM) image of the working electrode coated with the nitrate ion-selective membrane. The nitrate ISM is deliberately layered and folded to better visually distinguish the membrane from the conductive Ag electrode. The result shows the uniform dispersion of dibutyl phthalate / tetra-n-octylammonium bromide / polyvinyl chloride matrix on the electrode. Figure 2Figure e shows the SEM image of a reference electrode coated with a mixture of potassium chloride and polyvinyl chloride. Potassium chloride particles with a diameter of approximately 10 μm were finely ground and embedded in the polyvinyl chloride deposited on the Ag / AgCl electrode. The potassium chloride particles can dissolve in water, diffuse into the interior of the polyvinyl chloride in the sample solution, and generate a high-concentration chloride ion solution in the membrane to achieve a stable chloride ion concentration-dependent reference potential.

[0071] Figure 3 a- Figure 3 Figure c shows silver-based working electrodes (Ag-ISEs) with three different thicknesses of ion-selective membrane / functional membrane layers (ISM) prepared in Comparative Examples 1-3, showing the influence of the conductive electrode on the performance of ISEs. Among them, Figure 3 Figure a shows the time-dependent potential changes of Ag-ISEs with 50 μm (Comparative Example 1), 75 μm (Comparative Example 2), and 150 μm (Comparative Example 3) in 0.1 M KNO 3 solution within 96 hours. The 50-μm ISEs were tested 48 times repetitively, and each of the other samples was tested 36 times repetitively. The results show that Ag-ISEs with a thicker ISM require a longer activation time than those with a thinner ISM. For example, the 150-μm ISEs took more than 24 hours to reduce the potential drift from more than 2.9 mV / h to less than 2 mV / h, while the 50-μm ISEs reached this equilibrium within 15 hours, reducing the potential drift from more than 3.5 mV / h to 0.5 mV / h. Subsequently, the 150-μm ISEs exhibited a potential drift rate of 0.6 mV / h, slightly higher than 0.37 mV / h of the 50-μm ISEs during the period of 24 to 72 hours. Figure 3 Figures b and 3c show the steady-state Nernst response of ISEs after being immersed in 0.1 M KNO 3 solution for 24 hours. The slope of the 150-μm ISEs was -62.8 ± 0.56 mV / dec, while that of the thin-type ISEs was -58 ± 2.07 mV / dec, the latter being lower. This slightly super-Nernst response observed in the 150-μm ISEs may be attributed to incomplete activation within 24 hours. Overall, Ag-ISEs exhibited a standard potential change corresponding to a change in the ISM thickness of 170 mV per 100 μm, and the consistency between electrodes was 170 / 100 = 1.7 mV / μm, higher than 0.8 mV / μm in C-ISEs. In addition, the long-term stability of Ag-ISEs was worse than that of C-ISEs, and the potential drift of the latter was approximately 0.1 mV / h. The decrease in stability and reproducibility can be attributed to the lower chemical stability of silver compared to carbon nanomaterials.

[0072] Next, refer to Figure 5a-5c shows the differences between carbon and silver conductive electrodes analyzed by CV and EIS. In the CV analysis, the Ag electrode shows significantly larger oxidation and reduction peaks. The significant changes in the peaks in the Ag electrode indicate its higher electrochemical reactivity and potential instability. In contrast, the C electrode shows smaller peaks, indicating more stable electrochemical properties. The EIS results further confirm this stability. The double-layer capacitance of the C electrode (Cdl = 31.78 pF) is significantly larger than that of the Ag electrode, which is 4.29 pF, as shown in Table 1. Overall, these observations confirm that C-ISEs have greater chemical stability compared to Ag-ISEs and exhibit smaller potential drift.

[0073] Table 1 is a data table of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) for silver (Ag) electrodes and carbon (C) electrodes.

[0074]

[0075] Next, refer to Figure 6 a, the solid-state reference electrode (RE) was evaluated for activation, long-term stability, and reproducibility. The reference electrode was made by coating a KCl / PVC matrix on an Ag / AgCl electrode. The figure shows the time-dependent potential response of the RE at different ion concentrations, measured over 24 hours, involving 16 electrodes, for a total of 48 electrodes. The results show that the RE completes adjustment within 3 - 6 hours, and the stable potential in all solutions is approximately 40 mV. In addition, the potential drift of all electrodes is very small, indicating that the RE can provide a stable reference potential in a complex environment with different types and intensities of primary and interfering ions. As Figure 6 shown in b, the reproducibility of the RE was evaluated by plotting the steady-state potential of the electrode in each solution against the number of test electrodes. The reproducibility of the electrode in 0.1 M KNO 3 , 0.1 M KCl, and 0.5 M KCl solutions is excellent, with an EMF change of less than 5 mV. This change is rare in test environments such as soil and aquaculture waters, so the 5 mV difference can be ignored.

[0076] Therefore, it is best to use a carbon electrode (C-ISEs) as the working electrode and a silver / silver chloride electrode (Ag / AgCl-REs) as the reference electrode for the conductive substrate.

[0077] (2) The thickness and surface area of the functional film layer

[0078] Return to reference Figure 3 d- Figure 3 f shows the carbon-based working electrodes (C-ISEs) of four different thickness ion-selective membranes / functional film layers (ISM) prepared in Examples 1 - 4, where,Figure 3 d shows the time-dependent potential behavior of C-ISEs in 0.1 M KNO 3 solution over 60 hours. The results show that thicker ISEs have a higher standard potential, while thinner ISEs, although having a faster regulation speed, also have a larger potential drift. For example, after soaking in 0.1 M KNO 3 solution for 24 hours, the 200-μm ISEs showed the highest electromotive force (EMF) of 381 ± 3.87 mV, followed by the 150-μm, 100-μm, and 75-μm ISEs at 338.61 ± 3.26 mV, 296.27 ± 3.83 mV, and 285.96 ± 3.39 mV, respectively. The potential drift was large in the first few hours and then decreased, and this transition was the regulation or activation process of the ISE. For example, the potential drift of the 200-μm ISEs was -1.19 mV / h in the first 24 hours and decreased to -0.08 mV / h between 24 and 60 hours, indicating that the 200-μm ISEs required 24 hours of regulation time. A similar 24-hour regulation time was also observed for the 150-μm ISEs, while the 75-μm and 100-μm ISEs required a shorter regulation time. For example, in the first 12 hours, the potential drift of the 100-μm ISEs was -1.33 mV / h and then stabilized to -0.27 mV / h between 12 and 24 hours. The 75-μm ISEs also showed a similar 12-hour regulation time, with the potential drift decreasing from -1.03 mV / h to -0.13 mV / h.

[0079] Continuing to refer to Figure 3 d, the 200-μm film-thickness ISEs exhibited a potential drift rate of -0.08 mV / h, slightly lower than the -0.11 mV / h, -0.18 mV / h, and -0.13 mV / h of the 150-μm, 100-μm, and 75-μm ISEs, respectively, for an experimental time of 24 to 60 hours. After reaching equilibrium, solid-state ISEs may exhibit instability, usually due to the formation of a thin water layer between the electrode and the ISM. This internal water layer facilitates the ion transfer between it and the external sample solution, changes the composition of the solution, and causes potential drift after regulation. Therefore, thinner ISEs form a thicker water layer, resulting in a faster change in electromotive force within the same time range, while thicker ISEs change more slowly. Therefore, the long-term stability also shows a pattern related to the ISM geometry, with thicker ISEs showing a slower potential drift.

[0080] Continuing to refer to Figure 3 e, the Nernst response of the ISEs was carried out by measuring the electromotive force of the ISEs in the nitrate concentration range of 0.0001 M to 0.1 M with a ten-fold concentration interval in 0.1 M KNO 3Each measurement lasted 2 minutes after immersion in the solution for 24 hours. The step change in the electromotive force of the 200μm ISEs was about -60±0.14mV / dec, while the step change of the ISEs with functional membrane layer thickness of 75μm, 100μm and 150μm was about -57.5±0.16mV / dec to -58.7±0.33mV / dec. The slightly super-Nernst performance of the 200μm ISEs may be attributed to interface effects. Non-uniform charge distribution or non-ideal interface capacitance at the membrane / solution interface or solid contact interface may lead to the observed super-Nernst response. This phenomenon may be more obvious in thicker membranes because their slower internal diffusion can amplify the influence of interfacial effects. This leads to a larger difference in the concentration of major ions at the ISM / solution interface, which enhances the response. This suggests that more than 24 hours are required for the 200μm ISEs to be fully activated. Importantly, all ISEs showed a fast Nernst response within 10 seconds at all media concentrations tested.

[0081] Continue to refer Figure 3 f, which shows the Nernst calibration curves plotted under steady-state conditions, with nitrate concentration as the horizontal axis. The responses of all ISEs show that increasing the thickness of the ISEs requires longer adjustment time and exhibits smaller potential drift. In addition, the standard potential of the ISEs (i.e., 0.0001M KNO in the calibration curve) 3 The potential of the solution has a thickness-dependent change of about 100 mV for a thickness change of 125 μm. For the same thickness, the standard potential change of all ISEs is 15 mV, and each thickness test is repeated 10 times.

[0082] Next reference Figure 3 g, which shows that the film thickness of the ISEs in Examples 1, 5-7 is 75 μm, and different ISM surface areas are selected to test their potential performance within 60 hours, with a surface area range of 2×2 mm 2 Up to 5×5mm 2 It can be seen that 2×2mm 2 The activation time required for the ISEs of the 2×2 mm2 was significantly longer than that of the other three ISEs, with a potential drift of approximately -0.92 mV / h within 60 hours. The other ISEs only needed 12 hours, with the potential drift decreasing from -0.77 mV / h in the first 12 hours to -0.45 mV / h in the subsequent hours. 2 The ISEs showed a potential drift of -0.56 mV / h from 24 to 60 h, which is smaller than that of the 3×3 mm 2 , 4×4mm 2 and 5×5mm 2The ISEs of -0.49 mV / h, -0.29 mV / h, and -0.35 mV / h are more significant. Regarding the standard potential at equilibrium, the ISEs with different surface areas exhibit a similar pattern to those with different thicknesses, with larger surface areas showing higher standard potentials. As the surface area increases from 2×2 mm 2 to 5×5 mm 2 , the potential changes by approximately 50 mV. For the same surface area, the standard potential changes of the three larger-surface-area ISEs are within 15 mV, while the ISEs of 2×2 mm 2 show a potential change of 40 mV. The tests for all areas were repeated 10 times. Continuing to refer to Figure 3 h and 3i, which show that the ISEs exhibit a consistent steady-state Nernst response in the nitrate concentration range of 0.0001 M to 0.1 M, with a response of -56.59 ± 0.44 mV / dec and above, regardless of their surface area, which is expected since the steady-state Nernst response depends only on the interface between the ISM and the solution.

[0083] In summary, the geometry of the nitrate C-ISEs significantly affects their performance. Thicker ISEs require longer conditioning times and exhibit slower potential drifts, while thinner ISEs reach equilibrium faster but experience higher potential drifts. The surface area also plays a crucial role: smaller surface areas require longer activation times and exhibit larger potential drifts, while larger sensors reach equilibrium faster and exhibit lower drift rates. Additionally, changes in thickness or surface area affect the reproducibility of the electrodes, resulting in differences in the standard potential. Despite these differences, all ISEs exhibit a consistent Nernst response at different concentrations, indicating that the steady-state response is mainly affected by the interface between the ISM and the solution, rather than the geometry of the ISE.

[0084] As Figure 4 a and Figure 4 d show, which illustrate the ISEs with different ISM thicknesses in Examples 1-7 when immersed in 0.1 M KNO 3Cyclic voltammetry (CV) characteristics and electrochemical impedance spectroscopy (EIS) data after 24 hours of the solution showed that both Rm and Rct increased with the increase in membrane thickness. For example, when the ISM thickness was increased from 75 μm to 200 μm, Rm (defined as the diameter of the high-frequency EIS semicircle) increased from 34.05 kΩ to 134.55 kΩ, while Rct (defined as the charge transfer resistance at the C / ISM interface) increased from 24.09 kΩ to 69.08 kΩ. This can be explained by the fact that the resistance of the ISM is proportional to its thickness (i.e., the length l in the resistance definition, where ρ is the resistivity and A is the area). Therefore, thicker ISEs have a greater membrane resistance. At the same time, this change can also be attributed to the thin water layer formed between the ISM and the conductive electrode. Thinner ISEs form the water layer earlier than thicker ISEs. Therefore, the water layer facilitates the transport of ions at the C / ISM interface, resulting in a decrease in Rct. On the other hand, Cm, Cdl, and Ccv are inversely proportional to the ISM thickness. For example, when the ISM thickness was increased from 75 μm to 200 μm, Cm (the bulk capacitance of the ISM) decreased from 37.14 pF to 19.26 pF, Cdl (the electrical double layer capacitance at the C / ISM interface) decreased from 13.18 μF to 11.13 μF, and Ccv decreased from 38 μF to 4.17 μF. The changing trend of Cm can be explained by its defining formula, where ε is the dielectric constant and d is the thickness. With the increase in ISM thickness, the decrease in Cdl may also be due to the slower formation of the water layer between the ISM and the conductive electrode, resulting in a slower and smaller change in the dielectric constant. In addition, the CV capacitance (Ccv) can be defined as the area enclosed by the CV curve, and the formula is where A is the area of the enclosed CV curve, k is the scan rate, and V1 and V2 are the starting and ending scan voltages. It was observed that with the increase in membrane thickness, the decrease in Ccv can be explained by the increase in Rm. The increase in membrane thickness leads to an increase in resistance, thereby reducing the current and further decreasing the enclosed area of the CV curve, that is, the decrease in Ccv. In addition, the time constant τ also increases with the increase in thickness, indicating that thicker membranes require longer time to reach equilibrium (complete regulation).

[0085] Continue to refer to Figure 4 b and Figure 4 e, which show the CV characteristics and EIS data of ISEs with different surface areas. Different from the pattern observed when the ISM thickness changes, the increase in the ISM surface area results in a decrease in Rm and Rct, but an increase in Cm, Cdl, and Ccv. This indicates that a larger surface area facilitates ion flow and improves the charge transfer efficiency at the C / ISM interface. In addition, the time constant τ decreases with the increase in surface area. For 2×2 mm 2 ISEs, τ is 582.90, while for 5×5 mm 2For ISEs, τ is 317.50. These observations explain why ISEs with larger surface areas have faster activation speeds and better stability.

[0086] The time-dependent electrochemical performance of ISEs was evaluated at different time frames of 0.5, 1.5, 3, 6, 12, 24, 36, and 48 hours after soaking in 0.1 M KNO 3 solution for 48 hours. C-ISEs with a surface area of 5×5 mm 2 and a thickness of 75 μm were used in the test. As Figure 4 shown in Figure 4 c and 2 f, which show the CV and EIS data, Rm increased from 27.59 kΩ at 0.5 hour to 31.78 kΩ at 36 hours and decreased to 28.68 kΩ at 48 hours. In contrast, Cm decreased from 64.85 pF at 0.5 hour to 34.54 pF at 36 hours and then slightly increased to 36.13 pF at 48 hours. τ and Ccv also showed similar trends, with τ reaching a minimum value of 304.31 at 36 hours and Ccv reaching a maximum value of 39.92 μF at 36 hours. These results indicate that ISEs with a surface area of 5×5 mm

[0087] and a thickness of 75 μm were mainly dominated by regulation in the first 36 hours and showed a potential drift between 36 and 48 hours. The above data are shown in Table 2.

[0088]

[0089] Therefore, based on the above experimental analysis, the ISM with a thickness of 75 μm and an area of 5×5 mm 2 performed the best, i.e., Example 1.

[0090] (3) Performance Test

[0091] Based on the fact that the ISM with a thickness of 75 μm and an area of 5×5 mm 2 performed the best, Example 1 was selected for subsequent performance characterization. Figure 7a shows the anti-interference ability of an ion-selective electrode (ISE) under common ion interferences in soil. The results indicate that the selectivity coefficient of the ISE is proportional to the concentration of nitrate ions. Thus, as the nitrate concentration increases, the influence of interfering ions gradually decreases. Among various interfering ions, chloride ions have the most significant interference on the nitrate sensor, with its selectivity coefficient approaching 1, especially in low-concentration nitrate (such as 0.0001 M). This can be attributed to the structural similarity between nitrate and chloride ions at the molecular level. Additionally, the difference in the number of charges between nitrate and chloride ions may also exacerbate this interference. Considering these factors together, it leads to enhanced competitive adsorption between nitrate and chloride ions, thereby affecting the operation of the ISE. However, the impact of this interference on the ISE performance is not significant. Generally, the nitrate concentration range in agricultural soil is from 0.001 to 0.01 M, which can increase to 0.02 to 0.03 M after fertilization, while the chloride ion concentration is generally from 0.001 to 0.005 M. Assuming a typical nitrate concentration of 0.01 M and a chloride ion concentration of 0.005 M in the soil, the interference from chloride ions would cause a change in the interference potential of the ISE of approximately 1.37 mV. However, in the case of increasing nitrate concentration (usually exceeding 0.01 M), the interference from chloride ions becomes negligible, which ensures the reliable detection of nitrate by the ISE under different environmental conditions, highlighting its effectiveness in agricultural applications.

[0092] As Figure 7 shown in 3 b, it shows the steady-state Nernst performance of the ISE over a nitrate concentration range from 10^-9 M to 2.7 M (saturated KNO 3 solution). The activated ISE exhibits a long-term linear response with a detection limit as low as below 10^-6 M. In contrast, the unactivated ISE fails to produce an effective potential response when exposed to a nitrate concentration as high as 10^-5 M. Additionally, the activated ISE shows a rapid potential response when changing the concentration of the KNO Figure 7 solution. When the concentration changes from low to high and then back to low, continue to refer to 3 c, which shows that the potential difference in 0.1 M KNO 3 solution is 4.05 mV, while the potential differences in 0.001 M and 0.0001 M KNO Figure 7 solution are 11.48 mV and 10.28 mV respectively. In multiple tests, the potential difference always remains within 12 mV. It should be noted that the on-site application of the nitrate sensor requires the integration of the prepared working electrode and reference electrode. An 8-pair integrated nitrate sensor using 75 μm C-ISEs was tested in the differential mode of a data acquisition (DAQ) device. 3The instantaneous Nernst response of the sensor after 24 hours of activation in solution. The results show that the sensor rapidly transitions and reaches a stable potential within different nitrate concentration ranges (from 0.0001 M to 0.1 M). At equilibrium, the integrated sensor exhibits a Nernst slope of -56.34 ± 1.18 mV / dec, which is close to that of ISEs using commercial reference electrodes. In addition, compared with ISEs using commercial reference electrodes, the integrated sensor returns to reference Figure 3 f, and the standard potential change is 20 mV. This change is attributed to the difference in ion concentration between the prepared reference electrode and the commercial electrode, resulting in a change in the reference potential.

[0093] (4) In-situ and on-site characterization

[0094] As Figure 8 shown in a, the long-term accuracy of the C-ISEs based on Example 1 was tested in-situ. It was evaluated over 48 hours by mixing commercially available vegetable garden soil with deionized water and placing it in a large plastic container. Sixteen ISEs were firmly placed in the soil. The EMF between each ISE and the commercial reference electrode was measured using a NIDAQ device (NI USB-6211, National Instruments) and 16 analog channels. In the experimental setup, two bottles were placed at the edges of the container, designed to gradually release deionized water into the soil and extract nitrate ions for continuous detection. Before inserting into the soil, the ISEs were pre-treated in 0.1 M nitrate solution for 48 hours to ensure the stability of the EMF. Then, the results were compared with a commercial nitrate probe (Intellical TM ISENO3181, Hach).

[0095] As Figure 8 shown in d and Figure 8 e, within 48 hours, the humidity level of the soil rose from 0% to 30%. It rapidly rose to 30% in the first 9 hours and remained around 30% throughout the experiment. During this period, the measured EMF of the ISEs was stable at a nitrate concentration of 0.001 M. In contrast, the soil was sampled at a water-to-soil mass ratio of 50:30 (w / w) and analyzed using a commercial nitrate probe, and the result showed that the nitrate concentration was approximately 0.001 M. This result indicates that the integrated sensor can work effectively at a humidity level as low as 30%. After the experiment, the ISEs were taken out of the soil, and no peeling or damage was observed.

[0096] As Figure 8 shown in b and Figure 8 c, in Example 7, with a thickness of 75 μm and an area of 4 × 4 mm 2The C-ISE is paired with an SSRE of the same size to form a nitrate ion detection sensor. Then, four pairs of sensors are placed in a 3D printed polycarbonate frame (sized 7 cm x 3.5 cm) to ensure stability and durability. These sensors are connected to a reading device with a Bluetooth module via a flexible printed circuit (FPC) cable. To enhance waterproofness, a thermosetting silicone solution (SS-6002S) is applied. After assembly, a smartphone application is configured to convert the voltage signal into nitrate concentration via wireless communication and display it at predetermined intervals. As Figure 8 shown in Fig. f, the system was evaluated by real-time monitoring of the nitrate levels in a nitrifying bacteria reactor located at the South China Sea Fisheries Research Institute. The reactor simulates the nitrification process, in which bacteria convert ammonia to nitrate by controlling temperature, pH, oxygen level, and nutrient concentration. Three different pH environments were established for the growth of bacterial sludge and maintained for three days, and nitrate measurements were taken on the fourth day. Before testing, the ISE was activated in a 0.1 M nitrate solution for 12 hours and calibrated with a 10 mg / L standard solution for 5 minutes. Subsequently, six consecutive measurements (coarse filtration) of the reactor samples were taken to determine the nitrate concentration, once every 15 minutes. The results were verified using ultraviolet-visible spectroscopy. The integrated nitrate sensor exhibited high precision comparable to UV-Vis spectrophotometry (with a deviation within 2 mg / L), and good repeatability (with a deviation within 1 mg / L) for the same water sample in the pH range of 6 to 9, indicating the stability and reliability of the sensor in complex environments. As Figure 8 shown in Fig. g, the sensor was used to measure three different weights of water samples after treatment with nitrifying bacteria.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing a nitrate ion detection sensor based on screen-printed electrodes, characterized in that: The following steps are involved: (1) Preparation of conductive substrate: Printing the first slurry and the second slurry on the polyester substrate based on screen printing technology, and forming the conductive substrates of the working electrode and the reference electrode respectively after drying and cutting; (2) Insulation treatment: pasting an insulating layer on the non-working area of ​​the conductive substrate of the working electrode and the reference electrode; (3) Preparation of nitrate ion selective electrode: quaternary ammonium salt, polymer, plasticizer and organic solvent are mixed in proportion to prepare ion selective membrane precursor solution, and after stirring, the precursor solution is coated on the conductive substrate of the working electrode by slit coating technology to form a functional membrane layer, and after drying, a nitrate ion selective electrode is obtained; (4) Preparation of reference electrode: Potassium salt, polymer and organic solvent are mixed in proportion to prepare reference film precursor solution, which is then coated on the conductive substrate of the reference electrode using a slit coating technique after stirring to form a reference film layer, and then dried to obtain a reference electrode.

2. The method for preparing a nitrate ion detection sensor based on screen-printed electrodes according to claim 1, characterized in that: In step (1), the polyester substrate is a polybutylene terephthalate, polycarbonate, polypropylene or polyethylene terephthalate substrate, and the preferred polyester substrate is a polyethylene terephthalate substrate; the first slurry is a silver slurry or a carbon slurry, and the preferred first slurry is a carbon slurry, and the second slurry is a silver / silver chloride slurry.

3. The method for preparing a nitrate ion detection sensor based on screen-printed electrodes according to claim 1, characterized in that: The working electrode and the reference electrode in step (2) are both composed of a plurality of dumbbell-shaped structures, and the two ends of the dumbbell-shaped structures form square areas, which are respectively defined as active sensing areas and external connection areas, and the active sensing areas and the external connection areas are separated by the insulating layer.

4. The method for preparing a nitrate ion detection sensor based on screen-printed electrodes according to claim 3, characterized in that: In step (3), the polymer and the quaternary ammonium salt are added in a mass ratio of 10 to 20:1, the polymer and the plasticizer are added in a mass ratio of 1:1 to 3, the organic solvent and the polymer are added in a mass ratio of 3 to 6:1, and the stirring time is at least 24 hours; wherein the quaternary ammonium salt is tetra-n-octylammonium bromide, the polymer is polyvinyl chloride, the plasticizer is dibutyl phthalate, and the organic solvent is tetrahydrofuran; and the thickness of the functional film layer is 70 to 210 μm.

5. The method for preparing a nitrate ion detection sensor based on screen-printed electrodes according to claim 3, characterized in that: In step (3), the ion selective membrane precursor solution is coated on the active sensing area of ​​the conductive substrate; in step (4), the reference membrane precursor solution is coated on the active sensing area of ​​the conductive substrate.

6. The method for preparing a nitrate ion detection sensor based on screen-printed electrodes according to claim 1, characterized in that: The mass ratio of the potassium salt, the polymer and the organic solvent in step (3) is 1:1:9-11; wherein the potassium salt is potassium chloride, the polymer is polyvinyl chloride, and the organic solvent is tetrahydrofuran.

7. The method for preparing a nitrate ion detection sensor based on screen-printed electrodes according to claim 1, characterized in that: The insulating layer is a silicone patch with a thickness of 0.1-1.5 mm.

8. The method for preparing a nitrate ion detection sensor based on screen-printed electrodes according to claim 1, characterized in that: The coating parameters of the slit coating technology in step (3) are: shear speed of 10±2 mm / s, pumping speed of 0.05±0.01 m / s, distance between the coater and the insulating layer of 10±2 μm, and drying time after coating of at least 48 h.

9. A nitrate ion detection sensor based on screen-printed electrodes, characterized in that: The method is obtained by the preparation method described in any one of claims 1 to 8.

10. Application of the nitrate ion detection sensor based on screen-printed electrodes according to claim 9 in detecting nitrate concentration in environmental water or soil.

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