A planar optode fluorescence sensing film with an acidic pH response range, preparation method and application

Abstract

The application belongs to the field of environmental micro-interface two-dimensional pH monitoring technology, and discloses a planar optical electrode fluorescence sensing film with an acidic pH response range, a preparation method and application. Specifically, the application provides a pH-sensitive fluorescent indicator, the pH-sensitive fluorescent indicator is 2', 7'-dihalo-5(6)-N-octadecyl carboxamide fluorescein or 2', 4', 5', 7'-tetrahalo-5(6)-N-octadecyl carboxamide fluorescein, the molecular structural formula of the pH fluorescent indicator is shown as formula I; in the formula, X is selected from Cl or F; Y is selected from H, Cl or F; by introducing electron-withdrawing halogen atoms at the halogenated sites of fluorescein, the pKa value of the indicator is reduced, and the deficiency that the traditional planar optical electrode sensing film does not cover the acidic region is solved, wherein the tetrachloro derivative can realize the response in the strong acid interval of pH 1.70~4.96, and the tetrafluoro derivative can cover the acid-base interval of pH 2.75~7.68.

Description

Technical Field

[0001] This invention belongs to the field of planar optical technology for two-dimensional pH monitoring of environmental micro-interfaces. Specifically, it relates to a method for preparing a planar optical fluorescent sensing membrane that can cover a wide range of pH responses from acidic to neutral, and its application in in-situ two-dimensional pH monitoring of environmental micro-interfaces such as acidic soil and sediments. Background Technology

[0002] Acidified arable land is a core control target for arable land quality protection and food security in my country. The national strategy of "storing grain in the land and storing grain in technology," the national arable land quality improvement action, and the action plan for soil pollution prevention and control all list the comprehensive management of acidified soils in the south, the analysis of acidification process mechanisms, and the research and development of precise control technologies as key tasks. pH is a core physicochemical parameter that regulates the geochemical cycle of mineral nutrients, heavy metals, and other substances in the environment. Especially in micro-regions such as the rhizosphere and sediment-water interface, micro-scale spatial and micro-scale changes in pH directly affect the migration, transformation, biological activity, and bioabsorption of nutrients and pollutants. Traditional electrode methods can only achieve single-point measurement and have drawbacks such as strong invasiveness, easy damage to the micro-interface structure, and high cost for large-area deployment, which cannot meet the monitoring needs of two-dimensional dynamic distribution of pH in micro-regions.

[0003] Planar photoluminescence sensing technology, with its advantages of in-situ, real-time, non-invasiveness, and high spatiotemporal resolution, has become one of the core technologies for pH monitoring of environmental micro-interfaces. Its core lies in the construction of the fluorescence sensing membrane. Currently, publicly available pH fluorescence sensing membrane technologies still have the following shortcomings: (1) Fluorescent indicators have inherent defects that limit their application scenarios. Commonly used HPTS and fluorescein indicators have high pKa values, are not sensitive to pH changes in the acidic range, and unmodified water-soluble dyes are prone to leakage from the membrane matrix, resulting in poor stability and short service life of the sensing membrane.

[0004] (2) The membrane matrix relies on imported materials, which poses supply chain risks. The existing mainstream pH fluorescence sensing membranes all use imported polyurethane hydrogel Hydromed D4 as the embedding matrix. This material is relatively expensive and may pose supply chain risks, which limits the localization and large-scale application of related technologies.

[0005] (3) The pH response range is narrow, and there is a gap in the technology for adapting to the acidic range, making it unsuitable for monitoring the entire range of complex environments. Existing publicly available pH fluorescence sensing membranes use a single indicator, and the effective response range of a single indicator system is limited, mostly focusing on the neutral and weakly alkaline ranges. It is difficult to take into account the wide range of continuous detection from strongly acidic to neutral, and it cannot be adapted to the full-area monitoring scenarios of acidic soils, sediments, and other environments in my country. In summary, developing a domestically produced, cost-effective, wide response range covering strongly acidic to neutral, dye-leakage-resistant, highly stable, and highly sensitive planar photoluminescent sensing membrane is a technical challenge that urgently needs to be overcome in the field of in-situ two-dimensional pH monitoring of micro-interfaces in acidic environments. Summary of the Invention

[0006] This invention aims to provide a method for preparing and applying a planar photoluminescent sensing membrane with an acidic pH response range. Addressing the shortcomings of existing pH fluorescence sensing membranes, such as lack of acid detection capability, narrow response range in the acidic region, easy dye leakage, reliance on imported matrices, and high domestic production costs, this invention utilizes independently designed and synthesized multiple types of halogenated modified fluorescein indicators, combined with domestically produced polyurethane to construct a novel composite sensing system. This enables in-situ, real-time, and non-invasive two-dimensional pH monitoring across a wide pH range of 2.0–7.0 in environments such as acidic soils and weakly acidic sediments. It fills the gap in micro-interface imaging technology in the acidic region, achieves the localization of core raw materials and preparation processes for the sensing membrane, and provides independent technical support for the remediation of acidified farmland and the study of environmental mechanisms in acidic sediments in my country.

[0007] To achieve the aforementioned objectives, this invention employs the following technical solutions based on the core principles of halogenation modification to regulate the pKa of indicators, lipophilic modification to enhance membrane stability, multi-indicator compounding to broaden the response range, and domestic matrix substitution to overcome barriers: This invention provides a pH-sensitive fluorescent indicator, wherein the pH fluorescent indicator is 2',7'-dihalo-5(6)-N-octadecylcarboxamide fluorescein or 2',4',5',7'-tetrahalo-5(6)-N-octadecylcarboxamide fluorescein; the molecular structure of the pH fluorescent indicator is shown in Formula I; where X is selected from Cl or F; Y is selected from H, Cl or F; (Formula I).

[0008] In another aspect, the present invention provides a planar photoluminescent sensing membrane with an acidic pH response range, wherein the fluorescent sensing membrane is composed of polyurethane as the matrix material and encapsulating the pH-sensitive fluorescent indicator and the reference indicator.

[0009] Furthermore, the reference indicator is 9,10-diphenylanthracene.

[0010] Furthermore, the pH fluorescence indicator is at least one of 2',7'-dichloro-5(6)-N-octadecylcarboxamide fluorescein, 2',7'-difluoro-5(6)-N-octadecylcarboxamide fluorescein, 2',4',5',7'-tetrachloro-5(6)-N-octadecylcarboxamide fluorescein, or 2',4',5',7'-tetrafluoro-5(6)-N-octadecylcarboxamide fluorescein.

[0011] Furthermore, the pH fluorescence indicator is a combination of 2',4',5',7'-tetrachloro-5(6)-N-octadecylcarboxamide fluorescein and 2',7'-difluoro-5(6)-N-octadecylcarboxamide fluorescein.

[0012] Furthermore, the thickness of the fluorescent sensing film is 5~15 μm.

[0013] In another aspect, the present invention provides a planar optical sensor with an acidic pH response range, wherein the sensing layer of the sensor is prepared using any of the planar optical fluorescent sensing films described in the present invention.

[0014] The present invention provides the pH fluorescent indicator, or any of the planar photoluminescent sensing films, or the planar photoluminescent sensor, for the application of two-dimensional pH monitoring in environmental micro-interfaces, wherein the environment includes sediments, soil, wetlands, or water bodies.

[0015] The present invention also provides a method for preparing the planar photoluminescent sensing film according to any one of the claims, comprising the following steps: S1: Dissolve polyurethane in an organic solvent and stir until completely dissolved to prepare a polyurethane stock solution; S2: Dissolve the pH fluorescent indicator in an organic solvent, disperse it evenly, and prepare a fluorescent indicator stock solution; S3: Dissolve the pH-insensitive reference indicator in an organic solvent, disperse it evenly, and prepare a reference indicator stock solution; S4: Take the polyurethane stock solution, fluorescent indicator stock solution, and reference indicator stock solution and mix them evenly to obtain the casting solution; S5: The casting solution is coated onto the surface of the transparent substrate of the support layer, and after drying, the planar photoluminescent sensing film is obtained.

[0016] Furthermore, the preparation method described above, In step S1, the concentration of polyurethane in the polyurethane stock solution is 0.02~0.2 g / ml; In step S2, the total concentration of the fluorescent indicator stock solution is 5~20 mg / ml; In step S3, the concentration of the reference stock solution is 5~20 mg / ml; In step S4, the volume ratio of the polyurethane stock solution, the fluorescent indicator stock solution, and the reference indicator is 1:(0.02~0.2):(0.02~0.2).

[0017] In another aspect, the present invention provides a method for applying the planar optical sensor with the acidic pH response range, wherein the planar optical sensor is fixed to the interface to be measured, and under the illumination of an excitation light source, a fluorescence image of the interface to be measured is acquired using an image capture system, and the spatiotemporal distribution information of the pH of the interface to be measured is obtained after data processing; the excitation light source is an ultraviolet or visible light source of 360~450nm, and the image capture system includes a CCD / CMOS camera and an image processing terminal.

[0018] Compared with the prior art, the present invention has the following significant advantages: 1. A novel pH-sensitive fluorescent indicator was synthesized, achieving high sensitivity in the acidic range while simultaneously solving the dye leakage problem. This invention significantly reduces the pKa value of the indicator by introducing electron-withdrawing fluorine or chlorine atoms at the 2', 4', 5', and 7' positions of the fluorophore. Example 3, the tetrachloro derivative, has an apparent pKa of 3.33 and a dynamic range of pH 1.70–4.96, suitable for detection in strongly acidic environments. Example 4, the tetrafluoro derivative, has a dynamic range of pH 2.75–7.68, covering the acid-neutral transition range. The dichloro and difluoro derivatives of Examples 1 and 2 are suitable for the moderately acidic range. Comparative Example 1, a single commercially available indicator, only covers the weakly alkaline range of pH 6.67–9.2. This invention combines multiple modified indicators to form a wide-range acidic response system with pH 2.0~7.0, solving the technical problem that traditional sensing membranes cannot be adapted to the detection of micro-interfaces in acidic soils and filling the gap in existing acidic range detection technology. At the same time, a lipophilic octadecyl carboxamide group is introduced at position 5 (6) to improve the hydrophobicity of the indicator and improve its compatibility with polyurethane matrix. This solves the problem of easy dye leakage to a certain extent, and the sensing membrane can be used stably in the water environment for a long time.

[0019] 2. Domestic matrix substitution, high cost-effectiveness, and suitable for domestic mass production. This invention uses domestic polyurethane to replace the traditional imported Hydromed D4 polyurethane hydrogel, eliminating reliance on the import supply chain and reducing material procurement costs. At the same time, the domestic matrix has excellent film-forming properties, high mechanical strength, good water permeability, and good compatibility with modified indicators. The prepared sensing membrane has excellent uniformity and response rate, making it suitable for the needs of large-scale, industrialized mass production in China.

[0020] 3. A multi-indicator compound system is constructed to achieve a wide-range response of pH 2.0 to 7.0, covering the monitoring needs of soil and sediment in all scenarios. This invention combines self-synthesized tetrachloro and difluorinated derivatives in a certain proportion to achieve a continuous linear response across the entire range from strongly acidic to neutral, solving the problem of narrow response range of single indicators. A single sensing membrane can be adapted to the monitoring needs of environmental samples such as acidic to neutral soil and sediment.

[0021] 4. The preparation process is simple, the detection performance is excellent, and the application scenarios are highly adaptable. The synthetic route of the indicator in this invention is mild and the yield is stable. The preparation of the sensor membrane does not require demanding equipment or complex procedures. The prepared sensor membrane has a fast response speed (<60 s) and a spatial resolution of 74 μm, which can capture subtle changes in pH in the rhizosphere microzone. Verified by actual soil monitoring, it can clearly identify the rhizosphere acidification phenomenon in maize in acidic soil (rhizosphere pH is 0.3~1.1 units lower than that in non-rhizosphere). It can meet the needs of in-situ two-dimensional pH monitoring of micro-interfaces in multiple scenarios such as acidic soil and sediments, and has broad application prospects. Attached Figure Description

[0022] Figure 1 Synthetic routes of 2',7'-dihalo-5(6)-N-octadecylcarboxamide fluorescein and 2',4',5',7'-tetrahalo-5(6)-N-octadecylcarboxamide fluorescein; Figure 2. Fluorescence patterns of the sensing membranes of Examples 1-5 and Comparative Example 1 in gradient pH buffer solution; Figure 3. Standard curves of pH-fluorescence intensity ratio fitting for the sensing membranes of Examples 1-5 and Comparative Example 1; Figure 4. Spatial uniformity of the sensing membrane in Example 4; Figure 5. Two-dimensional pseudo-color map of pH distribution obtained by applying the sensing membrane of Example 7 to the rhizosphere pH characterization of maize in acidic red soil. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0024] The pH fluorescence indicator of the sensing membrane of this invention is a halogenated lipophilic modified fluorescein, and the synthesis route is as follows: Figure 1In the examples, all raw materials and reagents used in the synthesis process were analytically pure or had a purity of 98% or higher, and the methanesulfonic acid was anhydrous methanesulfonic acid. The reagent abbreviations are as follows: N-hydroxysuccinimide (NHS), tetrahydrofuran (THF), dicyclohexylcarbodiimide (DCC), and dicyclohexylurea (DCU). The emission light of the modified fluorophore can be recorded by the green channel of an RGB CMOS camera, and the fluorescence intensity increases with increasing pH. Using 9,10-diphenylanthracene as a reference indicator, its emission light can be recorded by the aforementioned camera's blue channel, and its fluorescence intensity does not easily change with pH. Both are embedded in a polyurethane film to prepare a fluorescent sensing membrane. The aforementioned green / blue channel fluorescence intensity ratio can be fitted with a known series of pH values, serving as a basis for quantitatively characterizing the two-dimensional pH distribution of environmental micro-interfaces.

[0025] Example 1: Synthesis of 2',7'-dichloro-5(6)-N-octadecylcarboxamide fluorescein and preparation of its fluorescent sensing film In this embodiment, a 2',7'-dichloro-5(6)-N-octadecylcarboxamide fluorescein fluorescent sensing membrane was prepared using the following steps: a) Synthesis of 2',7'-dichloro-5(6)carboxyfluorescein: 5.8 g (40 mmol) of 4-chlororesorcinol, 3.9 g (20 mmol) of trimellitic anhydride, and 5.5 g (20 mmol) of zinc chloride were weighed and placed in a round-bottom flask. Under nitrogen protection, the mixture was heated to 150 °C and stirred for 2 h. Then, the mixture was heated to 170 °C and stirred for 15 min. When cooled to 50-60 °C, 70 ml of 1 mol / L hydrochloric acid solution was poured in. The mixture was refluxed at 110 °C for 1 h. The mixture was filtered while hot. The filter cake was boiled with 200 ml of deionized water and refluxed for 1 h. The mixture was filtered again. The filter cake was washed three times with deionized water and dried at 60 °C to obtain 5.59 g of crude 2',7'-dichloro-5(6)carboxyfluorescein, which was an orange solid powder with a yield of 62.8%.

[0026] b) Preparation of NHS active lipid solution: Weigh 0.445 g (1 mmol) of crude 2',7'-dichloro-5(6)carboxyfluorescein and 0.138 g (1.2 mmol) of NHS, and dissolve them in 10 ml of THF. Then, dissolve 0.24 g (1.2 mmol) of DCC in 2 ml of THF and add it dropwise to the above reaction system under ice-water bath conditions. Stir and react under nitrogen protection for 30 min. Then remove the ice-water bath and react at room temperature for 12 h. Filter the solution through a 0.45 μm organic filter membrane to remove the solid DCU. The product is an orange solution.

[0027] c) Amide reaction: 0.32 g (1.2 mmol) of octadecylamine was dissolved in 2 ml of THF and added dropwise to the solution above. The mixture was stirred for 3 h at room temperature under nitrogen protection. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by silica gel column chromatography. The stationary phase was 60-100 mesh silica gel, and the mobile phase was 9:1 chloroform / methanol. 0.553 g of 2',7'-dichloro-5(6)-N-octadecylcarboxamide fluorescein was obtained as an orange-red solid with a yield of 79.3%.

[0028] d) Preparation of the sensing membrane: Take 5 g each of commercially available UBR Hydro-75A-30 and UBR Hydro-75A-100 polyurethane from Sichuan Youborui New Materials Co., Ltd., and dissolve them in 90 ml of THF under reflux stirring at 60 ℃ to prepare a polyurethane stock solution. Take 2 ml of the polyurethane stock solution, 200 μl of 10 mg / ml concentration 2',7'-dichloro-5(6)-N-octadecylcarboxamide fluorescein ethanol solution, and 50 μl of 10 mg / ml concentration 9,10-diphenylanthracene THF solution, vortex mix them, and then coat them onto the surface of a PET plastic film using a 125 μm thickness coater; after the solvent is dried, the fluorescent sensing membrane is obtained, and the membrane thickness after drying is about 10 μm.

[0029] Example 2: Synthesis of 2',7'-difluoro-5(6)-N-octadecylcarboxamide fluorescein and preparation of its fluorescent sensing film In this embodiment, a 2',7'-difluoro-5(6)-N-octadecylcarboxamide fluorescein fluorescent sensing membrane was prepared using the following steps: a) Synthesis of 2',7'-difluoro-5(6)carboxyfluorescein: 1.0 g (7.8 mmol) of 4-fluororesorcinol and 0.75 g (3.9 mmol) of trimellitic anhydride were weighed and dissolved in 7.8 ml of anhydrous methanesulfonic acid. The mixture was stirred at 80 °C for 16 h under nitrogen protection. Then, 60 ml of ice water was poured in, filtered, and the filter cake was washed three times with deionized water. The filter cake was dried at 60 °C to obtain 1.37 g of crude 2',7'-difluoro-5(6)carboxyfluorescein, which was an orange-yellow solid powder with a yield of 83.1%.

[0030] b) Preparation of NHS active lipid solution: Weigh 0.824 g (2 mmol) of crude 2',7'-difluoro-5 (6)-carboxyfluorescein and 0.276 g (2.4 mmol) of NHS, and dissolve them in 20 ml of THF. Then, dissolve 0.48 g (2.4 mmol) of DCC in 2 ml of THF and add it dropwise to the above reaction system under ice-water bath conditions. Stir and react under nitrogen protection for 30 min, then remove the ice-water bath and react at room temperature for 12 h. Filter through a 0.45 μm organic filter membrane to remove solid DCU, and the product is an orange-red solution.

[0031] c) Amide reaction: 0.64 g (2.4 mmol) of octadecylamine was dissolved in 2 ml of THF (a hot air gun could be used to promote dissolution), and added dropwise to the solution above. The mixture was stirred for 3 h at room temperature under nitrogen protection. The solvent was removed by rotary evaporation under reduced pressure, and the product was purified by silica gel column chromatography. The stationary phase was 60-100 mesh silica gel, and the mobile phase was 9:1 chloroform / methanol. 0.966 g of the product 2',7'-difluoro-5(6)-N-octadecylcarboxamide fluorescein was obtained as an orange-red solid with a yield of 72.9%.

[0032] d) Preparation of the sensing membrane: Take 5 g each of commercially available UBR Hydro-75A-30 and UBR Hydro-75A-100 polyurethane from Sichuan Youborui New Materials Co., Ltd., and dissolve them in 90 ml of THF under reflux stirring at 60℃ to prepare a polyurethane stock solution. Take 2 ml of the polyurethane stock solution, 200 μl of 10 mg / ml concentration 2',7'-difluoro-5(6)-N-octadecylcarboxamide fluorescein ethanol solution, and 50 μl of 10 mg / ml concentration 9,10-diphenylanthracene THF solution, vortex mix them, and then coat them onto the surface of a PET plastic film with a 125 μm thickness coating tool; after the solvent is dried, the fluorescent sensing membrane is obtained, and the membrane thickness after drying is about 10 μm.

[0033] Example 3: Synthesis of 2',4',5',7'-Tetrachloro-5(6)-N-octadecylcarboxamide fluorescein and preparation of its fluorescent sensing film. In this embodiment, a 2',4',5',7'-tetrachloro-5(6)-N-octadecylcarboxamide fluorescein fluorescent sensing membrane was prepared using the following steps: a) Synthesis of 2',4',5',7'-tetrachloro-5(6)carboxyfluorescein: 4.48 g (25 mmol) of 2,4-dichlororesorcinol and 2.41 g (12.5 mmol) of trimellitic anhydride were weighed and dissolved in 25 ml of anhydrous methanesulfonic acid. The mixture was stirred at 80 °C for 48 h under nitrogen protection. Then, 175 ml of ice water was poured in, filtered, and the filter cake was washed three times with deionized water. The filter cake was dried at 60 °C to obtain 4.01 g of crude 2',4',5',7'-tetrachloro-5(6)carboxyfluorescein, which was a dark red solid powder with a yield of 62.4%.

[0034] b) Preparation of NHS active lipid solution: Weigh 1.018 g (2 mmol) of crude 2',4',5',7'-tetrachloro-5(6)carboxyfluorescein and 0.276 g (2.4 mmol) of NHS, and dissolve them in 20 ml of THF. Then, dissolve 0.48 g (2.4 mmol) of DCC in 2 ml of THF and add it dropwise to the above reaction system under ice-water bath conditions. Stir and react under nitrogen protection for 30 min, then remove the ice-water bath and react at room temperature for 12 h. Filter through a 0.45 μm organic filter membrane to remove solid DCU, and the product is an orange-red solution.

[0035] c) Amide reaction: 0.64 g (2.4 mmol) of octadecylamine was dissolved in 2 ml of THF (a hot air gun could be used to promote dissolution), and added dropwise to the solution above. The mixture was stirred for 3 h at room temperature under nitrogen protection. The solvent was removed by rotary evaporation under reduced pressure, and the product was purified by silica gel column chromatography. The stationary phase was 60-100 mesh silica gel, and the mobile phase was 9:1 chloroform / methanol. 0.610 g of the product 2',4',5',7'-tetrachloro-5(6)carboxyfluorescein was obtained as a dark red solid with a yield of 39.9%.

[0036] d) Preparation of the sensing membrane: Take 5 g each of commercially available UBR Hydro-75A-30 and UBR Hydro-75A-100 polyurethane from Sichuan Youborui New Materials Co., Ltd., and dissolve them in 90 ml of THF under reflux stirring at 60℃ to prepare a polyurethane stock solution. Take 2 ml of the polyurethane stock solution, 300 μl of 10 mg / ml concentration 2',4',5',7'-tetrachloro-5(6)-N-octadecylcarboxamide fluorescein ethanol solution, and 50 μl of 10 mg / ml concentration 9,10-diphenylanthracene THF solution, vortex mix them, and then coat them onto the surface of a PET plastic film using a 125 μm thickness coater; after the solvent dries, the fluorescent sensing membrane is obtained, with a thickness of about 10 μm after drying.

[0037] Example 4: Synthesis of 2',4',5',7'-Tetrafluoro-5(6)-N-octadecylcarboxamide fluorescein and preparation of its fluorescent sensing film In this embodiment, a 2',4',5',7'-tetrafluoro-5(6)-N-octadecylcarboxamide fluorescein fluorescent sensing membrane was prepared using the following steps: a) Synthesis of 2',4',5',7'-tetrafluoro-5(6)carboxyfluorescein: 0.50 g (3.42 mmol) of 2,4-difluororesorcinol and 0.33 g (1.71 mmol) of trimellitic anhydride were weighed and dissolved in 4 ml of anhydrous methanesulfonic acid. The mixture was stirred at 80 °C for 16 h under nitrogen protection. Then, 30 ml of ice water was poured in, filtered, and the filter cake was washed three times with deionized water. The filter cake was dried at 60 °C to obtain 0.225 g of crude 2',4',5',7'-tetrafluoro-5(6)carboxyfluorescein, which was a brown solid powder with a yield of 29.3%.

[0038] b) Preparation of NHS active lipid solution: Weigh 0.225 g (0.5 mmol) of crude 2',4',5',7'-tetrafluoro-5(6)carboxyfluorescein and 0.069 g (0.6 mmol) of NHS, and dissolve them in 10 ml of THF. Then, dissolve 0.124 g (0.6 mmol) of DCC in 2 ml of THF and add it dropwise to the above reaction system under ice-water bath conditions. Stir and react under nitrogen protection for 30 min. Then remove the ice-water bath and react at room temperature for 12 h. Filter the solution through a 0.45 μm organic filter membrane to remove the solid DCU. The product is a deep magenta solution.

[0039] c) Amide reaction: 0.162 g (0.6 mmol) of octadecylamine was dissolved in 1 ml of THF (a hot air gun could be used to promote dissolution), and added dropwise to the solution above. The mixture was stirred for 3 h at room temperature under nitrogen protection. The solvent was removed by rotary evaporation under reduced pressure, and the product was purified by silica gel column chromatography. The stationary phase was 60-100 mesh silica gel, and the mobile phase was 9:1 chloroform / methanol. 0.122 g of the product 2',4',5',7'-tetrafluoro-5(6)carboxyfluorescein was obtained as a deep magenta solid, with a yield of 50.4%.

[0040] d) Preparation of the sensing membrane: Take 5 g each of commercially available UBR Hydro-75A-30 and UBR Hydro-75A-100 polyurethane from Sichuan Youborui New Materials Co., Ltd., and dissolve them in 90 ml of THF under reflux stirring at 60℃ to prepare a polyurethane stock solution. Take 2 ml of the polyurethane stock solution, 300 μl of 10 mg / ml concentration 2',4',5',7'-tetrafluoro-5(6)-N-octadecylcarboxamide fluorescein ethanol solution, and 50 μl of 10 mg / ml concentration 9,10-diphenylanthracene THF solution, vortex mix them, and then coat them onto the surface of a PET plastic film using a 125 μm thickness coater; after the solvent is dried, the fluorescent sensing membrane is obtained, and the membrane thickness after drying is about 10 μm.

[0041] Example 5: Preparation of a mixed fluorescent indicator sensing film In this embodiment, the fluorescent sensing film with mixed fluorescent indicator is prepared by the following steps: a) Preparation of the sensing membrane: Take 5 g each of commercially available UBR Hydro-75A-30 and UBR Hydro-75A-100 polyurethane from Sichuan Youborui New Materials Co., Ltd., and dissolve them in 90 ml of THF under reflux stirring at 60°C to prepare a polyurethane stock solution. Take 2 ml of the polyurethane stock solution, 100 μl of 10 mg / ml concentration 2',4',5',7'-tetrachloro-5(6)-N-octadecylcarboxamide fluorescein ethanol solution (synthesized in Example 3), 100 μl of 10 mg / ml concentration 2',7'-difluoro-5(6)-N-octadecylcarboxamide fluorescein ethanol solution (synthesized in Example 2), and 50 μl of 10 mg / ml concentration 9,10-diphenylanthracene THF solution, vortex mix them, and then coat them onto the surface of a PET plastic film with a 125 μm thickness coating tool; after the solvent is dried, the mixed fluorescent indicator fluorescent sensing membrane is obtained, and the membrane thickness after drying is about 10 μm.

[0042] Comparative Example 1: Preparation of 5-(octadecylamino)fluorescein fluorescent sensing film Preparation of the sensing membrane: 5 g each of commercially available UBR Hydro-75A-30 and UBR Hydro-75A-100 polyurethanes from Sichuan Youborui New Materials Co., Ltd. were dissolved in 90 ml of THF under reflux stirring at 60°C to prepare a polyurethane stock solution. A 10 mg / ml solution of commercially available 5-(octadecylamino)fluorescein was prepared by dissolving it in anhydrous ethanol. 2 ml of the polyurethane stock solution, 200 μl of the 10 mg / ml 5-(octadecylamino)fluorescein ethanol solution, and 50 μl of the 10 mg / ml 9,10-diphenylanthracene THF solution were vortexed and then coated onto the surface of a PET plastic film using a 125 μm thickness coater. After solvent drying, the fluorescent sensing membrane was obtained, with a thickness of 10 μm after drying.

[0043] Example 6: Calibration and Performance Testing of Fluorescent Sensing Membrane The sensing membranes prepared in Examples 1-5 and Comparative Example 1 were calibrated and their performance tested, and the steps are as follows: a) Preparation of standard buffer solutions: Using a 10 mmol concentration of 2-morpholine ethanesulfonic acid aqueous solution, adjust the pH with sodium hydroxide and hydrochloric acid to prepare a series of standard buffer solutions with pH 1.6~9.2 and an interval of about 0.5 pH units. Adjust the conductivity of the series of buffer solutions to 2000±10 μs / cm with sodium chloride solution. b) Image acquisition system setup: A 365 nm ultraviolet lamp was used as the excitation source (Xiniu ZF-23pro ultraviolet analyzer), and a CMOS camera (Hikvision MV-CE200-11UC industrial camera + 8 mm F1.4 one-inch target lens) was used. A 20 mm diameter 430 nm long-pass filter was configured between the lens and the sensor to build a planar optical imaging system. The parameters such as light source power, camera exposure time, white balance, focal length, and aperture were kept consistent. c) Calibration and Fitting: The sensing membrane was attached to the inner wall of the cuvette, and standard buffer solutions of different pH values ​​were added sequentially. After equilibration, fluorescence images were acquired. The average fluorescence intensity ratio of the green channel to the blue channel of the image was extracted using the R language raster package software. The pH-fluorescence intensity ratio data was fitted using the Boltzmann equation in the R language software to obtain the standard curve and fitting parameters. The dynamic range was calculated using 10% to 90% of the upper and lower bounds of the Boltzmann equation. d) Performance test results ( Figure 3 ): The sensing membrane of Example 1 has an apparent pKa of 6.79, a dynamic range of 5.80 to 7.79, a good signal-to-noise ratio within this range, and an adjusted R² of 0.9995 for the fitted curve.

[0044] The sensing membrane of Example 2 has an apparent pKa of 6.43, a dynamic range of 5.36 to 7.49, a good signal-to-noise ratio within this range, and an adjusted R² of 0.9967 for the fitted curve.

[0045] The sensing membrane of Example 3 has an apparent pKa of 3.33, a dynamic range of 1.70 to 4.96, a good signal-to-noise ratio within this range, and an adjusted R² of 0.9968 for the fitted curve.

[0046] The sensing membrane of Example 4 has an apparent pKa of 5.22, a dynamic range of 2.75–7.68, a good signal-to-noise ratio within this range, and an adjusted R² of 0.9962 for the fitted curve. The sensing membrane exhibits good uniformity; the average roughness Ra of the pH space grid (5 × 7 cm, 74 μm) characterized in a pH=4.72 buffer solution is 0.0890, and the coefficient of variation CV is 2.52%. Figure 4 ).

[0047] Example 5: The mixed fluorescent indicator sensing film was fitted using an additive Boltzmann equation, with apparent pKa1 = 3.45, apparent pKa2 = 5.84, and a dynamic range of 2.75~6.95. The signal-to-noise ratio was good in the ranges of 2.45~4.15 and 4.76~6.95, and the adjusted R2 of the fitted curve was 0.9970.

[0048] The sensing membrane of Comparative Example 1 has an apparent pKa of 7.93, a dynamic range of 6.67 to 9.2, a good signal-to-noise ratio within this range, and an adjusted R² of 0.9995 for the fitted curve.

[0049] In summary, Examples 4 and 5 prepared fluorescent sensing membranes covering a pH range from strongly acidic to neutral soils. The advantage of Example 4 is that it has a wider dynamic range, while the raw material cost of Example 5 is lower than that of Example 4, and the fluorescence intensity is higher than that of Example 4 at similar concentrations.

[0050] Example 7: Application of sensing membrane in two-dimensional pH distribution monitoring in rhizosphere of acidic red soil In this embodiment, the acidic zone sensing membrane prepared in Example 4 is applied to the two-dimensional pH distribution monitoring of maize rhizosphere in acidic red soil in southern China. The steps are as follows: a) Root box culture: Thick clayey, gray-muddy mountain red soil from Luliang County, Yunnan Province was collected as the test soil. The soil pH (1:5 H2O extraction) was 4.68, and the electrical conductivity (saturated soil slurry) was 2080 μs / cm. After air-drying, the soil samples were sieved through a 2 mm sieve and placed in a transparent root box with one side removable. The dimensions of the root box were 12×10×1.2 cm (width, height, and thickness). Two-day-old germinating maize seedlings were transplanted into the root box and cultured at a 45° angle to allow the roots to grow along one side of the removable plate. After 3 days of culture, the soil was watered to the maximum field capacity. After equilibration for 24 h, the two-dimensional distribution of rhizosphere pH was characterized by planar optical polarization. b) Sensor membrane installation: Remove the root box cover plate, and flatly attach the sensor membrane prepared in Example 4 to the soil and root surface, and equilibrate in the dark for 1 hour; c) In-situ imaging: The root box was moved to the UV analyzer of Example 5, and the imaging system built in Example 5 was used to acquire the fluorescence image of the sensing membrane, keeping the shooting parameters completely consistent with the calibration process. d) Data Processing: The green channel fluorescence intensity of each pixel in the image was extracted using the R language raster package. This intensity was then substituted into the calibrated standard curve to calculate the pH value corresponding to each pixel. A two-dimensional spatial distribution map of the maize rhizosphere pH was obtained through pseudo-color rendering. This map clearly identifies rhizosphere acidification in acidic soils, showing that the rhizosphere pH is 0.3–1.1 units lower than the non-rhizosphere pH, with a spatial resolution of 74 μm. Figure 5 ).

Claims

1. A pH fluorescent indicator, characterized in that, The pH fluorescent indicator is 2',7'-dihalo-5(6)-N-octadecylcarboxamide fluorescein or 2',4',5',7'-tetrahalo-5(6)-N-octadecylcarboxamide fluorescein; the molecular structure of the pH fluorescent indicator is shown in Formula I; where X is selected from Cl or F; Y is selected from H, Cl or F; (Formula I).

2. A planar photoluminescent sensing membrane with an acidic pH response range, wherein the fluorescent sensing membrane is composed of polyurethane as the matrix material and encapsulated with the pH fluorescent indicator and reference indicator as described in claim 1.

3. The fluorescent sensing membrane according to claim 2, characterized in that, The reference indicator is 9,10-diphenylanthracene.

4. The fluorescent sensing membrane according to claim 2, characterized in that, The pH fluorescence indicator is at least one of 2',7'-dichloro-5(6)-N-octadecylcarboxamide fluorescein, 2',7'-difluoro-5(6)-N-octadecylcarboxamide fluorescein, 2',4',5',7'-tetrachloro-5(6)-N-octadecylcarboxamide fluorescein, or 2',4',5',7'-tetrafluoro-5(6)-N-octadecylcarboxamide fluorescein.

5. The fluorescent sensing film according to claim 4, characterized in that, The pH fluorescent indicator is a combination of 2',4',5',7'-tetrachloro-5(6)-N-octadecylcarboxamide fluorescein and 2',7'-difluoro-5(6)-N-octadecylcarboxamide fluorescein.

6. The fluorescent sensing membrane according to any one of claims 2-5, characterized in that, The thickness of the fluorescent sensing film is 5~15 μm.

7. A planar optical sensor with an acidic pH response range, characterized in that, The sensing layer of the sensor is prepared using the planar photoluminescent sensing film as described in any one of claims 2-5.

8. The application of the pH fluorescent indicator of claim 1, or the planar photoluminescent sensing membrane of any one of claims 2-5, or the planar photoluminescent sensor of claim 7 in two-dimensional pH monitoring of environmental micro-interfaces, wherein the environment includes sediments, soil, wetlands, or water bodies.

9. A method for preparing the planar photoluminescent sensing film according to any one of claims 2 to 5, characterized in that, Includes the following steps: S1: Dissolve polyurethane in an organic solvent and stir until completely dissolved to prepare a polyurethane stock solution; S2: Dissolve the pH fluorescent indicator in an organic solvent, disperse it evenly, and prepare a fluorescent indicator stock solution; S3: Dissolve the pH-insensitive reference indicator in an organic solvent, disperse it evenly, and prepare a reference indicator stock solution; S4: Take the polyurethane stock solution, fluorescent indicator stock solution, and reference indicator stock solution and mix them evenly to obtain the casting solution; S5: The casting solution is coated onto the surface of the transparent substrate of the support layer, and after drying, the planar photoluminescent sensing film is obtained.

10. The preparation method according to claim 9, characterized in that, In step S1, the concentration of polyurethane in the polyurethane stock solution is 0.02~0.2 g / ml; In step S2, the total concentration of the fluorescent indicator stock solution is 5~20 mg / ml; In step S3, the concentration of the reference stock solution is 5~20 mg / ml; In step S4, the volume ratio of the polyurethane stock solution, the fluorescent indicator stock solution, and the reference indicator stock solution is 1 : (0.02~0.2) : (0.02~0.2).

11. A method for applying the planar optical sensor with the acidic pH response range as described in claim 7, characterized in that, The planar optical sensor is fixed to the interface to be measured. Under the illumination of the excitation light source, the fluorescence image of the interface to be measured is acquired by the image capture system. After data processing, the spatiotemporal distribution information of pH of the interface to be measured is obtained. The excitation light source is an ultraviolet or visible light source of 360~450nm. The image capture system includes a CCD / CMOS camera and an image processing terminal.

Images