Visual fluorescent probe for reducing free radicals as well as preparation method and device of visual fluorescent probe
By designing a detection device that combines a visual fluorescent probe for reducing free radicals with a gel layer, in-situ, real-time visual monitoring of reducing free radicals in soil was achieved. This solves the problems of inability to observe in situ and low sensitivity in traditional methods, and provides a more accurate detection method.
Patent Information
- Application Number
- CN202511087541.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-05
- Publication Date
- 2025-10-31
AI Technical Summary
Traditional free radical detection methods cannot visualize the spatial distribution of soil pores or solid-liquid interfaces in situ, cannot dynamically track the generation and transformation of free radicals, and have low sensitivity and selectivity in complex soil environments. They are easily affected by soil matrix interference and lack fluorescent quantitative probes for reducing free radicals.
A fluorescent probe for visualizing reducing free radicals and its detection device were designed. The fluorescent probe HPBD was combined with an agar gel layer, and the generation and distribution of reducing free radicals in the soil were monitored in real time by a confocal laser scanning microscope. A polyvinylidene fluoride filter membrane was used to isolate soil particles and avoid signal interference.
This method enables in-situ, real-time, and visual monitoring of reducing free radicals during soil remediation, revealing the regulatory role of organic matter on free radical pathways. It provides a more intuitive and reliable detection method, avoiding the biases of traditional methods.
Smart Images
Figure CN120865087A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of soil pollution detection technology, and in particular to a reducing free radical visualization fluorescent probe and its preparation method and apparatus. Background Technology
[0002] Traditional free radical detection methods, such as electron paramagnetic resonance (EPR) technology, require the use of specific spin trapping agents to capture free radicals in order to indirectly infer the presence and type of free radicals. They can only detect the overall concentration of free radicals in the solution and cannot reveal their spatial distribution in soil pores or solid-liquid interfaces, resulting in insufficient understanding of local reaction hotspots and mass transfer limitations.
[0003] Traditional detection methods rely on offline sampling and laboratory analysis (such as liquid chromatography-mass spectrometry and EPR), which can only sample and detect at specific time points and cannot dynamically track the instantaneous process of free radical generation and transformation (such as the initial burst stage or the later decay stage), and may miss key kinetic information.
[0004] Existing free radical detection methods may exhibit low sensitivity and selectivity in complex soil environments, and are easily affected by soil matrix interference, leading to decreased accuracy and reliability of detection results and making it difficult to accurately reflect the true behavior of free radicals under actual soil conditions. In complex soil systems, multiple free radicals and non-target reactive species coexist, and traditional scavengers (such as DMPO) are susceptible to cross-reaction interference (e.g., DMPO-·OH reacting with DMPO-SO4·) - Signal overlap and the potential generation of secondary free radicals (·CH2OH) by quenchers (such as methanol) can lead to errors in quantitative analysis.
[0005] Existing methods require removing samples from the reaction system, which may alter oxygen concentration, pH, or temperature. This is especially problematic in anaerobic remediation where maintaining in-situ conditions is difficult, affecting the reliability of test results.
[0006] Lack of in-situ visualization capabilities: It is impossible to visually demonstrate the differences in the distribution of free radicals on the surface of soil particles or in solution, making it difficult to assess the regulatory role of soil organic matter (SOM) or minerals on free radical pathways.
[0007] There is a lack of methods for characterizing reducing free radicals: Currently, various fluorescent quantitative probes such as 2',7'-dichlorofluorescein diacetate and SOSG have been developed for oxidizing reactive oxygen species such as hydroxyl radicals and singlet oxygen. However, there is still a lack of reports on fluorescent quantitative probes for reducing free radicals. Summary of the Invention
[0008] The purpose of this invention is to provide a reducing free radical visualization fluorescent probe and its preparation method and apparatus, in order to solve the above-mentioned technical problems.
[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0010] This invention provides a reducing radical visualization fluorescent probe, which comprises compounds with the following structure:
[0011]
[0012] This invention also provides a method for preparing a reducing free radical visualization fluorescent probe, comprising the following steps:
[0013] 1) Propylamine was mixed in an ethanol solution of 4-nitro-1,8-naphthoic anhydride and refluxed to obtain a rice-yellow solid product;
[0014] 2) Mix the rice-yellow solid product, zinc powder and NH4Cl and react them. Then add CH2Cl2 and methanol to the reaction product and continue the reaction. The resulting yellow solid is the reducing free radical visualization fluorescent probe, denoted as HPBD.
[0015] Furthermore, the reflux reaction temperature is 70–90°C, and the reflux reaction time is 12–36 h;
[0016] The ratio of 4-nitro-1,8-naphthoic anhydride in the ethanol solution of propylamine and 4-nitro-1,8-naphthoic anhydride is 400-410 μL: 500-650 mg; the volume ratio of the ethanol solution of propylamine and 4-nitro-1,8-naphthoic anhydride is 400-410 μL: 30-50 mL.
[0017] Furthermore, the ratio of propylamine, zinc powder, and NH4Cl is 400–410 μL: 300–350 mg: 120–150 mg; the ratio of propylamine, CH2Cl2, and methanol is 400–410 μL: 10 mL: 10 mL.
[0018] This invention also provides a visual detection device for reducing free radicals in soil based on a novel fluorescent probe, comprising a plastic substrate.
[0019] The plastic substrate has grooves, and the grooves are filled with an agar gel layer and a filter membrane.
[0020] A fluorescent probe for visualizing reducing free radicals is immobilized within an agarose gel layer.
[0021] Furthermore, the agar gel layer is laid to the bottom of the groove, and the filter membrane is laid on the agar gel layer;
[0022] The filter membrane is a polyvinylidene fluoride filter membrane, a nylon membrane, or a cellulose acetate membrane.
[0023] Furthermore, the agar gel layer is formed by mixing and solidifying a reducing free radical visualization fluorescent probe with agar.
[0024] Furthermore, the thickness of the filter membrane is 0.3–0.5 μm.
[0025] The beneficial effects of this invention are:
[0026] (1) This invention enables in-situ, real-time visual monitoring of reducing free radicals in the soil remediation process, and intuitively displays their generation and distribution.
[0027] (2) By comparing soils with different organic matter (SOM) contents, this device revealed that SOM competitively consumes SO4· - / ·OH free radicals or promote the generation of non-free radical species (such as 1O2), thereby inhibiting COO· - The generation efficiency of pollutants, which in turn affects their degradation kinetics.
[0028] (3) Semi-quantitative analysis of fluorescence signals provides a more intuitive and reliable detection method, avoiding the bias of traditional methods in assessing the contribution of free radicals in complex soil systems. Attached Figure Description
[0029] Figure 1 The fluorescent probe HPBD prepared for this invention 13 C NMR spectrum;
[0030] Figure 2 A schematic diagram of the assembled reductive free radical visualization detection device;
[0031] Figure 3 This is a schematic diagram of a fluorescent probe embedded in an agar gel layer, with the blue area representing the agar labeled by the probe.
[0032] Figure 4 The image shows the result after the reducing free radical visualization detection device is inserted into a degradation bottle containing soil. The yellow area represents the fluorescent product after the RFR reacts with the probe.
[0033] Figure 5 Distribution diagrams of RFR fluorescence signals and intensities in soil detected by fluorescent probes, including blank soil 1 (1), aerobic biochar / PDS / MA / soil 1 system (2), and anaerobic biochar / PDS / soil system (3-5). Detailed Implementation
[0034] This invention provides a reducing radical visualization fluorescent probe, which comprises compounds with the following structure:
[0035]
[0036] This invention also provides a method for preparing a reducing free radical visualization fluorescent probe, comprising the following steps:
[0037] 1) Propylamine was mixed in an ethanol solution of 4-nitro-1,8-naphthoic anhydride and refluxed to obtain a rice-yellow solid product;
[0038] 2) Mix the rice-yellow solid product, zinc powder and NH4Cl and react them. Then add CH2Cl2 and methanol to the reaction product and continue the reaction. The resulting yellow solid is the reducing free radical visualization fluorescent probe, denoted as HPBD.
[0039] In this invention, the temperature of the reflux reaction is 70-90°C, preferably 80°C; the time of the reflux reaction is 12-36 hours, preferably 24 hours.
[0040] The ratio of 4-nitro-1,8-naphthoic anhydride in the ethanol solution of propylamine and 4-nitro-1,8-naphthoic anhydride is 400-410 μL: 500-650 mg, preferably 406 μL: 600 mg; the volume ratio of the ethanol solution of propylamine and 4-nitro-1,8-naphthoic anhydride is 400-410 μL: 30-50 mL, preferably 406 μL: 40 mL.
[0041] In this invention, the ratio of propylamine, zinc powder and NH4Cl is 400-410 μL: 300-350 mg: 120-150 mg, preferably 406 μL: 320 mg: 140 mg; the ratio of propylamine, CH2Cl2 and methanol is 400-410 μL: 10 mL: 10 mL, preferably 406 μL: 10 mL: 10 mL.
[0042] This invention also provides a visual detection device for reducing free radicals in soil based on a novel fluorescent probe, comprising a plastic substrate.
[0043] The plastic substrate has grooves, and the grooves are filled with an agar gel layer and a filter membrane.
[0044] A fluorescent probe for visualizing reducing free radicals is immobilized within an agarose gel layer.
[0045] In this invention, the agar gel layer is laid to the bottom of the groove, and the filter membrane is laid on the agar gel layer;
[0046] The filter membrane is a polyvinylidene fluoride filter membrane, a nylon membrane, or a cellulose acetate membrane, preferably a polyvinylidene fluoride filter membrane.
[0047] In this invention, the agar gel layer is formed by mixing and solidifying a reducing free radical visualization fluorescent probe with agar.
[0048] In this invention, the thickness of the filter membrane is 0.3 to 0.5 μm, preferably 0.4 to 0.45 μm.
[0049] The device of this invention aims to capture and visualize the generation and spatial distribution of reducing free radicals (RFRs, such as carboxylic acid anion radicals COO-) during soil remediation in real time and in situ. Its core innovation lies in combining fluorescent probes, gel immobilization technology, and a filter membrane to achieve non-invasive, highly sensitive dynamic monitoring of free radicals, overcoming the limitations of traditional methods (such as EPR or chemical quenching) that cannot be observed in situ. The main structure of the device includes a plastic substrate (5cm long, 1.5cm wide) with grooves, an agar gel layer, and a polyvinylidene fluoride (PVDF) filter membrane. Figure 1 A 3cm long and 0.5cm wide groove was machined on the surface of the plastic substrate to fix the subsequent gel layer, ensuring uniform probe distribution and standardized reaction areas. The agar gel layer was formed by mixing the fluorescent probe HPBD with agar and pouring it into the groove to solidify, creating a three-dimensional network structure that both fixes the probe molecules and maintains a neutral environment to preserve probe activity. The 0.45μm pore size PVDF membrane covering the gel surface has the dual function of isolating soil particles and allowing free radicals to pass freely. Its chemical stability and low adsorption prevent free radicals from being consumed by the membrane material. Finally, the device was vertically inserted into the degradation bottle, and the fluorescence signal was detected using a confocal laser scanning microscope (excitation wavelength 544nm, emission wavelength 440nm), recording the spatiotemporal distribution of free radicals in real time.
[0050] In this invention, the key inventive points of the visualization detection device are reflected in three aspects: First, the probe-gel composite system captures reducing free radicals and generates fluorescent products through the specific hydroxyl amino groups of the HPBD probe, enabling in-situ observation without disrupting the reaction system; second, the innovative design of the PVDF membrane solves the problem of interference from the complex soil matrix on signal detection; and finally, the spatiotemporal dynamic visualization technology achieves in-situ spatial distribution analysis of reducing free radicals for the first time, providing a direct basis for optimizing remediation processes. For example, in low SOM soils, the device can detect high-intensity fluorescence signals (COO- dominant), while in high SOM soils, the weakened signal reflects the process of free radicals being competitively consumed by organic matter or converted into SQ-.
[0051] RFR (Reductive Free Radicals) refers to reducing free radicals, such as COO2. - SQ -The device comprises the following components: HPBD (hydroxylamine fluorescent probe) generates fluorescent products through a specific reaction; PVDF (polyvinylidene fluoride) is a hydrophobic polymer filter membrane; and SOM (Soil Organic Matter) significantly affects the generation and transformation of free radicals. Through the above technical solutions, those skilled in the art can accurately implement the device to achieve in-situ dynamic monitoring of reducing free radicals during soil remediation, providing crucial data support for optimizing the degradation process.
[0052] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0053] Example 1
[0054] 1) Synthesis of reducing radical probe: First, 406 μL of propylamine was added to 40 mL of ethanol solution containing 600 mg of 4-nitro-1,8-naphthenic anhydride, and the mixture was refluxed and stirred at 80 °C. After reacting overnight, the solvent was removed using a rotary evaporator to obtain a rice-yellow solid product. Then, the product was mixed with 320 mg of zinc powder and 140 mg of NH4Cl, and stirred at room temperature for 1 hour. Subsequently, 10 mL of CH2Cl2 and 10 mL of methanol were added, and the reaction continued for another hour. After filtering to remove the solid, the solvent was evaporated using a rotary evaporator, and the product was purified by column chromatography to finally obtain a yellow solid.
[0055] That is, 6-(hydroxyamino)-2-propyl-1Hbenzoisoquinoline-1,3(2H)-dione, named HPBD. HPBD's... 13 C NMR spectrum as follows Figure 1 As shown (101MHz, CD3OD: 163.59, 156.59, 135.65, 132.83, 130.75, 129.92, 125.14, 123.86, 108.74, 43.17, 21.80, 11.75).
[0056] 2) Construction of a visual detection device for reducing free radicals: The in-situ RFR capture device consists of a grooved plastic substrate (7×2 cm), a gel layer, and a filter membrane (e.g., Figure 2As shown. First, 0.2 g of agar powder was added to 20 mL of PBS buffer (10 mM, pH 7) and heated by microwave. After dissolution, 100 μM of the RFR capture probe HPBD was mixed with the agar solution, poured into a plastic substrate with grooves, and placed in the refrigerator to cool and solidify. After 2 h, the plastic substrate was taken out, and a polyvinylidene fluoride membrane with a pore size of 0.45 μm was covered on the gel surface. Subsequently, the plastic substrate was inserted into the degradation bottle to in-situ monitor the generation of RFR. After the degradation was completed, the plastic substrate was placed on a confocal laser scanning microscope (Nikon Eclipse Ti2) in a dark environment to observe the fluorescence signal obtained by capturing RFR. The excitation wavelength was set at 488 nm, and the emission wavelength was set at 525 nm.
[0057] In this invention, a capture device for RFR was assembled by embedding a self-made fluorescent probe into an agar gel layer ( Figure 3 ), and then it was vertically inserted into a degradation vial for in-situ RFR capture ( Figure 4 ). As far as we know, this is the first visualization of the spatial distribution of RFR in the advanced reduction remediation system.
[0058] As shown in the figure, compared with the control groups (i.e., blank soil 1 without additives, aerobic biochar / PDS / MA / soil 1 system), more obvious fluorescence signals were observed in the anaerobic biochar / PDS / soil system, indicating that a large amount of reducing active substances were formed in the soil slurry ( Figure 5 left). It is worth noting that the highest fluorescence intensity was obtained in the treatment group using Soil14, followed by Soil1, and finally Soil13. This means that the production of RFR increased in the order of Soil13 < Soil1 < Soil14 ( Figure 5 right).
[0059] Table 1 Information of the three selected soils in the experiment
[0060]
[0061]
[0062] Example 2
[0063] 1) Synthesis of reducing radical probe: First, 410 μL of propylamine was added to 40 mL of ethanol solution containing 600 mg of 4-nitro-1,8-naphthenic anhydride, and the mixture was refluxed and stirred at 80 °C. After reacting overnight, the solvent was removed using a rotary evaporator to obtain a rice-yellow solid product. Then, the product was mixed with 350 mg of zinc powder and 140 mg of NH4Cl, and stirred at room temperature for 1 hour. Subsequently, 10 mL of CH2Cl2 and 10 mL of methanol were added, and the reaction continued for another hour. After filtering to remove the solid, the solvent was evaporated using a rotary evaporator, and the product was purified by column chromatography to finally obtain a yellow solid.
[0064] 2) Same as Example 1.
[0065] Example 3
[0066] 1) Same as Example 1;
[0067] 2) Construction of a visual detection device for reducing free radicals: The in-situ RFR capture device consists of a grooved plastic substrate (7×2 cm), a gel layer, and a filter membrane (e.g., Figure 2 (As shown). First, 0.2 g of agar powder was added to 20 mL of PBS buffer (10 mM, pH 7) and microwaved. After dissolution, 100 μM of the RFR capture probe HPBD was mixed with the agar solution, poured into a grooved plastic substrate, and placed in a refrigerator to cool and solidify. After 2 hours, the plastic substrate was removed, and a 0.45 μm cellulose acetate membrane was applied to the gel surface. The plastic substrate was then inserted into a degradation bottle to monitor RFR generation in situ. After degradation, the plastic substrate was placed on a confocal laser scanning microscope (Nikon Eclipse Ti2) in a dark environment to observe the fluorescence signal obtained from capturing RFR. The excitation wavelength was set to 488 nm, and the emission wavelength was set to 525 nm.
[0068] Example 4
[0069] 1) Same as Example 1;
[0070] 2) Construction of a visual detection device for reducing free radicals: The in-situ RFR capture device consists of a grooved plastic substrate (7×2 cm), a gel layer, and a filter membrane (e.g., Figure 2(As shown). First, 0.2 g of agar powder was added to 20 mL of PBS buffer (10 mM, pH 7) and microwaved. After dissolution, 100 μM of the RFR capture probe HPBD was mixed with the agar solution, poured into a grooved plastic substrate, and placed in a refrigerator to cool and solidify. After 2 hours, the plastic substrate was removed, and a nylon membrane with a pore size of 0.45 μm was applied to the gel surface. The plastic substrate was then inserted into a degradation bottle to monitor RFR generation in situ. After degradation, the plastic substrate was placed on a confocal laser scanning microscope (Nikon Eclipse Ti2) in a dark environment to observe the fluorescence signal obtained from capturing RFR. The excitation wavelength was set to 488 nm, and the emission wavelength was set to 525 nm.
[0071] As can be seen from the above embodiments, the present invention provides a fluorescent probe for visualizing reducing free radicals, as well as its preparation method and apparatus. By comparing soils with different organic matter (SOM) contents, the present invention reveals that SOM competitively consumes SO4· - / ·OH free radicals or promote the generation of non-free radical species (such as 1O2), thereby inhibiting COO· - The generation efficiency of pollutants, which in turn affects their degradation kinetics.
[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A fluorescent probe for visualizing reducing free radicals, characterized in that, The reducing radical visualization fluorescent probe comprises compounds with the following structure:
2. The method for preparing the reducing free radical visualization fluorescent probe according to claim 1, characterized in that, Includes the following steps: 1) Propylamine was mixed in an ethanol solution of 4-nitro-1,8-naphthoic anhydride and refluxed to obtain a rice-yellow solid product; 2) Mix the rice-yellow solid product, zinc powder and NH4Cl and react them. Then add CH2Cl2 and methanol to the reaction product and continue the reaction. The resulting yellow solid is the reducing free radical visualization fluorescent probe, denoted as HPBD.
3. The method for preparing a reducing free radical visualization fluorescent probe according to claim 2, characterized in that, The reflux reaction temperature is 70–90°C, and the reflux reaction time is 12–36 h; The ratio of 4-nitro-1,8-naphthoic anhydride in the ethanol solution of propylamine and 4-nitro-1,8-naphthoic anhydride is 400-410 μL: 500-650 mg; the volume ratio of the ethanol solution of propylamine and 4-nitro-1,8-naphthoic anhydride is 400-410 μL: 30-50 mL.
4. The method for preparing a reducing free radical visualization fluorescent probe according to claim 2 or 3, characterized in that, The ratio of propylamine, zinc powder and NH4Cl used is 400-410 μL: 300-350 mg: 120-150 mg; the ratio of propylamine, CH2Cl2 and methanol used is 400-410 μL: 10 mL: 10 mL.
5. A visual detection device for reducing free radicals in soil based on a novel fluorescent probe, characterized in that, Including plastic substrates, The plastic substrate has grooves, and the grooves are filled with an agar gel layer and a filter membrane. The reducing free radical visualization fluorescent probe of claim 1 is immobilized in an agarose gel layer.
6. The visual detection device for reducing free radicals in soil based on a novel fluorescent probe according to claim 5, characterized in that, The agar gel layer is laid to the bottom of the groove, and the filter membrane is laid on the agar gel layer; The filter membrane is a polyvinylidene fluoride filter membrane, a nylon membrane, or a cellulose acetate membrane.
7. The visual detection device for reducing free radicals in soil based on a novel fluorescent probe according to claim 5, characterized in that, The agar gel layer is formed by mixing and solidifying a reducing free radical visualization fluorescent probe with agar.
8. The visual detection device for reducing free radicals in soil based on a novel fluorescent probe according to claim 5 or 6, characterized in that, The thickness of the filter membrane is 0.3 to 0.5 μm.