A method for in-situ detection of reactive oxygen species based on a ratiometric fluorescent probe

Abstract

The application discloses a kind of in-situ detection methods of active oxygen, comprising the following steps: active oxygen composite film based on ratio type fluorescent probe is prepared and is attached in the region to be detected and active oxygen response;Green light signal on active oxygen composite film is captured using digital camera equipped with 520-540nm narrow band filter, and picture A is obtained;Red light signal on active oxygen composite film is captured using digital camera equipped with 640-660nm filter, and picture B is obtained;Image processing software is used, and picture A and picture B are digitally processed, respectively obtain the value (G) of green light channel of picture A and the value (R) of red light channel of picture B, calculate relative fluorescence intensity (FI) as active oxygen response value;The active oxygen response value (FI) is substituted into active oxygen concentration quantitative standard curve, and the concentration of in-situ active oxygen is obtained.The application can improve the stability of fluorescent signal and solve the problem of environmental disturbance, while improving spatial resolution and soil universality.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of environmental monitoring, more specifically, relates to a reactive oxygen species in-situ detection method based on a ratio-type fluorescent probe. BACKGROUND

[0002] Reactive oxygen species (ROS) are important reactive species in the environment, including hydroxyl radicals (·OH), hydrogen peroxide (H2O2) and superoxide anion (O2· - ) and others, which are widely involved in biological, chemical and physical processes. Recent studies have shown that plant root systems are an important source of ROS and play a variety of key roles in rhizosphere environments, such as regulating heavy metal speciation. In the prior art, ROS detection methods mainly rely on ex situ analysis methods, such as high-performance liquid chromatography (HPLC) technology based on molecular probes and electron paramagnetic resonance (EPR) technology based on unpaired electron spins. Although these methods can provide relatively accurate quantitative analysis, their application is limited by the following factors: (a) limited sampling integrity: ex situ detection techniques require sample collection and processing, and the inevitable chemical and biological changes during the sampling process can alter the properties of the sample, thereby affecting the accuracy of the detection results. (b) Low spatial resolution: Traditional detection methods are difficult to capture ROS distribution at the micron or even smaller scale, which is particularly insufficient in studying the dynamic behavior of ROS in plant rhizosphere and other microenvironments. (c) Environmental interference: Fluorescent probes may be disturbed by background fluorescence in complex soil and sediment environments, resulting in decreased detection sensitivity and accuracy. In addition, the uneven distribution of fluorescent probes can also affect the results.

[0003] To address these challenges, in-situ detection techniques have gradually gained attention in recent years. In-situ detection methods can directly capture the dynamic distribution of ROS without destroying the original redox state of the sample. However, current in-situ detection methods still have significant limitations, such as insufficient fluorescence signal stability, significant background interference, and difficulty in achieving high-resolution detection of ROS. Moreover, the spatial resolution and soil universality of existing in-situ detection methods need to be further improved. SUMMARY

[0004] 1. Problems to be solved

[0005] In view of the technical problems of insufficient fluorescence signal stability, significant background interference, and the need for further improvement of spatial resolution and soil universality in the prior art in-situ detection methods, the present application provides a reactive oxygen species in-situ detection method based on a ratio-type fluorescent probe, which for the first time combines ratio-type fluorescent probes and planar optical electrode technology, significantly improves the stability of fluorescence signals and effectively solves the problem of environmental disturbance, while further improving the spatial resolution and soil universality based on existing in-situ detection methods.

[0006] 2. Technical solution

[0007] To solve the above problems, the technical scheme adopted by the present application is as follows:

[0008] [In-situ detection method of reactive oxygen species]

[0009] The first aspect of the present application provides an in-situ detection method of reactive oxygen species, comprising the following steps:

[0010] S1. A reactive oxygen composite film prepared based on a ratio type fluorescent probe is attached to the detection area and reacts with reactive oxygen;

[0011] S2. A 490-510 nm LED lamp is used as excitation light to irradiate the reactive oxygen composite film, and a digital camera equipped with a 520-540 nm narrow-band filter is used to capture the green light signal on the reactive oxygen composite film, obtaining a picture A recording the green light fluorescence intensity;

[0012] S3. A 520-540 nm LED lamp is used as excitation light to irradiate the reactive oxygen composite film, and a digital camera equipped with a 640-660 nm filter is used to capture the red light signal on the reactive oxygen composite film, obtaining a picture B recording the red light fluorescence intensity;

[0013] S4. Using image processing software, the picture A and picture B are digitally processed to obtain the numerical value (G) of the green light channel of picture A and the numerical value (R) of the red light channel of picture B, respectively, and the relative fluorescence intensity (FI) is calculated as the reactive oxygen response value by formula (1);

[0014]

[0015] S5. The reactive oxygen response value (FI) in S4 is substituted into the reactive oxygen concentration quantitative standard curve to obtain the concentration of in-situ reactive oxygen.

[0016] As a preferred embodiment of any one of the first aspect of the present application, the reaction time of the reactive oxygen composite film with reactive oxygen in S1 is 4-70 min.

[0017] The reaction time of the reactive oxygen composite film with reactive oxygen is 4-70 min, which can prevent the reaction time from being too short and the reactive oxygen from being difficult to be detected, and can also prevent the reaction time from being too long and the entire reactive oxygen composite film from reacting with reactive oxygen to make it difficult to distinguish the distribution of reactive oxygen in two-dimensional space.

[0018] As a preferred embodiment of any one of the first aspect of the present application, the preparation of the reactive oxygen composite film comprises the following steps:

[0019] Step 1: dissolve 2',7'-dichlorofluorescein in acetonitrile to obtain a 2',7'-dichlorofluorescein mother liquor with a concentration of 5-15 mmol / L; dissolve Nile red in acetonitrile to obtain a Nile red mother liquor with a concentration of 5-15 mmol / L;

[0020] Step 2: dissolve agarose in water by heating to obtain an agarose solution;

[0021] Step 3: cool the agarose solution to 50-60°C, then add the 2',7'-dichlorofluorescein mother liquor and the Nile red mother liquor to obtain a mixed gel solution, wherein the concentrations of 2',7'-dichlorofluorescein and Nile red are both 40-60 μmol / L;

[0022] Step 4: inject the mixed gel solution into a mold, and after cooling and solidification, obtain an active oxygen composite film.

[0023] Further preferably, the mass percentage of the agarose solution is 0.5wt%-1.5wt%; the thickness of the active oxygen composite film is 1-4 mm.

[0024] 2',7'-dichlorofluorescein (DCFH) is the hydrolysis product of 2',7'-dichlorofluorescein diacetate (DCFH-DA). When using DCFH-DA as a probe, due to the heterogeneity of sediments and plant roots, the hydrolysis process of DCFH-DA will differ spatially, thereby affecting the accuracy of reactive oxygen species (ROS) detection. Using DCFH can directly react with ROS, thereby achieving accurate capture of the spatial distribution of ROS.

[0025] The mass percentage of agarose in the agarose solution is preferably 0.5wt%-1.5wt%. By relatively reducing the water content in the active oxygen composite film, the transverse diffusion of 2',7'-dichlorofluorescein (DCF, a fluorescent substance produced by the reaction of DCFH with active oxygen) in the active oxygen composite film can be reduced, the interference of DCF with ROS detection can be reduced, and the spatial resolution of in-situ ROS detection can be improved.

[0026] As a preferred embodiment of any one of the first aspects of the present application, the establishment of the active oxygen concentration quantitative standard curve comprises the following steps:

[0027] Step a: react the active oxygen composite film with active oxygen of a known concentration for 4-70 min;

[0028] Step b: use a 490-510 nm LED lamp as an excitation light to irradiate the active oxygen composite film, use a digital camera equipped with a 520-540 nm narrow-band filter to capture the green light signal on the active oxygen composite film, and obtain a picture C recording the green light fluorescence intensity;

[0029] Step c: the active oxygen composite film was irradiated with LED light of 520-540 nm as excitation light, the red light signal on the active oxygen composite film was captured by a digital camera equipped with a 640-660 nm filter, and a picture D recording the red light fluorescence intensity was obtained;

[0030] Step d: the picture C and the picture D were digitally processed by using image processing software, the numerical value (G) of the green light channel of the picture C and the numerical value (R) of the red light channel of the picture D were obtained respectively, and the relative fluorescence intensity (FI) was calculated as the active oxygen response value by formula (1);

[0031]

[0032] Step e: the above steps a-d were repeated with different concentrations of active oxygen to obtain the corresponding active oxygen response values, and a relationship equation between X-Y was constructed with the active oxygen concentration as the abscissa X and the active oxygen response value as the ordinate Y, that is, Y=aX+b, to obtain the active oxygen concentration quantitative standard curve of the active oxygen composite film.

[0033] Further preferably, the R 2 of the relationship equation Y=aX+b is 0.980-0.996, and p<0.002.

[0034] The active oxygen concentration quantitative standard curve of the active oxygen composite film has a good linear relationship, and the corresponding active oxygen concentration can be calculated by measuring the relative fluorescence intensity (FI).

[0035] The second aspect of the present application provides the application of the in-situ detection method of active oxygen provided in the first aspect of the present application in the detection of active oxygen.

[0036] The third aspect of the present application provides the application of the in-situ detection method of active oxygen provided in the first aspect of the present application in the quantitative detection of active oxygen in soil.

[0037] As a preferred embodiment of the third aspect of the present application, the ionic strength in the soil is equivalent to 0-260 mmol / L of NaCl, and further preferably 0-200 mmol / L of NaCl.

[0038] As a preferred embodiment of the third aspect of the present application, the pH value in the soil is 2-11, and further preferably the pH value of the soil is 3.0-9.5.

[0039] The simulated environment of ionic strength (0-200 mmol / L of NaCl concentration) and pH value (3.0-9.5) has covered most of the natural environmental conditions. Therefore, the active oxygen composite film (ROS-CM) can meet the in-situ two-dimensional distribution detection of active oxygen (ROS) in different properties of soil and sediment environment.

[0040] 3. Advantages

[0041] Compared with the prior art, the advantages of the present application are:

[0042] (1) The present application first combines the ratio-type fluorescent probe and the planar light electrode technology to solve the quantitative error caused by environmental background fluorescence interference, fluorescence concentration change and uneven excitation light field of traditional single fluorescent probe. At the same time, more active oxygen complex film reaction points per square centimeter can be captured, and this high pixel can ensure accurate capture of the two-dimensional spatial distribution of ROS in plant roots and sediments, and ensure that the oxidation-reduction state is not destroyed. Experiments show that in the phosphate buffer solution, the ratio fluorescence signal (FI) of the active oxygen complex film (ROS-CM) has a significant linear correlation (R 2 = 0.980-0.996, p<0.002) with the concentration of hydroxyl radical (·OH), realizing high sensitivity quantitative detection of active oxygen concentration. In summary, the present application significantly improves the stability of the fluorescence signal and effectively solves the problem of environmental disturbance, while further improving the detection accuracy and spatial resolution on the basis of the existing in-situ detection method.

[0043] (2) The present application optimizes the deployment time in the rhizobox experiment, effectively avoids the interference of fluorescence product diffusion, and ensures the accuracy of the detection results. By selecting appropriate fluorescent probes, the difference in the spatial DCFH-DA hydrolysis process caused by the heterogeneity of sediments and plant roots is avoided, further improving the accuracy of the detection results. By relatively reducing the water content in the active oxygen complex film, the degree of lateral diffusion of the fluorescence product in the active oxygen complex film is reduced, the interference of the fluorescence product on the detection of ROS is reduced, and the spatial resolution of the in-situ detection of ROS is improved.

[0044] (3) The present application has wide applicability and can stably operate in various complex soil conditions. Experimental data show that the active oxygen complex film has a stable fluorescence response under different ion strengths (0-200 mmol / L NaCl) and pH values (3.0-9.5), indicating its universality in various soil environments.

[0045] (4) The detection technology of the present application can be widely applied to the research of ROS distribution in plant roots and sediment environment, realizing accurate capture of the spatial distribution of ROS in sediments and plant roots. This method provides strong tool support for exploring the mechanism of ROS in rhizosphere ecosystem, especially in the fields of pollutant migration and transformation, ecological environment protection and agricultural production optimization, and has important significance. It provides a new perspective for understanding the mechanism of ROS and pollutant transformation, and has wide ecological and environmental application potential.

[0046] (5) The present application belongs to the technical innovation in the cross field of environmental science and material science, and the active oxygen composite film has simple structure and reasonable in-situ detection method. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 :

[0048] (a) is a top view of the active oxygen composite film (ROS-CM) template (the unit in the figure is mm);

[0049] (b) is a cross-sectional view of the active oxygen composite film (ROS-CM) template (the unit in the figure is mm);

[0050] Figure 2 :

[0051] (a) is a schematic diagram of the fluorescence response of the ROS probe (DCFH) and the reference probe (Nile Red) in the active oxygen composite film (ROS-CM) prepared in Preparation Example 1 under different Fe 2+ concentrations;

[0052] (b) is a relationship between the Fe 2+ concentration and the ·OH generation amount in the Fenton solution of Test Example 2;

[0053] (c) is a schematic diagram of the calibration line between the ·OH concentration and the fluorescence intensity (FI) of the response of the active oxygen composite film (ROS-CM) in Test Example 2;

[0054] (d) is a schematic diagram of the fluorescence intensity of the response of the active oxygen composite film (ROS-CM) to active oxygen (ROS) under different ionic strength (calculated by NaCl) conditions in Test Example 3;

[0055] (e) is a schematic diagram of the fluorescence intensity of the response of the active oxygen composite film (ROS-CM) to active oxygen (ROS) under different pH conditions in Test Example 4;

[0056] Figure 3 :

[0057] (a) is a schematic diagram of the green fluorescence imaging of the active oxygen composite film (ROS-CM) in Test Example 5 under different deployment times in the rice roots;

[0058] (b) is a schematic diagram of the green fluorescence intensity of the active oxygen composite film (ROS-CM) in Test Example 5 under different deployment times in the red dashed line area; Figure 3 (a) is a schematic diagram of the green fluorescence intensity of the active oxygen composite film (ROS-CM) in Test Example 5 under different deployment times in the red dashed line area;

[0059] In which, the red box shows the nylon rope (diameter: 1.5mm) soaked in 20 μmol / L H2O2, the rest of the image is the real rice root; the excitation light and emission light wavelength (Ex / Em) are 500 nm and 530 nm respectively. DETAILED DESCRIPTION

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0061] Unless otherwise indicated, conventional methods or those described in the literature are employed in the examples. Unless otherwise indicated, the reagents or instruments used are commercially available conventional products.

[0062] As used herein, the term "about" is used to provide flexibility to a given term, measurement, or value. One of skill in the art can readily determine the degree of flexibility afforded by the term "about" for a particular variable.

[0063] Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be construed as having been followed to following the principle of including and disclosing every single numerical value and subrange within the open- ended range. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited values of 1 to about 4.5, but also the individual values and sub-ranges within the indicated range, for example, 2, 3, 4, and 1 to 3, 2 to 4, etc. The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be interpreted to include all of the above-noted values and ranges. In addition, this application should apply regardless of the breadth of the range or the characteristics being described.

[0064] The application is further described below in connection with specific embodiments.

[0065] Preparation Example

[0066] Preparation materials:

[0067] 2',7'-dichlorodihydrofluorescein (DCFH), ferrous chloride were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., Nile Red, acetonitrile solvent, agarose, ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma Company.

[0068] Preparation Example 1

[0069] The embodiment discloses a preparation method of a reactive oxygen species composite membrane (ROS-CM), comprising the following steps:

[0070] 1. 2', 7'-dichlorodihydrofluorescein (DCFH) was added to an acetonitrile solvent, and after ultrasonic treatment for 1 min, a 10 mmol / L DCFH solution 2 mL was obtained; Nile Red was added to an acetonitrile solvent, and after ultrasonic treatment for 1 min, a 10 mmol / L Nile Red solution 2 mL was obtained.

[0071] 2. Agarose was dissolved in ultrapure water by heating, and a 1 wt% agarose solution 400 mL was obtained by stirring.

[0072] 3. The above-mentioned 1 wt% agarose solution was cooled to 60°C, and then the above-mentioned 2 mL DCFH solution and 2 mL Nile Red solution were added, and rapid stirring and mixing were performed to obtain a mixed gel solution, wherein the concentration of DCFH and Nile Red in the mixed gel solution was 50 μmol / L.

[0073] 4. The above-mentioned mixed gel solution was injected into a mold as shown in Figure 1 , and after cooling and solidification, a reactive oxygen species composite membrane (ROS-CM) was obtained.

[0074] Test Example

[0075] Test Method:

[0076] The reactive oxygen species composite membrane (ROS-CM) prepared in Preparation Example 1 was reacted with reactive oxygen species (ROS) under specific conditions, and the fluorescence information on the reactive oxygen species composite membrane was obtained by planar photogalvanic technology, so as to detect the content of the reactive oxygen species. The specific steps are as follows:

[0077] 1. A 500 nm LED lamp was used as excitation light to irradiate the reactive oxygen species composite membrane, a digital camera (Canon 1300, Japan, aperture f / 2.8, shutter speed 1 / 4 second, ISO 400, resolution 5184x3456 pixels) equipped with a 530 nm narrow-band green light sheet was used to capture the green light signal on the reactive oxygen species composite membrane, and a picture A recording the green light fluorescence intensity was obtained;

[0078] 2. A 530 nm LED lamp was used as excitation light to irradiate the reactive oxygen species composite membrane, and a digital camera (Canon 1300, Japan, aperture f / 2.8, shutter speed 1 / 4 second, ISO 400, resolution 5184x3456 pixels) equipped with a 650 nm filter was used to capture the red light signal on the reactive oxygen species composite membrane, and a picture B recording the red light fluorescence intensity was obtained;

[0079] 3. Using ImageJ software, the picture A and picture B are digitally processed to obtain the value of the green light channel of picture A (G) and the value of the red light channel of picture B (R), respectively. The relative fluorescence intensity (FI) is calculated by formula (1) and used as the response value of the active oxygen composite film (ROS-CM) to the active oxygen (ROS). That is, the fluorescence ratio (G / R) of the ROS probe and the reference probe represents the relative fluorescence intensity (FI);

[0080]

[0081] Test Example 1

[0082] Ferrous chloride and ethylenediaminetetraacetic acid (EDTA) are mixed in a molar ratio of 1:1 in ultrapure water to prepare an EDTA-Fe complex solution with a concentration of 10 mmol / L. In a phosphate buffer solution with a pH of 7.4, 8 solutions with a volume of 150 mL and a hydrogen peroxide concentration of 200 μmol / L are prepared, and a certain amount of the aforementioned EDTA-Fe complex solution is taken and added to the aforementioned 8 hydrogen peroxide solutions, so that the Fe 2+ concentration in the mixed solution is 0, 5, 10, 20, 30, 50, 80, and 100 μmol / L, respectively. -1 Hydroxyl radicals (·OH) are generated by Fenton reaction to obtain Fenton solutions containing different concentrations of hydroxyl radicals.

[0083] Using steps 1 and 2 in the above test method, the emission intensity of DCFH (green fluorescence) and Nile Red (red fluorescence) of the active oxygen composite film (ROS-CM) after reaction is detected, and the results are shown in Figure 2 (a). From Figure 2 a, it can be seen that the green fluorescence intensity of ROS-CM increases with the increase of Fe 2+ concentration (i.e. active oxygen concentration) in the Fenton system, while the red fluorescence intensity remains stable.

[0084] Test Example 2

[0085] This test example is the calibration of the active oxygen composite film, and the specific operation is as follows:

[0086] Ferrous chloride and ethylenediaminetetraacetic acid (EDTA) are mixed in a molar ratio of 1:1 in ultrapure water to prepare an EDTA-Fe complex solution with a concentration of 10 mmol / L. In a phosphate buffer solution with a pH of 7.4, 8 solutions with a volume of 150 mL and a hydrogen peroxide concentration of 200 μmol / L are prepared, and a certain amount of the aforementioned EDTA-Fe complex solution is taken and added to the aforementioned 8 hydrogen peroxide solutions, so that the Fe 2+Concentrations of 0, 5, 10, 20, 30, 50, 80, and 100 μmol·L⁻¹ were given. -1 Hydroxyl radicals (·OH) were generated through the Fenton reaction, and Fenton solutions with hydroxyl radical concentrations of 0.00, 0.57, 1.17, 1.58, 2.83, 4.72, 7.96, and 8.96 μmol / L were obtained.

[0087] The reactive oxygen species composite membrane (ROS-CM) prepared in Preparation Example 1 was placed on the inner side of an acrylic box, and 70-mesh quartz sand was poured in as simulated soil. Then, Fenton solutions of different concentrations were poured into the acrylic box. The reactive oxygen species composite membrane (ROS-CM) reacted fully with hydroxyl radicals for 30 minutes. Finally, the acrylic box was placed in a planar photoelectric system and fluorescence excitation and capture were performed according to the above test method.

[0088] Fe in different Fenton solutions 2+ The relationship between concentration and ·OH generation is as follows: Figure 2 As shown in (b); the calibration results of the active oxygen composite membrane prepared in Preparation Example 1 are as follows. Figure 2 As shown in (c), the specific calibration curve is Y = 0.980X + 14.503, R 2 =0.99, p<0.001, the fluorescence ratio (FI=G / R) showed a good linear relationship with the ·OH concentration.

[0089] Test Example 3

[0090] The purpose of this test case is to test the stability of the reactive oxygen species composite membrane (ROS-CM) in simulated environments with different ionic intensities.

[0091] Ferrous chloride and ethylenediaminetetraacetic acid (EDTA) were mixed in an ultrapure water at a molar ratio of 1:1 to obtain Fe. 2+ A 10 mmol / L EDTA-Fe complex solution was prepared. 250 mL of 200 μmol / L hydrogen peroxide was prepared using phosphate buffer (pH 7.4). Five 50 mL aliquots of the above hydrogen peroxide solution were taken, and NaCl was added to each aliquot to obtain hydrogen peroxide solutions with NaCl concentrations of 0.5 mmol / L, 5 mmol / L, 50 mmol / L, 100 mmol / L, and 200 mmol / L. Then, 0.1 mL of the aforementioned EDTA-Fe complex solution was added to each aliquot to achieve the desired Fe concentration. 2+ Concentration of 20 μmol·L -1 The solution obtained contained approximately 1.58 μmol·L⁻¹ -1 Fenton solution of hydroxyl radicals.

[0092] The active oxygen composite membrane (ROS-CM) prepared in Preparation Example 1 was placed on the inner side of a plexiglass box, 70-mesh quartz sand was poured into the box as simulated soil, and then a Fenton solution containing a series of concentration gradient NaCl was poured into the plexiglass box, so that the active oxygen composite membrane (ROS-CM) was fully reacted with hydroxyl radicals under the condition that the concentration of NaCl was 0.5 mmol / L, 5 mmol / L, 50 mmol / L, 100 mmol / L, and 200 mmol / L, respectively, for 30 minutes. Finally, the plexiglass box was placed in a planar light polar system, and fluorescence excitation and capture were carried out according to the above test method.

[0093] The test results of the active oxygen composite membrane (ROS-CM) on the same concentration of hydroxyl radicals in different ionic strength simulated environments are shown in Figure 2 (d). As can be seen from Figure 2 (d), the test results of the active oxygen composite membrane (ROS-CM) on the same concentration of hydroxyl radicals in different ionic strength simulated environments are all within the range of ±10% of the average value, i.e. within the area between the two dotted lines in Figure 2 (d).

[0094] The results show that the fluorescence response of the active oxygen composite membrane (ROS-CM) is stable within the above-mentioned condition range, and can meet the detection of in-situ two-dimensional distribution of active oxygen (ROS) in soil and sediment environments under different ionic strength conditions.

[0095] Test Example 4

[0096] The purpose of this test example is to test the stability of the active oxygen composite membrane (ROS-CM) in different pH value simulated environments.

[0097] Chloride ferrous and ethylenediaminetetraacetic acid (EDTA) were mixed according to a molar ratio of 1:1 to obtain a Fe 2+ concentration of 10 mmol·L -1 EDTA-Fe complex solution. A series of 50 mL solutions with pH values of 3.0, 4.5, 6.0, 7.0, 8.5, and 9.5 were prepared by adding NaOH and HCl in ultrapure water, and hydrogen peroxide and the above-mentioned EDTA-Fe complex solution was added to make their concentrations 200 μmol·L -1 and 20 μmol·L -1 , respectively, to generate hydroxyl radicals (·OH) by Fenton reaction to obtain a Fenton solution containing a concentration of about 1.58 μmol·L -1 hydroxyl radicals.

[0098] The active oxygen composite membrane (ROS-CM) prepared in Preparation Example 1 was placed on the inner side of a plexiglass box, 70-mesh quartz sand was poured into the box as simulated soil, and then a series of Fenton solutions were poured into the plexiglass box, so that the active oxygen composite membrane (ROS-CM) was fully reacted with hydroxyl radicals at pH values of 3.0, 4.5, 6.0, 7.0, 8.5, and 9.5 for 30 minutes, respectively. Finally, the plexiglass box was placed in a planar light polar system, and fluorescence excitation and capture were carried out according to the above test method.

[0099] The test results of the active oxygen composite membrane (ROS-CM) on the same concentration of hydroxyl radicals in different pH value simulated environments are shown in Figure 2 (e). Figure 2 As can be seen from Figure 2 (e), the test results of the active oxygen composite membrane (ROS-CM) on the same concentration of hydroxyl radicals in different pH value simulated environments are all within the range of ±10% of the average value, i.e. within the area between the two dotted lines in

[0100] The results show that the fluorescence response of the active oxygen composite membrane (ROS-CM) is stable within the above-mentioned condition range, and can meet the detection of the in-situ two-dimensional distribution of active oxygen (ROS) in soil and sediment environments under different pH conditions.

[0101] In summary of Test Example 3 and Test Example 4, the simulated environments of different ionic strengths (NaCl concentrations of 0-200 mmol / L) and pH values (3.0-9.5) have covered most of the natural environmental conditions. Therefore, the active oxygen composite membrane (ROS-CM) can meet the detection of the in-situ two-dimensional distribution of active oxygen (ROS) in soil and sediment environments of different properties, and it also shows that the above-mentioned active oxygen in-situ detection method based on the ratio-type fluorescence probe technology can be used for the detection of the in-situ two-dimensional distribution of active oxygen (ROS) in soil and sediment environments of different properties.

[0102] Test Example 5

[0103] The purpose of this test example is to test the application of the active oxygen composite membrane (ROS-CM) in actual environments.

[0104] A flat longitudinal section was made on the root of a rice plant, and a nylon rope with a diameter of about 0.5 mm and a length of 20 mm, which was infected in a 200 μmol / L hydrogen peroxide solution, was placed in the non-rhizosphere region Figure 3(a) the red box area), the active oxygen composite film (ROS-CM) prepared in Preparation Example 1 was attached to the longitudinal section, and the surface of the ROS-CM should be avoided to be scratched during the deployment process. After the active oxygen composite film reacted with the rice roots and the simulated roots for 5 min, 60 min, 90 min, 120 min, and 240 min, respectively, each active oxygen composite film was taken out and placed in a planar optical electrode system for fluorescence excitation and capture according to the above test method.

[0105] At the same time, the diffusivity of the above five active oxygen composite films after reaction in this test example was tested using simulated roots. The fluorescence intensity of the cross-section of the simulated root system (a) in different deployment times was extracted by using imageJ software. Figure 3

[0106] The green fluorescence imaging of the active oxygen composite film after reacting with the rice roots for 5 min, 60 min, 90 min, 120 min, and 240 min is shown in Figure 3 (a); and the green fluorescence diffusion of the active oxygen composite film after reacting with the simulated roots for 5 min, 60 min, 90 min, 120 min, and 240 min is shown in Figure 3 (b).

[0107] From Figure 3 (a), it can be seen that as the time increases, the active oxygen is continuously generated, resulting in the gradual increase of the fluorescence intensity and the gradual increase of the fluorescence range. From Figure 3 (b), it can be seen that the coincidence degree of the rise and fall of the five curves is high, that is, the five curves all rise at about 3 mm and fall at about 7 mm, which indicates that the transverse diffusion degree of the fluorescent substance 2', 7'-dichlorofluorescein (DCF) produced by the reaction of DCFH and active oxygen in the composite film is relatively low, which also indicates that the transverse diffusion of 2', 7'-dichlorofluorescein (DCF) is not Figure 3 (a) the main reason for the expansion of the fluorescence range, that is, the detection interference caused by the diffusion of 2', 7'-dichlorofluorescein (DCF) in the ROS-CM is very limited, which indirectly indicates that Figure 3 (a) the main reason for the expansion of the fluorescence range is the continuous generation of active oxygen.

[0108] According to the clarity of each image, the detection time of 5-60 minutes is preferred, which can effectively detect the in situ distribution of ROS.

[0109] ​The above is a schematic description of the present application and its embodiments, which is not restrictive, and the embodiments shown are only one of the embodiments of the present application, and the actual embodiments are not limited thereto. Therefore, if a person skilled in the art is inspired thereby, without departing from the spirit of the present application, similar embodiments and embodiments are not created by creative design, which should belong to the protection scope of the present application.

Claims

1. A method for in situ detection of reactive oxygen species, characterized by, The method comprises the following steps: S1. An active oxygen composite film prepared based on a ratio type fluorescent probe is attached to the detection area and reacts with active oxygen for 4-70 min, wherein the preparation of the active oxygen composite film comprises the following steps: Step 1: 2',7'-dichlorodihydrofluorescein is dissolved in acetonitrile solvent to obtain a 5-15 mmol / L 2',7'-dichlorodihydrofluorescein mother liquor; Nile red is dissolved in acetonitrile solvent to obtain a 5-15 mmol / L Nile red mother liquor; Step 2: Agarose is dissolved in water by heating to obtain an agarose solution; Step 3: After the agarose solution is cooled to 50-60℃, the 2',7'-dichlorodihydrofluorescein mother liquor and the Nile red mother liquor are added to obtain a mixed gel solution, wherein the concentrations of 2',7'-dichlorodihydrofluorescein and Nile red are both 40-60 μmol / L; Step 4: The mixed gel solution is injected into a mold, and after cooling and solidification, an active oxygen composite film is obtained; S2. A 490-510 nm LED lamp is used as excitation light to irradiate the active oxygen composite film, and a digital camera equipped with a 520-540 nm narrowband filter is used to capture the green light signal on the active oxygen composite film to obtain a picture A recording the green light fluorescence intensity; S3. A 520-540 nm LED lamp is used as excitation light to irradiate the active oxygen composite film, and a digital camera equipped with a 640-660 nm filter is used to capture the red light signal on the active oxygen composite film to obtain a picture B recording the red light fluorescence intensity; S4. Image processing software is used to digitally process pictures A and B to obtain the value G1 of the green light channel of picture A and the value R1 of the red light channel of picture B, respectively, and the relative fluorescence intensity FI1 is calculated as the active oxygen response value by formula (1); S5. The active oxygen response value FI1 in S4 is substituted into the active oxygen concentration quantitative standard curve to obtain the concentration of in-situ active oxygen.

2. The method for in situ detection of reactive oxygen species according to claim 1, wherein, The mass percentage of agarose in the agarose solution is 0.5wt%-1.5wt%; The thickness of the active oxygen composite film is 1-4 mm.

3. The method for in situ detection of reactive oxygen species according to claim 2, wherein, The establishment of the active oxygen concentration quantitative standard curve comprises the following steps: Step a: The active oxygen composite film is reacted with active oxygen of a known concentration for 4-70 min; Step b: A 490-510 nm LED lamp is used as excitation light to irradiate the active oxygen composite film, and a digital camera equipped with a 520-540 nm narrowband filter is used to capture the green light signal on the active oxygen composite film to obtain a picture C recording the green light fluorescence intensity; Step c: A 520-540 nm LED lamp is used as excitation light to irradiate the active oxygen composite film, and a digital camera equipped with a 640-660 nm filter is used to capture the red light signal on the active oxygen composite film to obtain a picture D recording the red light fluorescence intensity; Step d: Image processing software is used to digitally process pictures C and D to obtain the value G2 of the green light channel of picture C and the value R2 of the red light channel of picture D, respectively, and the relative fluorescence intensity (FI2) is calculated as the active oxygen response value by formula (2); Step e: repeating steps a~d above with different concentrations of active oxygen to obtain the corresponding active oxygen response value, taking the active oxygen concentration as the abscissa X and the active oxygen response value as the ordinate Y, constructing the relationship equation between X and Y: Y=aX+b, and obtaining the active oxygen concentration quantitative standard curve of the active oxygen composite film.

4. The method for in situ detection of reactive oxygen species according to claim 3, wherein, The R² of the relationship equation: Y=aX+b is 0.980~0.996, p<0.

002.

5. The application of the in-situ detection method of active oxygen according to any one of claims 1~4 in active oxygen detection.

6. The application of the in-situ detection method of active oxygen according to any one of claims 1~4 in quantitatively detecting active oxygen in soil.

7. Use according to claim 6, characterized in that, The ionic strength in the soil is equivalent to 0~260 mmol / L of NaCl.

8. Use according to claim 7, characterized in that, The pH value in the soil is 2~11.

Images