In-situ characterization device and method for monitoring free radicals in rice field system during rainfall process in real time

Abstract

The application provides an in-situ characterization device and method for monitoring free radicals in a rice field system in real time during a rainfall process, and the device comprises: a carrier mold in a cuboid structure, a first groove being arranged on one side surface of the carrier mold in a thickness direction, the first groove being open on one side in a length direction of the cuboid, and a limiting frame being arranged in the first groove; a ROS capturing film being arranged in a space surrounded by the limiting frame and used for capturing ROS; a cover plate being located in the first groove and matching a shape of the first groove, a notch being arranged on the cover plate at a position corresponding to the limiting frame, the limiting frame being located in the notch, and a filter membrane being arranged on a side of the cover plate away from the carrier mold and capable of covering the notch.

Description

Technical Field

[0001] This invention relates to the field of rainfall monitoring, and in particular to an in-situ characterization device and method for real-time monitoring of free radicals in paddy field systems during rainfall. Background Technology

[0002] Rainwater commonly contains micromolar levels of hydrogen peroxide (H₂O₂) generated by photochemical reactions, with concentrations ranging from 2 μM to 40 μM. During natural rainfall, H₂O₂ is likely to react with ferrous ions (Fe₂O₂) in flooded environments such as paddy fields in South China. 2+ Contact occurs in Fe. 2+ Under the catalysis of [a specific substance], the in-situ Fenton effect can occur, generating highly oxidizing reactive oxygen species (ROS) – hydroxyl radicals (·OH). ·OH is a very active oxidant that can react with reducing substances in the paddy field soil and water environment, thus affecting ROS in the paddy field soil and water system. Furthermore, paddy fields have abundant multiphase interfaces, and their gas-liquid-solid-biological interfaces are hotspots for ROS accumulation and reaction. When natural rainfall causes changes in the water level of the overlying water in paddy fields, it leads to alternating aerobic and anoxic states in the soil, thereby promoting ROS formation, especially at the soil-water interface, where strong material exchange promotes the non-photochemical formation of ROS in the overlying water. However, due to the high activity, diverse types, and short lifespan of ROS, real-time monitoring and characterization of ROS processes in complex soil systems during rainfall is an international challenge, especially the urgent need to solve the black-box technical challenge of multi-interface ROS processes in paddy field systems during rainfall.

[0003] On the other hand, traditional ROS characterization methods and existing patents and technologies all have certain limitations. For example, electron paramagnetic resonance (EPR) cannot detect ROS in situ and is difficult to analyze the spatiotemporal distribution of soil ROS; chemical molecular probe methods require expensive reagents and equipment; quenching methods require destructive sampling of the soil, damaging the original soil structure. Existing patents and technologies, such as ordinary in-situ capture membranes, are easily damaged during sampling and extraction, simultaneously damaging the soil structure and affecting the measurement results and soil microorganisms; moreover, changes in the overlying water level of paddy fields during rainfall increase oxygen disturbance, affecting the changes in pH, dissolved oxygen (DO), and CO2 partial pressure (pCO2) in the water / sediment system, interfering with the measurement results at different interfaces. Therefore, there is an urgent need to develop in-situ visualized ROS monitoring methods and devices for real-time monitoring of paddy field soil during rainfall, to accurately stratify paddy field interfaces, and to reduce or eliminate the interference of sampling and rainfall processes on different interfaces in paddy fields. Summary of the Invention

[0004] The main objective of this invention is to provide an in-situ characterization device and method for real-time monitoring of free radicals in paddy field systems during rainfall, in order to solve the aforementioned technical problems.

[0005] To achieve the above objectives, the technical solution adopted by this invention is: an in-situ characterization device for real-time monitoring of free radicals in a paddy field system during rainfall, comprising:

[0006] The carrier mold is a cuboid structure with a first groove on one side surface along its thickness direction. The first groove opens along one side of the length direction of the cuboid, and a limit frame is provided in the first groove.

[0007] A ROS-capturing membrane is disposed within the space enclosed by the limiting frame for capturing ROS.

[0008] A cover plate is located in the first groove and its shape matches the first groove. A notch is provided on the cover plate at a position corresponding to the position of the limiting frame. The limiting frame is located in the notch. A filter membrane is provided on the side of the cover plate away from the carrier mold. The filter membrane can cover the notch.

[0009] Preferably, the device further includes a housing, on which a second groove extending along the length direction is provided, the cross-section of the second groove being an inverted T-shape; the cross-section of the carrier mold is also an inverted T-shape, one end of the second groove along the length direction is sealed, the carrier mold is inserted into the second groove from the unsealed end of the second groove, and along the width direction of the cuboid, both sides of the carrier mold are engaged in the second groove of the housing.

[0010] Preferably, the preparation method is as follows: Step 1: Prepare a certain amount of phosphate buffer, 2,7-dichlorodihydrofluorescein diacetate solution and agarose solution respectively, with the pH of the phosphate buffer being 7.4;

[0011] Step 2: Add the 2,7-dichlorodihydrofluorescein diacetate solution to the phosphate buffer and mix well;

[0012] Step 3: Heat the agarose using a microwave, add the mixed solution obtained in Step 2 to the agarose solution and mix well;

[0013] Step 4: Pour the product obtained in Step 3 into the space of the limiting frame and cool it. The solid in the space is the ROS capture membrane.

[0014] Step 5: Install the cover plate onto the carrier mold;

[0015] Step 6: Cover the filter membrane on the side of the cover plate away from the carrier mold to cover the ROS capture membrane.

[0016] This invention also proposes an in-situ characterization method for monitoring free radicals in a paddy field system during real-time rainfall, using the aforementioned in-situ characterization device, specifically including the following steps:

[0017] Step S1: During rainfall, the in-situ characterization device is inserted into the paddy field. The ROS capture membrane corresponds to the overlying water layer, soil-water layer, and soil layer of the paddy field in the vertical direction, respectively. Free radicals in the soil will react with 2,7-dichlorodihydrofluorescein diacetate on the ROS capture membrane to achieve in-situ capture of free radicals in different layers.

[0018] Step S2: After being inserted into the paddy field for a period of time, the in-situ characterization device is pulled out of the paddy field, and the ROS capture membrane is taken out from the in-situ characterization device. The ROS capture membranes corresponding to the overlying water layer, the soil-water layer, and the soil layer are then divided.

[0019] Step S3: Place the segmented ROS capture films on glass slides and perform fluorescence imaging using a dual-scanning laser confocal microscope at an excitation wavelength of 480 nm.

[0020] Step S4: By comparing the obtained fluorescence image with the standard value, the amount of ROS generated at different levels can be obtained, that is, the in-situ characterization data of free radicals can be obtained.

[0021] Preferably, it further includes:

[0022] Step S5: Using 3D visualization software, present the spatial distribution of free radicals at different soil depths based on in-situ characterization data, and construct a spatial 3D image of soil free radicals.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention enables in-situ, real-time monitoring of free radicals generated in paddy field systems during rainfall. It achieves precise stratification of the paddy field interface by segmenting the capture membrane. Furthermore, the materials used in the mold and cover plate exhibit better strength and dimensional retention characteristics in humid environments. The filter membrane material possesses sealing properties, and the outer shell prevents damage to the capture membrane and soil structure during sampling and extraction, minimizing interference from sampling and rainfall processes on different interfaces within the paddy field. This invention overcomes the bottleneck of achieving precise, in-situ, and real-time characterization of free radicals in paddy field soil during rainfall. Moreover, this invention is simple to operate, economical, efficient, and easy to promote and apply. Attached Figure Description

[0025] Figure 1 This is a perspective view of the device of the present invention;

[0026] Figure 2 yes Figure 1 Cross-sectional view;

[0027] Figure 3 and Figure 4 These are structural diagrams of the carrier mold from two angles;

[0028] Figure 5 This is a structural diagram of the cover plate;

[0029] Figure 6 It is an assembly drawing of the carrier mold and the cover plate;

[0030] Figure 7 This is a structural diagram of the outer shell;

[0031] Figure 8 This is a schematic diagram of the device of the present invention in use;

[0032] Figure 9 This is a comparison chart of fluorescence images and ROS content;

[0033] Figure 10 It is a three-dimensional spatial image of soil ROS;

[0034] Figure 11 These are ROS fluorescence images of a paddy field system during rainfall.

[0035] Figure 12 This is a graph showing the ROS concentration and percentage at each interface. Detailed Implementation

[0036] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0037] Example 1

[0038] like Figure 1-7 As shown, an in-situ characterization device for real-time monitoring of free radicals in a paddy field system during rainfall includes a shell 1, a carrier mold 2, a cover plate 3, and a ROS capture membrane. Viewed from above, the carrier mold 2 is rectangular, with a first groove 21 formed on one side. The first groove 21 opens along one side of the length of the rectangle. A limiting frame 22 is provided within the first groove 21, forming a space for placing the ROS membrane. The limiting frame 22 opens along the side opposite to the carrier mold 2. Nine protrusions 23 are respectively provided on the two inner sidewalls of the limiting frame 22 along the width direction of the rectangle. The protrusions 23 on each side are equally spaced, and the protrusions 23 on both sides correspond one-to-one. The nine pairs of protrusions 23 divide the ROS capture membrane into ten equal parts.

[0039] The cover plate 3 can be placed into and fit into the first groove 21. A notch 31 is provided on the cover plate 3 at a position corresponding to the limiting frame 22. When the cover plate 3 is placed into the first groove 21, the outer surface of the cover plate 3 is aligned with the corresponding surfaces of the carrier mold 2 and the limiting frame 22. A filter membrane is provided on the outer surface of the cover plate 3. The filter membrane has a pore size of 0.45 μm and is made of polyvinylidene fluoride (PVDF). The filter membrane can cover the ROS capture membrane located in the space of the limiting frame 22.

[0040] The preparation method of the in-situ characterization device is as follows:

[0041] Step 1: Prepare 10mM phosphate-buffered saline (PBS) (pH=7.4), 100μM 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) solution and agarose solution respectively. The agarose solution contains 2% agarose by mass.

[0042] Step 2: Add the 2,7-dichlorodihydrofluorescein diacetate solution to the phosphate buffer and mix well;

[0043] Step 3: Heat the agarose to about 43 degrees Celsius using a microwave. Add 400 μL of the mixed solution obtained in Step 2 to the agarose solution and mix well.

[0044] Step 4: Pour the product obtained in Step 3 into the space of the limiting frame and cool it. The solid in the space is the ROS capture membrane.

[0045] Step 5: Install the cover plate onto the carrier mold;

[0046] Step 6: Cover the filter membrane on the side of the cover plate away from the carrier mold to cover the ROS capture membrane.

[0047] Furthermore, a plurality of positioning posts 32 are provided on the inner surface of the cover plate 3, and a positioning hole 24 is provided on the carrier mold 2 corresponding to the position of each positioning post 32, with the positioning post 32 and the positioning hole 24 being interference fit.

[0048] The outer shell 1 is provided with a second groove 11 extending along the length direction, and the cross-section of the second groove 11 is inverted T-shaped. The cross-section of the carrier mold 2 is also inverted T-shaped. One end of the second groove 11 along the length direction is sealed, and the carrier mold 2 is inserted into the second groove 11 from the unsealed end. Along the width direction, both sides of the carrier mold 2 are engaged within the second groove 11 of the outer shell 1. Preferably, an interference fit can be provided at the engagement point between the carrier mold 2 and the outer shell 1 to prevent the carrier mold 2 from detaching from the outer shell 1 on its own.

[0049] The carrier mold 2, cover plate 3 and shell 1 are mainly composed of three-dimensional light-forming resin, specifically BESTY-4 material.

[0050] Preferably, a slope is provided at the lower end of the carrier template 2 and on the side away from the outer shell 1. The slope gradually approaches the outer shell 1 from top to bottom, and the slope facilitates the smooth insertion of the device into the soil.

[0051] Example 2

[0052] This embodiment describes the in-situ characterization method of the device in Embodiment 1, specifically including the following steps:

[0053] Step S1: During rainfall, the in-situ characterization device is inserted into the paddy field. The ROS capture membrane corresponds to the overlying water layer, soil-water layer, and soil layer in the vertical direction, respectively, to achieve in-situ capture of free radicals in different layers, such as... Figure 8 In situ fluorescence capture membranes were inserted into soil profiles at different depths.

[0054] Step S2: After being inserted into the paddy field for a period of time, the in-situ characterization device is pulled out of the paddy field, and the ROS capture membrane is taken out from the in-situ characterization device. The ROS capture membranes corresponding to the overlying water layer, the soil-water layer, and the soil layer are then divided.

[0055] Step S3: Place the segmented ROS capture films on glass slides and perform fluorescence imaging using a dual-scanning laser confocal microscope (Nikon A1) at an excitation wavelength of 480 nm.

[0056] Step S4: By comparing the obtained fluorescence images with standard values, the amount of ROS generated at different levels can be obtained, i.e., in-situ characterization data of free radicals can be obtained, such as... Figure 9 and Figure 11 .

[0057] Simultaneously, fluorescence images can be input into ImageJ software for analysis to obtain more accurate ROS concentration data, such as... Figure 12 Using 3D visualization software, the spatial distribution of free radicals at different soil depths was presented, constructing a spatial 3D image of soil free radicals, such as... Figure 10 .

[0058] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. An in-situ characterization method for an in-situ characterization device for real-time monitoring of free radicals in a paddy field system during rainfall, wherein the in-situ characterization device comprises: The carrier mold is a cuboid structure with a first groove on one side surface along its thickness direction. The first groove opens along one side of the length direction of the cuboid, and a limit frame is provided in the first groove. A ROS-capturing membrane is disposed within the space enclosed by the limiting frame for capturing ROS. A cover plate is located in the first groove and matches the shape of the first groove. A notch is provided on the cover plate at the position corresponding to the limiting frame. The limiting frame is located in the notch. A filter membrane is provided on the side of the cover plate away from the carrier mold. The filter membrane can cover the notch. Its features are, The in-situ characterization method includes the following steps: Step S1: During rainfall, the in-situ characterization device is inserted into the paddy field. The ROS capture membrane corresponds to the overlying water layer, soil-water layer, and soil layer of the paddy field in the vertical direction, respectively. Free radicals in the soil will react with 2,7-dichlorodihydrofluorescein diacetate on the ROS capture membrane to achieve in-situ capture of free radicals in different layers. Step S2: After being inserted into the paddy field for a period of time, the in-situ characterization device is pulled out of the paddy field, and the ROS capture membrane is taken out from the in-situ characterization device. The ROS capture membranes corresponding to the overlying water layer, the soil-water layer, and the soil layer are then divided. Step S3: Place the segmented ROS capture membranes on glass slides and perform fluorescence imaging using a dual-scanning laser confocal microscope at an excitation wavelength of 480 nm. Step S4: By comparing the obtained fluorescence image with the standard value, the amount of ROS generated at different levels can be obtained, that is, the in-situ characterization data of free radicals can be obtained.

2. The in-situ characterization method according to claim 1, characterized in that, The in-situ characterization device further includes a housing, on which a second groove extending along the length direction is provided, the cross-section of the second groove being inverted T-shaped; the cross-section of the carrier mold is also inverted T-shaped, one end of the second groove along the length direction is sealed, the carrier mold is inserted into the second groove from the unsealed end of the second groove, and along the width direction of the cuboid, both sides of the carrier mold are stuck in the second groove of the housing.

3. The in-situ characterization method according to claim 1, characterized in that, The preparation method of the in-situ characterization device is as follows: Step 1: Prepare a certain amount of phosphate buffer, 2,7-dichlorodihydrofluorescein diacetate solution, and agarose solution respectively. The pH of the phosphate buffer is 7.

4. Step 2: Add the 2,7-dichlorodihydrofluorescein diacetate solution to the phosphate buffer and mix well; Step 3: Heat the agarose using a microwave, add the mixed solution obtained in Step 2 to the agarose solution and mix well; Step 4: Pour the product obtained in Step 3 into the space of the limiting frame and cool it. The solid in the space is the ROS capture membrane. Step 5: Install the cover plate onto the carrier mold; Step 6: Cover the filter membrane on the side of the cover plate away from the carrier mold to cover the ROS capture membrane.

4. The in-situ characterization method according to claim 1, characterized in that, Also includes: Step S5: Using 3D visualization software, present the spatial distribution of free radicals at different soil depths based on in-situ characterization data, and construct a spatial 3D image of soil free radicals.

Images