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

Abstract

The invention provides an in-situ characterization device and method for monitoring free radicals of a rice field system in the rainfall process in real time, and the device comprises a carrier mold which is of a cuboid structure, a first groove is formed in the surface of one side in the thickness direction of the carrier mold, and an opening is formed in the side, in the length direction of the cuboid, of the first groove; a limiting frame is arranged in the first groove; the ROS capturing film is arranged in the space defined by the limiting frame and used for capturing ROS; the covering plate is located in the first groove and matched with the first groove in shape, a notch is formed in the position, corresponding to the limiting frame, of the covering plate, the limiting frame is located in the notch, a filter membrane is arranged on the side, away from the carrier mold, of the covering plate, and the filter membrane can cover the notch.

Description

Technical Field

[0001] The present invention relates to the field of rainfall monitoring, and particularly to an in-situ characterization device and method for free radicals in a paddy field system during real-time rainfall monitoring. Background Art

[0002] Rainwater generally contains micromolar levels of hydrogen peroxide (H2O2) generated by photochemical reactions, and its concentration ranges from 2 μM to 40 μM. During natural rainfall, H2O2 is very likely to come into contact with ferrous ions (Fe 2+ ) in flooded environments such as paddy fields in the South China region. Under the catalytic action of Fe 2+ , an in-situ Fenton effect can occur, generating a typical reactive oxygen species (ROS) with strong oxidizing properties - hydroxyl radical (·OH). ·OH is a very active oxidant that can undergo oxidation reactions with reducing substances in the paddy soil and water environment, thereby affecting the ROS in the paddy soil and water environment system. In addition, paddy fields have rich multiphase interfaces, and their gas-liquid-solid-living interfaces are hot spots for ROS aggregation and reaction. When natural rainfall causes changes in the water level of the overlying water in the paddy field, it will cause alternating aerobic and anaerobic states in the soil, thus promoting the generation of ROS. Especially at the soil-water interface, strong mass 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, how to real-time monitor and characterize the ROS process in the complex soil system during rainfall is an international problem. In particular, it is urgent to overcome the technical challenge of the black box of the ROS process at multiple interfaces in the paddy field system 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 be used for in-situ detection and it is difficult to analyze the spatial and temporal distribution characteristics of soil ROS; the chemical molecular probe method requires expensive reagents and equipment; the quenching method requires destructive sampling of the soil, which destroys the original soil structure. Existing patents and technologies, such as ordinary in-situ capture membranes, are extremely easy to damage the capture membrane during the sampling and extraction process, and at the same time destroy the soil structure, affecting the measurement results and soil microorganisms; moreover, the change in the overlying water level in the paddy field during rainfall will increase the oxygen disturbance, affecting the variation laws of pH, dissolved oxygen (DO), and carbon dioxide partial pressure (pCO2) in the water / sediment system, and interfering with the measurement results of different interfaces. Therefore, there is an urgent need to develop real-time monitoring means and devices for in-situ visualization of ROS in paddy field soil during rainfall, accurately stratify the paddy field interface, and weaken or eliminate the interference of sampling and rainfall processes on different interfaces of the paddy field. Summary of the Invention

[0004] The main purpose of the present invention is to provide an in-situ characterization device and method for real-time monitoring of free radicals in a rice field system during rainfall, so as to solve the above-mentioned technical problems.

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

[0006] The carrier mold is a rectangular parallelepiped structure, and a first groove is provided on one side surface along the thickness direction of the carrier mold. The first groove is open along one side along the length direction of the rectangular parallelepiped, and a limiting frame is provided in the first groove.

[0007] A ROS capture membrane is disposed in the space surrounded by the limiting frame and is used to capture ROS;

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

[0009] Preferably, it also includes a shell, which is provided with a second groove extending along the length direction, and the cross-section of the second groove is an inverted T-shape; the cross-section of the carrier mold is also an inverted T-shape, and one end of the second groove along the length direction is blocked, and the carrier mold is inserted into the second groove from the unblocked end of the second groove, and along the width direction of the rectangular parallelepiped, both sides of the carrier mold are stuck in the second groove of the shell.

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

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

[0012] Step 3, heating the agarose by microwave, adding the mixed solution obtained in step 2 to the agarose solution and mixing them evenly;

[0013] Step 4: pouring the product obtained in step 3 into the space of the limiting frame and cooling it, wherein the solid in the space is the ROS capture membrane;

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

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

[0016] The present invention also provides an in-situ characterization method for monitoring free radicals in a paddy field system during rainfall. Using the above in-situ characterization device, the method specifically includes the following steps:

[0017] Step S1: Insert the in-situ characterization device into the paddy field during rainfall. The ROS capture membrane corresponds to the overlying water layer, soil-water layer, and soil layer of the paddy field in the vertical direction. Free radicals in the soil 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 inserting the device into the paddy field for a period of time, pull out the in-situ characterization device from the paddy field, take out the ROS capture membrane from the device, and divide the ROS capture membrane corresponding to the overlying water layer, soil-water layer, and soil layer.

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

[0020] Step S4: Compare the obtained fluorescence images with the standard values to obtain the ROS generation amounts on different levels, that is, obtain the in-situ characterization data of free radicals.

[0021] Preferably, it further includes:

[0022] Step S5: Use three-dimensional visualization software to present the spatial distribution of free radicals at different soil depths according to the in-situ characterization data and construct a three-dimensional spatial image of soil free radicals.

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

[0024] The present invention realizes in-situ real-time monitoring of free radicals generated in the paddy field system during rainfall, accurately stratifies the paddy field interface by dividing the capture membrane, and the materials used for the mold and cover plate have better strength and dimensional stability characteristics in a humid environment. The material used for the filter membrane has sealing properties, and the use of the outer shell avoids damaging the capture membrane and destroying the soil structure during the sampling and extraction process, reducing the interference of sampling and rainfall processes on different interfaces of the paddy field, and breaking through the bottleneck that it is difficult to accurately, in-situ, and real-time characterize soil free radicals in the paddy field during rainfall. Moreover, the present invention is simple to operate, economical and efficient, and is easy to promote and apply. Description of the Drawings

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

[0026] Figure 2 is Figure 1 the cross-sectional view of

[0027] Figure 3 and Figure 4 It is a structural diagram of the carrier mold from two angles;

[0028] Figure 5 It is the structural diagram of the covering plate;

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

[0030] Figure 7 It is the structural diagram of the shell;

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

[0032] Figure 9 It is a comparison chart of fluorescence image and ROS content;

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

[0034] Figure 11 This is the ROS fluorescence image of the rice field system during rainfall;

[0035] Figure 12 It is a graph of ROS concentration and proportion at each interface. DETAILED DESCRIPTION

[0036] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0037] Embodiment 1

[0038] like Figure 1-7 As shown, an in-situ characterization device for real-time monitoring of free radicals in a rice field system during rainfall includes a housing 1, a carrier mold 2, a cover plate 3, and a ROS capture membrane. When viewed from above, the carrier mold 2 is rectangular, and a first groove 21 is formed on one side thereof, and the first groove 21 is open along one side of the length direction of the rectangle. A limiting frame 22 is arranged in the first groove 21, and a space for placing a ROS membrane is formed in the limiting frame 22, and the limiting frame 22 is open along the side away from the carrier mold 2. Nine convex strips 23 are respectively arranged on the two inner side walls of the limiting frame 22 along the width direction of the rectangle, and the convex strips 23 on each side are arranged at equal intervals, and the convex strips 23 on both sides correspond to each other one by one, and the 9 pairs of convex strips 23 divide the ROS capture membrane into 10 equal parts.

[0039] The cover plate 3 can be placed into the first groove 21 and fit with the first groove 21. A notch 31 is provided at a position of the cover plate 3 corresponding to the limit 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 limit frame 22. A filter membrane is provided on the outer side surface of the cover plate 3. The pore size of the filter membrane is 0.45 μm and it is made of polyvinylidene fluoride (PVDF). The ROS capture membrane located in the space of the limit frame 22 can be covered by the filter membrane.

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

[0041] Step 1: Prepare 10 mM phosphate buffer solution (PBS) (pH = 7.4), 100 μM 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) solution and agarose solution respectively. The mass ratio of agarose in the agarose solution is 2%;

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

[0043] Step 3: Heat the agarose to about 43 degrees by microwave, and add 400 μL of the mixed solution obtained in Step 2 to the agarose solution and mix evenly;

[0044] Step 4: Pour the product obtained in Step 3 into the space of the limit 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 facing away from the carrier mold to cover the ROS capture membrane.

[0047] Furthermore, a plurality of positioning posts 32 are provided on the inner side surface of the cover plate 3, and positioning holes 24 are provided at positions of the carrier mold 2 corresponding to each positioning post 32. The positioning posts 32 and the positioning holes 24 are in interference fit.

[0048] A second groove 11 extending along the length direction is provided on the housing 1. The cross-section of the second groove 11 is in an inverted T shape. The cross-section of the carrier mold 2 is also in an inverted T shape. One end of the second groove 11 along the length direction is blocked. The carrier mold 2 is inserted into the second groove 11 from the unblocked end of the second groove 11, and along the width direction, both sides of the carrier mold 2 are stuck in the second groove 11 of the housing 1. Preferably, an interference fit can be set at the engagement position between the carrier mold 2 and the housing 1 to prevent the carrier mold 2 from detaching from the housing 1 by itself.

[0049] The main components of the carrier mold 2, the cover plate 3, and the outer shell 1 are stereolithography resins, specifically, it can be BESTY-4 material.

[0050] Preferably, a slope is provided at the lower end of the carrier template 2 and on the side facing away from the outer shell 1. Along the direction from top to bottom, the slope gradually approaches the outer shell 1, which is beneficial for the device to be smoothly inserted into the soil through the slope.

[0051] Example Two

[0052] This example is an in-situ characterization method for the device in Example One, specifically including the following steps:

[0053] Step S1: Insert the in-situ characterization device into the paddy field during rainfall. The ROS capture membrane corresponds to the overlying water layer, the soil-water layer, and the soil layer of the paddy field in the vertical direction respectively to achieve in-situ capture of free radicals in different layers. For example, Figure 8 Insert the in-situ fluorescence capture membrane into different depths of the soil profile;

[0054] Step S2: After inserting the in-situ characterization device into the paddy field for a period of time, pull out the in-situ characterization device from the paddy field, take out the ROS capture membrane from the in-situ characterization device, and divide the ROS capture membranes corresponding to the overlying water layer, the soil-water layer, and the soil layer;

[0055] Step S3: Place the divided ROS capture membranes on glass slides respectively and perform fluorescence imaging with a dual-scanning laser confocal microscope (Nikon A1) at an excitation light wavelength of 480 nm;

[0056] Step S4: Compare the obtained fluorescence images with the standard values to obtain the ROS generation amounts on different levels, that is, obtain the in-situ characterization data of free radicals. For example, Figure 9 and Figure 11 .

[0057] Meanwhile, the fluorescence images can be input into Image J software for analysis to obtain more accurate ROS concentration data. For example, Figure 12 . Using three-dimensional visualization software, present the spatial distribution of free radicals at different soil depths and construct a three-dimensional spatial image of soil free radicals. For example, Figure 10 .

[0058] The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, various changes and improvements will occur to the present invention, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims

1. An in-situ characterization device for real-time monitoring of free radicals in a rice field system during rainfall, characterized in that: include: The carrier mold is a rectangular parallelepiped structure, and a first groove is provided on one side surface along the thickness direction of the carrier mold. The first groove is open along one side along the length direction of the rectangular parallelepiped, and a limiting frame is provided in the first groove. A ROS capture membrane is disposed in the space surrounded by the limiting frame and is used to capture ROS; A covering plate is located in the first groove and matches the shape of the first groove. A notch is provided on the covering plate at a position corresponding to the limiting frame. The limiting frame is located in the notch. A filter membrane is provided on the side of the covering plate facing away from the carrier mold, and the filter membrane can cover the notch.

2. The in-situ characterization device for real-time monitoring of free radicals in a rice field system during rainfall according to claim 1, characterized in that: It also includes a shell, which is provided with a second groove extending along the length direction, and the cross-section of the second groove is an inverted T-shape; the cross-section of the carrier mold is also an inverted T-shape, and one end of the second groove along the length direction is blocked, and the carrier mold is inserted into the second groove from the unblocked end of the second groove, and along the width direction of the rectangular parallelepiped, both sides of the carrier mold are stuck in the second groove of the shell.

3. The in-situ characterization device for real-time monitoring of free radicals in a rice field system during rainfall according to claim 1, characterized in that: The preparation method is as follows: Step 1: prepare a certain amount of phosphate buffer, 2,7-dichlorodihydrofluorescein diacetate solution, and agarose solution respectively, wherein the pH of the phosphate buffer is 7.4; Step 2: Add 2,7-dichlorodihydrofluorescein diacetate solution into phosphate buffer and mix well; Step 3, heating the agarose by microwave, adding the mixed solution obtained in step 2 to the agarose solution and mixing them evenly; Step 4: pouring the product obtained in step 3 into the space of the limiting frame and cooling it, wherein the solid in the space is the ROS capture membrane; Step 5: installing the cover plate onto the carrier mold; Step six: Cover the filter membrane on the side of the cover plate facing away from the carrier mold to cover the ROS capture membrane.

4. A method for in-situ characterization of free radicals in a rice field system during rainfall in real time, using an in-situ characterization device for real-time monitoring of free radicals in a rice field system during rainfall as claimed in any one of claims 1 to 3, specifically comprising the following steps: Step S1, inserting the in-situ characterization device into the paddy field during rainfall, the ROS capture membrane corresponds to the overlying water layer, the soil water layer and the soil layer of the paddy field in the upper and lower directions respectively, and the free radicals in the soil react with the 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 rice field for a period of time, the in-situ characterization device is pulled out from the rice field, and the ROS capture membrane is taken out from the in-situ characterization device, and the ROS capture membrane corresponding to the overlying water layer, the soil water layer and the soil layer is segmented; Step S3, placing the segmented ROS capture membranes on glass slides respectively and performing fluorescence imaging using a double scanning laser confocal microscope at an excitation light 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.

5. The in-situ characterization method according to claim 4, characterized in that: Also includes: Step S5: Using three-dimensional visualization software, the spatial distribution of free radicals at different soil depths is presented based on the in-situ characterization data, and a spatial three-dimensional image of soil free radicals is constructed.

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