A preparation method of a wearable sensor for dynamic monitoring of H2O2 in a plant body

Abstract

The application discloses a preparation method of a wearable sensor for dynamic monitoring of H2O2 in a plant body. The method comprises the following steps: preparing a laser-induced graphene electrode on the surface of a polyimide film by adopting a laser-induced graphene technology; cutting the polyimide adhesive tape by a laser micro-processing system to obtain an electrode mask, tearing off the mask and attaching the mask to the surface of the laser-induced graphene electrode; coating Ag / AgCl paste on a reference electrode area to obtain an integrated electrochemical three-electrode system; electrochemically depositing platinum nanoparticles on the surface of a working electrode as a sensing layer, and adding Nafion solution as an anti-interference protective layer, so as to obtain a non-enzyme electrochemical sensor for in-situ detection of H2O2 in the plant body. The sensor is used as a sensing module, a photovoltaic power supply module, a data acquisition module and a data transmission module are integrated, and a self-driven sensing system is formed. Therefore, long-term dynamic monitoring of the content change of H2O2 in the plant body is realized.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a method for preparing a wearable sensor for dynamic monitoring of H2O2 in plants. Background Technology

[0002] Monitoring changes in internal biochemical indicators of plants, especially key signaling molecules, can help detect changes in plant health status earlier. Transpiration, runoff changes, and the release of volatile organic compounds are merely surface phenomena of crop physiological activity. In fact, these surface phenomena are caused by the interaction mechanisms between key plant signaling molecules and biomarkers such as hormones. For example, when plants experience drought stress, reactive oxygen species (ROS), acting as second messengers, cause changes in the content of the plant hormone abscisic acid, further leading to stomatal closure. In plant stress and disease resistance processes, ROS is one of the earliest key signaling molecules besides calcium ions. ROS signals mainly include superoxide ions (O2). - ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) — Of the various reactive oxygen species (ROS), H2O2 has the longest half-life and can be transmitted over long distances and across membranes, making it a key ROS molecule mediating rapid systemic signals in plants. Therefore, accurate and real-time monitoring of H2O2 signaling molecules is crucial for monitoring plant health.

[0003] Currently, the main methods for detecting H2O2 in plants include tissue section staining and extracting H2O2 from ground tissue samples followed by quantitative analysis using ultraviolet spectrophotometry. These two methods cannot meet the requirements for high temporal resolution and continuous monitoring, are prone to missing the optimal detection window, and are susceptible to human error. Furthermore, larger wounds are easily infected by bacteria, leading to plant diseases. Directly monitoring H2O2 levels in plants using implantable sensing technology can avoid complex sample pretreatment processes, reduce matrix interference, and achieve in-situ dynamic monitoring. In recent years, photonic nanosensors that can be injected into plant leaves and implantable electrochemical sensors have been developed for monitoring H2O2 levels. These sensors can provide real-time spatiotemporal information on plant signaling molecules; however, due to limitations in energy supply and wearability, these studies can currently only be conducted in the laboratory. Therefore, there is an urgent need to develop a wearable sensing analysis tool and method for long-term, real-time monitoring of dynamic changes in H2O2 in plants within agricultural facilities. Summary of the Invention

[0004] To address the shortcomings of existing technologies for monitoring the dynamic level of H2O2 in plants, this invention provides a method for preparing a wearable sensor for dynamic monitoring of H2O2 in plants. This sensor can achieve in-situ continuous monitoring of changes in H2O2 content in plants, and when combined with a photovoltaic power supply module, it can achieve self-driven sensing, facilitating real-time dynamic monitoring of changes in H2O2 content in plants.

[0005] I. A method for preparing a wearable sensor for dynamic monitoring of H2O2 in plants

[0006] Includes the following steps:

[0007] Step S1: Prepare a laser-induced graphene electrode on the surface of a polyimide film; the specific method is as follows:

[0008] (1-1) The polyimide film was ultrasonically cleaned with ethanol and deionized water for 10 minutes respectively. After the cleaned polyimide film was dried with high-purity nitrogen, it was fixed on the surface of the heat-conducting copper plate for later use.

[0009] (1-2) Use CorelDraw drawing software to draw the required electrode pattern and design and determine the electrode dimensions. Import the designed electrode pattern into the computer, ready for laser printing;

[0010] (1-3) Place the polyimide film fixed on the surface of the copper plate at a specific position on the operating table of the laser micromachining system, and adjust the focal length using a laser focuser;

[0011] (1-4) Set the laser power to 2% to 16%, preferably 8%, the scanning rate to 5% to 30%, preferably 14%, and the pixel density to 500 to 1000 PPI, preferably 750 PPI, and start the laser-induced graphene program;

[0012] (1-5) Take out the laser-micro-processed polyimide film, rinse the laser-induced graphene area repeatedly with deionized water to remove residues with poor mechanical properties, and let it air dry at room temperature for later use.

[0013] Step S2: Take another polyimide film, ultrasonically clean it with deionized water, dry it with nitrogen, fix it on the surface of the thermally conductive copper plate, stick polyimide tape on the surface of the polyimide film, cut it with a laser micromachining system to obtain an electrode mask, peel off the mask and attach it to the electrode surface obtained in step S1.

[0014] Step S3: The laser-induced graphene electrode reference electrode region obtained in step S2 is coated with Ag / AgCl slurry and dried in an oven to obtain an integrated electrochemical three-electrode system with Ag / AgCl as the reference electrode;

[0015] Step S4: Electrochemically deposit platinum nanoparticles as a sensing layer on the surface of the working electrode in step S3;

[0016] Step S5: Add Nafion solution as an anti-interference protective layer to the working electrode area of ​​the electrode obtained in step S4, and allow it to dry naturally at room temperature to obtain a non-enzymatic electrochemical sensor for in-situ detection of H2O2 in plants.

[0017] In step S1, the laser-induced graphene electrode includes a reference electrode region, a working electrode region, and a counter electrode region.

[0018] In step S1, the thickness of the polyimide film can be selected from 50 to 100 μm, preferably 80 μm. The electrode working area is a circle with a diameter of 0.5 mm.

[0019] In step S2, the polyimide tape is 50 μm thick, and the mask covers the area excluding the working electrode, reference electrode, counter electrode, and conductive connection portion.

[0020] In step S3, the drying time is 30 minutes and the temperature is 60°C.

[0021] In step S4, the solution for depositing platinum nanoparticles is a 1–10 mM K₂PtCl₄ solution, and the electrolyte is a 0.1–0.5 M Na₂SO₄ solution. The electrochemical deposition method is cyclic voltammetry, with a potential range of -0.4 V to +0.5 V and a scan rate of 50–100 mV s⁻¹. -1 The number of scans is 5 to 15.

[0022] In step S5, the Nafion solution concentration is 0.1% to 1.0%, and the volume is 0.2 to 1.0 μL.

[0023] II. Self-powered sensing applications of wearable sensors for dynamic monitoring of H2O2 in plants

[0024] The self-powered sensing system of the wearable H2O2 sensor developed in this invention includes a sensing module, a photovoltaic power supply module, a data acquisition module, and a data transmission module. The sensing module is the core of the system and is a wearable sensor for dynamic monitoring of H2O2 within plants, prepared in the aforementioned sections. This electrochemical sensor consists of a polyimide substrate, a laser-induced graphene layer, a noble metal layer, and an anti-interference layer.

[0025] The photovoltaic power supply module mainly relies on solar panels to collect agricultural ambient light energy, convert it into electrical energy and store it in a rechargeable battery to power the electrochemical sensing module and the data acquisition and transmission module.

[0026] The working principle of the self-powered sensing system of the wearable H2O2 sensor in plants of this invention is as follows: The working area of ​​the laser-induced graphene electrochemical sensor is modified with platinum nanoparticles that have an electrochemical catalytic effect on H2O2. The surface of the platinum nanoparticles is covered with a Nafion polymer film that can block some negatively charged interferences in the plant fluid. This film serves as an anti-interference layer and a physical protective layer. When the sensor is implanted in the plant, when a specific oxidation potential is applied, the H2O2 in the plant is catalyzed by the platinum nanoparticles at the sensing interface, undergoing an oxidation reaction and generating an oxidation current. The magnitude of the current is related to the H2O2 concentration. The generated oxidation current is captured by the signal acquisition module and converted into a voltage signal. The converted electrical signal is sent to the receiving and reading interface via the data transmission module, and then converted into a readable current signal by software, allowing for remote reading of changes in the H2O2 content in the plant. The electrical energy used in the monitoring process comes from the electrical energy stored in the rechargeable battery after the solar panel converts light energy.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention constructs a wearable electrochemical sensor for dynamic monitoring of H2O2 in plants, as well as a self-powered sensing system. It can realize in-situ detection of H2O2 in plants, and with the help of the self-powered system, it makes full use of ambient light energy to provide continuous power to the sensor, thus realizing dynamic monitoring of H2O2 in plants. Attached Figure Description

[0029] Figure 1 (a) shows the laser-induced graphene electrode pattern prepared on the surface of the polyimide film, and (b) shows the actual laser-induced graphene electrode formed after the mask is attached and its size comparison.

[0030] Figure 2 In the figure, (a) is the chronoamperometry response curve of the sensor to 2-200 μM H2O2, and (b) is the corresponding standard curve;

[0031] Figure 3 Three graphene morphologies were prepared by adjusting laser parameters: fiber (3a), fiber-sheet mixture (3b), and graphene sheet (3c).

[0032] Figure 4 In the figure, (a) is the cyclic voltammetric response curve of the graphene fiber-based electrode to H2O2 solutions of different concentrations, and (b) is the cyclic voltammetric response curve of the graphene sheet-based electrode to H2O2 solutions of different concentrations.

[0033] Figure 5 (a) is the system architecture diagram of the wearable self-powered sensing system, and (b) is the circuit composition diagram of the electrochemical data acquisition module.

[0034] Figure 6 (a) in the image is a physical diagram of the sensor system application, and (b) is the monitoring result data.

[0035] Figure 7 The curve is obtained by ultraviolet spectrophotometry. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not constitute any limitation thereof.

[0037] Example 1

[0038] Step S1: Prepare a laser-induced graphene electrode on the surface of a polyimide (PI) film;

[0039] Step S1, the specific method is as follows:

[0040] (1-1) The polyimide film was ultrasonically cleaned with ethanol and deionized water for 10 minutes respectively. After the cleaned polyimide film was dried with high-purity nitrogen, it was fixed on the surface of the heat-conducting copper plate for later use.

[0041] (1-2) Use CorelDraw drawing software to draw the required electrode pattern and design and determine the electrode dimensions. Import the designed electrode pattern into the computer, ready for laser printing;

[0042] (1-3) Place the polyimide film fixed on the surface of the copper plate at a specific position on the operating table of the laser micromachining system, and adjust the focal length using a laser focuser;

[0043] (1-4) Set the laser power to 2% to 16%, preferably 8%, the scanning rate to 5% to 30%, preferably 14%, and the pixel density to 500 to 1000 PPI, preferably 750 PPI, and start the laser-induced graphene program;

[0044] (1-5) Take out the laser-micro-processed polyimide film, rinse the laser-induced graphene area repeatedly with deionized water to remove residues with poor mechanical properties, and let it air dry at room temperature for later use.

[0045] Step S2: Take another polyimide film, ultrasonically clean it with deionized water, dry it with nitrogen, and fix it on the surface of the thermally conductive copper plate. Adhere polyimide tape to the surface of the polyimide film, and cut it using a laser micromachining system to obtain an electrode mask. Peel off the mask and attach it to the electrode surface obtained in step S1. The resulting electrode is as follows... Figure 1 As shown, Figure 1 a represents the laser-induced graphene electrode pattern prepared on the surface of a polyimide film. Figure 1 b shows a physical image and size comparison of the laser-induced graphene electrode formed after the mask is attached.

[0046] Step S3: The laser-induced graphene electrode reference electrode region obtained in step S2 is coated with Ag / AgCl slurry and dried in an oven to obtain an integrated electrochemical three-electrode system with Ag / AgCl as the reference electrode;

[0047] Step S4: Electrochemically deposit platinum nanoparticles on the surface of the working electrode of the electrochemical electrode in step S3;

[0048] Step S5: Add Nafion solution to the working electrode area of ​​the electrode obtained in step S4 and allow it to dry naturally at room temperature to obtain a non-enzymatic electrochemical sensor for in-situ detection of H2O2 in plants.

[0049] The electrode obtained in Example 1 was connected to an electrochemical workstation, and H2O2 solutions of different concentrations were analyzed by chronoamperometry. A potential of +0.6V was applied, and the results are as follows: Figure 2 As shown, it exhibits a good linear relationship in the H2O2 concentration range of 2–200 μM, with a detection limit (LOD, S / N = 3) of 0.35 μM.

[0050] Example 2

[0051] The steps in Example 2 are the same as those in Example 1. The difference is:

[0052] In step 1, different laser powers and scanning rates were selected in Example 2 for laser-induced graphene electrode fabrication, and the electrode morphology was characterized by scanning electron microscopy (SEM). Figure 3 As shown, by adjusting the laser parameters, three graphene morphologies can be obtained: fibers (3a), a mixture of fibers and sheets (3b), and graphene sheets (3c).

[0053] The graphene fiber-based electrodes and graphene sheet-based electrodes obtained in Examples 1 and 2 were connected to an electrochemical workstation to analyze H2O2 solutions of different concentrations. The results are as follows: Figure 4 As shown, comparing the size of the oxidation peaks produced by the two electrodes at around +0.6V, it was found that the graphene-based electrode was more sensitive to the electrochemical response to H2O2. Therefore, the morphology of laser-induced graphene is crucial for the sensitivity of H2O2 detection sensors.

[0054] Self-powered sensing applications of wearable sensors for dynamic monitoring of H2O2 in plants:

[0055] Design an electrochemical data acquisition and transmission integrated circuit with a photovoltaic power supply module. The system architecture is as follows: Figure 5As shown in Figure a, the system consists of three parts: a photovoltaic power supply module (output voltage of 5.5V), a data acquisition module, and a data transmission module. Data can be transmitted to the user interface wirelessly via Wi-Fi or Bluetooth. The circuit composition of the electrochemical data acquisition module is as follows: Figure 5 As shown in b. According to the circuit design, the power management components of the power supply module and the data acquisition and data transmission module are integrated and fabricated into a flexible circuit board, and waterproof encapsulation is performed.

[0056] The sensor tip prepared in Example 1 was inserted into the stem of a tomato plant. The sensor was then connected to an integrated circuit board. A preset working potential was applied to the sensor through the circuit management module of the integrated circuit board, causing the H2O2 in the plant to be catalyzed by platinum nanoparticles at the sensing interface, resulting in an oxidation reaction and generating an oxidation current. The generated oxidation current was captured by the data acquisition module and converted into a voltage signal. The converted voltage signal was sent to the receiving and reading interface through the data transmission module, and then converted into a readable current signal by the software. The H2O2 content was calculated based on the calibration curve established by the current signal and the H2O2 concentration, and the changes in the H2O2 content in the plant could be read remotely.

[0057] like Figure 6 As shown, tomatoes subjected to osmotic stress will produce H2O2, and the sensor of the present invention can detect the generated H2O2 signal; as a control group, tomatoes not subjected to osmotic stress will not produce H2O2 warning signal, so the sensor will not detect H2O2 signal.

[0058] like Figure 7 As shown, the same test was performed using ultraviolet spectrophotometry, and the results obtained were consistent with those of the sensor of this invention.

Claims

1. A method for preparing a wearable sensor for dynamic monitoring of H2O2 in a plant body, characterized by, The method comprises the following steps: Step S1: preparing a laser-induced graphene electrode on the surface of a polyimide film, the laser-induced graphene electrode comprising a reference electrode region, a working electrode region and a counter electrode region; Step S2: taking another piece of polyimide film, cleaning it with ultrasonic deionized water, and then drying it with nitrogen, fixing one side on the surface of a heat-conducting copper plate, and then sticking a polyimide adhesive tape on the other side, and then cutting and preparing an electrode mask through a laser micro-processing system, tearing off the mask and attaching it to the surface of the electrode prepared in step S1; Step S3: coating Ag / AgCl paste on the reference electrode region of the laser-induced graphene electrode with the mask attached in step S2, and drying it in an oven to prepare an integrated electrochemical three-electrode system with Ag / AgCl as the reference electrode; Step S4: depositing platinum nanoparticles on the working electrode region of the integrated electrochemical three-electrode system prepared in step S3 by using an electrochemical method; Step S5: adding Nafion solution on the surface of the platinum nanoparticle layer deposited in step S4, and naturally drying it at room temperature to prepare a non-enzymatic electrochemical sensor for in-situ detection of H2O2 in plants; In step S4, the solution for depositing platinum nanoparticles is a 1-10 mM K2PtCl4 solution, and the electrolyte is 0.1-0.5 M Na2SO4; the electrochemical deposition method is cyclic voltammetry, the potential range is -0.4 V to +0.5 V, the scanning rate is 50-100 mV / s, and the scanning number is 5-15.

2. The preparation method of the wearable sensor for dynamic monitoring of H2O2 in plants according to claim 1, characterized in that: In steps S1 and S2, the thickness of the polyimide film is 50-100 μm; In step S1, the working electrode region of the laser-induced graphene electrode is a circular shape with a diameter of 0.5 mm.

3. The method of claim 1, wherein the wearable sensor is prepared by the following steps: 1) mixing the HRP and the ABTS in a solution; 2) mixing the solution with the cellulose fiber to form a mixture; 3) drying the mixture to obtain the wearable sensor. In step S1, one end of the polyimide film is triangular-shaped, which facilitates the penetration into plant tissues.

4. The method for preparing a wearable sensor for dynamic monitoring of H2O2 in plants according to claim 1, characterized in that: In step S2, the mask covers the regions on the surface of the laser-induced graphene electrode in step S1 other than the working electrode, the reference electrode, the counter electrode and the conductive connection part.

5. The method of claim 1, wherein the wearable sensor is prepared by the following steps: 1) mixing the HRP and the ABTS in a solution; 2) mixing the solution with the cellulose fiber to form a mixture; 3) drying the mixture to obtain the wearable sensor. In step S3, the drying time is 30 min, and the temperature is 60°C.

6. The method of claim 1, wherein the wearable sensor is prepared by the following steps: 1) mixing the HRP and the ABTS in a solution; 2) mixing the solution with the cellulose paper to form the wearable sensor; and 3) drying the wearable sensor. In step S5, the concentration of the Nafion solution is 0.1%-1%, and the volume is 0.2-1 μL.

7. A wearable sensor for dynamic monitoring of H2O2 in plants, characterized in that: The preparation method is obtained by using any one of claims 1-6.

8. A self-powered sensing application of a wearable sensor for dynamic monitoring of H2O2 in vivo in a plant according to claim 7, characterized in that: The application of the self-driven sensing system in monitoring the change of H2O2 content in plants; the self-driven sensing system comprises a sensor, and an integrated circuit board integrated with a photovoltaic power supply module, a data acquisition module and a data transmission module.

9. The self-powered sensing application of wearable sensor for in vivo dynamic monitoring of H2O2 in plants according to claim 8, wherein, The sensor front end is inserted into the inside of the plant stem, and then the sensor is connected with the integrated circuit board, a preset working potential is applied to the sensor through the circuit management module of the integrated circuit board, so that H2O2 in the plant body is catalyzed by the platinum nanoparticles at the sensor interface, an oxidation reaction occurs, an oxidation current is generated, the generated oxidation current is captured by the data acquisition module of the integrated circuit board and is converted into a voltage signal, the converted voltage signal is sent to the upper computer through the data transmission module of the integrated circuit board, and then is converted into a readable current signal, the H2O2 content is calculated according to the calibration curve established according to the current signal and the H2O2 concentration, and the change of the H2O2 content in the plant body can be remotely read.

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