A method for monitoring the growth state of crops in situ under environmental stress

Abstract

The present application belongs to the technical field of monitoring, and particularly relates to an electrochemical analysis method for in-situ monitoring of active oxygen of crop production under environmental stress. The present application combines copper picolinic acid complex Cu@PyCA with excellent peroxidase activity and reduced graphene with excellent conductivity to form an electrochemical sensor with a 4-Amin / PEDOT:PSS electrode, and completes the monitoring of crops through a self-constructed portable detection circuit. The present application uses Cu@PyCA instead of biological enzymes, and the constructed electrochemical sensor has high stability, strong catalytic activity and low cost. Moreover, the present application takes advantage of the biocompatibility of conductive hydrogel, and can directly monitor the in-situ hydrogen peroxide without liquid electrolyte, thereby realizing the accurate tracking of active oxygen in the growth process of crops.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of monitoring, and particularly relates to an electrochemical analysis method for in-situ monitoring of crop yield under environmental stress. BACKGROUND

[0002] Crops are often subjected to environmental stress such as drought, salinity and high temperature during growth. It is of great significance to obtain stress information of biological systems and monitor crop growth for improving crop yield and promoting the high-quality development of modern agriculture. Active oxygen produced by photosynthesis in plant systems, especially hydrogen peroxide as the most representative environmental stress signal molecule, is an effective bridge for obtaining stress information. However, due to the active nature and easy decomposition of hydrogen peroxide, the existing spectrophotometric method, chemiluminescence method and fluorescence method cannot meet the demand of online and rapid detection in plant systems. Therefore, it is particularly important to establish an effective hydrogen peroxide analysis platform for detecting the change of hydrogen peroxide content in crop systems.

[0003] Electrochemical analysis method has broad application prospects in in-situ analysis of hydrogen peroxide in crop systems due to its fast response speed, high sensitivity and easy portability. However, the existing electrochemical analysis technology still has limitations such as complex design of sensing interface, large trauma on surface of sample to be detected, and high dependence on liquid electrolyte and biological enzymes. Therefore, developing an analysis method with simple operation and in-situ micro-area perception is the key to realize online monitoring of hydrogen peroxide in crops. SUMMARY

[0004] In order to make up for the shortcomings of the prior art, the application provides a copper picolinic acid complex Cu@PyCA with peroxidase-like activity, which is combined with reduced graphene to construct an electrochemical sensing interface.

[0005] In the application, a hydrogel with excellent electrical conductivity and biocompatibility is also provided to replace the traditional liquid electrolyte, and then combined with a sensitive response type sensing interface to construct an electrochemical sensor for in-situ micro-area monitoring of hydrogen peroxide in crops.

[0006] In the application, a suitable portable detection device is also provided, which can be electrically connected with the electrochemical sensor to complete in-situ monitoring of crops.

[0007] In order to achieve the above technical purposes, the specific steps of the application are as follows:

[0008] (1) Construction of sensing interface:

[0009] Process one: preparation of Cu@PyCA;

[0010] Firstly, copper nitrate and 1H-pyrazole-4-carboxaldehyde are dissolved in a mixture of N,N-dimethylformamide, water and ethanol to obtain a mixed solution; then, the mixed solution is transferred into a reaction kettle for incubation, and a yellow single crystal product is obtained after incubation; finally, the obtained yellow single crystal is washed with water and acetone several times and then vacuum dried, and the obtained product is Cu@PyCA; the Cu@PyCA is dispersed in water to obtain a Cu@PyCA dispersion liquid;

[0011] Process two: reducing graphene (rGO) is dispersed in water to obtain an rGO solution; then, the Cu@PyCA dispersion liquid prepared in process one is mixed with the rGO solution to obtain a Cu@PyCA / rGO solution.

[0012] Further, in process one of step (1), the amount of copper nitrate, 1H-pyrazole-4-carboxaldehyde, N,N-dimethylformamide, water and ethanol is 0.5-1 mmol: 1-1.5 mmol: 5-10 mL: 5-10 mL: 5-10 mL; and the volume ratio of N,N-dimethylformamide, water and ethanol is 1:0.7:1.

[0013] Further, in process one of step (1), the incubation temperature is 100℃, and the incubation time is 12h; the washing is performed for 3-5 times; the vacuum drying temperature is 100-120℃, and the vacuum drying time is 10-16h; and the concentration of the Cu@PyCA dispersion liquid is 4mg / mL.

[0014] Further, in process two of step (1), the concentration of the rGO solution is 1mg / mL; and the volume ratio of the Cu3@PyCA dispersion liquid and the rGO solution is 1:1.

[0015] (2) Construction of an electrochemical sensor:

[0016] Process one: 4-aminoindole (4-Amin), water and PEDOT:PSS are mixed to obtain a mixed solution A;

[0017] Process two: ammonium sulfate is dissolved in water to obtain an ammonium sulfate aqueous solution;

[0018] Process three: the mixed solution A is taken to the three-electrode area of a screen-printed electrode, then the ammonium sulfate aqueous solution is added to the mixed solution A on the three-electrode area, and after sufficient mixing and reaction, the screen-printed electrode is immersed in a PBS solution for washing, and after washing, the screen-printed electrode is dried at room temperature to obtain a 4-Amin / PEDOT:PSS electrode;

[0019] Process four: the Cu@PyCA / rGO solution prepared in step (1) is added dropwise on the 4-Amin / PEDOT:PSS electrode, and an electrochemical sensor is obtained after drying;

[0020] Further, in process one of step (2), the amount ratio of 4-Amin, water and PEDOT:PSS is 15-20 mg: 100-150 μL: 50-100 μL.

[0021] Further, in process two of step (2), the concentration of the ammonium sulfate aqueous solution is 0.25-0.3 g / mL.

[0022] Further, in process three of step (2), the volume ratio of the mixed solution A to the ammonium sulfate aqueous solution is 2:1, the reaction time is 20-40 min, the concentration of the PBS solution is 0.1 M, and the pH is 7.

[0023] Further, in process four of step (2), the volume of the Cu@PyCA / rGO solution added dropwise is 8-12 μL.

[0024] (3) Construction of a portable detection device:

[0025] The portable detection device is composed of a power supply, a Bluetooth module, a microcontroller, a constant potential instrument, an electrochemical sensor, an IV converter, a low-pass filter and a smart phone. The smart phone is electrically connected to the microcontroller through the Bluetooth module to realize signal transmission and control. Meanwhile, the microcontroller is electrically connected to the low-pass filter and the constant potential instrument, and the low-pass filter is electrically connected to the IV converter. The electrochemical sensor is electrically connected to the IV converter and the constant potential instrument.

[0026] Further, in step (3), the power supply is ZMV2405D, the Bluetooth module is BG24, and the microcontroller is STC89C52.

[0027] (4) In-situ monitoring of hydrogen peroxide:

[0028] Process one: the smart phone is connected to the Bluetooth module and sends a detection instruction to the microcontroller. After receiving the detection instruction, the microcontroller applies a constant voltage to the electrochemical sensor through the constant potential instrument. The electrochemical sensor generates a signal in response to the stimulus, and the voltage signal is converted into a current signal through the IV converter and is transmitted back to the microcontroller after being processed by the low-pass filter. The microcontroller sends the processed data to the smart phone through the Bluetooth module.

[0029] Process two: observe the current curve on the smart phone, after the current is stable, add hydrogen peroxide standard solution to the electrochemical sensor every certain time to obtain a series of current step change curves of hydrogen peroxide standard solution with different concentrations; and a linear regression equation is established between the hydrogen peroxide concentration and the corresponding response current, the regression equation is y=ax+b, wherein x represents the concentration of hydrogen peroxide, y represents the response current value, and a and b are constants;

[0030] Process three: cut the plant surface to form a wound, place the electrochemical sensor on the wound surface of the plant, obtain the current value according to the operation of process one of step (4) one, and then bring the current value into the linear regression equation of process two of step (4) to realize the detection of the hydrogen peroxide concentration.

[0031] Further, in process one of step (4), the applied constant voltage is-0.3 V.

[0032] Further, in process two of step (4), the time for the current to be stable is 400-600 s, the time interval is 50-100 s, the concentration of the hydrogen peroxide standard solution is 1-10 μM, and the dropwise addition amount is 6-10 μL.

[0033] Further, in process three of step (4), the length of the wound is 2-5 mm.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] (1) The present application adopts copper picolinic acid complex with peroxidase-like activity to construct an electrochemical sensing interface with high stability, strong catalytic activity and low cost, and realizes sensitive detection of low content hydrogen peroxide.

[0036] (2) The present application utilizes the high conductivity and good biocompatibility of hydrogel to directly realize in-situ monitoring of hydrogen peroxide without liquid electrolyte.

[0037] (3) The present application constructs an electrochemical sensor by means of conductive hydrogel and efficient flexible electrochemical sensing interface, realizes online rapid analysis of hydrogen peroxide, does not need to perform complex pretreatment on the plant system to be measured, and effectively realizes in-situ and dynamic monitoring of hydrogen peroxide in crops. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a scanning electron microscope graph of Cu@PyCA in the examples.

[0039] Figure 2 It is a module block diagram of the portable detection device.

[0040] Figure 3 It is a schematic diagram of in-situ monitoring of green bean sprout growth state, wherein 1 is a portable detection device. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0044] The screen-printed electrode used in this embodiment was purchased from Qingdao Botan Technology Co., Ltd.

[0045] Example 1:

[0046] (1) Construction of the sensing interface:

[0047] Procedure 1: First, 0.2 g of copper nitrate and 0.1 g of 1H-pyrazole-4-carboxaldehyde were dissolved in a mixture of 6.7 mL of N,N-dimethylformamide, 5 mL of water, and 6.7 mL of ethanol to obtain a mixed solution. Then, the mixed solution was transferred to a reaction vessel and incubated at 100 °C for 12 h to obtain yellow single crystals. Finally, the obtained yellow single crystals were washed three times with water and acetone respectively, and then vacuum dried at 120 °C for 10 h to obtain Cu@PyCA. Cu@PyCA was dispersed in water to obtain a Cu@PyCA dispersion with a concentration of 4 mg / mL.

[0048] Figure 1 The image shows a scanning electron microscope (SEM) image of Cu@PyCA. As can be seen from the image, Cu@PyCA exhibits a cuboid structure of varying sizes, with a layered distribution on the surface of the structure.

[0049] Step 2: Disperse 4 mg of reduced graphene (rGO) in 4 mL of water to obtain an rGO solution; then, mix 1 mL of the Cu@PyCA dispersion prepared in Step 1 with 1 mL of the rGO solution to obtain a Cu@PyCA / rGO solution.

[0050] (2) Construction of electrochemical sensor:

[0051] Process one: 15 mg of 4-Amin was dissolved in 100 μL of water to obtain a mixed solution A after adding 50 μL of PEDOT:PSS;

[0052] Process two: Ammonium sulfate was dissolved in water to obtain an ammonium sulfate aqueous solution, and the concentration was 0.25 g / mL.

[0053] Process three: 2 μL of the mixed solution A was added to the three-electrode area of the screen-printed electrode, and then 1 μL of the ammonium sulfate aqueous solution was added to the mixed solution A on the three-electrode area, and the mixture was fully mixed and reacted for 30 min. Then, the screen-printed electrode was washed by immersing in a PBS solution (0.1 M, pH = 7), and dried at room temperature after washing to obtain a 4-Amin / PEDOT:PSS electrode.

[0054] Process four: 10 μL of the Cu@PyCA / rGO solution prepared in step (1) was dropped on the 4-Amin / PEDOT:PSS electrode, and an electrochemical sensor was obtained after drying.

[0055] (3) Construction of portable detection device:

[0056] The portable detection device mainly comprises a power supply (ZMV2405D), a Bluetooth module (BG24), a microcontroller (STC89C52), a constant potential instrument, an electrochemical sensor (prepared in step (2)), an IV converter, a low-pass filter and a smart phone. The smart phone is electrically connected to the microcontroller through the Bluetooth module to realize signal transmission and control. Meanwhile, the microcontroller is electrically connected to the low-pass filter and the constant potential instrument, and the low-pass filter is electrically connected to the IV converter. The electrochemical sensor is electrically connected to the IV converter and the constant potential instrument to form the connection relationship of the whole device.

[0057] Among them, the smart phone sends a detection command to the microcontroller through the Bluetooth module, and the microcontroller applies a constant voltage to the electrochemical sensor through the constant potential instrument after receiving the detection command. The electrochemical sensor generates a signal in response to the stimulus, and the voltage signal is converted into a current signal through the IV converter. The current signal is transmitted back to the microcontroller after being processed by the low-pass filter. The microcontroller sends the data analyzed and processed to the smart phone through the Bluetooth module.

[0058] (4) In-situ monitoring of hydrogen peroxide:

[0059] Process one: connect the smart phone to the Bluetooth module and send a detection instruction to the microcontroller, the microcontroller applies a constant voltage of-0.3V to the electrochemical sensor through the potentiostat after receiving the detection instruction, the electrochemical sensor generates a signal in response to the stimulus, the voltage signal is converted into a current signal through the IV converter and transmitted back to the microcontroller after being processed by the low-pass filter, and the microcontroller sends the analyzed and processed data to the smart phone through the Bluetooth module.

[0060] Process two: observe the current curve on the smart phone, after 500s, add 8μL of hydrogen peroxide standard solution(5μM) to the electrochemical sensor every 50s to obtain a series of current step change curves of hydrogen peroxide standard solution with different concentrations; establish a linear regression equation y=7.4747x+6.3407(R 2 =0.9953) between the concentration of hydrogen peroxide and the corresponding response current, where x represents the concentration of hydrogen peroxide and y represents the response current value.

[0061] Process three: make a 4-5mm wound on the surface of the cultivated mung bean sprouts, place the electrochemical sensor on the surface wound, and at the same time, connect the smart phone to the Bluetooth module and send a detection instruction to the microcontroller, the microcontroller applies a constant voltage of-0.3V to the electrochemical sensor through the potentiostat after receiving the detection instruction, the electrochemical sensor generates a signal in response to the stimulus, the voltage signal is converted into a current signal through the IV converter and transmitted back to the microcontroller after being processed by the low-pass filter, the microcontroller sends the analyzed and processed data to the smart phone through the Bluetooth, and the corresponding current value 6.393μA is detected, which is further brought into the linear regression equation established in process two of step(4) to automatically calculate the concentration of hydrogen peroxide, and the result is 7μM.

[0062] (5) Hydrogen peroxide standard addition recovery method determination:

[0063] Take 10g of mung bean sprout sample and add 5mL of water, grind it thoroughly with a mortar; collect the liquid and centrifuge, then take the supernatant for hydrogen peroxide standard addition recovery determination, set up 3 groups of experiments(No.1, No.2, No.3), and the corresponding results are shown in Table 1.

[0064] Table 1 Standard addition recovery experiment results

[0065] Sample No. Amount of spike (mM) Amount of detection (mM) Relative error (%) Recovery rate (%) No. 1 0.08 0.082 2.5 100 No. 2 0.51 0.54 5.9 106 No. 3 1 1.05 5.0 105

[0066] In summary, in this experiment, Cu@PyCA combined with reduced graphene to construct a sensing interface, and with the excellent conductivity and biocompatibility of hydrogel to construct an electrochemical sensor, realizing the in-situ micro-zone monitoring of hydrogen peroxide in plant system. The sensor overcomes the shortcomings of traditional biological enzyme sensors, and takes hydrogen peroxide as the research object and measurement index, providing a new way to obtain the stress information of biological system, so as to provide the most suitable growth conditions for crops, effectively improve the yield of crops, and help the high-quality development of modern agriculture.

[0067] It should be noted that the above examples are only used to illustrate the technical solutions described in the present application and are not intended to limit the present application; therefore, although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the present application can still be modified or replaced equivalently; and all technical solutions and improvements that do not deviate from the spirit and scope of the present application should be covered within the scope of the claims of the present application.

Claims

1. A method for in-situ monitoring of crop growth status under environmental stress, characterized in that, Includes the following steps: (1) Construction of the sensing interface: Process 1: Preparation of Cu@PyCA; First, copper nitrate and 1H-pyrazole-4-carboxaldehyde were dissolved in a mixture of N,N-dimethylformamide, water, and ethanol to obtain a mixed solution. Then, the mixed solution was transferred to a reaction vessel for incubation, and yellow single crystals were obtained after incubation. Finally, the obtained yellow single crystals were washed several times with water and acetone, and then dried under vacuum to obtain Cu@PyCA. Cu@PyCA was dispersed in water to obtain Cu@PyCA dispersion. Step 2: Disperse reduced graphene in water to obtain an rGO solution; then, mix the Cu@PyCA dispersion prepared in Step 1 with the rGO solution to obtain a Cu@PyCA / rGO solution. (2) Construction of electrochemical sensors: Process 1: Mix 4-aminoindole, water and PEDOT:PSS to obtain mixed solution A; Step 2: Dissolve ammonium sulfate in water to obtain an ammonium sulfate aqueous solution; Step 3: Take mixed solution A into the three-electrode region of the screen-printed electrode, then add ammonium sulfate aqueous solution to mixed solution A in the three-electrode region, mix and react thoroughly, then immerse the screen-printed electrode in PBS solution for washing, and dry it at room temperature to obtain the 4-Amin / PEDOT:PSS electrode. Step 4: Take the Cu@PyCA / rGO solution prepared in step (1) and drop it onto the 4-Amin / PEDOT:PSS electrode. After drying, the electrochemical sensor is obtained. (3) Construction of a portable detection device: The portable detection device consists of a power supply, a Bluetooth module, a microcontroller, a potentiostat, an electrochemical sensor, an IV converter, a low-pass filter, and a smartphone. The smartphone is electrically connected to the microcontroller via the Bluetooth module to achieve signal transmission and control. Simultaneously, the microcontroller is electrically connected to the low-pass filter and the potentiostat, and the low-pass filter is electrically connected to the IV converter. The electrochemical sensor is electrically connected to the IV converter and the potentiostat. (4) In-situ monitoring of hydrogen peroxide: Process 1: Connect the smartphone to the Bluetooth module and send a detection command to the microcontroller. After receiving the detection command, the microcontroller applies a constant voltage to the electrochemical sensor through a potentiostat. The electrochemical sensor generates a signal when stimulated. The voltage signal is converted into a current signal by an IV converter and processed by a low-pass filter before being sent back to the microcontroller. The microcontroller then sends the analyzed and processed data to the smartphone via the Bluetooth module. Step 2: Observe the current curve on the smartphone. After the current stabilizes, add hydrogen peroxide standard solution to the electrochemical sensor at regular intervals to obtain a series of current step change curves for hydrogen peroxide standard solutions of different concentrations. Establish a linear regression equation between the hydrogen peroxide concentration and the corresponding response current. The regression equation is y = ax + b, where x represents the concentration of hydrogen peroxide, y represents the response current value, and a and b are constants. Step 3: Make a cut on the plant surface to form a wound, place the electrochemical sensor on the wound, and obtain the current value according to the operation of Step (4) Step 1. Substitute the obtained current value into the linear regression equation of Step (4) Step 2 to realize the detection of hydrogen peroxide concentration.

2. The method for in-situ monitoring of crop growth status under environmental stress according to claim 1, characterized in that, In step (1), the amounts of copper nitrate, 1H-pyrazole-4-carboxaldehyde, N,N-dimethylformamide, water and ethanol are 0.5-1 mmol: 1-1.5 mmol: 5-10 mL: 5-10 mL: 5-10 mL; wherein the volume ratio of N,N-dimethylformamide, water and ethanol is 1:0.7:

1.

3. The method for in-situ monitoring of crop growth status under environmental stress according to claim 1, characterized in that, In step (1), the incubation temperature is 100℃ and the incubation time is 12h; the washing is performed 3-5 times; the vacuum drying temperature is 100-120℃ and the vacuum drying time is 10-16h; the concentration of the Cu@PyCA dispersion is 4mg / mL.

4. The method for in-situ monitoring of crop growth status under environmental stress according to claim 1, characterized in that, In step (1), the concentration of the rGO solution is 1 mg / mL; the volume ratio of the Cu@PyCA dispersion and the rGO solution is 1:

1.

5. The method for in-situ monitoring of crop growth status under environmental stress according to claim 1, characterized in that, In step (2), the ratio of 4-Amin, water and PEDOT:PSS is 15-20 mg: 100-150 μL: 50-100 μL.

6. The method for in-situ monitoring of crop growth status under environmental stress according to claim 1, characterized in that, In step (2), the concentration of the ammonium sulfate aqueous solution is 0.25-0.3 g / mL.

7. The method for in-situ monitoring of crop growth status under environmental stress according to claim 1, characterized in that, In step (2), the volume ratio of the mixed solution A to the ammonium sulfate aqueous solution is 2:1, the reaction time is 20-40 min, the concentration of the PBS solution is 0.1 M, and the pH is 7.

8. The method for in-situ monitoring of crop growth status under environmental stress according to claim 1, characterized in that, In step (2), the volume of Cu@PyCA / rGO solution added is 8-12 μL.

9. The method for in-situ monitoring of crop growth status under environmental stress according to claim 1, characterized in that, In step (3), the power supply is ZMV2405D, the Bluetooth is BG24, and the microcontroller is STC89C52.

10. The method for in-situ monitoring of crop growth status under environmental stress according to claim 1, characterized in that, In step (4), a constant voltage of -0.3V is applied; the current stabilizes for 400-600s, with intervals of 50-100s; the concentration of the hydrogen peroxide standard solution is 1-10μM, and the amount added is 6-10μL; the length of the wound is 2-5mm.

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