Flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants, preparation method thereof and detection method thereof

Abstract

The present invention relates to the technical field of γ-aminobutyric acid detection, and provides a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants, a manufacturing method thereof, and a detection method. The flexible molecularly imprinted sensor includes: a flexible substrate and a graphene electrode unit, and the graphene electrode unit is disposed on the flexible substrate; the graphene electrode unit includes a guide rail, a working electrode, a counter electrode, and a reference electrode. The working electrode sequentially has a first modification layer, a second modification layer, and a third modification layer from the inside to the outside. The first modification layer includes AuNPs material, the second modification layer includes Fc-Ni3(HITP)2-ZnFe-LDH material, and the third modification layer includes MIP material. The flexible molecularly imprinted sensor adheres to the surface of the plant leaf and can still maintain stable electrochemical performance under deformation conditions, has high sensitivity and high specificity, and can perform in-vivo and in-situ detection of the γ-aminobutyric acid content in the plant body on the surface of the plant leaf.

Description

Technical Field

[0001] The present invention relates to the technical field of γ-aminobutyric acid detection, and particularly to a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants, a manufacturing method thereof, and a detection method thereof. Background Art

[0002] Gamma-aminobutyric acid (GABA) is a non-protein amino acid that widely exists in various plants, including soybeans, potatoes, and lettuce. GABA promotes photosynthesis, vegetative growth, and reproductive growth of plants, thereby enhancing the resistance of plants to different stresses. In addition, GABA also plays an important role in the nitrogen metabolism of plants, participates in regulating signal transduction between cells, helps plants better utilize nitrogen resources, and promotes plant growth and development. Therefore, detecting the GABA content in plants is of great significance for crop breeding and nutritional value assessment.

[0003] Currently, the detection methods of GABA mainly include colorimetry, high-performance liquid chromatography, gas chromatography, etc. However, these methods have relatively high requirements for equipment and the technical level of operators, and it is difficult to achieve rapid and convenient detection. In addition, these methods are all in-vitro detections, and the collection process may cause great damage to plants, resulting in the loss of some important biological information and making it difficult to meet the real-time analysis requirements. At the same time, traditional rigid electrodes (such as glassy carbon electrodes, platinum electrodes, and gold electrodes) are difficult to adapt to the soft and curved surface of plants during plant monitoring, which affects the accuracy and stability of detection. In a dynamic environment (such as a plant leaf blown by the wind), rigid electrodes cannot maintain stable contact, easily cause mechanical damage or compression to the plant surface, thus limiting their application in continuous monitoring. In contrast, the substrate material of flexible electrodes is soft, which can better conform to the surface of plant leaves, making the measurement more accurate and reducing damage to plant tissues. Therefore, how to design a flexible sensor that can detect GABA in plants in-situ and in-vivo has become a technical problem urgently to be solved in this field. Summary of the Invention

[0004] The present invention provides a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants, a manufacturing method thereof, and a detection method thereof, so as to solve the problem that γ-GABA is difficult to detect rapidly and conveniently in the prior art.

[0005] In a first aspect, the present invention provides a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants, comprising: a flexible substrate and a graphene electrode unit, and the graphene electrode unit is disposed on the flexible substrate;

[0006] The graphene electrode unit includes a guide rail, a working electrode, a counter electrode, and a reference electrode. The counter electrode and the reference electrode are respectively located on both sides of the working electrode. The working electrode is sequentially provided with a first modification layer, a second modification layer, and a third modification layer from the inside to the outside. The first modification layer includes AuNPs material, the second modification layer includes Fc-Ni3(HITP)2-ZnFe-LDH material, and the third modification layer includes MIP material.

[0007] According to a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants provided by the present invention, an Ag / AgCl ink layer is provided on the reference electrode to form an Ag / AgCl reference electrode.

[0008] According to a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants provided by the present invention, a copolyester insulating layer is provided in the area where the guide rail is located.

[0009] According to a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants provided by the present invention, the flexible substrate is a copolyester flexible substrate.

[0010] In a second aspect, the present invention provides a method for fabricating a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants, including:

[0011] Using a computer-controlled laser direct writer to pattern the PI tape, and obtaining a graphene electrode unit body on the PI tape through induction;

[0012] Transferring the graphene electrode unit body on the PI tape to the flexible substrate;

[0013] Coating a set amount of Ag / AgCl ink on the exposed area of the reference electrode and heating and curing it to obtain an Ag / AgCl reference electrode;

[0014] Preparing a copolyester insulating layer in the area where the guide rail is located;

[0015] Modifying the working electrode to obtain a MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode.

[0016] According to a method for fabricating a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants provided by the present invention, the transferring the graphene electrode unit body on the PI tape to the flexible substrate includes:

[0017] Spin-coat the Ecoflex mixture with a set ratio onto the graphene electrode unit body located on the PI tape, and then perform vacuum drying and heat treatment.

[0018] Peel the graphene electrode unit body from the PI tape to obtain the graphene electrode unit located on the flexible substrate.

[0019] According to the present invention, a method for fabricating a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants is provided. Modifying the working electrode to obtain an MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode includes:

[0020] Immerse the electrode in a 0.5 - 2.5 mg / ml (preferably 1 mg / ml) HAuCl4 solution, and obtain AuNPs by chronoamperometry.

[0021] Dissolve Fc, Ni3(HITP)2, and ZnFe-LDH in a chitosan solution, and coat 1 - 5 μL (preferably 4 μL) of the Fc-Ni3(HITP)2-ZnFe-LDH solution on the working electrode to obtain Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex.

[0022] Prepare an MIP solution using β-cyclodextrin as the functional monomer and γ-aminobutyric acid as the template molecule.

[0023] Immerse the electrode in the MIP solution, and electro-polymerize to form MIP by cyclic voltammetry. Then, place the electrode in a 10 - 60 mM (preferably 50 mM) NaOH solution to wash off the template molecule, and finally obtain the eluted MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode.

[0024] According to the present invention, a method for fabricating a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants is provided. The conditions for electro-polymerization by cyclic voltammetry include: the voltage is -0.4 V - 1.0 V, the number of electro-polymerization cycles is 30 - 60 cycles (preferably 50 cycles), and the scanning rate is 25 - 120 mV / s (preferably 100 mV / s).

[0025] In a third aspect, the present invention provides a detection method based on a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants, including:

[0026] Punch holes at set positions on the surface of the plant leaf to be measured.

[0027] Attach the flexible molecularly imprinted sensor to the lower surface of the plant leaf to be measured, and align the working electrode with the punching position.

[0028] Drop the buffer solution at the punching position, then connect the electrochemical workstation, and detect the concentration of γ-aminobutyric acid by differential pulse voltammetry.

[0029] The flexible molecularly imprinted sensor provided by the present invention for in-situ and in-vivo detection of γ-aminobutyric acid content in plants, its manufacturing method and detection method. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] By integrating the graphene electrode unit on the flexible substrate, it has better conformability compared with the traditional rigid electrochemical sensor. This flexible molecularly imprinted sensor can closely adhere to the surface of the plant leaf and still maintain stable electrochemical performance under deformation. By sequentially arranging the first modification layer (AuNPs material), the second modification layer (Fc-Ni3(HITP)2-ZnFe-LDH material) and the third modification layer (MIP material) from the inside to the outside at the detection end of the working electrode, it has high sensitivity and high specificity, and can realize in-situ and in-vivo detection of γ-aminobutyric acid in plants. In this way, it not only has high sensitivity, a wider detection range, a simple process, but also strong adaptability and can work in a variety of environments. Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is a schematic diagram of the flexible molecularly imprinted sensor provided by the embodiment of the present invention for in-situ and in-vivo detection of γ-aminobutyric acid content in plants.

[0033] Figure 2 It is a flow schematic diagram of the preparation method of the flexible molecularly imprinted sensor provided by the embodiment of the present invention for in-situ and in-vivo detection of γ-aminobutyric acid content in plants.

[0034] Figure 3 It is a process diagram of the preparation of the LIG / Ecoflex part of the flexible molecularly imprinted sensor provided by the embodiment of the present invention.

[0035] Figure 4 It is a schematic diagram of the modification of the working electrode in the preparation method of the flexible molecularly imprinted sensor provided by the embodiment of the present invention.

[0036] Figure 5 It is a schematic diagram of the detection principle of the flexible molecularly imprinted sensor provided by the embodiment of the present invention.

[0037] Figure 6 It is a comparison diagram of the flexible molecularly imprinted sensor provided by the comparative example of the present invention.

[0038] Figure 7 It is a schematic flow diagram of the detection method of the flexible molecularly imprinted sensor for in-situ and in-vivo detection of the γ-aminobutyric acid content in plants provided by the embodiment of the present invention.

[0039] Reference numerals:

[0040] 10. Flexible substrate; 20. Graphene electrode unit; 210. Working electrode; 220. Counter electrode; 230. Reference electrode; 30. Guide rail. Detailed implementation manners

[0041] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0042] To solve the above problems, as Figure 1 shown, the flexible molecularly imprinted sensor for in-situ and in-vivo detection of the γ-aminobutyric acid content in plants according to the embodiment of the present invention includes: a flexible substrate 10 and a graphene electrode unit 20, and the graphene electrode unit 20 is disposed on the flexible substrate 10. Among them, the graphene electrode unit 20 includes a working electrode 210, a counter electrode 220, a reference electrode 230 and a guide rail 30. The counter electrode 220 and the reference electrode 230 are respectively located on both sides of the working electrode 210. The working electrode is sequentially provided with a first modification layer, a second modification layer and a third modification layer from the inside to the outside. The first modification layer includes AuNPs material, the second modification layer includes Fc-Ni3(HITP)2-ZnFe-LDH material, and the third modification layer includes MIP material.

[0043] The flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants according to the embodiments of the present invention integrates the graphene electrode unit 20 on the flexible substrate 10, and has better conformability compared with traditional rigid electrochemical sensors. This flexible molecularly imprinted sensor can closely adhere to the surface of plant leaves and still maintain stable electrochemical performance under deformed conditions. By sequentially arranging a first modification layer (AuNPs material), a second modification layer (Fc-Ni3(HITP)2-ZnFe-LDH material), and a third modification layer (MIP material) on the working electrode 210, it has high sensitivity and high specificity. In this way, not only is the detection range larger and the detection limit lower, but also it has strong adaptability and can work in a variety of environments, including field applications, etc. In addition, by combining with the application scenarios of smart agriculture, the flexible molecularly imprinted sensor can also support unmanned and intelligent real-time dynamic monitoring, providing an efficient and convenient solution for modern agriculture.

[0044] Specifically, the flexible substrate 10 is a copolyester flexible substrate (Ecoflex). The copolyester flexible substrate 10 has excellent flexibility and deformability, can remain stable under bending or stretching conditions, and is suitable for closely adhering to the plant surface. In addition, it has high mechanical strength and durability, can withstand external forces without damage, and at the same time has good chemical stability, can resist environmental influences such as acid-base corrosion and oxidation.

[0045] In some embodiments, the graphene electrode unit 20 includes a working electrode 210, a counter electrode 220, a reference electrode 230, and a guide rail 30. Among them, the working electrode 210 is the main electrode for electrochemical reactions. The third modification layer of this working electrode is a molecularly imprinted membrane, and the principle of the molecular imprinting method is used to realize the recognition and detection of GABA.

[0046] In specific implementation, the working electrode 210, the counter electrode 220, and the reference electrode 230 are usually connected to an external electrochemical workstation or a potential control system through wires, and precise control of voltage and current is required during operation. The external device provides current through the counter electrode 220, measures the potential difference through the reference electrode 230, and then an electrochemical reaction occurs between the working electrode 210 and the substances in the solution.

[0047] According to some embodiments of the present invention, an Ag / AgCl ink layer is provided on the reference electrode 230 to form the Ag / AgCl reference electrode 230.

[0048] By providing an Ag / AgCl ink layer on the reference electrode 230 and forming the Ag / AgCl reference electrode 230, it provides a stable potential, can maintain consistency for a long time, and ensures the reliability and high repeatability of the electrochemical experimental results.

[0049] SeeFigure 1 As shown, according to some embodiments of the present invention, a copolyester insulating layer is provided in the area where the guide rail 30 is located.

[0050] By providing a copolyester insulating layer in the area where the guide rail 30 is located, the insulating of the guide rail area of the graphene electrode unit 20 can be achieved.

[0051] The following describes the fabrication and modification process of the working electrode 210.

[0052] First, gold nanoparticles (AuNPs) are deposited in a chloroauric acid solution (HAuCl4) using chronoamperometry (i-t). Then, a mixture of ferrocene (Fc), metal-organic framework Ni3(HITP)2, and zinc-iron layered double hydroxide (ZnFe-LDH) is further modified. These nanomaterials can form a network structure with a large specific surface area and have excellent conductivity to amplify the electrical signal. Then, γ-aminobutyric acid (GABA) is used as the template molecule, and β-cyclodextrin (β-CD) is used as the functional monomer, and a molecularly imprinted polymer (MIP) is prepared using phosphate buffer solution (PBS). Then, after polymerizing the MIP film on the electrode surface by cyclic voltammetry (CV), the electrode is placed in an NaOH solution to wash off the GABA template molecule, obtaining MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex.

[0053] Holes with the same shape, structure, and size as GABA are formed on the surface of the MIP film. Using these holes, GABA can be specifically recognized and bound. After binding GABA, it will cause a decrease in the signal of the electroactive molecule Fc, and the decreased current signal is related to the concentration of GABA. Then, a standard curve for GABA detection is prepared. Finally, holes are punched on the leaf surface, and the prepared flexible molecularly imprinted sensor is attached to the lower surface of the plant leaf, and an appropriate amount of PBS is added dropwise so that the juice in the leaf can be released onto the working electrode. The real-time response signal of GABA is recorded by differential pulse voltammetry (DPV). This flexible molecularly imprinted sensor can specifically bind to GABA. After binding, the oxidation peak current of the electrode decreases within the potential range of the electroactive small molecule Fc, thereby realizing the highly sensitive detection of GABA. In addition, this method is used for GABA detection for the first time.

[0054] The following describes the preparation method of the flexible molecularly imprinted sensor provided by the present invention. The preparation method of the flexible molecularly imprinted sensor described below can be correspondingly referred to each other with the flexible molecularly imprinted sensor for detecting the content of GABA described above.

[0055] See Figure 2 、 Figure 3 、 Figure 4 and Figure 5As shown, the preparation method of the flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants provided by the embodiments of the present invention is used to prepare the flexible molecularly imprinted sensor described in the above embodiments, and includes the following steps.

[0056] S210. Pattern the PI tape using a computer-controlled laser direct writing instrument, and obtain a graphene electrode unit body on the PI tape through induction.

[0057] S220. Transfer the graphene electrode unit body on the PI tape to the flexible substrate.

[0058] S230. Coat a set amount of Ag / AgCl ink on the exposed area of the reference electrode and heat-cure it to obtain an Ag / AgCl reference electrode.

[0059] S240. Prepare a copolyester insulating layer in the area where the guide rail is located.

[0060] S250. Modify the working electrode to obtain a MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode.

[0061] Specifically, in step S210, paste the PI tape on a polytetrafluoroethylene mold. Wash the PI tape with distilled water and ethanol in sequence. Then pattern the PI tape using a computer-controlled laser direct writing instrument, and obtain a graphene electrode unit body (LIG electrode) with good conductivity through induction.

[0062] It should be noted that the graphene electrode unit body at this time only includes the working electrode 210, the reference electrode 230, the guide rail 240, the counter electrode 220, and the guide rail 30, and does not include the encapsulation layer on the above electrodes, as well as the material layers (the first modification layer, the second modification layer, and the third modification layer) modified on the working electrode 210.

[0063] In step S220, mix and stir the Ecoflex main agent Ecoflex 00-20 and the curing agent Ecoflex 00-20 at a ratio of 15:1 to 20:1 (preferably 10:1) for 10 min using a spin coater, spin coat at a speed of 50 to 200 rpm (preferably 100 rpm) for 20 to 80 s (preferably 60 s) to uniformly cover the PI tape with the graphene electrode unit body, and then heat it in a vacuum drying oven at 60 to 100 °C (preferably 80 °C) for 1 to 5 h (preferably 4 h). Then peel the graphene electrode unit body (LIG) from the PI tape to obtain the graphene electrode unit 20 (LIG / Ecoflex electrode) on the flexible substrate 10.

[0064] That is, the graphene electrode unit 20 is prepared by laser printing on a flexible PI tape according to a designed pattern and then transferred to copolyester Ecoflex.

[0065] In step S230, a certain amount of Ag / AgCl ink is evenly coated on the exposed area of the reference electrode 230, and after heating and curing at 80 °C for 10 - 50 min (preferably 30 min), the Ag / AgCl reference electrode 230 can be obtained.

[0066] In step S240, a certain amount of Ecoflex is coated on the area where the guide rail 30 is located and heated and cured to obtain a copolyester insulating layer, so that the area where the guide rail 30 is located is insulated. Thus, the LIG / Ecoflex three-electrode is constructed.

[0067] Then, the electrode can be placed in a 0.1 M dilute sulfuric acid solution and cyclic voltammetry scanning in the range of 0 to 1.5 V can be carried out to clean and activate the electrode surface.

[0068] Example 1: In step S250, a HAuCl4 solution with a concentration of 0.5 - 2.5 mg / ml (preferably 1 mg / ml) is prepared with phosphate buffer solution (PBS). AuNPs are deposited in the HAuCl4 solution by the i-t method (-1 V, 800 s) to obtain AuNPs / LIG / Ecoflex. Then, a composite material of 5 - 25 g / L (preferably 10 g / L) Fc, 0.5 - 2 mg / mL (preferably 1 mg / mL) Ni3(HITP)2, and 0.5 - 2 mg / mL (preferably 1 mg / mL) ZnFe-LDH is prepared with a 0.2% chitosan solution. After ultrasonic dispersion until uniform, 1 - 5 μL (preferably 4 μL) of Fc-Ni3(HITP)2-ZnFe-LDH is drop-coated on the surface of the working electrode to obtain Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex. MIP (GABA:β-CD molar ratio is 5:1, GABA is 10 - 30 mM (preferably 25 mM), β-CD is 1 - 10 mM (preferably 5 mM)) is prepared with PBS and electro-polymerized by the CV method (-0.4 V - 1 V, 100 mV / s, 50 cycles) to form MIP, obtaining uneluted MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex. Then, the electrode is placed in a 10 - 60 mM (preferably 50 mM) NaOH solution for 1 - 10 min (preferably 5 min) to wash off GABA, and finally, the eluted MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex can be obtained.

[0069] According to some embodiments of the present invention, the method for preparing a flexible molecularly imprinted sensor further comprises the following steps.

[0070] Example 2: Prepare a γ-aminobutyric acid solution with a set concentration to perform DPV detection on the flexible molecularly imprinted sensor; based on the relationship curve between the logarithm of the γ-aminobutyric acid solution concentration and the current difference, plot the standard curve of the flexible molecularly imprinted sensor.

[0071] Specifically, prepare GABA solutions with concentrations of 0, 1 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM using PBS (pH = 7.2 - 7.5, preferably pH = 7.4), and use the prepared flexible molecularly imprinted sensor to perform DPV detection (potential -0.2 V - 0.6 V, interval time of 0.5 s, amplitude potential of 0.025 V). Take the peak current of the oxidation peak obtained in the blank solution as I0, and record the peak currents obtained from standard γ-aminobutyric acid solutions with different concentrations as I1, I2, I3... respectively. Calculate ΔI1, ΔI2, ΔI3... respectively through the formula ΔI = I0 - I1 (I2, I3...). Take the logarithm of the GABA concentration (lgC GABA ) and perform linear fitting with the ΔI obtained at different concentrations to plot the standard curve of the GABA flexible molecularly imprinted sensor. Among them, the linear equation is ΔI = 0.6862lgC GABA + 10.572 (nM), and the linear range can reach 1×10 -9 ~1×10 -2 M, and the detection limit is 5.79×10 -10 M.

[0072] Example 3: In addition, perform a recovery experiment on the flexible molecularly imprinted sensor. Taking lettuce leaves as an example, the specific process is as follows: Squeeze lettuce leaves into juice, take the supernatant after centrifugation, and according to the established calibration curve, obtain that the concentration of GABA in lettuce juice is approximately 1.13 ± 0.26 μM. Add a GABA standard sample based on this as the matrix and calculate the recovery rate. The experimental results are shown in Table 1, and the spiked recovery rate of GABA is 96.97% - 103.41%. The results are also compared with the results obtained by HPLC. The relative error between the results of the prepared sensor and those obtained by HPLC is 12.4%, which is less than 15%, so it is considered acceptable. It shows that the sensor can be used for the accurate determination of GABA in actual samples.

[0073] Table 1 Spiked recovery rate results (n = 3)

[0074]

[0075] Example 4: Refer to Figure 7 As shown, the detection method of the flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants according to the embodiment of the present invention includes: S710, punching holes at a set position on the surface of the plant leaf to be measured; S720, attaching the flexible molecularly imprinted sensor to the lower surface of the plant leaf to be measured, and making the working electrode face the punching position; S730, dropping a buffer solution at the punching position, then connecting an electrochemical workstation, and detecting the concentration of γ-aminobutyric acid by differential pulse voltammetry. In the specific implementation process, the plant is any plant containing γ-aminobutyric acid, including but not limited to fruits, vegetables, flowers, crops, etc.

[0076] A live experiment was carried out on the flexible molecularly imprinted sensor. Taking lettuce leaves as an example, the specific process is as follows: First, the prepared flexible molecularly imprinted sensor was tested for stability in blank PBS. Then, holes were punched in the lettuce leaves, and the flexible molecularly imprinted sensor was fixed on the lower surface of the leaves, making its working electrode face the small holes. 20 μL of PBS was dropped on the holes to allow the juice in the leaves to be released onto the working electrode, and then DPV detection was carried out. The obtained current signal was used to calculate the instantaneous concentration of the measured sample through the calibrated working curve.

[0077] Two different varieties of lettuce plants, "Dasusheng" and "Hongshanhu", in the same period were selected, and the prepared electrode (MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex) was used to detect their GABA content. The detection results are shown in Table 2. The experimental results show that the flexible electrode sensor can detect GABA in live plants.

[0078] Table 2 Comparison table of test results

[0079]

[0080] Comparative Example 1: This comparative example provides a flexible molecularly imprinted sensor for detecting GABA. The preparation method is only different from that in Example 1 ( Figure 6 in a) in that: Ni3(HITP)2 is replaced with an equal amount of Cu(INA)2 to prepare a flexible molecularly imprinted sensor b: MIP / Fc-Cu(INA)2-ZnFe-LDH / AuNPs / LIG / Ecoflex. The detection performance of the sensor was tested by the method in Example 2, and the results are as Figure 6 shown in b. It can be seen that the linear detection range of the flexible molecularly imprinted sensor prepared in Comparative Example 1 is 1×10 -8 -1×10 -4 M, and the detection limit is 7.74×10 -9M, the detection effect is inferior to that of Example 1.

[0081] Comparative Example 2: This comparative example provides a flexible molecularly imprinted sensor for detecting GABA. The only difference in the preparation method from Example 1 is that ZnFe-LDH is replaced with an equal amount of CoFe-LDH to obtain a flexible molecularly imprinted sensor c: MIP / Fc-Ni3(HITP)2-CoFe-LDH / AuNPs / LIG / Ecoflex. The detection performance of the sensor was tested using the method in Example 2, and the results are as Figure 6 shown in c. It can be seen that the linear detection range of the flexible molecularly imprinted sensor prepared in Comparative Example 1 is 1×10 -8 -1×10 -5 M, and the detection limit is 3.21×10 -9 M, and the detection effect is inferior to that of Example 1.

[0082] Comparative Example 3: This comparative example provides a flexible molecularly imprinted sensor for detecting GABA. The only difference in the preparation method from Example 1 is that AuNPs / LIG / Ecoflex is replaced with LIG / Ecoflex to obtain a flexible molecularly imprinted sensor d: MIP / Fc-Ni3(HITP)2-ZnFe-LDH / LIG / Ecoflex. The detection performance of the sensor was tested using the method in Example 2, and the results are as Figure 6 shown in d. It can be seen that the linear detection range of the flexible molecularly imprinted sensor prepared in Comparative Example 1 is 1×10 -6 -1×10 -2 M, and the detection limit is 6.67×10 -7 M, and the detection effect is inferior to that of Example 1.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants, characterized in that, Comprising: A flexible substrate and a graphene electrode unit, the graphene electrode unit being disposed on the flexible substrate; The graphene electrode unit includes a guide rail, a working electrode, a counter electrode, and a reference electrode. The counter electrode and the reference electrode are respectively located on both sides of the working electrode. The working electrode is sequentially provided with a first modification layer, a second modification layer, and a third modification layer from the inside to the outside. The first modification layer includes AuNPs material, the second modification layer includes Fc-Ni3(HITP)2-ZnFe-LDH material, and the third modification layer includes MIP material.

2. The flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants according to claim 1, characterized in that, An Ag / AgCl ink layer is provided on the reference electrode to form an Ag / AgCl reference electrode.

3. The flexible molecularly imprinted sensor for in-situ and in-vivo detection of the γ-aminobutyric acid content in plants according to claim 1, wherein, A copolyester insulating layer is provided in the area where the guide rail is located.

4. The flexible molecularly imprinted sensor for in-situ and in-vivo detection of the γ-aminobutyric acid content in plants according to claim 1, characterized in that, The flexible substrate is a copolyester flexible substrate.

5. A method for fabricating a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants according to any one of claims 1 to 4, characterized in that, Comprising: Using a computer-controlled laser direct writer to pattern the PI tape, and obtaining a graphene electrode unit body on the PI tape through induction; Transferring the graphene electrode unit body on the PI tape to the flexible substrate; Coating a set amount of Ag / AgCl ink on the exposed area of the reference electrode and heating and curing it to obtain an Ag / AgCl reference electrode; Preparing a copolyester insulating layer in the area where the guide rail is located; Modifying the working electrode to obtain a MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode.

6. The method for fabricating a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants according to claim 5, characterized in that, The transferring the graphene electrode unit body on the PI tape to the flexible substrate includes: Coating a set ratio of Ecoflex mixture solution on the graphene electrode unit body located on the PI tape through a spin coater, and then performing vacuum drying and heat treatment; Peeling the graphene electrode unit body from the PI tape to obtain the graphene electrode unit located on the flexible substrate.

7. The manufacturing method of the flexible molecularly imprinted sensor for in-situ and in-vivo detection of the γ-aminobutyric acid content in plants according to claim 5, characterized in that, The modifying the working electrode to obtain a MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode includes: Immersing the electrode in a 0.5 - 2.5 mg / ml HAuCl4 solution, and obtaining AuNPs through chronoamperometry; Dissolving Fc, Ni3(HITP)2, and ZnFe-LDH in a chitosan solution, and coating 1 - 5 μL of Fc-Ni3(HITP)2-ZnFe-LDH solution on the working electrode to obtain Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex; Preparing a MIP solution using β-cyclodextrin as a functional monomer and γ-aminobutyric acid as a template molecule; Immersing the electrode in the MIP solution, and electro-polymerizing to form MIP through cyclic voltammetry. Then, placing the electrode in a NaOH solution to wash off the template molecule, and finally obtaining the eluted MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode.

8. The method for fabricating a flexible molecularly imprinted sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants according to claim 7, wherein The conditions for cyclic voltammetry electro-polymerization include: the voltage is -0.4V - 1.0V, the number of electro-polymerization cycles is 30 - 60 cycles, and the scanning rate is 25 - 120 mV / s.

9. A detection method for a flexible molecularly imprinted sensor for in-situ and in-vivo detection of the content of γ-aminobutyric acid in plants as described in any one of claims 1 to 4, characterized in that, including: Punch holes at a set position on the surface of the plant leaf to be measured; Attach the flexible molecularly imprinted sensor to the lower surface of the plant leaf to be measured, and make the working electrode face the punching position; Drop a buffer solution at the punching position, then connect an electrochemical workstation, and detect the concentration of γ-aminobutyric acid by differential pulse voltammetry.

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