Flexible molecular imprinting sensor for in-situ and in-vivo detection of content of gamma-aminobutyric acid in plant and manufacturing method and detection method of flexible molecular imprinting sensor

Abstract

The invention relates to the technical field of gamma-aminobutyric acid detection, and provides a flexible molecular imprinting sensor for in-situ and in-vivo detection of the content of gamma-aminobutyric acid in plants and a manufacturing method and a detection method of the flexible molecular imprinting sensor, the flexible molecular imprinting sensor comprises a flexible substrate and a graphene electrode unit, and the graphene electrode unit is arranged on the flexible substrate; the graphene electrode unit comprises a guide rail, a working electrode, a counter electrode and a reference electrode, the working electrode sequentially comprises a first modification layer, a second modification layer and a third modification layer from inside to outside, the first modification layer comprises an AuNPs material, the second modification layer comprises an Fc-Ni3 (HITP) 2-ZnFe-LDH material, and the third modification layer comprises an MIP material. The flexible molecular imprinting sensor is attached to the surface of a plant leaf, can still keep stable electrochemical performance under the condition of deformation, has high sensitivity and high specificity, and can perform in-vivo and in-situ detection on the content of gamma-aminobutyric acid in a plant body on the surface of the plant leaf.

Description

Technical Field

[0001] The present invention relates to the technical field of gamma-aminobutyric acid detection, and in particular to a flexible molecular imprinting sensor for in-situ and living detection of gamma-aminobutyric acid content in plants, and a manufacturing method and a detection method thereof. Background Art

[0002] Gamma-aminobutyric acid (GABA) is a non-protein amino acid that is widely found in many plants, including soybeans, potatoes, and lettuce. GABA promotes photosynthesis and plant vegetative and reproductive growth, thereby improving the plant's resistance to different adversities. In addition, GABA also plays an important role in plant nitrogen metabolism, participates in regulating intercellular signaling, 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] At present, the detection methods of GABA mainly include colorimetry, high performance liquid chromatography, gas chromatography, etc. However, these methods have high requirements on the technical level of equipment and operators, and it is difficult to achieve rapid and convenient detection. In addition, these methods are all in vitro detection, which may cause great damage to the plant during the collection process, resulting in the loss of some important biological information, and it is difficult to meet the needs of real-time analysis. 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 in plant monitoring, which affects the accuracy and stability of detection. In a dynamic environment (such as wind-blown plant leaves), rigid electrodes cannot maintain stable contact and are prone to mechanical damage or compression to the plant surface, thereby limiting their application in continuous monitoring. In contrast, the base material of the flexible electrode is soft and can better fit the surface of the plant leaves, making the measurement more accurate while 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 that needs to be solved in this field. Summary of the invention

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

[0005] In a first aspect, the present invention provides a flexible molecular imprinting sensor for in-situ and in vivo detection of γ-aminobutyric acid content in plants, comprising: a flexible substrate and a graphene electrode unit, wherein the graphene electrode unit is arranged on the flexible substrate; The graphene electrode unit includes a guide rail, a working electrode, a counter electrode and a reference electrode, wherein the counter electrode and the reference electrode are respectively located on both sides of the working electrode, and the working electrode is provided with a first modification layer, a second modification layer and a third modification layer from the inside to the outside, wherein 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.

[0006] According to the flexible molecular imprinting sensor for in-situ and living 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.

[0007] According to the flexible molecular imprinting sensor for in-situ and living detection of the content of γ-aminobutyric acid in plants provided by the present invention, a copolyester insulating layer is provided in the area where the guide rail is located.

[0008] According to the flexible molecular imprinting sensor for in-situ and living detection of the content of gamma-aminobutyric acid in plants provided by the present invention, the flexible substrate is a copolyester flexible substrate.

[0009] In a second aspect, the present invention provides a method for preparing a flexible molecular imprinting sensor for in situ and in vivo detection of γ-aminobutyric acid content in plants, comprising: The PI tape is patterned by using a computer-controlled laser direct writer, and a graphene electrode unit body is obtained on the PI tape by 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 heat-curing the ink to obtain an Ag / AgCl reference electrode; Prepare a copolyester insulation layer in the area where the guide rail is located; The working electrode was modified to obtain a MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode.

[0010] According to the present invention, a method for manufacturing a flexible molecular imprinting sensor for in-situ and living detection of γ-aminobutyric acid content in plants is provided, wherein the graphene electrode unit body on the PI tape is transferred to the flexible substrate, comprising: Applying the Ecoflex mixed solution of a set ratio to the graphene electrode unit body on the PI tape by a spin coater, and then performing a vacuum drying and heating treatment; The graphene electrode unit body is peeled off from the PI tape to obtain the graphene electrode unit located on the flexible substrate.

[0011] According to the present invention, a method for preparing a flexible molecular imprinting sensor for in-situ and in vivo detection of γ-aminobutyric acid content in plants is provided, wherein the working electrode is modified to obtain a MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode, comprising: The electrode is immersed in a 0.5-2.5 mg / ml (preferably 1 mg / ml) HAuCl4 solution, and AuNPs are obtained by chronoamperometry; Dissolve Fc, Ni3(HITP)2 and ZnFe-LDH in a chitosan solution, and coat the working electrode with 1-5 μL (preferably 4 μL) of the Fc-Ni3(HITP)2-ZnFe-LDH solution to obtain Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex; MIP solution was prepared using β-cyclodextrin as the functional monomer and γ-aminobutyric acid as the template molecule; The electrode is immersed in a MIP solution and electropolymerized by cyclic voltammetry to form a MIP. The electrode is then placed in a 10-60 mM (preferably 50 mM) NaOH solution to wash off the template molecules, and finally the washed MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode is obtained.

[0012] According to the present invention, a method for preparing a flexible molecular imprinting sensor for in-situ and living detection of γ-aminobutyric acid content in plants is provided, wherein the conditions for cyclic voltammetry electropolymerization include: a voltage of -0.4 V to 1.0 V, 30 to 60 electropolymerization cycles (preferably 50 cycles), and a scanning speed of 25 to 120 mV / s (preferably 100 mV / s).

[0013] In a third aspect, the present invention provides a detection method based on a flexible molecular imprinting sensor for in situ and in vivo detection of γ-aminobutyric acid content in plants, comprising: Punching holes at set positions on the surface of the plant leaf to be tested; The flexible molecular imprinting sensor is attached to the lower surface of the plant leaf to be tested, and the working electrode is aligned with the punching position; Buffer solution was added to the punched position, and then the electrochemical workstation was connected to detect the concentration of γ-aminobutyric acid by differential pulse voltammetry.

[0014] Compared with the prior art, the flexible molecular imprinting sensor for in-situ and in vivo detection of γ-aminobutyric acid content in plants provided by the present invention and its preparation method and detection method have the following beneficial effects: By integrating the graphene electrode unit on a flexible substrate, it has better fit than traditional rigid electrochemical sensors. The flexible molecular imprinting sensor can fit tightly to the surface of plant leaves and 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 of the detection end of the working electrode, it has high sensitivity and high specificity, and can realize in situ and living detection of γ-aminobutyric acid in plants. In this way, it not only has high sensitivity, a wider detection range, simple process, and strong adaptability, but also can work in a variety of environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 It is a schematic diagram of a flexible molecular imprinting sensor for in-situ and living detection of γ-aminobutyric acid content in plants provided by an embodiment of the present invention.

[0017] Figure 2 It is a schematic flow chart of a method for preparing a flexible molecular imprinting sensor for in-situ and living detection of γ-aminobutyric acid content in plants provided by an embodiment of the present invention.

[0018] Figure 3 1 is a diagram of the preparation process of the flexible molecular imprinting sensor LIG / Ecoflex provided in an embodiment of the present invention.

[0019] Figure 4 It is a schematic diagram of modification of the working electrode in the method for preparing a flexible molecular imprinting sensor provided in an embodiment of the present invention.

[0020] Figure 5 It is a schematic diagram of the detection principle of the flexible molecular imprinting sensor provided by an embodiment of the present invention.

[0021] Figure 6 It is a comparison diagram of the flexible molecular imprinting sensor provided in the comparative example of the present invention.

[0022] Figure 7It is a flow chart of a detection method of a flexible molecular imprinting sensor for in-situ and living detection of γ-aminobutyric acid content in plants provided by an embodiment of the present invention.

[0023] Reference numerals: 10. Flexible substrate; 20. Graphene electrode unit; 210. Working electrode; 220. Counter electrode; 230. Reference electrode; 30. Guide rail. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In order to solve the above problems, Figure 1 As shown, the flexible molecular imprinting sensor for in-situ and living detection of γ-aminobutyric acid content in plants according to an embodiment of the present invention comprises: a flexible substrate 10 and a graphene electrode unit 20, wherein the graphene electrode unit 20 is arranged on the flexible substrate 10. The graphene electrode unit 20 comprises a working electrode 210, a counter electrode 220, a reference electrode 230 and a guide rail 30, wherein the counter electrode 220 and the reference electrode 230 are respectively located on both sides of the working electrode 210, and the working electrode is provided with a first modification layer, a second modification layer and a third modification layer in sequence from the inside to the outside, wherein the first modification layer comprises AuNPs material, the second modification layer comprises Fc-Ni3(HITP)2-ZnFe-LDH material, and the third modification layer comprises MIP material.

[0026] The flexible molecular imprinting sensor for in-situ and live detection of γ-aminobutyric acid content in plants according to the embodiment of the present invention has better fit than traditional rigid electrochemical sensors by integrating the graphene electrode unit 20 on the flexible substrate 10. The flexible molecular imprinting sensor can fit closely to the surface of plant leaves and 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) on the working electrode 210 from the inside to the outside, it has high sensitivity and high specificity. In this way, not only the detection range is larger, the detection limit is lower, but also the adaptability is strong, and it can work in a variety of environments, including field applications. In addition, the flexible molecular imprinting sensor can also support unmanned and intelligent real-time dynamic monitoring by combining with the application scenarios of smart agriculture, providing an efficient and convenient solution for modern agriculture.

[0027] Specifically, the flexible substrate 10 is a copolyester flexible substrate (Ecoflex), which has excellent flexibility and deformability, can remain stable when bent or stretched, and is suitable for close contact with the surface of plants. In addition, it has high mechanical strength and durability, can withstand external forces without damage, and has good chemical stability, and can resist environmental influences such as acid and alkali corrosion and oxidation.

[0028] 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. The working electrode 210 is the main electrode for electrochemical reaction, and the third modified layer of the working electrode is a molecular imprinting membrane, which uses the principle of molecular imprinting method to realize the recognition and detection of GABA.

[0029] 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 the voltage and current need to be precisely controlled during operation. The external device provides current through the counter electrode 220, measures the potential difference through the reference electrode 230, and then generates an electrochemical reaction with the substance in the solution through the working electrode 210.

[0030] According to some embodiments of the present invention, an Ag / AgCl ink layer is disposed on the reference electrode 230 to form an Ag / AgCl reference electrode 230 .

[0031] By disposing an Ag / AgCl ink layer on the reference electrode 230 and forming the Ag / AgCl reference electrode 230 , a stable potential is provided and can be kept consistent for a long time, thereby ensuring the reliability and high repeatability of the electrochemical experimental results.

[0032] See also Figure 1 As shown, according to some embodiments of the present invention, a copolyester insulation layer is provided in the area where the guide rail 30 is located.

[0033] By providing a copolyester insulating layer in the area where the guide rail 30 is located, the guide rail area of ​​the graphene electrode unit 20 can be insulated.

[0034] The following is an explanation of the manufacturing and modification process of the working electrode 210.

[0035] First, gold nanoparticles (AuNPs) were deposited in hypochloroauric acid solution (HAuCl4) using chronoamperometry (IT), and then a mixture of ferrocene (Fc), metal organic framework Ni3(HITP)2 and zinc-iron layered double hydroxide (ZnFe-LDH) was further modified. These nanomaterials can form a network structure with a large specific surface area and excellent conductivity, amplifying electrical signals. Then, γ-aminobutyric acid (GABA) was used as a template molecule, β-cyclodextrin (β-CD) was used as a functional monomer, and MIP was prepared using phosphate buffer solution (PBS). Then, after polymerizing the molecular imprinting polymer (MIP) film on the electrode surface by cyclic voltammetry (CV), the electrode was placed in a NaOH solution to wash off the GABA template molecule, and MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex was obtained.

[0036] The surface of the MIP membrane forms holes that are consistent with the shape, structure, and size of GABA. These holes can be used to specifically identify and bind to GABA. After binding to GABA, the signal of the electroactive molecule Fc will be reduced, and the reduced current signal is related to the concentration of GABA. Then a standard curve for GABA detection is prepared. Finally, holes are punched on the surface of the leaf, the prepared flexible molecular imprinting sensor is attached to the lower surface of the plant leaf, and an appropriate amount of PBS is added so that the juice in the leaf can be released to the working electrode. The real-time response signal of GABA is recorded by DPV. The flexible molecular imprinting sensor can specifically bind to GABA. After binding, the oxidation peak current of the electrode decreases in the potential range of the electroactive small molecule Fc, thereby achieving highly sensitive detection of GABA. In addition, this method is used for GABA detection for the first time.

[0037] The following is a description of the method for preparing the flexible molecular imprinting sensor provided by the present invention. The method for preparing the flexible molecular imprinting sensor described below and the flexible molecular imprinting sensor for detecting the content of GABA described above can be referred to each other.

[0038] See also Figure 2 , Figure 3 , Figure 4 and Figure 5 As shown, the method for preparing a flexible molecular imprinting sensor for in-situ and living detection of γ-aminobutyric acid content in plants provided in an embodiment of the present invention is used to prepare the flexible molecular imprinting sensor described in the above embodiment, and includes the following steps.

[0039] S210, patterning the PI tape using a laser direct writer controlled by a computer, and obtaining a graphene electrode unit body on the PI tape through induction; S220, transferring the graphene electrode unit body on the PI tape to the flexible substrate; S230, coating a set amount of Ag / AgCl ink on the exposed area of ​​the reference electrode and heating and curing the ink to obtain an Ag / AgCl reference electrode; S240, preparing a copolyester insulation layer in the area where the guide rail is located; S250, modifying the working electrode to obtain a MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode.

[0040] Specifically, in step S210, the PI tape is pasted on the polytetrafluoroethylene mold. The PI tape is cleaned with distilled water and ethanol in turn. The PI tape is then patterned with a computer-controlled laser writer, and a graphene electrode unit body (LIG electrode) with good conductivity is obtained through induction.

[0041] It should be noted that the main body of the graphene electrode unit 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 packaging layer on the above-mentioned electrodes, and the material layers modified on the working electrode 210 (the first modified layer, the second modified layer and the third modified layer).

[0042] In step S220, Ecoflex main agent Ecoflex 00-20 and curing agent Ecoflex 00-20 are mixed and stirred at 15:1~20:1 (preferably 10:1) by a spin coater for 10 min, and spin coated at a speed of 50~200rpm (preferably 100rpm) for 20~80s (preferably 60s) to evenly cover the PI tape with the graphene electrode unit body, and then heated in a vacuum drying oven at 60~100℃ (preferably 80℃) for 1~5h (preferably 4h). Then, the graphene electrode unit body (LIG) is peeled off from the PI tape to obtain the graphene electrode unit 20 (LIG / Ecoflex electrode) located on the flexible substrate 10.

[0043] That is, the graphene electrode unit 20 is prepared by laser printing on a flexible PI tape according to a design pattern and then transferring it to the copolyester Ecoflex.

[0044] In step S230 , a certain amount of Ag / AgCl ink is evenly coated on the exposed area of ​​the reference electrode 230 , and heated and cured at 80° C. for 10 to 50 min (preferably 30 min), thereby obtaining the Ag / AgCl reference electrode 230 .

[0045] 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 insulation layer, thereby insulating the area where the guide rail 30 is located. At this point, the LIG / Ecoflex three-electrode construction is completed.

[0046] Then, the electrode can be placed in a 0.1 M dilute sulfuric acid solution and a cyclic voltammetry scan in the range of 0 to 1.5 V can be performed to clean and activate the electrode surface.

[0047] Example 1: In step S250, 0.5-2.5 mg / ml (preferably 1 mg / ml) HAuCl4 solution is prepared with phosphate buffer solution (PBS), and AuNPs are deposited in the HAuCl4 solution using the it method (-1V, 800s) 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 using 0.2% chitosan solution, and after being ultrasonically dispersed, 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 was prepared with PBS (GABA:β-CD molar ratio of 5:1, GABA of 10 ~30 mM (preferably 25 mM), β-CD of 1~10 mM (preferably 5 mM), and MIP was formed by electropolymerization by CV method (-0.4 V-1 V, 100 mV / s, 50 cycles) to obtain uneluted MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex. The electrode was then placed in 10~60 mM (preferably 50 mM) NaOH solution for 1~10 min (preferably 5 min) to elute GABA, and finally the eluted MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex was obtained.

[0048] According to some embodiments of the present invention, the method for preparing a flexible molecular imprinting sensor further includes the following steps.

[0049] Example 2: A γ-aminobutyric acid solution of a set concentration is configured to perform DPV detection on the flexible molecular imprinting sensor; Based on the relationship curve between the logarithm of the concentration of the γ-aminobutyric acid solution and the current difference, a standard curve of the flexible molecular imprinting sensor is drawn.

[0050] Specifically, PBS was used to prepare GABA solutions with concentrations of 0, 1 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM (pH = 7.2-7.5, preferably pH = 7.4), and the prepared flexible molecular imprinting sensor was used for DPV detection (potential -0.2 V-0.6 V, interval time of 0.5 s, amplitude potential of 0.025 V). The peak current of the oxidation peak obtained in the blank solution was taken as I0, and the peak currents obtained from the standard γ-aminobutyric acid solutions of different concentrations were recorded as I1, I2, I3, etc., and ΔI1, ΔI2, ΔI3, etc. were calculated by the formula ΔI = I0-I1 (I2, I3, etc.). Taking the logarithm of the GABA concentration (lgC GABA ), and linearly fit it with the ΔI obtained at different concentrations to draw the standard curve of GABA flexible molecular imprinting sensor, where the linear equation is ΔI = 0.6862lgC GABA + 10.572 (nM), linear range up to 1×10 -9 ~1×10 -2 M, the detection limit is 5.79×10 -10 M.

[0051] Example 3: In addition, the flexible molecular imprinting sensor was subjected to a recovery experiment, and lettuce leaves were used as an example for illustration. The specific process was as follows: the lettuce leaves were squeezed into juice, and the supernatant was taken after centrifugation. According to the established calibration curve, the concentration of GABA in the lettuce juice was about 1.13±0.26 μM. The GABA standard sample was added with this as the matrix, and the recovery rate was calculated. The experimental results are shown in Table 1, and the spiked recovery rate of GABA was 96.97%-103.41%. The results were also compared with the results obtained by HPLC. The relative error between the prepared sensor and the results obtained by HPLC was 12.4%, which was less than 15%, and therefore was considered acceptable. It shows that the sensor can be used for the accurate determination of GABA in actual samples.

[0052] Table 1 Spike recovery results (n=3)

[0053] Example 4: See Figure 7As shown, the detection method of the flexible molecular imprinting sensor for in-situ and live detection of the content of γ-aminobutyric acid in plants according to the embodiment of the present invention includes: S710, punching a hole at a set position on the surface of the plant leaf to be tested; S720, attaching the flexible molecular imprinting sensor to the lower surface of the plant leaf to be tested, and making the working electrode face the punching position; S730, dripping a buffer solution at the punching position, and then connecting an electrochemical workstation to detect 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.

[0054] The flexible molecular imprinting sensor was subjected to an in vivo experiment, and lettuce leaves were used as an example. The specific process was as follows: First, the prepared flexible molecular imprinting sensor was tested for stability in blank PBS, and then a hole was punched in the lettuce leaf, and the flexible molecular imprinting sensor was fixed on the lower surface of the leaf so that the working electrode was facing the hole. 20 μL PBS was dropped on the hole so that the juice in the leaf could be released onto the working electrode, and then DPV detection was performed. The obtained current signal was used to calculate the instantaneous concentration of the sample being tested through the calibrated working curve.

[0055] Two different varieties of lettuce plants, "Da Su Sheng" and "Hong Shan Hu", were selected from the same period, and their GABA content was detected using the prepared electrode (MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex). The test results are shown in Table 2. The experimental results show that the flexible electrode sensor can detect GABA in living plants.

[0056] Table 2 Test results comparison table

[0057] Comparative Example 1: This comparative example provides a flexible molecular imprinting sensor for detecting GABA, and the preparation method is only the same as that of Example 1 ( Figure 6 The difference in a) is that Ni3(HITP)2 is replaced by an equal amount of Cu(INA)2 to obtain a flexible molecular imprinting sensor b: MIP / Fc-Cu(INA)2-ZnFe-LDH / AuNPs / LIG / Ecoflex. The detection performance of the sensor was tested using the method in Example 2, and the results are shown in FIG. Figure 6 b. It can be seen that the linear detection range of the flexible molecular imprinting sensor prepared in Comparative Example 1 is 1×10 -8 -1×10 -4 M, detection limit is 7.74×10 -9 M, the detection effect is not as good as that of Example 1.

[0058] Comparative Example 2: This comparative example provides a flexible molecular imprinting sensor for detecting GABA. The preparation method is different from that of Example 1 in that ZnFe-LDH is replaced with an equal amount of CoFe-LDH to prepare a flexible molecular imprinting sensor c: MIP / Fc-Ni3(HITP)2-CoFe-LDH / AuNPs / LIG / Ecoflex. The detection performance of the sensor is tested by the method in Example 2. The results are shown in FIG. Figure 6 As shown in c. It can be seen that the linear detection range of the flexible molecular imprinting sensor prepared in Comparative Example 1 is 1×10 -8 -1×10 -5 M, the detection limit is 3.21×10 -9 M, the detection effect is not as good as that of Example 1.

[0059] Comparative Example 3: This comparative example provides a flexible molecular imprinting sensor for detecting GABA. The preparation method is different from that of Example 1 in that AuNPs / LIG / Ecoflex is replaced by LIG / Ecoflex to prepare a flexible molecular imprinting sensor d: MIP / Fc-Ni3(HITP)2-ZnFe-LDH / LIG / Ecoflex. The detection performance of the sensor is tested by the method in Example 2. The results are as follows: Figure 6 As shown in d. It can be seen that the linear detection range of the flexible molecular imprinting sensor prepared in Comparative Example 1 is 1×10 -6 -1×10 -2 M, the detection limit is 6.67×10 -7 M, the detection effect is not as good as that of Example 1.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A flexible molecular imprinting sensor for in situ and in vivo detection of γ-aminobutyric acid content in plants, characterized in that: include: A flexible substrate and a graphene electrode unit, wherein the graphene electrode unit is arranged on the flexible substrate; The graphene electrode unit includes a guide rail, a working electrode, a counter electrode and a reference electrode, wherein the counter electrode and the reference electrode are respectively located on both sides of the working electrode, and the working electrode is provided with a first modification layer, a second modification layer and a third modification layer from the inside to the outside, wherein 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 molecular imprinting 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 disposed on the reference electrode to form an Ag / AgCl reference electrode.

3. The flexible molecular imprinting sensor for in-situ and in vivo detection of γ-aminobutyric acid content in plants according to claim 1, characterized in that: The area where the guide rail is located is provided with a copolyester insulation layer.

4. The flexible molecular imprinting sensor for in-situ and in vivo detection of 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 preparing a flexible molecular imprinting sensor for in-situ and in-vivo detection of γ-aminobutyric acid content in plants as claimed in any one of claims 1 to 4, characterized in that: include: The PI tape is patterned by using a computer-controlled laser direct writer, and a graphene electrode unit body is obtained on the PI tape by 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 heat-curing the ink to obtain an Ag / AgCl reference electrode; Prepare a copolyester insulation layer in the area where the guide rail is located; The working electrode was modified to obtain a MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode.

6. The method for preparing the flexible molecular imprinting sensor for in-situ and in vivo detection of γ-aminobutyric acid content in plants according to claim 5, characterized in that: The step of transferring the graphene electrode unit body on the PI tape to the flexible substrate comprises: Applying the Ecoflex mixed solution of a set ratio to the graphene electrode unit body on the PI tape by a spin coater, and then performing a vacuum drying and heating treatment; The graphene electrode unit body is peeled off from the PI tape to obtain the graphene electrode unit located on the flexible substrate.

7. The method for preparing the flexible molecular imprinting sensor for in-situ and in vivo detection of γ-aminobutyric acid content in plants according to claim 5, characterized in that: The working electrode is modified to obtain a MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode, comprising: The electrode is immersed in a 0.5-2.5 mg / ml (preferably 1 mg / ml) HAuCl4 solution, and AuNPs are obtained by chronoamperometry; Dissolve Fc, Ni3(HITP)2 and ZnFe-LDH in a chitosan solution, and coat the working electrode with 1-5 μL (preferably 4 μL) of the Fc-Ni3(HITP)2-ZnFe-LDH solution to obtain Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex; MIP solution was prepared using β-cyclodextrin as the functional monomer and γ-aminobutyric acid as the template molecule; The electrode was immersed in a MIP solution and electropolymerized by cyclic voltammetry to form a MIP. The electrode was then placed in a NaOH solution to wash off the template molecules, and finally the washed MIP / Fc-Ni3(HITP)2-ZnFe-LDH / AuNPs / LIG / Ecoflex electrode was obtained.

8. The method for preparing the flexible molecular imprinting sensor for in-situ and in vivo detection of γ-aminobutyric acid content in plants according to claim 7, characterized in that: The conditions for the cyclic voltammetry electropolymerization include: a voltage of -0.4 V to 1.0 V, 30 to 60 electropolymerization cycles (preferably 50 cycles), and a scan rate of 25 to 120 mV / s (preferably 100 mV / s).

9. A detection method based on the flexible molecular imprinting 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: include: Punching holes at set positions on the surface of the plant leaf to be tested; The flexible molecular imprinting sensor is attached to the lower surface of the plant leaf to be tested, and the working electrode is aligned with the punching position; Buffer solution was added to the punched hole, and then the electrochemical workstation was connected to detect the concentration of γ-aminobutyric acid by differential pulse voltammetry.

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