Molecularly imprinted sensor for in-situ detection of gamma-aminobutyric acid in plant leaves and preparation method thereof

Abstract

The invention relates to the technical field of analysis and detection, in particular to a molecular imprinting sensor for in-situ detection of gamma-aminobutyric acid in plant leaves and a preparation method of the molecular imprinting sensor. The molecular imprinting sensor for detecting gamma-aminobutyric acid is prepared by assembling Fc-hBN-WSe2-MXene, PSS and MIP on the surface of an electrode layer by layer, and high-selectivity and high-sensitivity detection of target molecules is realized. The technology can effectively reduce the detection cost, simplify the operation process and improve the performance of the sensor. By using the molecular imprinting sensor, gamma-aminobutyric acid in plant leaves can be analyzed in vivo and in situ, rapid and reliable quantitative analysis can be realized in complex samples, and the molecular imprinting sensor has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a molecularly imprinted sensor for in-situ detection of γ-aminobutyric acid in plant leaves and a preparation method thereof. Background Art

[0002] γ-aminobutyric acid (GABA) is a free non-protein amino acid widely present in animals and plants. In plants, GABA is distributed in various organs and tissues. GABA not only acts as a metabolite, but also can act as an endogenous signal molecule to regulate plant growth and development, maintain carbon-nitrogen balance, and respond to biotic and abiotic stresses.

[0003] In view of the important functions of GABA in plants, accurate and rapid detection of it is particularly crucial. Common detection methods for GABA include the Berthelot colorimetric method, amino acid analyzer method, high performance liquid chromatography method, gas chromatography-mass spectrometry method, etc. However, these analytical methods are usually complex in operation, time-consuming, rely on large-scale instrument equipment and professional technical personnel, and have relatively high detection costs. In addition, these methods are easily interfered by other components in the sample, especially in samples with complex components, and their selectivity is poor. More importantly, these detection methods are all in vitro analysis, which may cause great damage to plant tissues during the sample collection process and are difficult to meet the requirements of in-situ detection. In contrast, electrochemical methods have significant application potential in in-situ live detection of plants due to their advantages such as simple operation, fast response speed, high sensitivity, good selectivity, and low cost. Molecular imprinting technology constructs a three-dimensional cavity with specific recognition sites for the target molecule, enabling the sensor to specifically recognize the target molecule. This effectively reduces the interference of other components (such as amino acids, sugars, etc.) in the sample to the detection and significantly improves the selectivity of the detection.

[0004] Plant leaves are important organs of plants. However, for the leaves of most crops, such as wheat, corn, etc., the leaves are extremely thin and flat, and it is difficult to implant micro-needle electrodes; moreover, the juice content in the leaves is extremely small, which is difficult to meet the requirements of electrochemical testing. Therefore, how to design a molecularly imprinted sensor capable of in-situ live detection of GABA in plant leaves has become a technical problem urgently to be solved in this field. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention conforms to the flat structure of plants, uses a screen-printed electrode as the base electrode, fits it to the plant leaves, and provides a preparation method for a molecularly imprinted sensor for detecting γ-aminobutyric acid, including: (1) Modifying a ferrocene (Fc)-hexagonal boron nitride (hBN)-tungsten diselenide (WSe2)-MXene composite material on the surface of the screen-printed electrode to obtain a first modified electrode; (2) Modify the surface of the first modified electrode with sodium polystyrene sulfonate (PSS) to obtain a second modified electrode; (3) Using β-cyclodextrin (β-CD) as a monomer and γ-aminobutyric acid as a template molecule, prepare a molecularly imprinted polymer film (MIP) on the surface of the second modified electrode, and obtain the molecularly imprinted sensor after eluting the template molecule.

[0006] Among them, the ferrocene (Fc)-hexagonal boron nitride (hBN)-tungsten diselenide (WSe2)-MXene composite material is a probe molecule composite nanomaterial composed of Fc, hBN, WSe2, and MXene, which has high conductivity, good dispersibility, and a large surface area. The SPE electrode modified with such a composite material can provide stronger electron transfer ability and higher stability, providing a good foundation for subsequent molecular imprinting.

[0007] In the acidic environment of plants, the sulfonic acid groups in PSS enrich positively charged GABA through electrostatic adsorption, and this electrostatic adsorption and enrichment effect further enhances the binding efficiency of GABA on the electrode surface, thus significantly improving the detection sensitivity. On the surface of the PSS-modified electrode, β-CD acts as a monomer and interacts with the GABA template molecule to form a MIP film with specific recognition ability for GABA. When the template molecule is eluted, binding sites highly matching the target molecule are formed on the surface of the MIP film, which can efficiently recognize and bind GABA.

[0008] Through the dual recognition effects of PSS and MIP, the organic combination of electrostatic adsorption and specific recognition is realized. At the same time, PSS can also effectively reduce the non-specific adsorption caused by the functional groups outside the MIP cavity, further improving the selectivity and reliability of the detection.

[0009] Preferably, the chemical formula of MXene is Ti3C2.

[0010] Preferably, the electrode is a screen-printed electrode (SPCE / SPE).

[0011] Preferably, step (1) specifically includes: dissolving ferrocene, hexagonal boron nitride, tungsten diselenide, and MXene in a chitosan solution to prepare the composite material, and then covering the composite material on the surface of the electrode to obtain the first modified electrode.

[0012] Preferably, the concentration of ferrocene is 5-10 mg / mL; and / or, the concentration of hexagonal boron nitride is 0.5-2 mg / mL; and / or, the concentration of tungsten diselenide is 0.5-2 mg / mL; and / or, the concentration of Mxene is 0.5-2 mg / mL; and / or, the concentration of the chitosan solution is 0.2%-1%.

[0013] Preferably, step (2) specifically includes: covering the surface of the first modified electrode with a sodium polystyrene sulfonate solution.

[0014] Preferably, the concentration of the sodium polystyrene sulfonate solution is 1 - 5 mg / mL.

[0015] Preferably, step (3) specifically includes: mixing β-cyclodextrin, γ-aminobutyric acid and a PBS solution to obtain a mixed solution; then placing the second modified electrode in the mixed solution for electro-polymerization to obtain a molecularly imprinted polymer film; and preparing the molecularly imprinted sensor after eluting the template molecules.

[0016] Preferably, the molar ratio of γ-aminobutyric acid to β-cyclodextrin is (1 - 5):1; and / or, the concentration of β-cyclodextrin in the mixed solution is 0.5 - 1 mM; and / or, the electro-polymerization uses cyclic voltammetry; and / or, a NaOH solution is used to elute the template molecules.

[0017] Preferably, the voltage of the electro-polymerization is -0.4 V to 1 V; and / or, the number of cycles of the electro-polymerization is 10 - 50 cycles.

[0018] Furthermore, the present invention provides a molecularly imprinted sensor for detecting γ-aminobutyric acid prepared by the above preparation method.

[0019] Furthermore, the present invention provides a method for in-situ live detection of γ-aminobutyric acid in plants, including: attaching a modified screen-printed electrode to a plant leaf, punching holes in the surface of the plant leaf to release plant sap onto the working electrode surface, dropping (a small amount of) phosphate buffer solution, and then connecting an electrochemical workstation to detect the concentration of γ-aminobutyric acid by differential pulse voltammetry.

[0020] In the specific implementation process, the plant is any plant containing γ-aminobutyric acid, including but not limited to fruits, vegetables, flowers, crops, etc.

[0021] In the specific implementation process, the punching site is mainly the plant leaf.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: By using layer-by-layer assembly of Fc-hBN-WSe2-MXene, PSS, and MIP on the electrode surface, the present invention prepares a molecularly imprinted sensor for detecting γ-aminobutyric acid, realizing high-selectivity and high-sensitivity detection of the target molecule. This technology can effectively reduce the detection cost, simplify the operation process, and improve the performance of the sensor. Using the molecularly imprinted sensor of the present invention can perform in-vivo and in-situ analysis of γ-aminobutyric acid in plant leaves, achieving rapid and reliable quantitative analysis in complex samples, and having broad application prospects. Description of the Drawings

[0023] Figure 1 It is the process flow chart of the molecularly imprinted sensor of Example 1.

[0024] Figure 2 It is the comparison chart of the detection performance of different molecularly imprinted sensors. Detailed implementation manners

[0025] 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. Apparently, 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. In the embodiments provided in this specification, those not specifying specific techniques or conditions shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0026] Example 1 This example provides a molecularly imprinted sensor for detecting γ-aminobutyric acid, and the process flow chart is as Figure 1 shown, and the preparation steps are as follows: (1) The SPE electrode (purchased from Weihai Botan Technology Co., Ltd.) is placed in 0.01 M phosphate buffer solution (pH = 7.2 - 7.4), and is activated for 180 s by the potentiostatic method (1.7 V) to remove impurities on the electrode surface.

[0027] (2) 50 mg of Fc (Shanghai Macklin Biochemical Co., Ltd., product number F809617), 5 mg of hBN (purchased from Shanghai Macklin Biochemical Co., Ltd., product number B917279), 5 mg of WSe2 (purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., product number 101261) and 5 mg of MXene (Ti3C2, purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., product number 104661) are dissolved in 5 mL of 0.2% chitosan solution and mixed. After ultrasonic treatment until uniformly dispersed, an Fc-hBN-WSe2-MXene composite material is obtained. Take 5 μL and drop it on the surface of the working electrode and dry it to obtain Fc-hBN-WSe2-MXene / SPE.

[0028] (3) Prepare a 5 mg / mL PSS solution, take 5 μL and drop it on the surface of the working electrode and dry it to obtain PSS / Fc-hBN-WSe2-MXene / SPE.

[0029] (4)Prepare the molecularly imprinted polymer solution with PBS, where the molar ratio of GABA:β-CD is 5:1 and β-CD is 1 mM. Electro-polymerize MIP for 50 cycles on the electrode surface by cyclic voltammetry (-0.4 V to 1 V) to obtain the uneluted MIP / PSS / Fc-hBN-WSe2-MXene / SPE. Then, immerse the electrode in a 50 mM NaOH solution for 5 min to wash off the template molecules, obtaining the molecularly imprinted sensor MIP / PSS / Fc-hBN-WSe2-MXene / SPE.

[0030] Example 2 In this example, the detection performance of the molecularly imprinted sensor for detecting γ-aminobutyric acid prepared in Example 1 was tested, and the steps are as follows: (1)Prepare a series of γ-aminobutyric acid-phosphate buffer solutions (pH = 7.2 - 7.4) with concentrations of 0, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM respectively. Connect the molecularly imprinted sensor of Example 1 to an electrochemical workstation (CHI 760E) and detect by differential pulse voltammetry (potential -0.2~0.6V, potential increment 0.004 V, amplitude 0.05 V, pulse width 0.05 s, pulse period 0.5 s, sampling width 0.02 s). As the concentration of the γ-GABA solution increases, the oxidation peak of Fc gradually decreases. Take the peak current of the oxidation peak obtained in the blank solution as I0, and record the peak currents obtained from the standard γ-GABA solutions with different concentrations as I x (x = 1, 2, 3…), and calculate ΔI through the formula ΔI = I0 - I x , and calculate ΔI respectively x , thus obtaining a relationship curve between the logarithm of the γ-GABA concentration and ΔI. As Figure 2 shown, the linear detection range is 1 nM - 10 mM, and the detection limit is 0.426 nM (S / N = 3).

[0031] (2)Determination of the recovery rate of the added standard Juice the lettuce, filter it, centrifuge it at a speed of 4000 r / min for 5 min, and take the supernatant. Based on this, add a γ-GABA standard sample for the recovery experiment.

[0032] The results are shown in Table 1. The recovery rate of the added γ-GABA is between 98.63% and 105.90%, and the response current values at the same concentration are relatively consistent (n = 3). The results show that the detection results of the molecularly imprinted sensor in Example 1 are accurate and reliable.

[0033] Table 1 Determination of the recovery rate of the added standard of the sensor (n = 3)

[0034] Example 3 In this example, the molecularly imprinted sensor of Example 1 was used to detect GABA in the leaves of potted lettuce in situ in vivo, and the steps were as follows: Make several small holes in the leaves of potted lettuce to release γ-aminobutyric acid. The sensor was fixed on the lower surface of the leaf, and its working electrode was facing the small holes. Drop 5 μL of PBS on the holes to promote the chemical connection between the electrode and the leaf. Then connect the electrochemical workstation (Chenhua, Shanghai, CHI760E), and record the concentration of γ-aminobutyric acid in living lettuce by differential pulse voltammetry (the test conditions are the same as those in Example 2). The results are shown in Table 2.

[0035] Table 2 Detection of GABA levels in lettuce leaves (μmol / L)

[0036] Comparative Example 1 This comparative example provides a molecularly imprinted sensor for detecting γ-aminobutyric acid. The only difference in the preparation method from Example 1 is that: WSe2 was replaced with an equal amount of molybdenum disulfide (MoS2).

[0037] The detection performance of the sensor was tested by the method in Example 2, and the results are as Figure 2 shown. The linear detection range of the molecularly imprinted sensor prepared in Comparative Example 1 is 1 nM~10 mM, but the current and sensitivity are not as good as those in Example 1.

[0038] Comparative Example 2 This comparative example provides a molecularly imprinted sensor for detecting γ-aminobutyric acid. The only difference in the preparation method from Example 1 is that: PSS was replaced with an equal amount of cetyltrimethylammonium bromide (CTAB).

[0039] The detection performance of the sensor was tested by the method in Example 2, and the results are as Figure 2 shown. The linear detection range of the molecularly imprinted sensor prepared in Comparative Example 2 is 1 nM~1 mM, which is not as good as that in Example 1.

[0040] Finally, it should be noted that the above examples 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 examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A preparation method of a molecularly imprinted sensor for detecting γ-aminobutyric acid, characterized in that, Comprising: (1) Modifying a ferrocene - hexagonal boron nitride - tungsten diselenide - MXene composite material on the surface of a screen - printed electrode to obtain a first modified electrode; (2) Modifying sodium polystyrene sulfonate on the surface of the first modified electrode to obtain a second modified electrode; (3) Using β - cyclodextrin as a monomer and γ - aminobutyric acid as a template molecule, preparing a molecularly imprinted polymer film on the surface of the second modified electrode, and obtaining the molecularly imprinted sensor after eluting the template molecule.

2. The preparation method according to claim 1, characterized in that, Step (1) specifically includes: Dissolving ferrocene, hexagonal boron nitride, tungsten diselenide, and MXene in a chitosan solution to prepare the composite material, and then covering the composite material on the electrode surface to obtain a first modified electrode.

3. The preparation method according to claim 2, characterized in that, The concentration of ferrocene is 5 - 10 mg / mL; and / or, the concentration of hexagonal boron nitride is 0.5 - 2 mg / mL; and / or, the concentration of tungsten diselenide is 0.5 - 2 mg / mL; and / or, the concentration of Mxene is 0.5 - 2 mg / mL; and / or, the concentration of the chitosan solution is 0.2% - 1%.

4. The preparation method according to claim 1, characterized in that, Step (2) specifically includes: Covering the sodium polystyrene sulfonate solution on the surface of the first modified electrode.

5. The preparation method according to claim 4, wherein The concentration of the sodium polystyrene sulfonate solution is 1 - 5 mg / mL.

6. The preparation method according to claim 1, characterized in that, Step (3) specifically includes: Mixing β - cyclodextrin, γ - aminobutyric acid, and PBS solution to prepare a mixed solution; then placing the second modified electrode in the mixed solution for electro - polymerization to obtain a molecularly imprinted polymer film; and obtaining the molecularly imprinted sensor after eluting the template molecule.

7. The preparation method according to claim 6, characterized in that, The molar ratio of γ - aminobutyric acid to β - cyclodextrin is (1 - 5):1; and / or, the concentration of β - cyclodextrin in the mixed solution is 0.5 - 1 mM; and / or, the electro - polymerization uses cyclic voltammetry; and / or, NaOH solution is used to elute the template molecule.

8. The preparation method according to claim 7, characterized in that, The voltage of the electro - polymerization is - 0.4 V - 1 V; and / or, the number of cycles of the electro - polymerization is 10 - 50 cycles.

9. A molecularly imprinted sensor for detecting γ - aminobutyric acid prepared by the preparation method according to any one of claims 1 - 8.

10. A method for in-situ and in-vivo detection of γ-aminobutyric acid in plants, characterized in that, Comprising: After punching holes on the surface of the plant body, pasting the molecularly imprinted sensor according to claim 9 to the punched holes, dropping phosphate buffer solution, then connecting an electrochemical workstation, and detecting the concentration of γ - aminobutyric acid by differential pulse voltammetry.

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