A molecular imprinting sensor for detecting gamma-aminobutyric acid in plant leaves in situ and a preparation method thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a molecular imprinting sensor for in-situ detection of gamma-aminobutyric acid in plant leaves and a preparation method thereof. Background Art

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

[0003] Given the crucial functions of GABA in plants, accurate and rapid detection is crucial. Common methods for GABA detection include the Berthelot colorimetric method, amino acid analyzer, high-performance liquid chromatography, and gas chromatography-mass spectrometry. However, these analytical methods are often complex and time-consuming, relying on extensive instrumentation and specialized personnel, resulting in high costs. Furthermore, these methods are susceptible to interference from other components in the sample, and their selectivity is poor, particularly in complex samples. Furthermore, these detection methods are in vitro analyses, which can cause significant damage to plant tissues during sample collection, making them difficult to meet the requirements of in situ detection. In contrast, electrochemical methods, with their advantages of simplicity, rapid response, high sensitivity, good selectivity, and low cost, hold significant potential for in situ detection in plants. Molecular imprinting technology enables sensors to specifically recognize target molecules by constructing three-dimensional cavities with specific recognition sites for the target molecule. This effectively reduces interference from other components in the sample, such as amino acids and sugars, significantly improving detection selectivity.

[0004] Plant leaves are vital organs, but the leaves of most crops, such as wheat and corn, are extremely thin and flat, making microneedle electrodes difficult to implant. Furthermore, the leaf sap content is extremely low, making it difficult to meet the requirements of electrochemical testing. Therefore, designing a molecularly imprinted sensor capable of in situ detection of GABA in living plant leaves has become a pressing technical challenge in this field. Summary of the Invention

[0005] To solve the above technical problems, the present invention conforms to the flat structure of plants, uses screen-printed electrodes as base electrodes, adheres to plant leaves, and provides a method for preparing a molecular imprinting sensor for detecting γ-aminobutyric acid, comprising:

[0006] (1) Modifying the surface of the screen-printed electrode with a ferrocene (Fc)-hexagonal boron nitride (hBN)-tungsten diselenide (WSe2)-MXene composite material to obtain the first modified electrode;

[0007] (2) Modifying the surface of the first modified electrode with sodium polystyrene sulfonate (PSS) to obtain a second modified electrode;

[0008] (3) Using β-cyclodextrin (β-CD) as a monomer and γ-aminobutyric acid as a template molecule, a molecular imprinting polymer (MIP) film is prepared on the surface of the second modified electrode, and the molecular imprinting sensor is prepared after eluting the template molecule.

[0009] Among them, the ferrocene (Fc)-hexagonal boron nitride (hBN)-tungsten diselenide (WSe2)-MXene composite material is a probe molecular composite nanomaterial composed of Fc, hBN, WSe2 and MXene, which has high conductivity, good dispersibility and 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.

[0010] In the acidic environment of plants, the sulfonic acid groups in PSS electrostatically attract positively charged GABA. This electrostatic adsorption and accumulation further enhance GABA binding efficiency on the electrode surface, significantly improving detection sensitivity. On the PSS-modified electrode surface, β-CD, as a monomer, interacts with the GABA template molecule, forming a membrane-initiated protein (MIP) membrane with specific recognition for GABA. After the template molecule is washed away, binding sites are formed on the MIP membrane surface that are highly compatible with the target molecule, enabling efficient recognition and binding of GABA.

[0011] Through the dual recognition effects of PSS and MIP, an organic combination of electrostatic adsorption and specific recognition is achieved. At the same time, PSS can effectively reduce the nonspecific adsorption caused by functional groups outside the MIP cavity, further improving the selectivity and reliability of detection.

[0012] Preferably, the chemical formula of MXene is Ti3C2.

[0013] Preferably, the electrode is a screen printed electrode (SPCE / SPE).

[0014] Preferably, step (1) specifically comprises: dissolving ferrocene, hexagonal boron nitride, tungsten diselenide and MXene in a chitosan solution to prepare the composite material, and then covering the surface of the electrode with the composite material to obtain a first modified electrode.

[0015] Preferably, the concentration of the ferrocene is 5-10 mg / mL; and / or, the concentration of the hexagonal boron nitride is 0.5-2 mg / mL; and / or, the concentration of the tungsten diselenide is 0.5-2 mg / mL; and / or, the concentration of the Mxene is 0.5-2 mg / mL; and / or, the concentration of the chitosan solution is 0.2%-1%.

[0016] Preferably, the step (2) specifically comprises: covering the sodium polystyrene sulfonate solution on the surface of the first modified electrode.

[0017] Preferably, the concentration of the sodium polystyrene sulfonate solution is 1-5 mg / mL.

[0018] Preferably, the step (3) specifically comprises: mixing the beta-cyclodextrin, gamma-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 preparing the molecularly imprinted sensor after eluting the template molecule.

[0019] Preferably, the molar ratio of the gamma-aminobutyric acid to the beta-cyclodextrin is (1-5):1; and / or, the concentration of the beta-cyclodextrin in the mixed solution is 0.5-1 mM; and / or, the electro-polymerization adopts a cyclic voltammetry method; and / or, the template molecule is eluted by using a NaOH solution.

[0020] Preferably, 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.

[0021] Further, the present application provides a molecularly imprinted sensor for detecting gamma-aminobutyric acid, which is prepared by the preparation method.

[0022] Further, the present application provides a method for in-situ and in-vivo detecting gamma-aminobutyric acid in a plant, which comprises: attaching the modified screen-printed electrode to a plant leaf, punching a hole on the surface of the plant leaf to release plant juice to the surface of the working electrode, and adding (a small amount of) a phosphate buffer solution, and then connecting an electrochemical workstation to detect the concentration of the gamma-aminobutyric acid by a differential pulse voltammetry method.

[0023] In the specific implementation process, the plant is any plant containing gamma-aminobutyric acid, including but not limited to fruits, vegetables, flowers, crops, etc.

[0024] In the specific implementation process, the punching site is mainly the plant leaf.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] This invention fabricates a molecularly imprinted sensor for detecting γ-aminobutyric acid (GABA) by assembling Fc-hBN-WSe2-MXene, PSS, and MIP layer by layer on an electrode surface, achieving highly selective and sensitive detection of the target molecule. This technology can effectively reduce detection costs, simplify the operation process, and improve sensor performance. The molecularly imprinted sensor of this invention can be used to analyze GABA in living plant leaves in situ, achieving rapid and reliable quantitative analysis in complex samples, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a process flow chart of the molecular imprinting sensor of Example 1.

[0028] Figure 2 This is a comparison chart of the detection performance of different molecular imprinting sensors. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, those without specifying specific techniques or conditions are described in accordance with the techniques or conditions described in the literature in this area, or are carried out according to product specifications. Reagents or instruments used are not specified by manufacturer and are conventional products that can be purchased through regular channels.

[0030] Example 1

[0031] This embodiment provides a molecular imprinting sensor for detecting γ-aminobutyric acid. The process flow chart is as follows: Figure 1 As shown, the preparation steps are as follows:

[0032] (1) The SPE electrode (purchased from Weihai Botan Technology Co., Ltd.) was placed in 0.01 M phosphate buffer (pH = 7.2-7.4) and activated using a constant potential method (1.7 V) for 180 s to remove impurities on the electrode surface.

[0033] (2) 50 mg of Fc (Shanghai McLean Biochemical Technology Co., Ltd., Product No. F809617), 5 mg of hBN (purchased from Shanghai McLean Biochemical Technology Co., Ltd., Product No. B917279), 5 mg of WSe2 (purchased from Jiangsu Xianfeng Nanomaterial Technology Co., Ltd., Product No. 101261), and 5 mg of MXene (Ti3C2, purchased from Jiangsu Xianfeng Nanomaterial Technology Co., Ltd., Product No. 104661) were dissolved in 5 mL of 0.2% chitosan solution and mixed. After ultrasonication until uniform dispersion, Fc-hBN-WSe2-MXene composite material was obtained. 5 μL was dropped on the surface of the working electrode and dried to obtain Fc-hBN-WSe2-MXene / SPE.

[0034] (3) Prepare a 5 mg / mL PSS solution, take 5 μL and drop it on the working electrode surface and dry it to obtain PSS / Fc-hBN-WSe2-MXene / SPE.

[0035] (4) A molecular imprinting polymer solution was prepared in PBS with a molar ratio of GABA to β-CD of 5:1 and a β-CD concentration of 1 mM. MIP was electropolymerized on the electrode surface for 50 cycles by cyclic voltammetry (-0.4 V to 1 V) to obtain an uneluted MIP / PSS / Fc-hBN-WSe2-MXene / SPE. The electrode was then placed in a 50 mM NaOH solution for 5 min to elute the template molecules, resulting in a molecular imprinting sensor MIP / PSS / Fc-hBN-WSe2-MXene / SPE.

[0036] Example 2

[0037] In this example, the detection performance of the molecular imprinting sensor for detecting γ-aminobutyric acid prepared in Example 1 was tested, and the steps were as follows:

[0038] (1) A series of γ-aminobutyric acid-phosphate buffer solutions (pH = 7.2-7.4) with the concentrations of 0, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM were prepared, and the molecular imprinting sensor of Example 1 was connected to an electrochemical workstation (CHI 760E). The Fc was detected by differential pulse voltammetry (potential -0.2-0.6 V, 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 γ-GABA solution increased, the oxidation peak of Fc gradually decreased. 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-GABA solutions of different concentrations were recorded as I. x (x=1,2,3…), by the formula ΔI=I0-Ix respectively, and then the difference of the current (ΔI) was calculated. x Thus a set of curves of the logarithm of the concentration of γ-GABA versus ΔI was obtained. As shown in Figure 2, the linear detection range was 1 nM-10 mM, and the detection limit was 0.426 nM (S / N=3). Figure 2

[0039] (2) Determination of the recovery rate

[0040] The juice of the lettuce was extracted, filtered, and centrifuged at a speed of 4000 r / min for 5 min, and the supernatant was taken. The recovery rate experiment was performed by adding the γ-GABA standard sample to the supernatant.

[0041] The results are shown in Table 1, and the recovery rate of γ-GABA was between 98.63-105.90%, and the response current values of the same concentration were relatively consistent (n=3). The results showed that the detection results of the molecular imprinting sensor of Example 1 were accurate and reliable.

[0042] Table 1 Determination of the recovery rate of the sensor (n=3)

[0043]

[0044] Example 3

[0045] In this example, the molecular imprinting sensor of Example 1 was used to detect GABA in the leaves of the potted lettuce in situ, and the steps were as follows:

[0046] Several small holes were punched on the leaves of the potted lettuce to release γ-aminobutyric acid, and the sensor was fixed on the lower surface of the leaves, with the working electrode facing the small hole. 5 μL of PBS was dropped on the hole to promote the chemical connection between the electrode and the leaves. Then, an electrochemical workstation (Shanghai Chenhua, CHI760E) was connected, and the concentration of γ-aminobutyric acid in the living lettuce was recorded by differential pulse voltammetry (the test conditions were the same as in Example 2). The results are shown in Table 2.

[0047] Table 2 Detection of the level of GABA in the leaves of the lettuce (μmol / L)

[0048]

[0049] Comparative Example 1

[0050] This comparative example provides a molecular imprinting sensor for detecting γ-aminobutyric acid, and the only difference in the preparation method is that:

[0051] WSe2 is replaced with an equal amount of molybdenum disulfide (MoS2).

[0052] The detection performance of the sensor was tested by the method in Example 2, and the results are shown in​ Figure 2 The linear detection range of the molecular imprinting 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.

[0053] Comparative Example 2

[0054] This comparative example provides a molecular imprinting sensor for detecting γ-aminobutyric acid. The preparation method is different from that of Example 1 only in that:

[0055] PSS was replaced with an equal amount of cetyltrimethylammonium bromide (CTAB).

[0056] The detection performance of the sensor was tested using the method in Example 2, and the results were as follows: Figure 2 The linear detection range of the molecular imprinting sensor prepared in Comparative Example 2 is 1 nM~1 mM, which is not as good as that in Example 1.

[0057] 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 various embodiments of the present invention.

Claims

1. A method for preparing a molecular imprinting sensor for detecting γ-aminobutyric acid, characterized in that: include: (1) Modifying the surface of the screen-printed electrode with a ferrocene-hexagonal boron nitride-tungsten diselenide-MXene composite material to obtain a first modified electrode; (2) modifying the surface of the first modified electrode with sodium polystyrene sulfonate to obtain a second modified electrode; (3) Using β-cyclodextrin as a monomer and γ-aminobutyric acid as a template molecule, a molecular imprinting polymer film is prepared on the surface of the second modified electrode, and the molecular imprinting sensor is obtained 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 surface of the electrode to obtain a first modified electrode.

3. The preparation method according to claim 2, characterized in that The concentration of the ferrocene is 5-10 mg / mL; and / or the concentration of the hexagonal boron nitride is 0.5-2 mg / mL; and / or the concentration of the tungsten diselenide is 0.5-2 mg / mL; and / or the concentration of the 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 surface of the first modified electrode with a sodium polystyrene sulfonate solution.

5. The preparation method according to claim 4, characterized in that 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 electropolymerization to obtain a molecularly imprinted polymer film; and eluting the template molecules to obtain the molecularly imprinted sensor.

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 electropolymerization is performed using cyclic voltammetry; and / or the template molecule is eluted using a NaOH solution.

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

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

10. A method for in situ detection of γ-aminobutyric acid in plants, characterized in that: include: After punching a hole on the plant surface, the molecular imprinting sensor according to claim 9 is attached to the hole, phosphate buffer is added dropwise, and then connected to an electrochemical workstation to detect the concentration of γ-aminobutyric acid by differential pulse voltammetry.

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