A molecular imprinting sensor for in-situ detection of epicatechin in plant leaves and a preparation and application method thereof

Abstract

The present application relates to the technical field of analytical detection, in particular to a molecular imprinting sensor for in-situ detection of epicatechin in plant leaves and a preparation and application method thereof.The present application utilizes a zeolite imidazolate framework-amide functionalized single-walled carbon nanotube composite material, amino-formyl imidazole modified reduced graphene, and a MIP film to be assembled layer by layer on the surface of an electrode, thereby preparing a molecular imprinting sensor for detecting epicatechin, and realizing high selectivity and high sensitivity detection of the target molecule epicatechin.This technology can effectively reduce the detection cost, simplify the operation process, and improve the performance of the sensor.Especially, the introduction of MIP enables the sensor to have the ability of in-situ detection and high-precision molecular recognition, and can realize rapid and reliable quantitative analysis in complex samples, thus having a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and in particular to a molecular imprinting sensor for in-situ detection of epicatechin in plant leaves, and a preparation and application method thereof. BACKGROUND

[0002] Epicatechin (EC) is a kind of plant polyphenol widely existing in tea plants, especially in the growing active parts such as leaf tips and leaves. Its main functions include regulating nutrient distribution in plants, resisting cold, resisting ultraviolet rays, resisting oxidation, resisting diseases and pests, and helping plants cope with drought, light and other plant stresses by adjusting stomatal opening and density.

[0003] Given the important functions of EC in plants, it is particularly important to accurately and quickly detect it. Common detection methods for EC include high-performance liquid chromatography and gas chromatography-mass spectrometry. However, these analytical methods usually take a long time, require large instruments and professional personnel, are complex to operate, and have high detection costs. More importantly, these detection methods are all in vitro analysis, which may cause great damage to plant tissues during sample collection, and even cause the death of plants. In contrast, electrochemical methods have significant application potential in in-situ live detection of plants due to their advantages of simple operation, fast response, high sensitivity, good selectivity, and low cost.

[0004] Plant leaves have different shapes, but most of the leaves of crops are extremely thin and flat, and have very little water content, which is difficult to meet the needs of electrochemical testing. Therefore, how to design an electrochemical sensor that can in-situ detect epicatechin in plant leaves has become a technical problem that needs to be solved in the field. SUMMARY

[0005] To solve the above technical problems, the present application provides a molecular imprinting sensor for in-situ detection of epicatechin in plant leaves, and a preparation method thereof, which comprises:

[0006] (1) modifying zeolite imidazolate framework and amide functionalized single-walled carbon nanotubes (f-SWCNT) on the surface of an electrode to obtain a first modified electrode;

[0007] (2) modifying amino-carbonyl imidazole modified reduced graphene (rGO-NH-Carboimidazole) on the surface of the first modified electrode to obtain a second modified electrode;

[0008] (3) using chitosan (CS) as a monomer and epicatechin (EC) as a template molecule to prepare a molecular imprinting polymer film (MIP) on the surface of the second modified electrode, and then eluting the template molecule to obtain the molecular imprinting sensor.

[0009] The composite nanomaterial composed of a zeolite imidazolate framework and an amide functionalized single-walled carbon nanotube has high conductivity, good dispersibility and a large surface area, and an electrode modified by the composite nanomaterial can provide stronger electron transfer capacity and higher stability, thereby providing a good foundation for subsequent molecular imprinting. The rGO-NH-Carboimidazole is a strong catalytic material, and the catalytic material further enhances the response efficiency of EC on the electrode surface, thereby significantly improving the detection sensitivity. On the electrode surface modified by the rGO-NH-Carboimidazole, CS as a monomer interacts with the EC template molecule to form a MIP film having specific recognition ability for EC. After the template molecule is eluted, the MIP film surface forms a binding site highly matched with the target molecule, which can efficiently recognize and combine EC, effectively reduces the interference of other components (such as flavonoids, amino acids, sugars and the like) in the sample on the detection, and significantly improves the selectivity of the detection. Through the synergistic effect of the rGO-NH-Carboimidazole and the MIP, the specific recognition and catalytic performance are enhanced, and the selectivity and sensitivity of the detection of epicatechin are further improved.

[0010] Preferably, the zeolite imidazolate framework is ZIF-8; and / or, the electrode is a screen-printed electrode (SPCE).

[0011] Preferably, the step (1) specifically comprises: mixing a zeolite imidazolate framework solution, an amide functionalized single-walled carbon nanotube solution and a chitosan solution to obtain a composite material; and then covering the composite material on the electrode surface.

[0012] Preferably, the concentration of the zeolite imidazolate framework solution is 1-10 mg / mL; and / or, the concentration of the amide functionalized single-walled carbon nanotube solution is 1-10 mg / mL; and / or, the concentration of the chitosan solution is 0.1%-1%.

[0013] Preferably, the step (2) specifically comprises: covering an amino-carbonyl imidazole modified reduced graphene oxide solution on the surface of the first modified electrode.

[0014] Preferably, the concentration of the amino-carbonyl imidazole modified reduced graphene oxide solution is 1-5 mg / mL.

[0015] Preferably, the step (3) specifically comprises: mixing chitosan, epicatechin and a PBS solution to prepare a mixed solution; and 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.

[0016] Preferably, the molar ratio of epicatechin to chitosan is (1-5):1; and / or, the concentration of chitosan in the mixed solution is 5-10 mM; and / or, the electro-polymerization is performed by chronoamperometry; and / or, the electro-polymerization time is 60-150 s; and / or, the template molecules are eluted by NaOH solution.

[0017] Preferably, the elution of the template molecules by the NaOH solution is performed for 5-15 min.

[0018] Preferably, the electrode is activated before use.

[0019] More preferably, the step of activation comprises: placing the electrode in a phosphate buffer solution (pH=7.2-7.4) and activating by constant potential method (1.7 V) for 180-300 s to remove impurities on the surface of the electrode.

[0020] Further, the application provides a molecular imprinting sensor for detecting epicatechin prepared by the preparation method.

[0021] Further, the application provides a method for in-situ and in-vivo detection of epicatechin in plant leaves, comprising: releasing plant juice by punching the surface of the plant leaves, attaching the molecular imprinting sensor to the punched part, then adding (a small amount of) buffer solution (such as phosphate buffer solution), connecting an electrochemical workstation, and detecting the concentration of epicatechin by differential pulse voltammetry.

[0022] In specific implementation, the plant is any plant containing epicatechin, including but not limited to fruits, vegetables, flowers, crops, etc.

[0023] In specific implementation, the punched part is mainly the plant leaves.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] The application realizes high selectivity and high sensitivity detection of the target molecule epicatechin by using the zeolite imidazolate framework-amide functionalized single-walled carbon nanotube composite material, the amino-formyl imidazole modified reduced graphene, and the MIP film to layer-by-layer assemble on the surface of the electrode. This technology can effectively reduce the detection cost, simplify the operation process, and improve the performance of the sensor. In particular, the introduction of MIP enables the sensor to have the ability of in-situ detection and high-precision molecular recognition, and can be used for in-situ and in-vivo detection of epicatechin in plant leaves, which has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a process flow chart of the molecular imprinting sensor of Example 1.

[0027] Figure 2 Figure 4 is a contrastive diagram of detection performance of different molecular imprinting sensors. DETAILED DESCRIPTION

[0028] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. In the embodiments provided in the present specification, the specific techniques or conditions not mentioned are performed according to the techniques or conditions described in the literature in the art or according to the product manual. The reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased through a regular channel.

[0029] Embodiment 1

[0030] The present embodiment provides a molecular imprinting sensor for detecting epicatechin, and a process flow chart is shown in Figure 1, and the preparation steps are as follows: Figure 1

[0031] (1) The SPCE electrode (POTEN, Weihai Wave Technology Co., Ltd.) was placed in 0.01 M phosphate buffer (pH = 7.2-7.4), and activated by constant potential method (1.7 V) for 180 s to remove impurities on the surface of the electrode.

[0032] (2) 25 mg of zeolite imidazolate framework ZIF-8 (XFNANO-102723, Nanjing Xianfeng Nanometer Material Technology Co., Ltd.) and 25 mg of amide functionalized single-walled carbon nanotube f-SWCNT (MERCK-685380, Sigma-Aldrich, Germany) were mixed in 5 mL of 0.2% chitosan solution, and then ultrasonically dispersed to obtain a ZIF8-SWCNT composite material. 3 μL of the composite material was dropped on the surface of the working electrode and dried to obtain a ZIF8-SWCNT / SPCE.

[0033] (3) 3 mg of amino-carbonyl imidazole modified reduced graphene rGO-NH-Carboimidazole (XFNANO-100396, Nanjing Xianfeng Nanometer Material Technology Co., Ltd.) was mixed in 3 mL of ultrapure water, and then ultrasonically dispersed to obtain a rGO-NH-Carboimidazole / ZIF8-SWCNT / SPCE working electrode.

[0034] ​(4) The molecularly imprinted polymer solution was prepared with PBS, and the molar ratio of EC to CS was 2:1, and the CS was 10 mM. The MIP was electropolymerized on the electrode surface for 120 seconds by chronoamperometry (-1 V) to obtain the uneluted MIP / rGO-NH-Carboimidazole / ZIF8-SWCNT / SPCE, and then the electrode was placed in a 100 mM NaOH solution for 10 min to elute the template molecule to obtain the molecularly imprinted sensor MIP / rGO-NH-Carboimidazole / ZIF8-SWCNT / SPCE.

[0035] Example 2

[0036] In this example, the detection performance of the molecularly imprinted sensor for detecting epicatechin prepared in Example 1 was tested, and the steps were as follows:

[0037] Epicatechin-phosphate buffer (pH = 7.2-7.4) solutions with concentrations of 10 μmoL / L, 50 μmoL / L, 100 μmoL / L, 200 μmoL / L, 500 μmoL / L, 1000 μmoL / L, and 4000 μmoL / L were prepared, respectively, and the molecularly imprinted sensor for detecting epicatechin prepared in Example 1 was used for differential pulse voltammetry detection (voltage range was -0.2-0.5 V, interval time was 0.5 s, modulation time was 0.05 s, and amplitude potential was 0.025 V). As the concentration of the EC solution increased, the oxidation peak of EC gradually increased, and the peak current obtained from the standard epicatechin solutions with different concentrations was recorded as I1, I2, I3, …, respectively, so as to obtain a set of concentration logarithm and current I relationship curves, and a standard curve of epicatechin was prepared. As shown in FIG. 2, the linear detection range was 10 μM-4 mM, and the detection limit was 0.325 μM (S / N = 3). Figure 2

[0038] Example 3

[0039] In this example, the recovery rate of the molecularly imprinted sensor of Example 1 was detected, and the steps were as follows: the tea seedling leaves were broken in a high-efficiency sample breaking system, and then buffer solution with the same mass as the broken sample was added. Then, the sample was centrifuged in a high-speed centrifuge, the speed of which was set to 10000 rpm, the time was set to 20 min, and the temperature was -4℃. After centrifugation, the supernatant was collected by a pipette. Through linear regression equation, it was calculated that the content of tea polyphenols in the original tea seedling leaves was 2.17±0.035 mmol / L. Then, the standard addition method was used, and known concentrations of EC (1, 3, 5, and 10 mmol / L) were added, respectively, and then the molecularly imprinted sensor of Example 1 was used for DPV detection, and finally the recovery rate was calculated. The results of the recovery rate test are shown in Table 1. The results show that the molecularly imprinted sensor of Example 1 has potential for practical application.​

[0040] Table 1. Recovery of EC content in tea seedling leaves (mmol / L)

[0041]

[0042] Example 4

[0043] In this example, the molecular imprinting sensor of Example 1 was used to detect EC in the leaves of potted tea seedlings in situ in vivo, and the steps were as follows:

[0044] The experimental material was the leaves of potted tea seedlings. A puncher was used to punch a hole with a diameter of 1 mm on the leaf, and the molecular imprinting sensor of Example 1 was attached to the lower surface of the leaf, with the working electrode facing the hole. 5 μL of 10 mM phosphate (PBS) buffer was added at the punched hole to facilitate the chemical connection between the electrode and the leaf. Then, an electrochemical workstation (CHI 1040c) was connected, and the EC concentration in the tea seedling leaves in vivo was calculated by differential pulse voltammetry (test conditions same as Example 2) combined with the standard curve in Example 2. The results are shown in Table 2.

[0045] Table 2. Detection of EC level in tea seedling leaves (μmol / L)

[0046]

[0047] Comparative Example 1

[0048] This comparative example provides a molecular imprinting sensor for detecting epicatechin, and the only difference in the preparation method from Example 1 is that:

[0049] The amide-functionalized single-walled carbon nanotubes were replaced with an equal amount of carboxylated single-walled carbon nanotubes to obtain the molecular imprinting sensor MIP / rGO-Carboimidazole / ZIF8-SWCNT-COOH / SPCE.

[0050] The detection performance of the sensor was tested by the method in Example 2, and the results are shown in Table 2. Figure 2 The linear detection range of the molecular imprinting sensor prepared in Comparative Example 1 was 10 μM to 980 μM, the detection limit was 8.5 μM, and the current decreased, and the detection effect was not as good as that of Example 1.

[0051] Comparative Example 2

[0052] This comparative example provides a molecular imprinting sensor for detecting epicatechin, and the only difference in the preparation method from Example 1 is that:

[0053] The rGO-NH-Carboimidazole was replaced with an equal amount of porous graphene to obtain the molecular imprinting sensor MIP / PG / ZIF8-SWCNT / SPCE.

[0054] The detection performance of the sensor was tested by the method in Example 2, and the results are shown in Table 1. Figure 2 The linear detection range of the molecularly imprinted sensor prepared in Comparative Example 2 was 10 μM to 1 mM, and the detection limit was 7.7 μM. The current decreased again, and the detection effect was not as good as that of Example 1.

[0055] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part 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 application.

Claims

1. A method for preparing a molecularly imprinted sensor for in situ detection of epicatechin in a plant leaf, characterized by, The preparation method comprises the following steps: (1) obtaining a first modified electrode by modifying a zeolite imidazolate framework and an amide functionalized single-walled carbon nanotube on the surface of an electrode; (2) obtaining a second modified electrode by modifying an amino-formyl imidazole modified reduced graphene on the surface of the first modified electrode; (3) preparing a molecularly imprinted polymer film on the surface of the second modified electrode by using chitosan as a monomer and epicatechin as a template molecule, and then preparing the molecularly imprinted sensor after eluting the template molecule; Step (1) specifically comprises: mixing a zeolite imidazolate framework solution, an amide functionalized single-walled carbon nanotube solution and a chitosan solution to obtain a composite material; and then covering the composite material on the surface of an electrode; Step (2) specifically comprises: covering an amino-formyl imidazole modified reduced graphene solution on the surface of the first modified electrode; Step (3) specifically comprises: mixing chitosan, epicatechin 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 then preparing the molecularly imprinted sensor after eluting the template molecule.

2. The production method according to claim 1, characterized by, The zeolite imidazolate framework is ZIF-8; and / or the electrode is a screen-printed electrode.

3. The preparation method according to claim 1, characterized in that, The concentration of the zeolite imidazolate framework solution is 1-10 mg / mL; and / or the concentration of the amide functionalized single-walled carbon nanotube solution is 1-10 mg / mL; and / or the concentration of the chitosan solution is 0.1%-1%.

4. The production method according to claim 1, characterized by, The concentration of the amino-formyl imidazole modified reduced graphene solution is 1-5 mg / mL.

5. The method of claim 1, wherein, The molar ratio of epicatechin to chitosan is (1-5):1; and / or the concentration of chitosan in the mixed solution is 5-10 mM; and / or the electro-polymerization adopts a chronoamperometry method; and / or the electro-polymerization time is 60-150 s; and / or the template molecule is eluted by using a NaOH solution.

6. The molecularly imprinted sensor for detecting epicatechin prepared by the preparation method in any one of claims 1-5.

7. A method for detecting epicatechin in a plant leaf in situ in a living organism, characterized by, The preparation method comprises the following steps: After punching a hole on the surface of a plant leaf, the molecularly imprinted sensor in claim 6 is attached to the punched hole, phosphate buffer solution is added dropwise, and then an electrochemical workstation is connected to detect the concentration of epicatechin by a differential pulse voltammetry method.

Images