Molecularly imprinted sensor for in-situ detection of epicatechin in plant leaves and preparation and application methods thereof

Abstract

The invention relates to the technical field of analysis and detection, in particular to a molecularly imprinted sensor for in-situ detection of epicatechin in plant leaves and a preparation and application method of the molecularly imprinted sensor. The molecular imprinting sensor for detecting epicatechin is prepared by assembling a zeolite imidazate skeleton-amide functionalized single-walled carbon nanotube composite material, amino-formyl imidazole modified reduced graphene and an MIP film on the surface of an electrode layer by layer. The high-selectivity and high-sensitivity detection on the target molecule epicatechin is realized. The technology can effectively reduce the detection cost, simplify the operation process and improve the performance of the sensor. Particularly, due to the introduction of the MIP, the sensor has the capabilities of in-situ detection and high-precision molecular recognition, can realize rapid and reliable quantitative analysis in a complex sample, and 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 epicatechin in plant leaves, and a preparation and application method thereof. Background Art

[0002] Epicatechin (EC) is a plant polyphenol widely present in tea plants, especially in the actively growing parts such as the leaf tips and leaves of plants. Its main functions include regulating the nutrient distribution in plants, resisting cold, ultraviolet rays, oxidation and pests and diseases, and can help plants cope with plant stresses such as drought and light by regulating behaviors such as stomatal aperture and density.

[0003] In view of the important functions of EC in plants, accurate and rapid detection of it is particularly crucial. Common detection methods for EC include high performance liquid chromatography, gas chromatography-mass spectrometry, etc. 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, and may cause great damage to plant tissues during the sample collection process, and even cause the death of plants. In contrast, electrochemical methods have significant application potential in in-situ living detection of plants due to their advantages such as simple operation, fast response speed, high sensitivity, good selectivity and low cost.

[0004] Plant leaves have different shapes, but the leaves of most crops are extremely thin and flat, and have very little water content, making it difficult to meet the requirements of electrochemical testing. Therefore, how to design an electrochemical sensor capable of in-situ living detection of epicatechin 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 provides a molecularly imprinted sensor for in-situ detection of epicatechin in plant leaves, and its preparation method includes: (1) After modifying the surface of the electrode with zeolitic imidazolate framework and amide-functionalized single-walled carbon nanotubes (f-SWCNT), a first modified electrode is obtained; (2) Modifying the surface of the first modified electrode with amino-carbamoyl imidazole modified reduced graphene oxide (rGO-NH-Carboimidazole) to obtain a second modified electrode; (3) Using chitosan (CS) as a monomer and epicatechin (EC) as a template molecule, a molecularly imprinted polymer membrane (MIP) is prepared on the surface of the second modified electrode, and the template molecule is eluted to obtain the molecularly imprinted sensor.

[0006] Among them, the composite nanomaterial composed of zeolitic imidazolate framework and amide-functionalized single-walled carbon nanotubes has high conductivity, good dispersibility and a large surface area. The electrode modified with this composite nanomaterial can provide stronger electron transfer ability and higher stability, providing a good foundation for subsequent molecular imprinting. And rGO-NH-Carboimidazole is a strong catalytic material, which further enhances the response efficiency of EC on the electrode surface, thus significantly improving the detection sensitivity. On the surface of the electrode modified with rGO-NH-Carboimidazole, CS acts as a monomer and interacts with the EC template molecule to form a MIP film with specific recognition ability for EC. 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 EC, effectively reducing the interference of other components in the sample (such as flavonoids, amino acids, sugars, etc.) on the detection, and significantly improving the selectivity of the detection. Through the synergistic effect of rGO-NH-Carboimidazole and MIP, the present invention enhances the specific recognition and catalytic performance, and further improves the selectivity and sensitivity of epicatechin detection.

[0007] Preferably, the zeolitic imidazolate framework is ZIF-8; and / or, the electrode is a screen-printed carbon electrode (SPCE).

[0008] Preferably, step (1) specifically includes: mixing a zeolitic imidazolate framework solution, an amide-functionalized single-walled carbon nanotube solution and a chitosan solution to obtain a composite material; then covering the composite material on the surface of the electrode.

[0009] Preferably, the concentration of the zeolitic 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%.

[0010] Preferably, step (2) specifically includes: covering the surface of the first modified electrode with an amino-carbamoyl imidazole modified reduced graphene oxide solution.

[0011] Preferably, the concentration of the amino-carbamoyl imidazole modified reduced graphene oxide solution is 1-5 mg / mL.

[0012] Preferably, step (3) specifically includes: 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 preparing the molecularly imprinted sensor after eluting the template molecule.

[0013] 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, chronoamperometry is used for the electropolymerization; and / or, the time of the electropolymerization is 60~150 s; and / or, a NaOH solution is used to elute the template molecules.

[0014] Preferably, the time for eluting the template molecules with a NaOH solution is 5~15 min.

[0015] Preferably, the electrode is activated before use.

[0016] More preferably, the activation step includes: placing the electrode in a phosphate buffer solution (pH = 7.2~7.4), and activating it for 180~300 s by the potentiostatic method (1.7 V) to remove impurities on the electrode surface.

[0017] Furthermore, the present invention provides a molecularly imprinted sensor for detecting epicatechin prepared by the above preparation method.

[0018] Furthermore, the present invention provides a method for in-situ and in-vivo detection of epicatechin in plant leaves, including: punching holes on the surface of plant leaves to release plant sap, attaching the above-mentioned molecularly imprinted sensor to the punched holes, then dropping 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.

[0019] In the specific implementation process, the plant is any plant containing epicatechin, including but not limited to fruits, vegetables, flowers, crops, etc.

[0020] In the specific implementation process, the punching site is mainly the plant leaves.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: By using a zeolitic imidazolate framework-functionalized single-walled carbon nanotube composite material, amino-carbamoyl imidazole-modified reduced graphene oxide, and layer-by-layer assembly of an MIP film on the electrode surface, the present invention prepares a molecularly imprinted sensor for detecting epicatechin, achieving 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. In particular, the introduction of MIP enables the sensor to have the ability of in-situ detection and high-precision molecular recognition, and can perform in-situ and in-vivo detection of epicatechin in plant leaves, with broad application prospects. Description of the Drawings

[0022] Figure 1 It is the process flow chart of the molecularly imprinted sensor in Example 1.

[0023] Figure 2 It is a comparison chart of the detection performance of different molecularly imprinted sensors. Specific implementation manners

[0024] 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. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the scope of protection 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. The reagents or instruments not indicating the manufacturer can all be obtained as conventional products through regular channels.

[0025] Example 1 This example provides a molecularly imprinted sensor for detecting epicatechin. The process flow chart is as Figure 1 shown, and the preparation steps are as follows: (1) The SPCE electrode (Wave Detection Technology (Weihai) Co., Ltd., model POTEN) was placed in 0.01 M phosphate buffer solution (pH = 7.2 - 7.4), and was activated by the potentiostatic method (1.7 V) for 180 s to remove impurities on the electrode surface.

[0026] (2) 25 mg of zeolitic imidazolate framework ZIF-8 (Nanjing Xianfeng Nano Materials Technology Co., Ltd., XFNANO-102723) and 25 mg of amide-functionalized single-walled carbon nanotube f-SWCNT (Sigma Co., Ltd., Germany, MERCK-685380) were dissolved in 5 mL of 0.2% chitosan solution for mixing. After ultrasonic treatment until uniformly dispersed, a ZIF8-SWCNT composite material was obtained. 3 μL was taken and dropped on the surface of the working electrode and dried to obtain ZIF8-SWCNT / SPCE.

[0027] (3) 3 mg of amino-carbamoyl imidazole modified reduced graphene oxide rGO-NH-Carboimidazole (Nanjing Xianfeng Nano Materials Technology Co., Ltd., XFNANO-100396) was dissolved in 3 mL of ultrapure water for mixing. After ultrasonic treatment until uniformly dispersed, 3 μL was taken and dropped on the surface of the ZIF8-SWCNT / SPCE working electrode and dried to obtain rGO-NH-Carboimidazole / ZIF8-SWCNT / SPCE.

[0028] (4) Molecular imprinting polymer solution was prepared with PBS, the molar ratio of EC to CS was 2:1, and CS was 10 mM. MIP was electropolymerized on the electrode surface by chronoamperometry (-1 V) for 120 seconds to obtain uneluted MIP / rGO-NH-Carboimidazole / ZIF8-SWCNT / SPCE. The electrode was then placed in 100 mM NaOH solution for 10 min to elute the template molecules, and the molecular imprinting sensor MIP / rGO-NH-Carboimidazole / ZIF8-SWCNT / SPCE was obtained.

[0029] Example 2 In this example, the detection performance of the molecular imprinting sensor for detecting epicatechin prepared in Example 1 was tested, and the steps were as follows: Prepare epicatechin-phosphate buffer (pH=7.2-7.4) solutions with concentrations of 10μmoL / L, 50μmoL / L, 100μmoL / L, 200μmoL / L, 500μmoL / L, 1000μmoL / L, and 4000μmoL / L, respectively, and use the molecular imprinting sensor for detecting epicatechin prepared in Example 1 to perform differential pulse voltammetry detection (voltage range is -0.2-0.5V, interval time is 0.5s, modulation time is 0.05s, amplitude potential is 0.025V). As the concentration of EC solution increases, the oxidation peak of EC gradually becomes higher, and the peak currents obtained from standard epicatechin solutions of different concentrations are recorded as I1, I2, I3..., respectively, thereby obtaining a set of relationship curves between the logarithm of the concentration and the current I, and making a standard curve for epicatechin. Figure 2 As shown, the linear detection range is 10μM-4mM and the detection limit is 0.325 μM (S / N=3).

[0030] Example 3 In this embodiment, the recovery rate of the molecular imprinting sensor of Example 1 is detected, and the steps are as follows: the tea seedlings and leaves are cracked in an efficient sample crushing system, and then a buffer solution of the same mass as the crushed leaves is added. Then centrifuge in a high-speed centrifuge, the speed of the centrifuge is set to 10000rpm, the time is set to 20min, and the temperature is -4°C. After the centrifugation, the supernatant is aspirated with a pipette. According to the linear regression equation, the tea polyphenol content in the original leaves of the tea seedlings is calculated to be 2.17±0.035mmol / L. Then, the standard addition method is used to add known concentrations of EC (1, 3, 5, 10mmol / L), and then the molecular imprinting sensor of Example 1 is used for DPV detection, and finally the spiked recovery rate is calculated. The results of the recovery test are shown in Table 1 below. The results show that the molecular imprinting sensor of Example 1 has the potential for practical application.

[0031] Table 1 Recovery rate of EC content in tea seedling leaves (mmol / L)

[0032] Example 4 In this example, the molecularly imprinted sensor of Example 1 was used to detect EC in the leaves of potted tea seedlings in situ in vivo. The steps are as follows: The experimental material was the leaves of potted tea seedlings. A hole with a diameter of 1 mm was punched in the leaves using a hole puncher. The molecularly imprinted sensor of Example 1 was attached to the lower surface of the leaf, with its working electrode facing the small hole. 5 μL of 10 mM phosphate (PBS) buffer was added dropwise at the punched hole to promote the chemical connection between the electrode and the leaf. Then, an electrochemical workstation (CHI 1040c) was connected, and the EC concentration in the leaves of living tea seedlings was calculated by differential pulse voltammetry (test conditions were the same as in Example 2) in combination with the standard curve in Example 2. The results are shown in Table 2.

[0033] Table 2 Detection of EC level in tea seedling leaves (μmol / L)

[0034] Comparative Example 1 This comparative example provided a molecularly imprinted sensor for detecting epicatechin. The only difference in the preparation method from Example 1 was that: The amide-functionalized single-walled carbon nanotubes were replaced with an equal amount of carboxylated single-walled carbon nanotubes to obtain the molecularly imprinted sensor MIP / rGO-Carboimidazole / ZIF8-SWCNT-COOH / SPCE.

[0035] The detection performance of the sensor was tested using the method in Example 2, and the results were as Figure 2 shown. The linear detection range of the molecularly imprinted sensor prepared in Comparative Example 1 was 10 μM - 980 μM, the detection limit was 8.5 μM, and the current decreased, with the detection effect being inferior to that of Example 1.

[0036] Comparative Example 2 This comparative example provided a molecularly imprinted sensor for detecting epicatechin. The only difference in the preparation method from Example 1 was that: rGO-NH-Carboimidazole was replaced with an equal amount of porous graphene to obtain the molecularly imprinted sensor MIP / PG / ZIF8-SWCNT / SPCE.

[0037] The detection performance of the sensor was tested using the method in Example 2, and the results were as Figure 2 shown. The linear detection range of the molecularly imprinted sensor prepared in Comparative Example 2 was 10 μM - 1 mM, the detection limit was 7.7 μM, and the current decreased again, with the detection effect being inferior to that of Example 1.

[0038] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for 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 in-situ detection of epicatechin in plant leaves, characterized in that, Comprising: (1) After modifying the surface of the electrode with zeolitic imidazolate framework and amide-functionalized single-walled carbon nanotubes, a first modified electrode is obtained; (2) Modifying the surface of the first modified electrode with amino-carbamoyl imidazole modified reduced graphene oxide to obtain a second modified electrode; (3) Using chitosan as a monomer and epicatechin as a template molecule, a molecularly imprinted polymer film is prepared on the surface of the second modified electrode, and after eluting the template molecule, the molecularly imprinted sensor is obtained.

2. The preparation method according to claim 1, wherein The zeolitic 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, Step (1) specifically includes: mixing a zeolitic imidazolate framework solution, an amide-functionalized single-walled carbon nanotube solution and a chitosan solution to obtain a composite material; then covering the composite material on the surface of the electrode.

4. The preparation method according to claim 3, characterized in that, The concentration of the zeolitic 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%.

5. The preparation method according to claim 1, characterized in that, Step (2) specifically includes: covering the amino-carbamoyl imidazole modified reduced graphene oxide solution on the surface of the first modified electrode.

6. The preparation method according to claim 5, characterized in that, The concentration of the amino-carbamoyl imidazole modified reduced graphene oxide solution is 1-5 mg / mL.

7. The preparation method according to claim 1, characterized in that, Step (3) specifically includes: mixing chitosan, epicatechin and 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; after eluting the template molecule, the molecularly imprinted sensor is obtained.

8. The preparation method according to claim 7, characterized in that, 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 chronoamperometry; and / or, the time of the electro-polymerization is 60-150 s; and / or, NaOH solution is used to elute the template molecule.

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

10. A method for in-situ and in-vivo detection of epicatechin in plant leaves, characterized in that, Comprising: After punching holes in the surface of the plant leaf, the molecularly imprinted sensor according to claim 9 is pasted at the punched holes, phosphate buffer solution is added dropwise, and then an electrochemical workstation is connected, and the concentration of epicatechin is detected by differential pulse voltammetry.

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