Molecularly imprinted sensor for in-situ detection of brassinolide 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 brassinolide in plant leaves and a preparation method of the molecular imprinting sensor. The molecular imprinting sensor for detecting brassinolide is prepared by assembling a 4-MPBA-Fc-GO-metal polynitrogen azole framework composite material 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, brassinolide 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 brassinolide in plant leaves and a preparation method thereof. Background Art

[0002] Brassinolide (BR) is a natural plant hormone widely present in plant organs. Different concentrations of BR exist in different tissue parts of plants such as stems, leaves, flowers, and fruits. BR can promote cell elongation and division and improve the resistance of plants to stresses such as salinity and drought.

[0003] The concentration of BR in plant tissues is extremely low, about between nM and μM. Therefore, detection is very difficult, and there is an urgent need to develop a sensitive and accurate BR detection method. Traditional BR content detection methods mainly include evaporative light scattering method and high performance liquid chromatography. The above methods are all ex vivo analyses, which may cause great damage to plant tissues during sample collection, making it difficult to meet the requirements of in-situ detection. And complex sample pretreatment is required, and small molecule substances in the sample will undergo hydrolysis or oxidation during the treatment process. In contrast, electrochemical methods have the advantages of fast response speed, high sensitivity, good selectivity, wide detection range, etc., and show excellent application potential in in-situ live detection of plants. However, BR has no electroactivity and cannot be directly detected by electrochemical methods.

[0004] As a key organ, plant leaves present an extremely thin and flat morphology in crops such as wheat and corn, resulting in difficult effective implantation of micro-needle electrodes. In addition, the leaf juice is scarce, making it difficult to meet the requirements of electrochemical detection. Therefore, developing a molecularly imprinted sensor that can in-situ detect BR in plant leaves has become a technical challenge that needs to be urgently broken through. Summary of the Invention

[0005] In order to solve the above technical problems, in line with the flat structure of plants, the present invention uses a screen-printed electrode as a base electrode, which is attached to plant leaves, and provides a preparation method of a molecularly imprinted sensor for detecting brassinolide, including: (1) Modifying a 4-mercaptophenylboronic acid (4-MPBA)-ferrocene (Fc)-graphene oxide (GO)-metal polyazole framework composite material on the surface of the screen-printed electrode to obtain a composite material modified electrode; (2) Using β-cyclodextrin (β-CD) as a functional monomer and brassinolide as a template molecule, preparing a molecularly imprinted polymer film on the surface of the composite material modified electrode, and obtaining the molecularly imprinted sensor after eluting the template molecule.

[0006] Among them, GO has the characteristics of high conductivity while being prone to aggregation, while the metal polyazole framework composite material has good dispersibility. Therefore, introducing the metal polyazole framework composite material into GO can effectively improve the conductivity of the sensor and avoid the aggregation of GO. At the same time, the metal polyazole framework composite material has a large specific surface area and high porosity, providing a large number of active sites, which not only facilitates the introduction of boric acid groups (-B(OH)2), but also the regularly arranged pore structure is conducive to the transmission of electrical signals, enabling the sensor to quickly respond to the concentration change of the target substance and further improving the detection ability of the sensor. The Fc therein provides an internal reference signal for the sensor. The electrode modified with such a composite material can provide stronger electron transfer ability and higher stability, improving the sensitivity of the sensor and providing a good basis for subsequent polymerization of the molecularly imprinted membrane.

[0007] The -B(OH)2 in 4-MPBA has the characteristic of selectively recognizing molecules containing 1,2-diols and forming borate esters, thereby significantly improving the specific recognition ability of the sensor for brassinolide. Further combined with the molecular imprinting technology, cavities with the same size and structure as the target molecule are formed on the surface of the MIP, which can efficiently recognize and bind BR. Through the dual recognition effects of 4-MPBA and MIP in the present invention, the specific recognition ability of the sensor for BR molecules is further enhanced. At the same time, the synergistic effect of 4-MPBA and MIP effectively reduces the interference caused by the isomers of the target molecule to the sensor, further improving the selectivity and reliability of BR detection.

[0008] Preferably, the electrode is a screen-printed electrode (SPE).

[0009] Preferably, the metal polyazole framework composite material is MAF-5; and / or, the graphene oxide is HOOC-GO.

[0010] Preferably, step (1) specifically includes: dissolving 4-mercaptophenylboronic acid, ferrocene, graphene oxide and the metal polyazole framework material in a chitosan solution to prepare the composite material, and then covering the composite material on the surface of the electrode to obtain a composite material modified electrode.

[0011] Preferably, the concentration of 4-mercaptophenylboronic acid is 1-5 mg / mL; and / or, the concentration of ferrocene is 5-10 mg / mL; and / or, the concentration of graphene oxide is 0.5-2 mg / mL; and / or, the concentration of the metal polyazole framework material is 0.5-2 mg / mL.

[0012] Preferably, step (2) specifically includes: mixing β-cyclodextrin, brassinolide and PBS solution to obtain a mixed solution; then placing the composite material 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.

[0013] Preferably, the molar ratio of brassinolide to β-cyclodextrin is (2-5):1; and / or, the concentration of brassinolide in the mixed solution is 0.5-1 mM; and / or, the electro-polymerization adopts cyclic voltammetry; and / or, NaOH solution is used to elute the template molecules, and the concentration of NaOH is 10-50 mM.

[0014] 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.

[0015] Furthermore, the present invention provides a molecularly imprinted sensor for detecting brassinolide prepared by the above preparation method.

[0016] Furthermore, the present invention provides a method for in-situ live detection of brassinolide in plant leaves, including: attaching the modified screen-printed electrode to the plant leaf, punching holes on the surface of the plant leaf to release plant juice to the surface of the working electrode, dropping (a small amount of) phosphate buffer solution, then connecting an electrochemical workstation, and detecting the concentration of brassinolide by differential pulse voltammetry.

[0017] In the specific implementation process, the plant is any plant containing brassinolide, including but not limited to fruits, vegetables, flowers, crops, etc.

[0018] In the specific implementation process, the detection part is mainly the plant leaf.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention prepares a molecularly imprinted sensor for detecting brassinolide by layer-by-layer assembly of 4-MPBA-Fc-GO-MAF-5 and MIP on the electrode surface, 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 analyze brassinolide in plants in vivo and in situ, and achieve rapid and reliable quantitative analysis in complex samples, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the process flow chart of the molecularly imprinted sensor in Example 1.

[0021] Figure 2 is the comparison chart of the detection performance of different molecularly imprinted sensors. Detailed implementation manners

[0022] 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 part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. In the embodiments provided in this specification, for those without specific technical or conditions noted, the technologies or conditions described in the literature in the field are followed, or the product specifications are followed. For the reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through regular channels of commercial suppliers.

[0023] Example 1 This example provides a molecularly imprinted sensor for detecting brassinolide. The process flow chart is as Figure 1 shown, and the preparation steps are as follows: (1) The SPE electrode is placed in a 0.01 M phosphate buffer solution (pH = 7.2 - 7.4), and activated by the potentiostatic method (1.7 V) for 180 s to remove impurities on the electrode surface.

[0024] (2) 100 mg of Fc (Aladdin, F108389), 10 mg of HOOC-GO (XFNANO 100009), 10 mg of MAF-5 (XFNANO 105350), and 10 mg of 4-MPBA (Sigma Aldrich 524018) are dissolved in 10 mL of a 0.2% chitosan solution for mixing. After ultrasonic treatment until uniformly dispersed, a 4-MPBA-Fc-GO-MAF-5 composite material is obtained.

[0025] (3) Take 5 μL of the composite material and drop it on the surface of the working electrode and dry it to obtain 4-MPBA-Fc-GO-MAF-5 / SPE.

[0026] (4) Prepare a molecularly imprinted polymer solution with PBS, the molar ratio of BR:β-CD is 2:1, and BR is 0.5 mM. Electro-polymerize MIP 50 cycles on the electrode surface by cyclic voltammetry (-0.4 V - 1 V) to obtain the uneluted MIP / 4-MPBA-Fc-GO-MAF-5 / SPE, and then place the electrode in a 50 mM NaOH solution for 5 min to wash off the template molecules to obtain the molecularly imprinted sensor MIP / 4-MPBA-Fc-GO-MAF-5 / SPE.

[0027] Example 2 In this example, the detection performance of the molecularly imprinted sensor for detecting brassinolide prepared in Example 1 was tested. The steps are as follows: (1) Brassinolide-phosphate buffer solutions (pH = 7.2 - 7.4) with concentrations of 0, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 20 μM were prepared respectively. The molecularly imprinted sensor of Example 1 was connected to an electrochemical workstation (CHI 760E), and detected by differential pulse voltammetry (potential -0.2~0.6 V, potential increment 0.004 V, amplitude 0.05V, pulse width 0.05 s, pulse period 0.5 s, sampling width 0.02 s). As the concentration of the BR 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 standard BR solutions with different concentrations were denoted as I x (x = 1, 2, 3…), and ΔI was calculated respectively through the formula ΔI = I0 - I x , and a set of relationship curves between the logarithm of BR concentration and ΔI were obtained. As x shown, the linear detection range was 10 pM - 20 μM, and the detection limit was 7.45 pM (S / N = 3). Figure 2

[0028] (2) Determination of spike recovery Lettuce was juiced, filtered, and centrifuged at 4000 r / min for 5 min, and the supernatant was taken. Based on this, a BR standard sample was added for the recovery experiment.

[0029] The results are shown in Table 1. The spike recoveries of BR were between 94.35% and 102.35%, and the response current values at the same concentration were relatively consistent (n = 3). The results indicate that the detection results of the molecularly imprinted sensor in Example 1 are accurate and reliable.

[0030] Table 1 Determination of spike recovery of the sensor (n = 3)

[0031] Example 3 In this example, the molecularly imprinted sensor of Example 1 was used to perform in-situ live detection of BR in the leaves of potted lettuce. The steps are as follows: Several small holes were made in the leaves of potted lettuce to release BR. The sensor was fixed on the lower surface of the leaf, and its working electrode was facing the small holes. 20 μL of PBS was dropped on the holes to promote the chemical connection between the electrode and the leaf. Then it was connected to an electrochemical workstation (CHI760E Shanghai Chenhua), and the concentration of BR in live lettuce was recorded by differential pulse voltammetry (the test conditions were the same as in Example 2). The results are shown in Table 2.​

[0032] Table 2 Detection of BR levels in lettuce leaves (nM)

[0033] Comparative Example 1 This comparative example provides a molecularly imprinted sensor for detecting BR. The only difference in the preparation method from Example 1 is that 4-MPBA is replaced with an equal amount of 3-aminophenylboronic acid (3-APBA), as Figure 2 shown. It was found that the linear detection range was 10 pM to 10 μM, and the detection effect was inferior to that of Example 1.

[0034] Comparative Example 2 This comparative example provides a molecularly imprinted sensor for detecting BR. The only difference in the preparation method from Example 1 is that β-CD is replaced with an equal amount of polypyrrole (PPy), as Figure 2 shown. It was found that the linear detection range was 10 nM to 20 μM, and the detection effect was inferior to that of Example 1.

[0035] 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 recorded in the foregoing embodiments, or perform equivalent replacements for some 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 various embodiments of the present invention.

Claims

1. A preparation method of a molecularly imprinted sensor for detecting brassinolide, characterized in that, Comprising: (1) Modifying the surface of a screen-printed electrode with a 4-mercaptophenylboronic acid-ferrocene-graphene oxide-metal polyazole framework composite material to obtain a composite material-modified electrode; (2) Using β-cyclodextrin as a functional monomer and brassinolide as a template molecule, preparing a molecularly imprinted polymer film on the surface of the composite material-modified electrode, and obtaining the molecularly imprinted sensor after eluting the template molecule.

2. The preparation method according to claim 1, wherein The electrode is a screen-printed electrode.

3. The preparation method according to claim 1, characterized in that, The metal polyazole framework composite material is MAF-5; and / or, the graphene oxide is HOOC-GO.

4. The preparation method according to claim 1, characterized in that, Step (1) specifically includes: dissolving 4-mercaptophenylboronic acid, ferrocene, graphene oxide and a metal polyazole framework material in a chitosan solution to prepare the composite material, and then covering the composite material on the surface of the electrode to obtain a composite material-modified electrode.

5. The preparation method according to claim 4, characterized in that, The concentration of 4-mercaptophenylboronic acid is 1-5 mg / mL; and / or, the concentration of ferrocene is 5-10 mg / mL; and / or, the concentration of graphene oxide is 0.5-2 mg / mL; and / or, the concentration of the metal polyazole framework material is 0.5-2 mg / mL.

6. The preparation method according to claim 1, characterized in that, Step (2) specifically includes: mixing β-cyclodextrin, brassinolide and a PBS solution to prepare a mixed solution; then placing the composite material-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 brassinolide to β-cyclodextrin is (2-5):1; and / or, the concentration of brassinolide 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 to 1 V; and / or, the number of cycles of the electro-polymerization is 10-50 cycles.

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

10. A method for in-situ live detection of brassinolide in plant leaves, characterized in that, Comprising: After punching holes in the surface of a plant leaf, attaching the molecularly imprinted sensor according to claim 9 to the punched holes, dropping a phosphate buffer solution, and then connecting an electrochemical workstation to detect the concentration of brassinolide by differential pulse voltammetry.

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