A molecular imprinting sensor for detecting brassinosteroids 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 brassinosteroids in plant leaves and a preparation method thereof.The present application utilizes 4-MPBA-Fc- GO-metal polyazole framework composite material and MIP to perform layer-by-layer assembly on the surface of an electrode, thereby preparing a molecular imprinting sensor for detection of brassinosteroids, and realizing high selectivity and high sensitivity detection of target molecules.This technology can effectively reduce detection cost, simplify the operation process, and improve the performance of the sensor.The molecular imprinting sensor of the present application can be used for in-vivo and in-situ analysis of brassinosteroids in plant leaves, and can realize rapid and reliable quantitative analysis in complex samples, and has a wide application prospect.

Description

TECHNICAL FIELD

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

[0002] Brassinosteroid (BR) is a natural plant hormone widely existing 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 stress such as salt, alkali and drought.

[0003] The concentration of BR in plant tissues is extremely low, about nM to μM, so it is very difficult to detect. Therefore, a sensitive and accurate BR detection method is urgently needed. Traditional BR content detection methods mainly include evaporation light scattering method and high performance liquid chromatography. The above methods are in-vitro analysis, which may cause great damage to plant tissues during sample collection, and are difficult to meet the needs of in-situ detection, and need complex sample pretreatment, which may cause hydrolysis or oxidation of small molecules in the sample during the treatment process. In comparison, the electrochemical method has the advantages of fast response, high sensitivity, good selectivity and wide detection range, and has excellent application potential in in-situ live detection of plants. However, BR is not electrically active and cannot be directly detected by electrochemical methods.

[0004] Plant leaves, as key organs, present an extremely thin and flat morphology in crops such as wheat and corn, making it difficult for microneedle electrodes to be effectively implanted. In addition, the leaf juice is scarce, which is difficult to meet the needs of electrochemical detection. Therefore, it is a technical challenge to develop a molecular imprinting sensor for in-situ live detection of BR in plant leaves. SUMMARY

[0005] In order to solve the above technical problems, the present application conforms to the flat structure of plants, uses a screen-printed electrode as a base electrode, and is attached to the plant leaves, and provides a preparation method of a molecular imprinting sensor for detecting brassinosteroid, comprising:

[0006] (1) modifying a 4-mercapto phenyl boronic acid (4-MPBA)-ferrocene (Fc)-graphene oxide (GO)-metal polyazole framework composite material on the surface of a screen-printed electrode to obtain a composite material modified electrode;

[0007] (2) preparing a molecular imprinting polymer film on the surface of the composite material modified electrode by using β-cyclodextrin (β-CD) as a functional monomer and brassinosteroid as a template molecule, and preparing the molecular imprinting sensor after eluting the template molecule.

[0008] The GO has the characteristics of high conductivity and easy aggregation, and the metal polyazolate framework composite material has good dispersity, so that the introduction of the metal polyazolate framework composite material into the GO can effectively improve the conductivity of the sensor and avoid the aggregation of the GO. Meanwhile, the metal polyazolate framework composite material has a large specific surface area and high porosity, so that a large number of active sites are provided, the introduction of the boronic acid group (-B(OH)2) is facilitated, and the regular arrangement of the pore structure is also beneficial to the transmission of the electrical signal, so that the sensor can quickly respond to the concentration change of the target substance and further improve the detection capability of the sensor. The Fc provides an internal reference signal for the sensor, the electrode modified by the composite material can provide stronger electron transfer capability and higher stability, and the sensitivity of the sensor is improved, which provides a good foundation for the subsequent polymerization of the molecularly imprinted film.

[0009] The -B(OH)2 in the 4-MPBA has the characteristics of selectively recognizing molecules containing 1,2-diol and forming borate ester, so that the specific recognition capability of the sensor for brassinosteroids is significantly improved. Further combined with the molecular imprinting technology, the cavities with the same size and structure as the target molecules are formed on the surface of the MIP, which can efficiently recognize and combine the BR. Through the dual recognition of the 4-MPBA and the MIP, the specific recognition capability of the sensor for the BR molecules is further enhanced, and the synergistic effect of the 4-MPBA and the MIP effectively reduces the interference of the isomers of the target molecules on the sensor, and further improves the selectivity and reliability of the BR detection.

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

[0011] Preferably, the metal polyazolate framework composite material is MAF-5; and / or, the graphene oxide is HOOC-GO.

[0012] Preferably, the step (1) specifically comprises: dissolving 4-mercaptophenyl boronic acid, ferrocene, graphene oxide and a metal polyazolate framework material in a chitosan solution to obtain the composite material, and then covering the composite material on the surface of the electrode to obtain a composite material modified electrode.

[0013] Preferably, the concentration of the 4-mercaptophenyl boronic acid is 1-5 mg / mL; and / or, the concentration of the ferrocene is 5-10 mg / mL; and / or, the concentration of the graphene oxide is 0.5-2 mg / mL; and / or, the concentration of the metal polyazolate framework material is 0.5-2 mg / mL.

[0014] Preferably, step (2) specifically comprises: mixing the beta-cyclodextrin, brassinolide and PBS solution to prepare a mixed solution; and then placing the composite material modified electrode in the mixed solution for electro-polymerization to obtain a molecularly imprinted polymer film; and eluting the template molecule to prepare the molecularly imprinted sensor.

[0015] Preferably, the molar ratio of brassinolide to beta-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, the template molecule is eluted by using a NaOH solution with a concentration of 10-50 mM.

[0016] 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.

[0017] Further, the present application provides a molecularly imprinted sensor for detecting brassinolide prepared by the preparation method.

[0018] Further, the present application provides a method for in-situ and in-vivo detection of brassinolide in plant leaves, comprising: attaching the modified screen-printed electrode to the plant leaves, and punching holes on the surface of the plant leaves to release plant juice to the surface of the working electrode, and then adding (a small amount of) phosphate buffer, and then connecting an electrochemical workstation to detect the concentration of brassinolide by differential pulse voltammetry.

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

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

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] The present application uses 4-MPBA-Fc-GO-MAF-5 and MIP to layer-by-layer assemble on the surface of the electrode to prepare a molecularly imprinted sensor for detecting brassinolide, which realizes 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. The molecularly imprinted sensor of the present application can be used for in-vivo and in-situ analysis of brassinolide in plants, and can realize rapid and reliable quantitative analysis in complex samples, which has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

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

[0024] Figure 2are different molecular imprinting sensor detection performance comparison chart. DETAILED DESCRIPTION

[0025] 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, rather than 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 noted are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not noted by the manufacturer are conventional products that can be purchased through a regular channel.

[0026] Example 1

[0027] The present embodiment provides a molecular imprinting sensor for detecting brassinolide, and a process flow chart is shown as follows: Figure 1 The preparation steps are as follows:

[0028] (1) The SPE electrode is 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.

[0029] (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 mixed in 10 mL of 0.2% chitosan solution, and ultrasonically dispersed to obtain a 4-MPBA-Fc-GO-MAF-5 composite material.

[0030] (3) 5 μL of the composite material is dropped on the surface of the working electrode and dried to obtain a 4-MPBA-Fc-GO-MAF-5 / SPE.

[0031] (4) The molecular imprinting polymer solution is prepared with PBS, the molar ratio of BR:β-CD is 2:1, and BR is 0.5 mM. The MIP is electro-polymerized on the surface of the electrode by cyclic voltammetry (-0.4 V-1 V) for 50 cycles to obtain a non-eluted MIP / 4-MPBA-Fc-GO-MAF-5 / SPE, and then the electrode is placed in a 50 mM NaOH solution for 5 min to elute the template molecule to obtain a molecular imprinting sensor MIP / 4-MPBA-Fc-GO-MAF-5 / SPE.

[0032] Example 2

[0033] The detection performance of the brassinolide detection molecular imprinting sensor prepared in Example 1 was tested in this example, and the steps were as follows:

[0034] (1) 0, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 20 μM of brassinolide-phosphate buffer (pH = 7.2-7.4) were prepared respectively. The molecular imprinting sensor of Example 1 was connected to an electrochemical workstation (CHI 760E), and the differential pulse voltammetry method was used for detection (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 BR solution increased, the oxidation peak of Fc gradually decreased. The peak current of the oxidation peak in the blank solution was taken as I0, and the peak currents of the standard BR solutions of different concentrations were taken as I x (x = 1, 2, 3…), respectively. ΔI x was calculated by the formula ΔI = I0-I x , and a set of curves of the logarithm of BR concentration and ΔI was obtained. As shown in Figure 2 , the linear detection range was 10 pM-20 μM, and the detection limit was 7.45 pM (S / N = 3).

[0035] (2) Determination of recovery rate

[0036] The juice of lettuce was squeezed, filtered, and centrifuged at a speed of 4000 r / min for 5 min, and the supernatant was taken. The recovery rate experiment was carried out by adding BR standard sample to the supernatant.

[0037] The results are shown in Table 1. The recovery rate of BR was between 94.35 % and 102.35 %, and the response current values of the same concentration were consistent (n = 3). The results showed that the detection results of the molecular imprinting sensor of Example 1 were accurate and reliable.

[0038] Table 1 Determination of recovery rate of sensor (n = 3)

[0039]

[0040] Example 3

[0041] In this example, the molecular imprinting sensor of Example 1 was used to detect BR in the leaves of potted lettuce in situ and in vivo, and the steps were as follows:

[0042] Several small holes were punched on the leaves of the potted lettuce to release BR, and the sensor was fixed on the lower surface of the leaves, with the working electrode facing the small holes. 20 μL PBS was dropped on the holes to promote the chemical connection between the electrode and the leaves. Then, an electrochemical workstation (CHI760E, Shanghai Chenhua) was connected, and the concentration of BR in the living lettuce was recorded by differential pulse voltammetry (test conditions were the same as in Example 2), and the results are shown in Table 2.

[0043] Table 2. Detection of BR levels in lettuce leaves (nM)

[0044]

[0045] Comparative Example 1

[0046] This comparative example provides a molecular imprinting sensor for detecting BR, and the preparation method is only different from Example 1 in that 4-MPBA is replaced with an equal amount of 3-aminophenylboronic acid (3-APBA), as shown in Figure 2 The results show that the linear detection range is 10 pM to 10 μM, and the detection effect is not as good as that of Example 1.

[0047] Comparative Example 2

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

[0049] β-CD is replaced with an equal amount of polypyrrole (PPy), as shown in Figure 2 The results show that the linear detection range is 10 nM to 20 μM, and the detection effect is not as good as that of Example 1.

[0050] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions 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 detecting brassinosteroids, characterized in that, The preparation method comprises the following steps: (1) modifying the surface of a silk screen printing electrode with a 4-mercaptophenyl boronic acid-ferrocene-graphene oxide-metal azolate framework composite material to obtain a composite material modified electrode; (2) preparing a molecularly imprinted polymer film on the surface of the composite material modified electrode by using β-cyclodextrin as a functional monomer and brassinolide as a template molecule, and then preparing the molecularly imprinted sensor after eluting the template molecule; Step (1) specifically comprises the following steps: dissolving 4-mercaptophenyl boronic acid, ferrocene, graphene oxide and a metal azolate framework material in a chitosan solution to obtain the composite material, and then covering the composite material on the surface of the electrode to obtain the composite material modified electrode; the metal azolate framework material is MAF-5; and the graphene oxide is HOOC-GO.

2. The production method according to claim 1, characterized by, The concentration of the 4-mercaptophenyl boronic acid is 1-5 mg / mL; and / or, the concentration of the ferrocene is 5-10 mg / mL; and / or, the concentration of the graphene oxide is 0.5-2 mg / mL; and / or, the concentration of the metal azolate framework material is 0.5-2 mg / mL.

3. The production method according to claim 1, characterized by, Step (2) specifically comprises the following steps: mixing β-cyclodextrin, brassinolide and a 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 then preparing the molecularly imprinted sensor after eluting the template molecule.

4. The production method according to claim 3, characterized by, 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 is performed by using a cyclic voltammetry method; and / or, the template molecule is eluted by using a NaOH solution.

5. The preparation method according to claim 4, 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.

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

7. A method for detecting brassinosteroids in situ in a leaf of a plant, comprising contacting the leaf with a compound of claim 1. 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 brassinolide by using a differential pulse voltammetry method.

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