A flexible molecularly imprinted sensor for detecting zeatin riboside, its preparation method and application

Abstract

The present invention relates to the field of analytical detection technology, and particularly to a flexible molecularly imprinted sensor for detecting zeatin riboside, and a preparation method and application thereof. The preparation method includes obtaining a laser-induced graphene pattern on a polyimide tape through laser direct writing technology, and transferring it onto polydimethylsiloxane. Then, a composite material of zinc-aluminum layered double metal hydroxide and iron-based metal-organic framework is modified onto the working electrode. Then, using o-phenylenediamine as a monomer and zeatin riboside as a template molecule, a molecularly imprinted membrane is prepared on the electrode surface. After eluting the template molecule, a flexible molecularly imprinted sensor is obtained. The flexible molecularly imprinted sensor of the present invention can realize in-situ in-vivo detection of zeatin riboside in plants, is beneficial to adapting to the irregular surface of plant tissues, and does not cause essential damage to the detection site, providing a low-cost and highly accurate detection tool for in-vivo detection of plants and providing a basis for understanding the physiological state of plants.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a flexible molecularly imprinted sensor for detecting zeatin riboside, a preparation method thereof, and an application thereof. Background Art

[0002] Cytokinins play an important role in regulating plant growth. Among them, zeatin riboside (ZR) is an endogenous purine cytokinin in plants, which widely exists in higher plants, especially in actively growing parts such as the root tips and leaves of plants. Its main functions include promoting cell division, regulating cell differentiation, delaying leaf senescence, and promoting lateral bud growth, etc., and it can help plants cope with plant stresses such as drought and light by regulating behaviors such as stomatal aperture and density.

[0003] The ZR level in plants is generally low. Traditionally, the methods for ZR determination are mostly chromatography and immunoassay. These methods have low detection efficiency, mainly adopt in vitro detection forms, and have high costs for detection equipment and complex operations. Compared with other methods, electrochemical sensors have low costs, simple operations, are convenient for portability, have high detection accuracy, and fast response speed, and can be applied to the in vivo detection field of small molecules in plants.

[0004] However, the tissue parts of plants are very soft and mostly have irregular shapes. Traditional sensors based on rigid electrodes (such as glassy carbon electrodes, gold electrodes, etc.) cannot effectively fit the plant surface, reducing the reliability and accuracy of detection results. Therefore, how to design a flexible electrode capable of in situ in vivo detecting zeatin riboside in plants has become a technical problem urgently to be solved in this field. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a preparation method of a flexible molecularly imprinted sensor for detecting zeatin riboside, including: modifying a ZnAl-LDH and MIL-101(Fe) composite material on the surface of a LIG / PDMS electrode to obtain a ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode, and then using o-phenylenediamine as a monomer and zeatin riboside as a template molecule to prepare a molecularly imprinted membrane (MIP) on the surface of the ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode, and obtaining the flexible molecularly imprinted sensor after eluting the template molecule.

[0006] Among them, ZnAl-LDH is a layered double metal hydroxide (LDHs) composed of zinc (Zn) and aluminum (Al), which has good stability and catalytic properties. In the present invention, it is compounded and co-modified with an iron-based metal-organic framework (MOFs) material (MIL-101(Fe)) on the surface of a flexible LIG / PDMS electrode, so that the electrode has excellent stability and electroactivity when detecting zeatin riboside, provides a stable response current for the sensor, and further combines with the molecular imprinting technology, so that the flexible molecular imprinting sensor has excellent detection effects during in-situ in-vivo detection, realizing the highly selective detection of ZR.

[0007] Preferably, the step of modifying the ZnAl-LDH and MIL-101(Fe) composite material on the surface of the LIG / PDMS electrode includes: dissolving ZnAl-LDH and MIL-101(Fe) in water, dispersing evenly to obtain a ZnAl-LDH-MIL-101(Fe) composite solution, and then dropping the composite solution on the working electrode surface of the LIG / PDMS electrode to obtain a ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode.

[0008] Preferably, ZnAl-LDH and MIL-101(Fe) are dissolved in water according to a mass ratio of 1:(2-10), and dispersed evenly to obtain a ZnAl-LDH-MIL-101(Fe) composite solution.

[0009] Preferably, the step of preparing the molecularly imprinted membrane includes: mixing a zeatin riboside solution and an o-phenylenediamine solution to obtain a mixed solution; then placing the ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode in the mixed solution, and performing electro-polymerization by cyclic voltammetry. After washing and drying, the template molecules are eluted to prepare the flexible molecularly imprinted sensor.

[0010] Preferably, the concentration of zeatin riboside in the zeatin riboside solution is 1-5 mM; the concentration of o-phenylenediamine in the o-phenylenediamine solution is 1-10 mM.

[0011] Preferably, the conditions for the electro-polymerization include: the voltage is -0.2 V to 1.0 V, the number of electro-polymerization cycles is 10-30 cycles, and the scanning rate is 25-100 mV / S.

[0012] Preferably, acetic acid solution is used to elute the template molecules. Preferably, the concentration of the acetic acid solution is 5%-15%.

[0013] Preferably, the preparation steps of the LIG / PDMS electrode include: preparing laser-induced graphene on a polyimide tape (PI) using laser direct writing technology, and further transferring it to polydimethylsiloxane (PDMS). A reference electrode is coated with Ag / AgCl paste, and after encapsulating the wire and the passivation region, the LIG / PDMS electrode is obtained.

[0014] The LIG / PDMS electrode is a laser-induced graphene (LIG) electrode with good flexibility and stretching properties.

[0015] The LIG / PDMS electrode includes a working electrode, a counter electrode, and a reference electrode.

[0016] Furthermore, the present invention provides a flexible molecularly imprinted sensor prepared by the preparation method in any of the above embodiments.

[0017] Furthermore, the present invention provides the application of the flexible molecularly imprinted sensor in in-situ live detection of zeatin riboside in plants.

[0018] In the specific implementation process, the plant is any plant containing zeatin riboside, including but not limited to fruits, vegetables, flowers, crops, etc.

[0019] Furthermore, the present invention provides a method for in-situ live detection of zeatin riboside in plants, including: after drilling a hole on the plant surface, attaching the flexible molecularly imprinted sensor to the drilled hole, dropping a buffer solution at the drilled hole, then connecting an electrochemical workstation, and detecting the concentration of zeatin riboside by differential pulse voltammetry.

[0020] In the specific implementation process, the drilling site can be the stem, leaf, fruit, etc. of the plant.

[0021] In the specific implementation process, the concentration of zeatin riboside can be obtained by establishing a standard curve.

[0022] In the specific implementation process, a relationship curve between current and the concentration of zeatin riboside is established using zeatin riboside-phosphate buffer solutions (pH = 7.2 - 7.4) with different concentrations, thereby obtaining the standard curve.

[0023] In the specific implementation process, the detection parameters of differential pulse voltammetry include: potential -0.2 - 0.6 V, pulse width 0.02 s, amplitude 0.05 V, pulse period 1 s, sampling width 0.02 s.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The present invention provides a flexible molecularly imprinted sensor for detecting zeatin riboside. This flexible molecularly imprinted sensor can achieve in-situ and in-vivo detection of zeatin riboside in plants, avoiding the mechanical mismatch problem caused by the rigid sensor and the soft tissue of plants in in-vivo detection. The flexible molecularly imprinted sensor of the present invention can still retain stable detection performance within a moderate deformation range, which is beneficial to adapting to the irregular surface of plant tissues and does not cause essential damage to the detection site, providing a low-cost and highly accurate detection tool for in-vivo detection of plants and providing a basis for understanding the physiological state of plants. Description of the Drawings

[0026] Figure 1 is the process flow chart of Embodiment 1 of the present invention.

[0027] Figure 2 is the comparison chart of the detection performance of three sensors: MIP / ZnAl-LDH-MIL-101(Fe) / LIG / PDMS, MIP / MgAl-LDH-MIL-101(Fe) / LIG / PDMS, and MIP / ZnAl-LDH-Cu-MOF / LIG / PDMS. Detailed Embodiments

[0028] 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. Apparently, 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 creative efforts shall fall within the protection scope of the present invention.

[0029] In the embodiments provided in this specification, for those without specific technical or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels. ZnAl-LDH (product number 103063) and MIL-101(Fe) (product number 103320) are purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.

[0030] At the endpoints and any values within the ranges disclosed in this specification, these ranges or values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0031] Embodiment 1

[0032] This embodiment provides a flexible molecularly imprinted sensor for detecting zeatin riboside. The process flow chart is as Figure 1 shown, and the preparation method is as follows:

[0033] (1) Stick a polyimide (PI) tape on a 6×6 cm 2 polytetrafluoroethylene plate. Use CAD to design the electrode pattern, including the working electrode, counter electrode and reference electrode. Load the derived electrode pattern into the software of the laser printing system. The printing power is 53%, and the printing depth is 10 μm. Pattern the PI tape.

[0034] (2) Use a spin coater to coat liquid polydimethylsiloxane (PDMS) on the polytetrafluoroethylene plate. The coating speed is 100 rpm and the time is 90 s. Then put it into a vacuum drying oven at 100 °C and heat it for 12 h under vacuum. Then, peel the PI film from the PDMS to obtain the LIG / PDMS electrode.

[0035] (3) Apply an appropriate amount of PDMS to the non-working area of the electrode and heat it for curing to avoid short circuit. Finally, coat Ag / AgCl silver paste on the exposed reference electrode and heat it for curing.

[0036] (4) Place the LIG / PDMS electrode in a phosphate buffer solution (0.1 mol / L, pH = 7.2 - 7.4) and activate it for 180 s by the potentiostatic method (-1.7 V) to remove impurities on the electrode surface.

[0037] (5) Weigh 1 mg of ZnAl-LDH and 5 mg of MIL-101(Fe) respectively and place them in 1 ml of ultrapure water. Ultrasonic for 2 hours to make them disperse evenly to obtain a ZnAl-LDH-MIL-101(Fe) composite solution. Take 5 μL and drop it on the surface of the working electrode and dry it to obtain ZnAl-LDH-MIL-101(Fe) / LIG / PDMS.

[0038] (6) Mix a 2 mM zeatin riboside solution and a 4 mM o-phenylenediamine solution in equal volume. Then place the electrode in it and perform electro-polymerization for 20 cycles by cyclic voltammetry (CV). The voltage range is -0.2 V to 1.0 V, and the scanning speed is 50 mV / S. After polymerization, wash away the residual polymerization solution with ultrapure water. After drying, place it in a 10% acetic acid solution for 50 s to elute the template molecules. After washing and drying, obtain the flexible molecularly imprinted sensor MIP / ZnAl-LDH-MIL-101(Fe) / LIG / PDMS for detecting zeatin riboside.

[0039] Example 2

[0040] In this example, the detection performance of the flexible molecularly imprinted sensor for detecting zeatin riboside prepared in the example was tested, and the steps are as follows:

[0041] (1) Prepare zeatin riboside-phosphate buffer (pH = 7.2 - 7.4) solutions with concentrations of 0, 1 nmol / L, 50 nmol / L, 100 nmol / L, 500 nmol / L, 50 μmol / L, 100 μmol / L, and 500 μmol / L respectively. Connect the flexible molecularly imprinted sensor of Example 1 to an electrochemical workstation (CHI 1040c) and detect by differential pulse voltammetry (potential -0.2~0.6V, pulse width 0.02 s, amplitude 0.05 V, pulse period 1s, sampling width 0.02 s). As the concentration of the ZR solution increases, the oxidation peak of Fc gradually decreases. Take the peak current of the oxidation peak obtained in the blank solution as I0, and record the peak currents obtained from standard zeatin riboside solutions with different concentrations as I1, I2, I3... respectively. Calculate ΔI1, ΔI2, ΔI3... respectively through the formula ΔI = I0 - I1 (I2, I3...), so as to obtain a relationship curve between the logarithm of the ZR concentration and ΔI, and make a standard curve of zeatin riboside, as Figure 2 shown. The linear detection range is 1 nmol / L~500 μmol / L, and the detection limit is 0.387 nmol / L (S / N = 3).

[0042] (2) After grinding and centrifuging the leaves of strawberry seedlings, the supernatant was taken to detect the zeatin riboside level by combining with the standard curve. Using this as a matrix, ZR standard samples (10 nmol / L, 50 nmol / L, 100 nmol / L) were gradually added, and the recovery rate was calculated. At the same time, liquid chromatography - mass spectrometry (LC - MS) was used for comparison. The chromatographic conditions were as follows: the chromatographic column was Eclipse XDB - C18 (250 mm×4.6 mm, 5 μm); the mobile phase was 0.1% formic acid + 5 mmol / L ammonium acetate aqueous solution (A) and methanol (B), and the gradient elution program was: from 0 to 2.0 min, the proportion of phase A was maintained at 85%, from 2.0 to 5.0 min, the proportion of phase A dropped to 70%, from 5.0 to 7.0 min, the proportion of phase A dropped to 1%, and from 7.0 to 8.0 min, the proportion of phase A was maintained at 1%; the flow rate was 0.3 mL / min; the injection volume was 1 μL; the column temperature was 40°C. The mass spectrometry conditions were as follows: the AJS EIS source was used, and the multi - reaction monitoring mode (MRM) was used for detection (positive ion scan, EMV plus 400 V, negative ion scan, EMV plus 500 V, positive and negative ions were scanned simultaneously), the nebulizing gas pressure was 310.3 kPa (40 psi); the drying gas temperature and flow rate were 300°C and 12 L / min respectively; the capillary voltage was: 3000 V for positive ions and 3500 V for negative ions.

[0043] The detection results are shown in Table 1.

[0044] Table 1 Determination of the recovery rate of the sensor with added standard in Example 1 (n = 3)

[0045]

[0046] Example 3

[0047] In this example, the flexible molecularly imprinted sensor of Example 1 was used to perform in - situ in - vivo detection of zeatin riboside in strawberry leaves. The steps were as follows:

[0048] The experimental material was the leaves of potted strawberry seedlings. A puncher was used to punch holes with a diameter of 1 mm on the leaves. The flexible molecularly imprinted sensor of Example 1 was attached to the leaves. 20 μL of 10 mM phosphate (PBS) buffer was added dropwise at the punched holes. The electrochemical workstation (CHI 1040c) was connected. The concentration of zeatin riboside in the in - vivo strawberry leaves was calculated by differential pulse voltammetry (the 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.

[0049] Table 2 Detection of ZR level in strawberry leaves (nmol / L)

[0050]

[0051] Comparative Example 1

[0052] This comparative example provides a flexible molecularly imprinted sensor for detecting zeatin riboside. The only difference in the preparation method from Example 1 is that:

[0053] ZnAl-LDH was replaced with an equal amount of MgAl-LDH to prepare a flexible molecularly imprinted sensor MIP / MgAl-LDH-MIL-101(Fe) / LIG / PDMS.

[0054] The detection performance of the sensor was tested using the method in Example 2, and the results are as Figure 2 shown.

[0055] It can be seen that the linear detection range of the flexible molecularly imprinted sensor prepared in Comparative Example 1 is 50 nmol / L to 100 μmol / L, and the detection limit is 2.17 nmol / L. The detection effect is not as good as that of Example 1.

[0056] Comparative Example 2

[0057] This comparative example provides a flexible molecularly imprinted sensor for detecting zeatin riboside. The only difference in the preparation method from Example 1 is that:

[0058] MIL-101(Fe) was replaced with an equal amount of Cu-MOF to prepare a flexible molecularly imprinted sensor MIP / ZnAl-LDH-Cu-MOF / LIG / PDMS.

[0059] The detection performance of the sensor was tested using the method in Example 2, and the results are as Figure 2 shown.

[0060] It can be seen that the linear detection range of the flexible molecularly imprinted sensor prepared in Comparative Example 2 is 1 nmol / L to 50 μmol / L, and the detection limit is 0.417 nmol / L. The detection effect is not as good as that of Example 1.

[0061] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, 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 flexible molecularly imprinted sensor for detecting zeatin riboside, characterized in that, Comprising: Modify the surface of the LIG / PDMS electrode with a ZnAl-LDH and MIL-101(Fe) composite material to obtain a ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode. Then, using o-phenylenediamine as a monomer and zeatin riboside as a template molecule, prepare a molecularly imprinted membrane on the surface of the ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode. After eluting the template molecule, the flexible molecularly imprinted sensor is obtained.

2. The preparation method according to claim 1, characterized in that, The steps of modifying the surface of the LIG / PDMS electrode with a ZnAl-LDH and MIL-101(Fe) composite material include: dissolving ZnAl-LDH and MIL-101(Fe) in water, dispersing evenly to obtain a ZnAl-LDH-MIL-101(Fe) composite solution, and then dropping the composite solution onto the working electrode surface of the LIG / PDMS electrode to obtain a ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode.

3. The preparation method according to claim 1, characterized in that, The steps of preparing the molecularly imprinted membrane include: mixing a zeatin riboside solution and an o-phenylenediamine solution to obtain a mixed solution; then placing the ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode in the mixed solution, performing electro-polymerization using cyclic voltammetry, washing and drying, and then eluting the template molecule to obtain the flexible molecularly imprinted sensor.

4. The preparation method according to claim 3, characterized in that, The concentration of zeatin riboside in the zeatin riboside solution is 1 - 5 mM; the concentration of o-phenylenediamine in the o-phenylenediamine solution is 1 - 10 mM.

5. The preparation method according to claim 3, wherein, The conditions for the electro-polymerization include: the voltage is -0.2V - 1.0 V, the number of electro-polymerization cycles is 10 - 30 cycles, and the scanning rate is 25 - 100 mV / S.

6. The preparation method according to claim 3, wherein, Elute the template molecule with a 5% - 15% acetic acid solution.

7. The preparation method according to claim 1, characterized in that The preparation steps of the LIG / PDMS electrode include: preparing laser-induced graphene on a polyimide tape using laser direct writing technology, and further transferring it to polydimethylsiloxane, coating the reference electrode with Ag / AgCl paste, and encapsulating the wire and the passivation area to obtain the LIG / PDMS electrode.

8. A flexible molecularly imprinted sensor for detecting zeatin riboside, characterized in that, It is prepared by the preparation method described in any one of claims 1 - 7.

9. Application of the flexible molecularly imprinted sensor described in claim 8 in in-situ in-vivo detection of zeatin riboside in plants.

10. A method for in-situ and in-vivo detection of zeatin riboside in plants, characterized in that, Comprising: After punching holes in the plant surface, attach the flexible molecularly imprinted sensor described in claim 8 to the punched holes, drop a buffer solution at the punched holes, then connect an electrochemical workstation, and detect the concentration of zeatin riboside by differential pulse voltammetry.

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