Flexible molecular imprinting sensor for detecting zeatin nucleoside as well as preparation method and application of flexible molecular imprinting sensor

Abstract

The invention relates to the technical field of analysis and detection, in particular to a flexible molecular imprinting sensor for detecting zeatin nucleoside as well as a preparation method and application of the flexible molecular imprinting sensor. The preparation method comprises the following steps: obtaining a laser-induced graphene pattern on a polyimide adhesive tape through a laser direct writing technology, transferring the laser-induced graphene pattern to polydimethylsiloxane, and then modifying a working electrode with a composite material of zinc and aluminum-based layered double hydroxides and an iron-based metal organic framework. And preparing a molecularly imprinted membrane on the surface of the electrode by taking o-phenylenediamine as a monomer and zeatin nucleoside as a template molecule, and eluting the template molecule to prepare the flexible molecularly imprinted sensor. The flexible molecular imprinting sensor disclosed by the invention can realize in-situ in-vivo detection of zeatin nucleoside in a plant body, is beneficial to adapting to irregular surfaces of plant tissues, does not cause essential damage to a detection part, provides a low-cost and high-accuracy detection tool for in-vivo detection of the plant, and has a wide application prospect. And a basis is provided for knowing the physiological status of the plant.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a flexible molecular imprinting sensor for detecting zeatin riboside and a preparation method and 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 is widely present in higher plants, especially in active growth parts such as 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. It can also help plants cope with plant stresses such as drought and light by regulating stomatal opening and density.

[0003] The level of ZR in plants is generally low. Traditionally, the methods used to measure ZR are mostly chromatography and immunoassays. These methods have low detection efficiency and mainly use in vitro detection. The detection equipment is expensive and the operation is complicated. Compared with other methods, electrochemical sensors are low in cost, simple to operate, portable, have high detection accuracy, and fast response speed. They can be applied to the field of in vivo detection of small molecules in plants.

[0004] However, plant tissues are very soft and irregular in shape. Traditional sensors based on rigid electrodes (such as glassy carbon electrodes and gold electrodes) cannot effectively adhere to the plant surface, reducing the reliability and accuracy of the detection results. Therefore, how to design a flexible electrode that can detect zeatin riboside in plants in situ has become a technical problem that needs to be solved in this field. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing a flexible molecular imprinting sensor for detecting zeatin riboside, comprising: 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, then preparing a molecular imprinting membrane (MIP) on the surface of the ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode using o-phenylenediamine as a monomer and zeatin riboside as a template molecule, and obtaining the flexible molecular imprinting sensor after eluting the template molecule.

[0006] Among them, ZnAl-LDH is a layered double hydroxide (LDHs) composed of zinc (Zn) and aluminum (Al), which has good stability and catalytic properties. The present invention co-modifies it 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, providing a stable response current for the sensor, and further combining molecular imprinting technology, so that the flexible molecular imprinting sensor has excellent detection effect in in situ living body detection, realizing high 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 comprises: dissolving ZnAl-LDH and MIL-101(Fe) in water, dispersing them 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 at 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 molecular imprinting membrane includes: mixing a zeatin riboside solution with an o-phenylenediamine solution to obtain a mixed solution; then placing a ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode in the mixed solution, performing electropolymerization using cyclic voltammetry, washing and drying, and eluting the template molecules to obtain the flexible molecular imprinting 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 electropolymerization include: a voltage of -0.2 V to 1.0 V, a number of electropolymerization cycles of 10 to 30 cycles, and a scanning speed of 25 to 100 mV / S.

[0012] Preferably, the template molecule is eluted with an acetic acid solution. Preferably, the concentration of the acetic acid solution is 5% to 15%.

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

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

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

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

[0017] Furthermore, the present invention provides the use of the flexible molecular imprinting sensor in in situ live detection of zeatin riboside in plants.

[0018] In a 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, comprising: after punching a hole on the surface of the plant body, attaching the flexible molecular imprinting sensor to the hole, dripping a buffer solution at the hole, and then connecting an electrochemical workstation to detect the concentration of zeatin riboside by differential pulse voltammetry.

[0020] In the specific implementation process, the punching parts can be the stems, leaves, fruits, etc. of the plants.

[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, different concentrations of zeatin riboside-phosphate buffer solutions (pH=7.2~7.4) were used to establish a relationship curve between current and zeatin riboside concentration, thereby obtaining a standard curve.

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

[0024] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a flexible molecular imprinting sensor for detecting zeatin riboside. The flexible molecular imprinting sensor can realize in-situ and living detection of zeatin riboside in plants, avoiding the mechanical mismatch problem caused by the rigid sensor and the soft tissue of the plant in the living plant detection. The flexible molecular imprinting sensor of the present invention can still retain stable detection performance within a moderate deformation range, is conducive to adapting to the irregular surface of the plant tissue, does not cause essential damage to the detection part, provides a low-cost and high-accuracy detection tool for plant living detection, and provides a basis for understanding the physiological state of the plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a process flow chart of Example 1 of the present invention.

[0026] Figure 2 This is a 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 DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In the examples provided in this specification, if no specific technology or conditions are specified, the technology or conditions described in the literature in this field or the product instructions are used. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels. ZnAl-LDH (Cat. No. 103063) and MIL-101(Fe) (Cat. No. 103320) were purchased from Jiangsu Xianfeng Nanomaterial Technology Co., Ltd.

[0029] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] Example 1 This embodiment provides a flexible molecular imprinting sensor for detecting zeatin riboside. The process flow chart is as follows: Figure 1 As shown, the preparation method is as follows: (1) Apply polyimide (PI) tape to a 6×6 cm 2 The electrode pattern, including the working electrode, the counter electrode and the reference electrode, was designed using CAD on a polytetrafluoroethylene plate. The exported electrode pattern was loaded into the software of the laser printing system with a printing power of 53% and a printing depth of 10 μm to pattern the PI tape.

[0031] (2) Liquid polydimethylsiloxane (PDMS) was coated on a polytetrafluoroethylene plate using a coating machine at a coating speed of 100 rpm for 90 s, and then placed in a vacuum drying oven at 100 °C and heated under vacuum for 12 h. The PI film was then peeled off from the PDMS to obtain a LIG / PDMS electrode.

[0032] (3) Apply an appropriate amount of PDMS to the non-working area of ​​the electrode and heat-cure it to avoid short circuit. Finally, apply Ag / AgCl silver paste on the exposed reference electrode and heat-cure it.

[0033] (4) The LIG / PDMS electrode was placed in a phosphate buffer solution (0.1 mol / L, pH = 7.2-7.4) and activated using a constant potential method (-1.7 V) for 180 s to remove impurities on the electrode surface.

[0034] (5) Weigh 1 mg of ZnAl-LDH and 5 mg of MIL-101(Fe) respectively, place them in 1 ml of ultrapure water, and ultrasonicate for 2 h to disperse them 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.

[0035] (6) A 2 mM zeatin riboside solution and a 4 mM o-phenylenediamine solution were mixed in equal volumes, and then an electrode was placed in the mixture. Cyclic voltammetry (CV) was used to perform electropolymerization for 20 cycles in a voltage range of -0.2 V to 1.0 V at a scan rate of 50 mV / s. After polymerization, the residual polymer solution was washed away with ultrapure water. After drying, the mixture was placed in a 10% acetic acid solution for 50 s to elute the template molecule. After washing and drying, a flexible molecular imprinting sensor MIP / ZnAl-LDH-MIL-101(Fe) / LIG / PDMS for detecting zeatin riboside was obtained.

[0036] Example 2 In this example, the detection performance of the flexible molecular imprinting sensor for detecting zeatin riboside prepared in the example is tested, and the steps are as follows: (1) 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 were prepared, respectively, and the flexible molecular imprinting sensor of Example 1 was connected to an electrochemical workstation (CHI 1040c) and detected by differential pulse voltammetry (potential -0.2-0.6 V, pulse width 0.02 s, amplitude 0.05 V, pulse period 1 s, sampling width 0.02 s), with the increase of ZR solution concentration, 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 the standard zeatin riboside solutions of different concentrations were recorded as I1, I2, I3..., respectively. By using the formula ΔI=I0-I1(I2, I3...), ΔI1, ΔI2, ΔI3... were calculated respectively, thereby obtaining a set of relationship curves between the logarithm of ZR concentration and ΔI, and making the standard curve of zeatin riboside, as shown in Figure 2 As shown in the figure, the linear detection range is 1 nmol / L~500 μmol / L, and the detection limit is 0.387 nmol / L (S / N=3).

[0037] (2) After the leaves of strawberry seedlings were ground and centrifuged, the supernatant was collected and combined with the standard curve to detect the level of zeatin riboside. ZR standard samples (10 nmol / L, 50 nmol / L, and 100 nmol / L) were gradually added to the supernatant as a matrix to calculate the recovery rate. Liquid chromatography-mass spectrometry (LC-MS) was used for comparison. The chromatographic conditions were as follows: the chromatographic column was EclipseXDB-C18 (250 mm×4.6 mm, 5 μm); 0.1 formic acid + 5 mmol / L ammonium acetate aqueous solution (A) and methanol (B) were used as the mobile phases. The gradient elution program was as follows: 0-2.0 min, the proportion of phase A was maintained at 85%, 2.0-5.0 min, the proportion of phase A was reduced to 70%, 5.0-7.0 min, the proportion of phase A was reduced to 1%, and 7.0-8.0 min, the proportion of phase A was maintained at 1%; the flow rate was 0.3 mL / min; the injection volume was 1 μL; and the column temperature was 40°C. The mass spectrometry conditions were as follows: AJS EIS source, multiple reaction monitoring mode (MRM) detection (EMV plus 400 V for positive ion scanning, EMV plus 500 V for negative ion scanning, and simultaneous scanning of positive and negative ions), nebulizer gas pressure of 310.3 kPa (40 psi); drying temperature and flow rate of 300 °C and 12 L / min, respectively; capillary voltage: 3000 V for positive ions and 3500 V for negative ions.

[0038] The test results are shown in Table 1.

[0039] Table 1 Determination of sensor spike recovery rate in Example 1 (n=3)

[0040] Example 3 In this embodiment, the flexible molecular imprinting sensor of embodiment 1 is used to perform in situ live detection of zeatin riboside in strawberry leaves, and the steps are as follows: Potted strawberry seedling leaves were selected as experimental materials. Holes with a diameter of 1 mm were punched on the leaves using a hole puncher. The flexible molecular imprinting sensor of Example 1 was attached to the leaves. 20 µL of 10 mM phosphate (PBS) buffer was dripped at the punched hole. The sensor was connected to an electrochemical workstation (CHI 1040c). The concentration of zeatin riboside in the living strawberry leaves was calculated by differential pulse voltammetry (test conditions were the same as those in Example 2) combined with the standard curve in Example 2. The results are shown in Table 2.

[0041] Table 2 ZR level detection in strawberry leaves (nmol / L)

[0042] Comparative Example 1 This comparative example provides a flexible molecular imprinting sensor for detecting zeatin riboside, and the preparation method is different from that of Example 1 only in that: By replacing ZnAl-LDH with an equal amount of MgAl-LDH, a flexible molecular imprinting sensor MIP / MgAl-LDH-MIL-101(Fe) / LIG / PDMS was prepared.

[0043] The detection performance of the sensor was tested using the method in Example 2. The results are as follows: Figure 2 shown.

[0044] It can be seen that the linear detection range of the flexible molecular imprinting sensor prepared in Comparative Example 1 is 50 nmol / L~100 μmol / L, and the detection limit is 2.17 nmol / L, and the detection effect is not as good as that of Example 1.

[0045] Comparative Example 2 This comparative example provides a flexible molecular imprinting sensor for detecting zeatin riboside, and the preparation method is different from that of Example 1 only in that: By replacing MIL-101(Fe) with an equal amount of Cu-MOF, the flexible molecular imprinting sensor MIP / ZnAl-LDH-Cu-MOF / LIG / PDMS was prepared.

[0046] The detection performance of the sensor was tested using the method in Example 2. The results are as follows: Figure 2 shown.

[0047] It can be seen that the linear detection range of the flexible molecular imprinting sensor prepared in Comparative Example 2 is 1 nmol / L~50 μmol / L, and the detection limit is 0.417 nmol / L, and the detection effect is not as good as that of Example 1.

[0048] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a flexible molecular imprinting sensor for detecting zeatin riboside, characterized in that: include: The ZnAl-LDH and MIL-101(Fe) composite materials were modified on the surface of LIG / PDMS electrode to obtain ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode, and then a molecular imprinting membrane was prepared on the surface of ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode using o-phenylenediamine as a monomer and zeatin riboside as a template molecule. After eluting the template molecules, the flexible molecular imprinting sensor was obtained.

2. The preparation method according to claim 1, characterized in that: 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 them 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.

3. The preparation method according to claim 1, characterized in that: The steps of preparing the molecular imprinting membrane include: mixing a zeatin nucleoside solution with an o-phenylenediamine solution to obtain a mixed solution; then placing a ZnAl-LDH-MIL-101(Fe) / LIG / PDMS electrode in the mixed solution, performing electropolymerization by cyclic voltammetry, washing and drying, and eluting the template molecules to obtain the flexible molecular imprinting 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, characterized in that: The conditions of the electropolymerization include: a voltage of -0.2 V to 1.0 V, a number of electropolymerization cycles of 10 to 30 cycles, and a scanning speed of 25 to 100 mV / S.

6. The preparation method according to claim 3, characterized in that: The template molecules were eluted with 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 a laser direct writing technique, and further transferring it to polydimethylsiloxane, coating a reference electrode with an Ag / AgCl slurry, and encapsulating the wires and the passivation area to obtain the LIG / PDMS electrode.

8. A flexible molecular imprinting sensor for detecting zeatin riboside, characterized in that: It is prepared by the preparation method described in any one of claims 1 to 7.

9. Use of the flexible molecular imprinting sensor according to claim 8 in in situ live detection of zeatin riboside in plants.

10. A method for in situ live detection of zeatin riboside in plants, characterized in that: include: After punching holes on the plant surface, the flexible molecular imprinting sensor according to claim 8 is attached to the holes, a buffer solution is dripped into the holes, and then the electrochemical workstation is connected to detect the concentration of zeatin riboside by differential pulse voltammetry.

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