Immune microelectrode, immune electrochemical sensor and preparation method and application of immune microelectrode and immune electrochemical sensor

Abstract

The invention discloses an immune microelectrode, an immune electrochemical sensor and a preparation method and application of the immune microelectrode and the immune electrochemical sensor. An immune microelectrode prepared by modifying a micron-level metal electrode wire with a carbon-based nano material and a plant hormone antibody is used as a working electrode, and the electrochemical sensor taking the working electrode as a substrate is used for detecting plant hormones in a plant tissue micro-area. A sensor based on the electrode shows good electrode stability in the process of detecting plant hormones in a plant tissue microcell, and the detection range is large. For other interfering substances such as auxin, salicylic acid and the like in a plant body, generated response signals are greatly different from the auxin and the salicylic acid or basically have no response signals, the electrode shows good specificity, and the method for rapidly detecting the plant hormones in the plant tissue microcell through the electrode is convenient and simple. On the basis of plant tissue micro-area detection, in-situ detection can be carried out on plants.

Description

Technical Field

[0001] This invention relates to the electrochemical detection of plant signal molecules using an immunomicroelectrode, specifically to an immunomicroelectrode, an immunoelectrochemical sensor, its preparation method, and its application. Background Technology

[0002] Electrochemical sensors are sensing devices based on three-electrode or two-electrode systems. Due to their high sensitivity, rapid response, low cost, integration, and portability, electrochemical sensors show great potential for widespread application in fields such as environmental monitoring, food safety, medical diagnostics, and smart agriculture. In the field of in vivo plant detection, in vitro detection methods such as chromatography-mass spectrometry have long dominated due to their superior separation and analysis capabilities, while the direct application of immunoelectrochemical sensing to in vivo, in-situ detection is relatively rare. Considering the stability and specificity issues of antibodies in the in vivo environment, most researchers have made limited attempts in this direction. In the complex in vivo environment, endogenous substances in plant microregions can induce severe non-specific adsorption and background interference, leading to signal drift and distortion.

[0003] Methyl jasmonate and abscisic acid, as key members of the plant hormone family, participate in important physiological processes in plants. Accurate and real-time quantitative analysis of the content of these electrochemically inactive plant hormones in plant cells is a crucial problem that needs to be solved. Because hormones are present in extremely low concentrations within plants and are unstable, and are easily interfered with by other compounds during detection, the requirements for detection methods are high; otherwise, it is difficult to detect hormones present in low concentrations in plants. Currently, many plant hormones are detected using techniques such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS). However, these methods have some problems. On the one hand, they require complex and destructive treatments of the plant, leading to the loss of some unstable plant hormones and affecting the accuracy of the results. On the other hand, these treatment processes are time-consuming and cannot reflect the hormone content in plants in a timely manner. Most existing technologies are limited to in vitro detection and cannot track the dynamic changes of key signaling molecules within plants in situ in real time. Summary of the Invention

[0004] Objectives of the Invention: The first objective of this invention is to provide an immunomicroelectrode with a simple structure suitable for the detection of micro-regions in plant tissues; the second objective is to provide an immunoelectrochemical sensor based on the immunomicroelectrode; the third objective is to provide a method for preparing the above-mentioned immunomicroelectrode and sensor; and the fourth objective is to provide its application in the in situ detection of plant hormones, so as to solve the problems of complex detection process and inability to monitor in situ in real time in the prior art.

[0005] Technical solution: The immune microelectrode of the present invention comprises: a micron-sized metal wire; a hollow sleeve through which the micron-sized metal wire passes; insulating adhesive for bonding the micron-sized metal wire to the hollow sleeve; conductive tape attached to one end of the micron-sized metal wire outside the hollow sleeve; the other end of the micron-sized metal wire is modified with carbon-based nanomaterials and plant hormone antibodies (e.g., methyl jasmonate antibody, brassinolide antibody, abscisic acid antibody, etc.).

[0006] Preferably, the micron-sized metal wire is stainless steel wire or platinum wire, with a diameter of 80~120 μm. More preferably, it is 100 μm.

[0007] Preferably, the hollow sleeve is one of a glass tube, a silicone tube, a fiberglass tube, or a ceramic tube; and the conductive tape is one of a copper foil tape, a carbon conductive tape, or a copper-nickel fiber tape.

[0008] The immunoelectrochemical sensor of the present invention includes the immunomicroelectrode as the working electrode; a reference electrode; and a counter electrode; wherein the working electrode, the reference electrode, and the counter electrode constitute a three-electrode system.

[0009] Preferably, the reference electrode is an Ag / AgCl electrode.

[0010] The method for preparing the immune microelectrode of the present invention includes passing a micron-sized metal wire through a hollow sleeve and fixing it with insulating adhesive; attaching conductive tape to one end of the metal wire; and modifying the other end with carbon-based nanomaterials and plant hormone antibodies to form an immune microelectrode.

[0011] Preferably, the immune microelectrode is used as the working electrode and assembled with a reference electrode and a counter electrode into a three-electrode system.

[0012] The application of the immunomicroelectrode or immunoelectrochemical sensor described in this invention in the detection of plant hormones in microregions of plant tissues.

[0013] Preferably, the plant hormone includes at least one of methyl jasmonate, abscisic acid, brassinolide, and gibberellin.

[0014] Preferably, the application method is as follows: a series of plant hormone standard solutions of different concentrations are prepared, and the differential pulse voltammetry (DPV) is used for determination to establish a standard curve of current response value versus plant hormone concentration; the immunomicroelectrode is placed on the surface of the micro-region of the plant tissue to be tested as the working electrode, and the reference electrode and the counter electrode are arranged near the working electrode without contacting each other; buffer solution is added to the surface of the micro-region of the tissue; an electrochemical workstation is connected for signal acquisition, and the concentration of plant hormone in the micro-region of the tissue is calculated according to the standard curve.

[0015] Preferably, the concentration of the plant hormone standard solution is 1 nM-100 μM, the buffer solution is 0.18-0.22 M PBS solution with pH 6.8-7.2; the differential pulse voltammetry is used for detection, the detection potential range is -0.5 V to 0.4 V, the amplitude is 0.045-0.055 V, the sampling interval is 0.020-0.030 s, and the resting time is 1.5-2.5 s.

[0016] Further preferredly, plant hormone solutions of different concentrations were prepared using 0.2 M PBS buffer (pH=7.0). Differential pulse voltammetry was used to obtain a series of plant hormone concentration variation curves, thereby qualitatively and quantitatively identifying the plant hormones and obtaining a linear relationship between current value and plant hormone concentration. Micrometer-scale metal wires were modified with nanomaterials and plant hormone antibodies to form an immunomicroelectrode, which served as the working electrode and was placed on a micro-region of the plant tissue. Simultaneously, the reference and counter electrodes were placed near the working electrode without touching each other. 10 μL of buffer solution was added to the detection site in the micro-region of the tissue. The initial addition amount was used to achieve a stabilizing effect, and subsequent additions were fixed to the same amount as the initial addition. A single-channel electrochemical workstation was connected to a computer to detect plant hormones in the micro-region of the plant tissue, obtaining the concentration of plant hormones in the plant cells. Taking methyl jasmonate as an example, the initial detection potential was set to -0.5 V, the termination potential to 0.4 V, the amplitude to 0.05 V, the sampling interval to 0.025 s, and the resting time to 2 s. The concentration of methyl jasmonate in the microregion of the plant hypocotyl was obtained.

[0017] This invention is the first to integrate antibodies onto microelectrodes for the detection of plant hormones. It can stably record the concentration curves of plant hormones in microregions of plant tissue at different times, revealing changes that were difficult to accurately observe using discrete sampling methods. These effects are not merely linear improvements to existing technologies, but rather represent a leap from "static, in vitro, and destructive detection" to "dynamic, in situ, and non-invasive monitoring."

[0018] From a technical perspective, this is not simply miniaturization, but a complex interdisciplinary integration involving deep intersections of materials science, electrochemistry, bioengineering, and plant physiology, presenting certain technical hurdles. For example, it requires addressing issues of biocompatibility and interface stability. The materials, size, and mechanical strength of the immunomicroelectrode must be sufficiently robust to penetrate plant tissue without breaking, while its surface chemical modifications must be sufficiently stable to maintain and immobilize the antibody's bioactivity. Furthermore, it necessitates resolving issues such as signal specificity, environmental interference, and reliable extraction of weak signals. Only by solving these problems can the stability of the immunomicroelectrode in actual detection be guaranteed. This invention breaks away from the traditional approach of relying on physical isolation to non-selectively shield interference, instead utilizing antibody-antigen specific recognition mechanisms to construct a bioselective interface. Through the high selectivity and affinity of molecular recognition, endogenous interfering substances are effectively excluded at the molecular level. This strategy shifts from "combating complex environments" to "achieving molecular resolution using biological intelligence," representing a profound reconstruction of the essence of the anti-interference problem in in vivo sensing. Its technical approach cannot be derived through simple improvements or combinations of existing non-selective barrier technologies. Therefore, by specifically binding antigens and antibodies, the interference of endogenous substances in plant microregions on electrochemical sensors can be reduced, enabling more accurate detection.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) This invention uses a three-electrode system to detect changes in plant hormone content in plant tissue microregions based on the tissue microregion level. It can realize fixed-point in vivo monitoring and collect dynamic change information of plant hormones in plant tissues from the tissue microregion level, providing a new method for studying hormone changes in plants from the tissue microregion level. At the same time, it eliminates the need for complex and time-consuming sample pretreatment, reduces measurement errors, and reflects the changes in plant hormone content in plant tissue microregions more timely. It can continuously track the dynamic change process of plant hormone concentration in the microregion for more than 4 hours, which is of great significance for subsequent in-depth research on plant hormones.

[0020] (2) The sensor exhibits good stability and a wide detection range (1 nM to 100 μM) in detection. It shows significant differences or almost no response to interfering substances such as auxin and salicylic acid, and has high specificity. The method is convenient and simple. In addition to detecting micro-regions of plant tissues such as hypocotyl, it can also perform in-situ and continuous detection of plants.

[0021] (3) By amplifying the signal through the nanomaterials on the surface, the complex interference problem of the living environment is cleverly solved, so that the sensor can still output a stable and reliable signal in complex matrices such as plant juice and extracellular fluid. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the fabrication process of the electrode for this invention.

[0023] Figure 2 A schematic diagram is created for the standard curve; where, Figure 2 A represents the qualitative and quantitative detection of different concentrations of plant hormones, using methyl jasmonate as an example, by DPV in this invention; Figure 2 B is a linear relationship graph between current and plant hormone concentration prepared in this invention, using methyl jasmonate as an example.

[0024] Figure 3 This diagram illustrates and shows the results of in-situ detection in micro-regions of plant tissue; among them, Figure 3 A represents the detection of plant hormones at the hypocotyl wound site of tomato seedlings in this invention, using methyl jasmonate as an example; Figure 3 B is the DPV detection curve for detecting the binding of plant hormone antigen and antibody at the hypocotyl wound site of tomato seedlings in this invention, taking methyl jasmonate as an example; Figure 3 C is a graph showing the changes in plant hormone current response values ​​at different time points in the early stage of tomato bud regeneration, using methyl jasmonate as an example. Figure 3 D is a graph showing the changes in plant hormone concentration at different time points during the early stage of tomato bud regeneration, using methyl jasmonate as an example.

[0025] Figure 4 A schematic diagram is created for the standard curve; where, Figure 4 A represents the qualitative and quantitative detection of different concentrations of plant hormones, taking abscisic acid as an example, using DPV in this invention; Figure 4 B is a linear relationship graph between current and abscisic acid concentration generated in this invention, taking abscisic acid as an example.

[0026] Figure 5 This diagram illustrates and shows the results of in-situ detection in micro-regions of plant tissue; among them, Figure 5 A represents the detection of abscisic acid at wound sites on detached Arabidopsis leaves in this invention, using abscisic acid as an example; Figure 5 B is the DPV detection curve for detecting the binding of ABA antigen and antibody at the wound site of isolated Arabidopsis thaliana leaves in this invention, taking abscisic acid as an example; Figure 5 C is a graph showing the change in ABA current response at the wound site of the detached leaf in the experimental group and the control group, taking abscisic acid as an example. Figure 5 D is a graph showing the change in ABA concentration at the wound site of detached leaves in the experimental and control groups, using abscisic acid as an example. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Example 1

[0029] In this embodiment, the hypocotyl part of tomato is selected as the experimental object. The method for rapidly detecting methyl jasmonate in the tissue micro-region of tomato hypocotyl based on an immunosensing microelectrode includes the following steps: Step 1: Preparation of the immunosensing microelectrode: The immunosensing microelectrode consists of four parts, namely a hollow sleeve, a micron-scale metal wire, insulating glue, and conductive tape. Among them, the diameter of the micron-scale metal wire is 100 μm. The micron-scale metal wire is passed through the lead-out interface of the hollow sleeve, and both ends of the micron-scale metal wire are adhered to the hollow sleeve with insulating glue (it should be noted that appropriate lengths must be left at both ends of the micron-scale metal wire for subsequent operations). A conductive tape is attached to one end of the micron-scale metal wire outside the hollow sleeve as the electrode pin, and the other end is trimmed to an appropriate length and modified with a composite prepared from carbon-based nanomaterials such as conductive carbon paste, graphene oxide (GO), ferrocene (Fc), etc. and methyl jasmonate antibody to obtain an immunosensing microelectrode formed by modifying the micron-scale metal electrode wire, as Figure 1 shown.

[0030] Step 2: Construction of the electrochemical sensor: The immunosensing microelectrode is used as the working electrode, and an electrochemical sensor is composed of the working electrode, reference electrode, and counter electrode. Among them, the reference electrode is Ag / AgCl, and the counter electrode is an unmodified micron-scale metal electrode wire.

[0031] Step 3: Detection of methyl jasmonate: Preparation of the methyl jasmonate standard curve: First, methyl jasmonate is dissolved in 0.2 M PBS buffer (pH = 7.0) to prepare a stock solution, and then methyl jasmonate solutions with different concentrations (1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM) are prepared with the buffer. The three-electrode system composed of the working electrode (Pt, d = 100 μm), counter electrode (Pt, d = 100 μm), and reference electrode (Ag / AgCl, d = 0.1 mm) is placed into the prepared methyl jasmonate solutions with different concentrations, and differential pulse voltammetry (DPV) is used to qualitatively and quantitatively determine methyl jasmonate with different concentrations, as Figure 2 shown in A. A linear relationship graph of current vs. methyl jasmonate concentration is made using the obtained data, as Figure 2 shown in B.

[0032] Detection of MeJA at the wound site of tomato seedling hypocotyl: The microelectrode formed by modifying the micron-scale metal wire is used as the working electrode and placed on the tissue micro-region of the tomato hypocotyl part. At the same time, the reference electrode and counter electrode need to be placed near the working electrode and cannot touch each other; 10 μL of buffer solution is dropped at the detected position so that the working electrode contacts the detected tissue micro-region, as Figure 3 shown in A.

[0033] A CHI1240C single-channel electrochemical workstation was connected to a computer to detect methyl jasmonic acid in plant hypocotyl cells. The initial detection potential for methyl jasmonic acid was -0.5 V, the termination potential was 0.4 V, the amplitude was 0.05 V, the sampling interval was 0.025 s, and the resting time was 2 s. The concentration changes of methyl jasmonic acid in plant root cells were obtained, such as... Figure 3 As shown in B. Figure 3 C represents the change in ΔI of MeJA at the wound site. It can be clearly observed that it first increases and then decreases, with the MeJA level fluctuation reaching its peak at 2 hours. Figure 3 D represents the change in MeJA content at the wound site; MeJA essentially disappeared after 4 hours.

[0034] This invention uses a modified immunomicroelectrode at the micrometer level as the working electrode and an immunoelectrochemical sensor based on the working electrode to detect methyl jasmonic acid in plant tissue microregions. Compared with other detection methods, it eliminates the complex and time-consuming processing steps, reduces the possible impact on the measurement results, and reflects the changes in the content of methyl jasmonic acid in plant tissue microregions more promptly. This is of great significance for further in-depth research on methyl jasmonic acid.

[0035] Example 2

[0036] This embodiment uses detached Arabidopsis thaliana leaf tissue as the experimental subject. The method for rapid detection of abscisic acid in microregions of detached Arabidopsis thaliana leaf tissue based on microelectrodes includes the following steps: Step 1: Fabrication of microelectrodes: The microelectrode consists of four parts: a hollow sleeve, a micron-sized metal wire, insulating adhesive, and conductive tape. The micron-sized metal wire has a diameter of 100 μm. The micron-sized metal wire is passed through the hollow sleeve and led out to the interface. Both ends of the micron-sized metal wire are bonded to the hollow sleeve with insulating adhesive (it should be noted that the ends of the micron-sized metal wire must be left with an appropriate length for subsequent operations). Conductive tape is attached to one end of the micron-sized metal wire outside the hollow sleeve, and the other end is trimmed to an appropriate length. It is then modified with a composite of carbon-based nanomaterials, such as conductive carbon adhesive, graphene oxide (GO), and ferrocene (Fc), and abscisic acid antibody, to obtain an immuno-microelectrode formed by modifying the micron-sized metal electrode wire.

[0037] Step 2: Construction of the electrochemical sensor: The microelectrode is used as the working electrode, and the electrochemical sensor is composed of the working electrode, the reference electrode, and the counter electrode. The reference electrode is Ag / AgCl, and the counter electrode is an unmodified micron-sized metal electrode wire.

[0038] Step 3: Detection of abscisic acid: Preparation of abscisic acid standard curve: First, abscisic acid was dissolved in 0.2 M PBS buffer (pH=7.0) to prepare a stock solution. Then, abscisic acid solutions of different concentrations (1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM) were prepared using buffer. A three-electrode system consisting of a working electrode (Pt, d=100 μm), a counter electrode (Pt, d=100 μm), and a reference electrode (Ag / AgCl, d=0.1 mm) was placed into the prepared abscisic acid solutions of different concentrations. Differential pulse voltammetry (DPV) was used for qualitative and quantitative determination of abscisic acid at different concentrations. Figure 4 As shown in Figure A, a linear relationship graph between current and abscisic acid concentration was plotted using the obtained data, as follows: Figure 4 As shown in B.

[0039] Detection of ABA at wound sites on detached Arabidopsis leaves: A microelectrode formed from modified micron-sized metal wires was used as the working electrode and placed on a micro-region of tissue in a detached Arabidopsis leaf. Simultaneously, the reference and counter electrodes were placed near the working electrode but not in contact with each other. 10 μL of buffer solution was added to the detection site to ensure the working electrode made contact with the detected leaf tissue micro-region. Figure 5 As shown in Figure A.

[0040] A CHI1240C single-channel electrochemical workstation was connected to a computer to detect abscisic acid (BAS) in micro-regions of detached Arabidopsis leaf tissue. The initial potential for ASA detection was -0.5 V, the termination potential was 0.4 V, the amplitude was 0.05 V, the sampling interval was 0.025 s, and the resting time was 2 s. The BSA curves and ABA curves of the experimental and control groups were compared. Figure 5 As shown in B. Figure 5 C is a graph showing the changes in ABA current response values ​​at the wound site of the detached leaf in the experimental group and the control group. Figure 5 Figure D shows the changes in ABA concentration at the wound site of the detached leaf in the experimental group and the control group.

[0041] This invention uses a modified immunomicroelectrode at the micrometer level as the working electrode and an immunoelectrochemical sensor based on the working electrode to detect abscisic acid in plant tissue microregions. Compared with other detection methods, it eliminates the complex and time-consuming processing steps, reduces the possible impact on the measurement results, and reflects the changes in abscisic acid content in plant tissue microregions more promptly, which is of great significance for further in-depth research on abscisic acid.

Claims

1. An immune microelectrode, characterized in that, include: Micron-sized metal wires; A hollow sleeve through which the micron-sized metal wire passes; Insulating adhesive is used to bond the micron-sized metal wire to the hollow sleeve. A conductive tape is attached to one end of the micron-sized metal wire outside the hollow sleeve; the other end of the micron-sized metal wire is modified with carbon-based nanomaterials and plant hormone antibodies.

2. The immunomicroelectrode according to claim 1, characterized in that, The micron-sized metal wire is made of stainless steel or platinum and has a diameter of 80-120 μm.

3. The immunomicroelectrode according to claim 1, characterized in that, The hollow sleeve is one of glass tube, silicone tube, fiberglass tube or ceramic tube; the conductive tape is one of copper foil tape, carbon conductive tape or copper-nickel fiber tape.

4. An immunoelectrochemical sensor, characterized in that, include: The immune microelectrode according to any one of claims 1 to 3 is used as the working electrode; a reference electrode; Counter electrode; the working electrode, reference electrode, and counter electrode constitute a three-electrode system.

5. A method for preparing the immune microelectrode according to claim 1, characterized in that, The process involves threading a micron-sized metal wire through a hollow sleeve and securing it with insulating adhesive; attaching conductive tape to one end of the metal wire; and modifying the other end with carbon-based nanomaterials and plant hormone antibodies to form an immunomicroelectrode.

6. A method for preparing the immunoelectrochemical sensor according to claim 4, characterized in that, This includes using the immune microelectrode described in claim 1 as the working electrode, and assembling it with a reference electrode and a counter electrode to form a three-electrode system.

7. The application of the immunomicroelectrode of claim 1 or the immunoelectrochemical sensor of claim 4 in detecting plant hormones in microregions of plant tissue.

8. The application according to claim 7, characterized in that, The plant hormones include at least one of methyl jasmonate, abscisic acid, brassinolide, and gibberellin.

9. The application according to claim 7, characterized in that, The specific method of the application is as follows: a series of plant hormone standard solutions of different concentrations are prepared, and the differential pulse voltammetry (DPV) is used for measurement to establish a standard curve of current response value and plant hormone concentration; the immunomicroelectrode is used as the working electrode and placed on the surface of the micro-region of the plant tissue to be tested, and the reference electrode and the counter electrode are arranged near the working electrode and do not contact each other. A buffer solution is dropped onto the surface of the tissue microregion; an electrochemical workstation is connected for signal acquisition, and the concentration of plant hormones in the tissue microregion is calculated based on the standard curve.

10. The application according to claim 9, characterized in that, The concentration of the plant hormone standard solution is 1 nM-100 μM, and the buffer solution is 0.18-0.22 M PBS solution with pH 6.8-7.2; The differential pulse voltammetry method was used for detection, with a detection potential range of -0.5 V to 0.4 V, an amplitude of 0.045 to 0.055 V, a sampling interval of 0.020 to 0.030 s, and a resting time of 1.5 to 2.5 s.

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