A wearable electrochemical sensor and its preparation method and application

Abstract

This invention discloses a wearable electrochemical sensor, its preparation method, and its applications. The wearable electrochemical sensor uses an ultrathin flexible stainless steel sheet modified with conductive carbon adhesive as the working electrode, combined with a counter electrode and a reference electrode to form a three-electrode system. It is used to detect plant hormones in microregions of plant tissue, achieving simultaneous detection of three plant hormones—indole-3-acetic acid, salicylic acid, and zeatin—in plant tissue using differential pulse voltammetry. This sensor exhibits good electrode stability during the detection of plant hormones in microregions of plant tissue, has a large detection range, and the detection method is convenient and simple. In addition to detecting plant hormones in microregions of plant tissue, it also enables in-situ detection of plants.

Description

Technical Field

[0001] This invention belongs to the field of plant physiological detection and electrochemical sensing technology, specifically relating to a wearable electrochemical sensor, its preparation method, and its application. Background Technology

[0002] Plant hormones such as indole-3-acetic acid (IAA), salicylic acid (SA), and zeatin (ZT) play important roles in regulating plant growth, development, and stress response. IAA, as one of the most important auxins, regulates multiple developmental stages from seed germination to fruit ripening, and its levels change significantly in stress response. SA is a key signaling molecule in plant immune defense, participating in the establishment of local and systemic acquired resistance. ZT, as a natural cytokinin, primarily stimulates cell division and controls the morphogenesis of roots and lateral branches. These three hormones synergistically regulate plant growth and stress adaptation through a complex interactive network. Due to these important biological functions, developing sensitive and accurate quantitative detection methods for IAA, SA, and ZT is crucial for plant science research and precision agriculture.

[0003] Electrochemical detection, due to its advantages of high sensitivity, fast response, and ease of miniaturization, has been proven to be a feasible method for in-situ determination of plant signal molecules. However, current technology still lacks an electrochemical sensing platform capable of simultaneously detecting IAA, SA, and ZT in situ in real time. This is mainly because expanding the detection targets to three presents significant technical challenges: First, the molecular structures and electrochemical active sites of IAA, SA, and ZT differ significantly, and their oxidation peak potentials are close to or even overlap in conventional neutral buffer solutions, making accurate differentiation and quantification of the three signals difficult, especially against the background of complex plant tissue extracts. Second, ZT exhibits a weak electrochemical response and poor stability in neutral buffer solutions, making it highly susceptible to severe interference from electroactive substances such as malic acid, citric acid, and abscisic acid, which coexist abundantly in plant tissues. Therefore, achieving simultaneous in-situ detection of these three hormones requires more than just the simple superposition of single detection methods; it necessitates overcoming the technical challenge of synergistic optimization of multi-target signal separation and anti-interference capabilities. Summary of the Invention

[0004] Objective of this invention: The first objective of this invention is to provide a wearable electrochemical sensor with a simple structure suitable for micro-area detection in plant tissues, overcoming the technological gap in the simultaneous, in-situ, and real-time detection of indole-3-acetic acid (IAA), salicylic acid (SA), and zeatin (ZT) in plant tissues. The second objective of this invention is to provide a method for preparing this wearable electrochemical sensor and its application in the in-situ detection of plant hormones.

[0005] Technical solution: The wearable electrochemical sensor of the present invention includes: a working electrode, which is made of an ultra-thin flexible stainless steel sheet modified with conductive carbon adhesive; a counter electrode; a reference electrode; and a paper-based detection interface, wherein the paper-based detection interface is used to carry electrolyte and is placed between the working electrode and the plant tissue to achieve contact with the plant tissue.

[0006] Preferably, the counter electrode is a platinum wire; the reference electrode is an Ag / AgCl electrode.

[0007] Preferably, the thickness of the ultrathin flexible stainless steel sheet is 10~50 μm, and more preferably 30 μm.

[0008] Preferably, the conductive carbon adhesive is formed by mixing conductive carbon adhesive stock solution and its diluent at a volume ratio of 1:4 to 6 and then curing, preferably 1:5.

[0009] Preferably, the paper-based detection interface is used to carry the electrolyte and is placed between the working electrode and the sample to be tested, and the diameter of the paper-based detection interface is 4~6mm.

[0010] Further preferably, the ultra-thin flexible stainless steel sheet also includes an insulating mask layer, which is an adhesive tape with circular through holes, the diameter of which is 3~5 mm, preferably 4 mm.

[0011] The method for preparing the wearable electrochemical sensor of the present invention includes the following steps: Step 1: After cutting and cleaning the ultra-thin flexible stainless steel sheet, cover it with transparent tape with round holes of 3-5 mm in diameter to define the effective detection area; Step 2: Drop 3~5 μL of conductive carbon adhesive mixture onto the exposed stainless steel sheet surface to form a decorative layer; Step 3: Use the modified stainless steel sheet as the working electrode, and assemble it with the platinum wire counter electrode and the Ag / AgCl reference electrode to form a three-electrode system.

[0012] Preferably, the conductive carbon adhesive mixture is obtained by mixing the conductive carbon adhesive stock solution and its diluent at a volume ratio of 1:4 to 6, and the application amount is 3 to 5 μL, preferably 4 μL.

[0013] The present invention relates to the application of the wearable electrochemical sensor in the simultaneous detection of indole-3-acetic acid, salicylic acid and zeatin in microregions of plant tissues.

[0014] Preferably, the application method is as follows: a series of mixed standard solutions containing indole-3-acetic acid, salicylic acid, and zeatin are prepared, and the solutions are measured using differential pulse voltammetry in an acidic buffer solution with pH 2.2-3.0 (preferably pH 2.6). Standard curves of current response values ​​versus concentrations for each hormone are established. The working electrode is placed in contact with or close to the surface of the micro-region of the plant tissue to be tested, and the reference electrode and counter electrode are arranged near the working electrode without contacting each other. A paper-based detection interface is added between the working electrode and the plant tissue, and an acidic buffer solution with pH 2.2-3.0 (preferably pH 2.6) is added. The electrochemical workstation is connected for signal acquisition, and each hormone is identified based on its characteristic peak potentials at 0.75 V-0.85 V, 0.95 V-1.05 V, and 1.10 V-1.20 V. The concentrations of each hormone in the micro-region of the tissue are calculated based on the corresponding standard curves.

[0015] Preferably, differential pulse voltammetry is used for detection, with the following key parameter combination: detection potential range of 0.1 V to 1.5 V, initial potential of 0.18–0.22 V, termination potential of 1.38–1.42 V, potential increment of 0.004–0.006 V, amplitude of 0.020–0.030 V, pulse width of 0.015–0.025 s, and settling time of 25–35 s. This specific parameter combination, combined with a strongly acidic detection environment, can effectively suppress the background current of endogenous interfering substances in plant tissues, significantly improve the signal-to-noise ratio, and thus achieve simultaneous high-sensitivity detection of three hormones.

[0016] Further preferred embodiment involves preparing mixed standard solutions of indole-3-acetic acid, salicylic acid, and zeatin at different concentrations using a phosphate buffer solution at pH 2.6; obtaining a series of concentration variation curves using differential pulse voltammetry to qualitatively and quantitatively analyze each hormone and obtain the linear relationship between the current response value and concentration of each hormone; placing the working electrode in contact with the micro-region of plant tissue, while simultaneously placing the reference electrode and the counter electrode near the working electrode without contacting each other; adding 7 μL of pH 2.6 PBS buffer solution between the working electrode and the tissue through the paper-based detection interface; connecting the electrochemical workstation to the computer, and setting the detection parameters as follows: initial potential 0.2 V, termination potential 1.4 V, potential increment 0.005 V, amplitude 0.025 V, pulse width 0.02 s, and settling time 30 s, to perform detection, thereby achieving simultaneous determination of the concentrations of indole-3-acetic acid, salicylic acid, and zeatin in micro-regions such as plant root tips or bud tips.

[0017] This invention is the first to propose the use of an ultra-thin flexible stainless steel substrate combined with conductive carbon adhesive to modify the interface, thereby constructing a flat panel sensor that can be directly attached to the surface of plants. This enables simultaneous, in-situ, and real-time electrochemical detection of three key signaling molecules—indole-3-acetic acid, salicylic acid, and zeatin—in living plant tissues. At the technical level, the innovation of this invention lies in the following: It abandons the traditional selective construction strategy that relies on biorecognition elements such as antibodies and enzymes. By precisely controlling the physicochemical properties of the conductive carbon gel modification layer and employing a strongly acidic detection system (pH 2.6), it was unexpectedly discovered that this system can enable three hormones to exhibit characteristic oxidation peaks with good potential separation on a single electrode. This achieves simultaneous resolution of multiple targets based on differences in material interfaces and intrinsic electrochemical behavior, overcoming the challenges of signal overlap and weak ZT response under neutral conditions. Structurally, a flexible stainless steel sheet tens of micrometers thick is used as the substrate, possessing excellent conductivity, mechanical flexibility, and environmental tolerance. It can closely adhere to uneven plant surfaces such as root tips and bud tips, achieving stable and reliable in-situ contact and overcoming the problems of poor adaptability of rigid electrodes and easy tissue damage from microneedles. In terms of application, for the first time, quantitative information on auxin, defense hormones, and cytokinins within the same plant microregion can be obtained simultaneously without relying on chromatographic separation and complex pretreatment, making it possible to directly study the immediate response and interactive dialogue of hormone networks under stress. This invention constructs a new paradigm for in-situ sensing of plant hormones that is non-biologically dependent, adaptable to living surfaces, and capable of simultaneous monitoring of multiple indicators through synergistic innovation of flexible substrate-carbon-based interface-acidic system. It provides a simpler, more comprehensive, and more realistic analytical method for plant physiological research and precision agriculture.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The sensor constructed in this invention is based on an ultrathin flexible stainless steel substrate and an integrated three-electrode system, which can directly and closely adhere to the surface of non-smooth tissues such as plant root tips and bud tips, realizing true in-situ, continuous, and non-destructive in vivo monitoring. This method does not require complex and time-consuming sample pretreatment, avoids hormone degradation and spatial information loss caused by in vitro sampling, and can acquire dynamic change information of endogenous hormones in plant microregions in real time, providing a breakthrough technical means for studying the spatiotemporal distribution and regulation mechanism of plant hormones at the tissue microregion level.

[0019] (2) By optimizing the conductive carbon gel-modified interface and the acidic detection system, this sensor achieves, for the first time, the simultaneous detection and accurate quantification of three important plant hormones—indole-3-acetic acid, salicylic acid, and zeatin—on a single flexible electrode. It exhibits a wide linear range of 2.5–30 μM, high sensitivity, and good reproducibility. This multi-index simultaneous detection capability helps to directly reveal the interaction networks and synergistic patterns of different hormones in development and stress responses, overcoming the limitations of traditional methods that can only perform single or sequential detection.

[0020] (3) The sensor exhibits good stability and anti-interference ability in complex plant matrices. Under selected potential windows and pH conditions, common endogenous substances such as abscisic acid and succinic acid do not produce significant interference signals, ensuring the specificity and reliability of the detection results. The sensor has a simple structure, low cost, and is easy to prepare and operate in batches. It is not only suitable for basic research on plant hormones in laboratory environments, but can also be extended to the dynamic tracking of various hormones during biotic and abiotic stress processes such as salt stress and pathogen infection, providing a powerful in-situ sensing tool for precision agriculture and plant phenotypic analysis. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the establishment of IAA, SA, and ZT standard curves based on wearable electrochemical sensors; where, Figure 1 A shows the differential pulse voltammetry (DPV) detection curves of mixed solutions of IAA, SA and ZT at different concentrations (2.5 ~ 30 μM) in pH 2.6 PBS buffer; Figure 1 B Figure 1 C and Figure 1 D represents the linear relationship between the current response values ​​of IAA, SA, and ZT and their corresponding concentrations.

[0022] Figure 2 A schematic diagram and results of in-situ simultaneous detection of IAA, SA, and ZT in micro-regions of maize seedling tissue; among which, Figure 2 A is a schematic diagram of the detection sites at the root tip and shoot tip of corn seedlings; Figure 2 B is a typical DPV curve actually measured at the root tip and shoot tip, and the characteristic oxidation peaks of IAA, SA and ZT are clearly shown in the figure; Figure 2 C represents a comparison of the current response values ​​of IAA, SA, and ZT in the root tip and shoot tip; Figure 2 D represents a comparison of the actual contents of IAA, SA, and ZT in root tips and shoot tips calculated based on the standard curve. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings, embodiments, and comparative examples.

[0024] Example 1

[0025] This embodiment uses the root tip and shoot tip of maize seedlings as experimental subjects. A method based on an ultrathin flexible stainless steel wearable sensor to simultaneously detect indole-3-acetic acid (IAA), salicylic acid (SA), and zeatin (ZT) in these tissue microregions includes the following steps: Step 1: Fabrication of the flexible flat working electrode: The flexible flat plate working electrode mainly consists of an ultra-thin flexible stainless steel sheet substrate, an insulating mask layer, and a conductive carbon adhesive modification layer. A 30 μm thick ultra-thin flexible stainless steel sheet is cut into strips of 8 mm × 20 mm, and then ultrasonically cleaned and dried sequentially with acetone, ethanol, and deionized water. Subsequently, a layer of transparent insulating tape with a 4 mm diameter circular through-hole is adhered to the sheet surface to precisely define the effective detection area. The conductive carbon adhesive (CCC) stock solution and its dedicated diluent are uniformly mixed at a ratio of 1:5 (v / v). 4 μL of this mixture is accurately transferred using a micropipette and dropped onto the exposed stainless steel sheet surface within the circular through-hole. The mixture is dried and cured at room temperature to form a uniform conductive carbon adhesive modification layer, thus obtaining the flexible flat plate working electrode.

[0026] Step 2: Construction of wearable electrochemical sensors: The flexible flat plate electrode prepared above is used as the working electrode and integrated with the Ag / AgCl electrode wire (reference electrode) and platinum wire (counter electrode) through a flexible clamping device to form a wearable sensor with a three-electrode system.

[0027] Step 3: Screening for the optimal detection pH: IAA, SA, and ZT standards were accurately weighed separately, and 30 μM single hormone solutions with pH values ​​of 2.6, 5.0, and 7.0 were prepared using 0.1 M phosphate-buffered saline (PBS). The assembled sensor working area (covered with a 4 mm diameter circular filter paper, i.e., the paper-based detection interface as the liquid phase mass transfer layer) was placed in 7 μL of the above solutions, and detection was performed using differential pulse voltammetry (DPV) on an electrochemical workstation. The DPV parameters were set as follows: initial potential 0.2 V, termination potential 1.4 V, potential increment 0.005 V, amplitude 0.025 V, pulse width 0.02 s, and settling time 30 s. DPV curves were recorded at different pH values. Under neutral conditions (pH 7.0), the oxidation peak potentials of IAA and SA were close together, and the ZT response signal was weak, making it difficult to distinguish all three simultaneously. As the pH value decreased, the oxidation peak potentials of both IAA and SA shifted positively. Unexpectedly, under strongly acidic conditions at pH 2.6, the oxidation peaks of IAA, SA, and ZT exhibited characteristic peaks with good potential separation, sharp peak shapes, and stability at approximately 0.80 V, 1.05 V, and 1.20 V, respectively.

[0028] Step 4: Simultaneous detection of IAA, SA, and ZT: Preparation of the standard curve: IAA, SA, and ZT standards were accurately weighed separately and dissolved in 0.1 M phosphate-buffered saline (PBS) at pH 2.6 to prepare single stock solutions. These stock solutions were then diluted and mixed to prepare a series of mixed standard solutions containing the three hormones, with concentration gradients of 2.5, 5, 10, 15, 20, 25, and 30 μM for each hormone. The assembled sensor working area (covered with 4 mm diameter circular filter paper, i.e., the paper-based detection interface as the liquid phase mass transfer layer) was placed in 7 μL of the above mixed standard solution droplets. Detection was performed using differential pulse voltammetry (DPV) on an IGS 1130 electrochemical workstation. The DPV parameters were set as follows: initial potential 0.2 V, termination potential 1.4 V, potential increment 0.005 V, amplitude 0.025 V, pulse width 0.02 s, and settling time 30 s. Recording the DPV curves at each concentration reveals independent and well-separated oxidation peaks corresponding to IAA, SA, and ZT at approximately 0.80 V, 1.05 V, and 1.20 V, respectively (e.g., ...). Figure 1 As shown in Figure A). The current response values ​​of each characteristic peak were extracted and linearly fitted with the corresponding hormone concentrations to establish standard curves for IAA, SA, and ZT (as shown in Figure A). Figure 1 (As shown in B, 1C, and 1D), the linear regression equation and correlation coefficient are obtained.

[0029] In situ detection of hormones in root tip and shoot tip tissues of maize seedlings: Maize seedlings that had grown normally to day 4 were selected. The flexible flat working electrode of the sensor was gently attached to the surface of the root tip or shoot tip tissue of the maize seedling to be tested, ensuring close contact between the electrode and the tissue without mechanical damage. 7 μL of pH 2.6 PBS buffer was added to a pre-placed circular filter paper between the electrode and the tissue. The reference electrode and counter electrode were placed near the working electrode, ensuring contact with the tissue or solution but not touching each other (see schematic diagram for detection). Figure 2 A).

[0030] Connect to an electrochemical workstation and perform in-situ detection using the aforementioned DPV parameters. Typical DPV curves obtained are shown below. Figure 2 As shown in Figure B, the characteristic peaks of IAA, SA, and ZT can be clearly distinguished. By substituting the current values ​​of each characteristic peak into the established standard curve, the real-time concentrations of IAA, SA, and ZT in the micro-region of this tissue can be calculated respectively.

[0031] The test results show (e.g.) Figure 2 (As shown in C and 2D), under normal growth conditions, the contents of IAA, SA, and ZT in the shoot tip tissue of maize seedlings were significantly higher than those in the root tip tissue. This embodiment achieves rapid, in-situ, and simultaneous quantitative analysis of three key hormones in microregions of different parts of the same plant sample, intuitively revealing the basic differences in hormone distribution in different plant organs.

[0032] To verify the structural advantages of the ultra-thin flexible stainless steel sheet selected in this invention as the working electrode substrate, the following comparative experiment was conducted: Comparative Example 1: Comparison of Construction and Detection Results of Rigid Substrate Electrodes The working electrode was constructed following the same steps as in Example 1, but the substrate material was replaced with a conventional rigid conductive material, namely glassy carbon electrode (GCE) and indium tin oxide conductive glass (ITO). The specific construction method is as follows: Glassy carbon electrode assembly: The surface of the glassy carbon electrode was polished to a mirror finish with 0.3 μm and 0.05 μm alumina powders in sequence, then ultrasonically cleaned in ethanol and deionized water in sequence, and dried. After drying, 4 μL of conductive carbon adhesive mixture (same as in Example 1) was drop-coated onto the electrode surface and dried at room temperature to form a modification layer.

[0033] ITO electrode assembly: ITO conductive glass is cut into strips of 8 mm × 20 mm, ultrasonically cleaned with acetone, ethanol and deionized water in sequence, dried, and then an insulating tape with a 4 mm diameter hole is attached to the surface. 4 μL of conductive carbon adhesive mixture is added and dried at room temperature.

[0034] The two rigid substrate electrodes were assembled into a three-electrode system with a platinum wire counter electrode and an Ag / AgCl reference electrode, respectively, and then attached to the root tip and bud tip of corn seedlings for in-situ detection as described in Example 1.

[0035] Experimental results: Glassy carbon electrode: Due to its rigid rod-like structure, the electrode cannot form a stable and tight contact with non-flat curved surfaces such as plant root tips and bud tips. During the detection process, gaps easily form between the electrode and the plant surface, preventing the electrolyte from effectively wetting the detection interface.

[0036] ITO electrodes: Although they are planar, due to their brittle and inflexible material, they are difficult to conform to the curved structure of plant tissues. Even slight contact can easily cause damage to the plant tissues or displacement of the electrodes, making it impossible to achieve stable in-situ contact.

[0037] Conclusion: In comparison, the ultra-thin flexible stainless steel sheet substrate with a thickness of 10-50 μm used in this invention possesses excellent flexibility and mechanical strength, enabling it to closely conform to uneven surfaces such as plant root tips and bud tips, achieving stable and non-destructive in-situ detection. The above comparative examples further confirm the irreplaceable role of the flexible substrate in wearable plant sensors, which is the key structural basis for achieving in-situ, simultaneous detection of multiple hormones in this invention.

[0038] Example 2: Detection of IAA, SA, and ZT in root tips and shoot tips of maize seedlings using a wearable electrochemical sensor with parameter range endpoints. In this embodiment, sensors were prepared using the lower and upper limits of the parameter range defined in the claims, and in situ detection of IAA, SA, and ZT in the root tips and shoot tips of corn seedlings was performed according to the method of Example 1 to verify the feasibility and stability of the technical solution of the present invention within the parameter range.

[0039] (a) Use the lower limit value of the parameter: Thickness of ultra-thin flexible stainless steel sheet: 10μm; The mixing ratio of conductive carbon adhesive is as follows: the conductive carbon adhesive stock solution and its diluent are mixed at a ratio of 1:4 (v / v); Paper-based detection interface diameter: 4mm; The diameter of the circular hole in the detection area is limited to 3mm; Dosage of conductive carbon adhesive mixture added: 3 μL; The pH of the PBS buffer used for testing is 2.2. Differential pulse voltammetry (DPV) parameters: initial potential 0.18V, termination potential 1.38V, potential increment 0.004V, amplitude 0.020V, pulse width 0.015s, resting time 25s.

[0040] (ii) Use the upper limit value of the parameter: Thickness of ultra-thin flexible stainless steel sheet: 50μm; The mixing ratio of conductive carbon adhesive is as follows: the conductive carbon adhesive stock solution and its diluent are mixed at a ratio of 1:6 (v / v); Paper-based detection interface diameter: 6mm; The diameter of the circular hole in the detection area is limited to 5mm; Dosage of conductive carbon adhesive mixture added: 5 μL; The pH of the PBS buffer used for the test is 3.0. Differential pulse voltammetry (DPV) parameters: initial potential 0.22V, termination potential 1.42V, potential increment 0.006V, amplitude 0.030V, pulse width 0.025s, resting time 35s.

[0041] The working electrode was prepared, the sensor was constructed, and in-situ detection was performed according to the method in Example 1. The results showed that, under the lower and upper limits of the parameters, the characteristic oxidation peaks of IAA, SA, and ZT were clearly visible at 0.75 V–0.85 V, 0.95 V–1.05 V, and 1.10 V–1.20 V, respectively, with sharp peak shapes and stable baselines. Based on the standard curves established using the mixed standard solutions, IAA, SA, and ZT all exhibited good linearity in the concentration range of 2.5–30 μM (R0). 2 (>0.99), the detection results are consistent with those of Example 1. This example demonstrates that, within the parameter range defined in the claims, the sensor can stably and accurately achieve simultaneous in-situ detection of three hormones.

[0042] Example 3: Wearable electrochemical sensor for in-situ detection of IAA, SA and ZT in tomato leaves In this embodiment, the constructed wearable electrochemical sensor was applied to tomato leaf tissue to verify its applicability in other plant species and tissue parts.

[0043] Experimental subjects: Tomato plants that have grown normally for 6 weeks, with mature leaves selected as the test sites.

[0044] Sensor fabrication: The working electrode (stainless steel sheet thickness 30 μm, conductive carbon adhesive ratio 1:5, circular hole diameter 4 mm, drop volume 4 μL) was prepared according to the method of Example 1, and assembled with Ag / AgCl reference electrode and platinum wire counter electrode to form a three-electrode system.

[0045] Detection method: Gently attach the flexible working electrode to the lower surface of a tomato leaf (avoiding the midrib). Add 7 μL of pH 2.6 PBS buffer to a pre-placed circular filter paper (4 mm in diameter). Place the reference electrode and counter electrode near the leaf to ensure contact with the solution. In situ detection was performed using the same DPV parameters as in Example 1 (initial potential 0.2 V, termination potential 1.4 V, potential increment 0.005 V, amplitude 0.025 V, pulse width 0.02 s, settling time 30 s).

[0046] Results: Three distinct oxidation peaks were detected in tomato leaves, corresponding to IAA (0.75 V–0.85 V), SA (0.95 V–1.05 V), and ZT (1.10 V–1.20 V), respectively. The concentrations of IAA, SA, and ZT in the leaves were calculated using a standard curve, and the results were consistent with the normal physiological levels reported in the literature. Furthermore, the relative standard deviation (RSD) of the three parallel detections was less than 5%, indicating that the sensor has good adhesion to the tomato leaf surface and good detection stability.

[0047] This embodiment demonstrates that the wearable electrochemical sensor is not only suitable for corn root tips and shoot tips, but can also be extended to other plant species and tissues such as tomato leaves, enabling simultaneous in situ detection of multiple hormones, and has good versatility and application potential.

[0048] This invention employs a wearable sensor based on an ultra-thin flexible stainless steel sheet to detect multiple plant hormones in microregions of plant tissues. It eliminates the need for complex tissue grinding, extraction, and separation steps, thus preserving the in-situ distribution and active state of hormones to the greatest extent. It can more realistically and conveniently reflect the spatial distribution characteristics of endogenous plant hormones, providing a powerful in-situ analysis tool for in-depth research on the physiological functions and interactions of plant hormones.

Claims

1. A wearable electrochemical sensor, characterized in that, include: The working electrode is made of an ultra-thin flexible stainless steel sheet modified with conductive carbon adhesive; the counter electrode; Reference electrode; And a paper-based detection interface, which is used to carry the electrolyte and is placed between the working electrode and the plant tissue.

2. The wearable electrochemical sensor according to claim 1, characterized in that, The counter electrode is a platinum wire; the reference electrode is an Ag / AgCl electrode.

3. The wearable electrochemical sensor according to claim 1, characterized in that, The thickness of the ultra-thin flexible stainless steel sheet is 10–50 μm.

4. The wearable electrochemical sensor according to claim 1, characterized in that, The conductive carbon adhesive is prepared by mixing conductive carbon adhesive stock solution and its diluent at a volume ratio of 1:4 to 6.

5. The wearable electrochemical sensor according to claim 1, characterized in that, The diameter of the paper-based detection interface is 4~6mm.

6. A method for preparing the wearable electrochemical sensor according to claim 1, characterized in that, Includes the following steps: Step 1: After cutting and cleaning the ultra-thin flexible stainless steel sheet, cover it with transparent tape with round holes of 3-5 mm in diameter to define the effective detection area; Step 2: Drop 3~5 μL of conductive carbon adhesive mixture onto the exposed stainless steel sheet surface to form a decorative layer; Step 3: Use the modified stainless steel sheet as the working electrode, and assemble it with the platinum wire counter electrode and the Ag / AgCl reference electrode to form a three-electrode system.

7. The application of the wearable electrochemical sensor of claim 1 in the simultaneous detection of indole-3-acetic acid (IAA), salicylic acid (SA), and zeatin (ZT) in plant tissues.

8. The application according to claim 7, characterized in that, The plant tissues include root tips, bud tips, leaves, or stem segments.

9. The application according to claim 7, characterized in that, The detection method includes: The sensor is attached to the surface of the part of the plant to be tested. Add PBS buffer with pH 2.2-3.0 to the paper substrate interface; The electrochemical workstation was connected, and differential pulse voltammetry was used for detection. Qualitative and quantitative analysis was performed by the characteristic peaks of IAA, SA, and ZT at 0.75 V~0.85 V, 0.95 V~1.05 V, and 1.10 V~1.20 V.

10. The application according to claim 9, characterized in that, The detection parameters of the differential pulse voltammetry method are as follows: detection potential range 0.1 V~1.5 V, initial potential 0.18~0.22 V, termination potential 1.38~1.42 V, potential increment 0.004~0.006 V, amplitude 0.020~0.030 V, pulse width 0.015~0.025 s, rest time 25~35 s, sampling width 0.0067s~0.0070s, and period 0.1s~0.2s.

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