WO2022148114A1 - 一种检测植物活性小分子的传感器和制备方法 - Google Patents

一种检测植物活性小分子的传感器和制备方法 Download PDF

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WO2022148114A1
WO2022148114A1 PCT/CN2021/128361 CN2021128361W WO2022148114A1 WO 2022148114 A1 WO2022148114 A1 WO 2022148114A1 CN 2021128361 W CN2021128361 W CN 2021128361W WO 2022148114 A1 WO2022148114 A1 WO 2022148114A1
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microneedle array
electrode
transparent flexible
array electrode
solution
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PCT/CN2021/128361
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English (en)
French (fr)
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谢曦
刘繁茂
谢致平
胡宁
杭天
陈惠琄
陶军
李湘凌
黄新烁
刘星星
刘子淇
周灵菲
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中山大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Definitions

  • the invention belongs to the field of plant sensors, and in particular relates to a sensor for detecting plant active small molecules and a preparation method.
  • Rhizosphere microorganisms are of great significance in the process of plant growth, and a considerable variety of rhizosphere microorganisms are beneficial to plant growth.
  • legume vegetables can perform symbiotic nitrogen fixation with the corresponding rhizobia, thereby reducing the input of nitrogen fertilizer, making legume vegetables not only occupy an important position in vegetable supply, bringing huge economic value to farmers, but also reducing agricultural non-point sources. pollution makes a substantial contribution.
  • the purpose of the present invention is to provide a flexible micro-needle array electrode sensor for plant stems and leaves, which can be attached to the surfaces of leaves and stems of plants to sense and fix It has dual functions and can make visible light shine through the sensor to the plant surface.
  • the present invention provides a sensor for detecting plant active small molecules, comprising:
  • Microneedle array electrodes which are used to detect the concentration of plant active small molecules
  • a plurality of signal conduction circuits are arranged between the transparent flexible substrate and the transparent flexible film, and each of the signal conduction circuits is connected to the microneedle array electrodes through the transparent flexible substrate.
  • the microneedle array electrode includes a working electrode, a counter electrode and a reference electrode that constitute an electrochemical three-electrode sensing system.
  • the present invention also provides a method for preparing the sensor according to claim 1, wherein the microneedle array electrode is prepared by the following method:
  • the zinc oxide microspheres are removed by acid etching to obtain porous microneedle array electrodes;
  • the porous microneedle array electrode is modified to obtain a microneedle array electrode including a working electrode, a counter electrode and a reference electrode.
  • the zinc oxide microspheres are prepared by the following method:
  • the preparation concentration is 20 ⁇ 80mmol/L hexahydrate zinc nitrate solution and 20 ⁇ 80mmol/L urotropine solution, and mix them in a volume ratio of 1:2 ⁇ 2:1;
  • the mixed solution is mixed with a sodium citrate solution with a concentration of 20-60 mmol/L in a volume ratio of 1:1-3:1 to obtain zinc oxide microspheres.
  • microneedle array electrode is specifically prepared by the following method:
  • the microneedle array electrode master mold has the same shape as the microneedle array electrode;
  • the dimethylsiloxane and the curing agent are mixed in a volume ratio of 9:1 to 11:1, and cast on the microneedle array electrode master mold, and after curing, the mold is demolded to obtain the microneedle array electrode mold;
  • the microneedle array electrode is put into hydrochloric acid with a concentration of 0.5-1.5 mol/L to remove the zinc oxide microspheres to obtain a porous microneedle array electrode.
  • the working electrode is prepared by the following method:
  • bovine serum albumin solution with a mass fraction of 0.5-1.5% and a glutaraldehyde solution or L-lysine solution with a mass fraction of 0.2-0.8% in a volume ratio of 8:1-10:1;
  • the final mixed solution was dripped onto the microneedle array electrode to complete the modification of the microneedle array electrode to obtain the working electrode.
  • the enzymes or sensing receptor molecules required for the sensing are glucose oxidase required for the detection of glucose, ferrocene derivatives required for the detection of salicylic acid, and superoxide required for the detection of reactive oxygen species one or more of dismutases.
  • the counter electrode and the reference electrode are prepared by the following methods:
  • a platinum layer is sputtered on the surface of the microneedle array electrode to complete the modification of the microneedle array electrode to obtain a counter electrode;
  • the microneedle array electrode was immersed in the Ag/AgCl slurry and pulled out to dry to complete the modification of the microneedle array electrode to obtain a reference electrode.
  • the transparent flexible substrate is prepared by the following method:
  • the polydimethylsiloxane and the curing agent are mixed in a volume ratio of 9:1 to 10:1, they are cast on a mold, and after curing, the mold is released to obtain a flexible substrate with a thickness of 0.5 mm to 0.8 mm.
  • the signal conduction circuit is prepared by the following method:
  • the transparent flexible film and the transparent flexible substrate are fixed with polydimethylsiloxane.
  • the present invention pierces the epidermis of the plant and inserts it into the plant tissue through the microneedle array electrode, so that the sensor can be attached to the surface of the leaves, stems, etc. of the plant, thereby achieving dual functions of sensing and fixing;
  • the transparent flexible substrate integrates the microneedle array electrodes and the signal conduction circuit, so that when the sensor is fixed on the plant, the visible light can still be irradiated to the surface of the plant through the sensor, so that the photosynthesis of the plant can not be affected.
  • the flexible transparent and flexible Substrates and transparent flexible films can adapt to parts such as thin leaves and curved stems of plants.
  • FIG. 1 is a schematic structural diagram illustrating a sensor for detecting plant active small molecules according to an embodiment of the present invention
  • FIG. 2 is a schematic three-dimensional structure diagram illustrating a sensor according to an embodiment of the present invention
  • FIG. 3 is a schematic diagram showing an explosion structure of a sensor according to an embodiment of the present invention.
  • FIG. 4 is a schematic diagram showing a three-dimensional structure of a sensor according to an embodiment of the present invention.
  • FIG. 5 is a schematic diagram showing an explosion structure of a sensor according to an embodiment of the present invention.
  • FIG. 6 is a schematic diagram illustrating the preparation of microneedle array electrodes according to an embodiment of the present invention.
  • FIG. 7 is a schematic diagram illustrating a sensing system for detecting plant active small molecules according to an embodiment of the present invention.
  • first, second, third, etc. may be used to describe . . . in the embodiments of the present invention, these . . . should not be limited to these terms. These terms are only used to distinguish ...
  • the first... may also be referred to as the second..., and similarly, the second... may also be referred to as the first... without departing from the scope of the embodiments of the present invention.
  • the present invention is a porous microneedle array electrode based on a biocompatible polymer material, obtains a working electrode, a counter electrode and a reference electrode, which are then integrated into a transparent flexible substrate and integrated into a three-electrode electrochemical sensor, which is attached to a plant leaf , stem and other surfaces, so that the microneedle electrode pierces the plant epidermis and inserts into the plant tissue, which can play the dual function of sensing and fixing; the sensor is connected with the electrochemical measuring instrument through the wire, which can realize in-situ, real-time, minimally invasive and continuous Plant active small molecule concentration detection.
  • the active small molecule indicators that can be detected in the present invention can be roughly divided into three categories: plant nutrients (such as glucose), plant hormones (such as abscisic acid ABA, salicylic acid SA, auxin IAA) and plant stress response indicators (such as reactive oxygen species (ROS).
  • plant nutrients such as glucose
  • plant hormones such as abscisic acid ABA, salicylic acid SA, auxin IAA
  • plant stress response indicators such as reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • an embodiment of the present invention provides a sensor 10 for detecting plant active small molecules, including:
  • Microneedle array electrode 11 which is used to detect the concentration of plant active small molecules
  • the transparent flexible substrate 12 has a groove 121 on one side for fixing the microneedle array electrodes 11;
  • a transparent flexible film 13 which is fixed on the other side of the transparent flexible substrate 12 away from the groove 121;
  • a plurality of signal conduction circuits 14 are arranged between the transparent flexible substrate 12 and the transparent flexible film 13 , and each of the signal conduction circuits 14 passes through the transparent flexible substrate 12 and the microneedle array electrodes 11 connect.
  • this implementation may also include the following content:
  • the microneedle array electrode 11 includes a microneedle array fixed on a plant and a substrate on which the microneedle array is fixed, and the microneedle array is formed by arranging a plurality of microneedles.
  • the groove 121 of the transparent flexible substrate 12 has a structural arrangement for fixing the substrate.
  • the cross section of the groove 121 of the transparent flexible substrate 12 is T-shaped, and the microneedle array is provided in the middle of one side surface of the base.
  • the bottom of the groove 121 of the transparent flexible substrate 12 has a through hole, so that the two sides of the transparent flexible substrate 12 are connected through the through hole, and the cross-section of the base corresponding to the structure is I-shaped.
  • the flexible substrate 12 is flexible, so the microneedle array electrodes 11 can be installed and fixed directly through the flexible properties of the transparent flexible substrate 12 .
  • the corresponding structures of the transparent flexible substrate 12 and the microneedle array electrodes 11 may be limited.
  • the base of the microneedle array electrode 11 is square, triangular or other irregular shape
  • the corresponding groove 121 of the transparent flexible substrate 12 is a matching square, triangle or other irregular shape.
  • the microneedles of the microneedle array of the microneedle array electrode 11 are arranged in a square, triangular or other irregular shape (for example, the entire microneedle array consists of 9 microneedles arranged in a 3 ⁇ 3 square)
  • the cross-section of the side wall of the groove 121 corresponding to the transparent flexible substrate 12 near the notch is a square, a triangle or other irregular shapes.
  • a single microneedle of the microneedle array can be in the shape of a quadrangular pyramid or a cone, and its length is 650 ⁇ m, the bottom (the part where the microneedle is connected to the substrate) has a side length or diameter of 200 ⁇ m, and the distance between the centers of adjacent microneedles is 200 ⁇ m. about 500 ⁇ m.
  • the microneedle array electrode 11 of the embodiment of the present invention includes a working electrode, a counter electrode and a reference electrode that constitute an electrochemical three-electrode sensing system.
  • this embodiment may also include the following content:
  • the embodiment of the present invention provides a method for preparing the above sensor, and the microneedle array electrode 11 is prepared by the following method:
  • the zinc oxide microspheres are removed by acid etching to obtain porous microneedle array electrodes;
  • the porous microneedle array electrode is modified to obtain a microneedle array electrode including a working electrode, a counter electrode and a reference electrode.
  • this embodiment may also include the following content:
  • the zinc oxide microspheres are prepared by the following methods:
  • the preparation concentration is 20 ⁇ 80mmol/L hexahydrate zinc nitrate solution and 20 ⁇ 80mmol/L urotropine solution, and mix them in a volume ratio of 1:2 ⁇ 2:1;
  • the mixed solution is mixed with a sodium citrate solution with a concentration of 20-60 mmol/L in a volume ratio of 1:1-3:1 to obtain zinc oxide microspheres.
  • zinc nitrate hexahydrate solution [Zn(NO 3 ) 2 ⁇ 6H 2 O] and urotropine (HTMA) solution with a concentration of 50 mmol/L can be prepared and mixed in a volume ratio of 1:1 ;
  • the mixed solution with a sodium citrate (Na 3 C 6 H 5 O 7 ) solution with a concentration of 38 mmol/L in a volume ratio of 2:1, and react at 90 ° C for a certain period of time (90 min) to obtain zinc oxide microspheres,
  • the solution was concentrated for later use.
  • the diameter of the zinc oxide microspheres can be adjusted by the solution mixing ratio and the reaction time, so that the finally obtained microneedle array electrode can meet different needs and improve its adaptability.
  • this embodiment may include the following contents:
  • microneedle array electrode is specifically prepared by the following method:
  • the microneedle array electrode master mold has the same shape as the microneedle array electrode;
  • the dimethylsiloxane and the curing agent are mixed in a volume ratio of 9:1 to 11:1, and cast on the microneedle array electrode master mold, and after curing, the mold is demolded to obtain the microneedle array electrode mold;
  • the microneedle array electrode is put into hydrochloric acid with a concentration of 0.5-1.5 mol/L to remove the zinc oxide microspheres to obtain a porous microneedle array electrode.
  • microneedle array electrodes based on polyethylene glycol diacrylate materials have high rigidity, which can ensure the efficiency of piercing plant stems and leaf epidermis.
  • the microneedle array electrodes can be prepared by the following methods:
  • a microneedle array master mold made of SU-8 photoresist material was prepared by photolithography technology (the structure shown in the upper left corner of Figure 6);
  • Dimethyl siloxane (PDMS) and curing agent are mixed in a volume ratio of 10:1, and then centrifuged to degas after uniform stirring, so as to avoid the existence of air bubbles in the final formed mold and affect the forming quality of microneedle array electrodes;
  • step 1 in Figure 6 where the mixed solution and the microneedle array master mold are located in the container), and placed side by side.
  • a heating device at 40°C, demolding is carried out after curing for a certain period of time (24 hours) (step 2 in Figure 6) to obtain a microneedle array mold;
  • the concentrated solution of zinc oxide microspheres was mixed with the polyethylene glycol diacrylate solution after mixing the photocrosslinking agent in a volume ratio of 1:8, and it was fully mixed for a certain period of time (30min) by ultrasonic, so as to make the oxidation template as a template.
  • the zinc microspheres are uniformly distributed in the polyethylene glycol diacrylate solution; among them, the different porosity of the microneedle array electrodes can be realized by adjusting the proportion of the zinc oxide microspheres.
  • step 3 in Figure 6 After the ultrasonic stops (that is, after sufficient mixing), immediately fill the finally prepared mixed solution into the mold formed by dimethylsiloxane through the curing agent (step 3 in Figure 6). 100 ⁇ L), then centrifuge it (step 4 in Figure 6), perform UV curing immediately after centrifugation to fill the mold (step 5 in Figure 6), and then demold to obtain a microneedle array electrode (step 6 in Figure 6).
  • microneedle array electrode into hydrochloric acid with a concentration of 1 mol/L and sonicate for a certain period of time (60 min) to corrode the zinc oxide microspheres in the microneedle array electrode, and take out the microneedle array electrode after ultrasonication. After being thoroughly cleaned with deionized water, it was put into a vacuum tank for full drying, and finally a porous microneedle array was obtained (step 7 in Figure 6).
  • zinc oxide microspheres are used as sacrificial materials and structures, which are pre-mixed with the mother solution for preparing microneedle array electrodes, and then the zinc oxide microspheres are acid-etched after the microneedle array electrodes are cured and demolded. Dissolving, thereby obtaining the channel formed by the continuous micron-scale holes in the microneedle array electrode structure (including the microneedle and the substrate).
  • These micro-scale holes and channels formed provide abundant attachment sites for sensing modification materials (different electrodes have different sensing modification materials), and at the same time increase the specific surface area of the microneedle array electrodes, and the capillary effect of the channel itself.
  • the microneedle sensing electrode can be fully infiltrated by the plant tissue fluid.
  • the combined effect of these three aspects can improve the detection sensitivity of the sensor and the stability of the attachment of the sensing modification material, and ensure that the sensor can sensitively and continuously monitor the concentration of active small molecules in plants in situ.
  • this embodiment may include the following contents:
  • the microneedle array electrode is prepared by a corresponding method to obtain a working electrode, a counter electrode and a reference electrode that constitute an electrochemical three-electrode sensing system.
  • the working electrode is prepared by the following method:
  • bovine serum albumin solution with a mass fraction of 0.5-1.5% and a glutaraldehyde solution or L-lysine solution with a mass fraction of 0.2-0.8% in a volume ratio of 8:1-10:1;
  • the final mixed solution is dripped onto the microneedle array electrode to complete the modification of the microneedle array electrode to obtain the working electrode.
  • the counter electrode and the reference electrode are prepared by the following methods:
  • a platinum layer is sputtered on the surface of the microneedle array electrode to complete the modification of the microneedle array electrode to obtain a counter electrode;
  • microneedle array electrodes are immersed in the Ag/AgCl slurry and pulled out to dry to complete the modification of the microneedle array electrodes to obtain a reference electrode, wherein Ag/AgCl indicates that the slurry contains Ag and AgCl.
  • the working electrode can be prepared by the following methods:
  • sensing modification materials required for sensing in a mass fraction of about 1%, for example, glucose oxidase required for the detection of glucose, and dimethylocene required for the detection of salicylic acid Iron derivative redox microneedle array electrodes, and one or more of materials such as superoxide dismutase required for the detection of reactive oxygen species;
  • the counter electrode can be prepared by the following methods:
  • the counter electrode can be prepared by a radio frequency magnetron sputtering process.
  • the sputtering material is platinum
  • the sputtering power is 80W
  • the sputtering time is 10 minutes. According to the sputtering parameters, a layer of platinum is uniformly sputtered on the surface of the microneedle array, thereby obtaining a modified counter electrode;
  • the reference electrode can be prepared in the following ways:
  • the reference electrode can be prepared by immersion method. Specifically, the microneedle array electrodes can be immersed in a commercial Ag/AgCl slurry for 30 minutes, then taken out and placed in a heating device (oven) at 40° C. for a certain period of time (12 hours), so that the slurry is dried and fixed in the micro on the needle array electrode to obtain a modified reference electrode.
  • a heating device Oven
  • this embodiment may include the following contents:
  • the transparent flexible substrate 12 is prepared by the following method:
  • the polydimethylsiloxane and the curing agent are mixed in a volume ratio of 9:1 to 10:1, they are cast on a mold, and after curing, the mold is released to obtain a flexible substrate with a thickness of 0.5 mm to 0.8 mm.
  • a flexible dimethylsiloxane material is used to make a transparent flexible substrate of the microneedle array electrode in an embodiment of the present invention, so as to play a supporting and fixing role.
  • the transparent flexible substrate can be prepared by the following methods:
  • the transparent flexible substrate includes three grooves 121 for fixing the microneedle array electrodes, and the microneedle array electrodes are clamped (or fixed) by the elasticity of dimethylsiloxane (or other fixing structures). in the groove 121 .
  • the other side of the transparent flexible substrate (the side away from the groove 121 ) has openings (which can be square openings or circular openings, etc.), and the openings are used for signal conduction circuits and microneedles on the transparent flexible film.
  • the backside of the array electrodes (the side of the substrate away from the microneedles) is connected.
  • this embodiment may include the following contents:
  • the signal conduction circuit is prepared by the following method:
  • the transparent flexible film and the transparent flexible substrate are fixed with polydimethylsiloxane.
  • the embodiment of the present invention prepares a patterned signal conduction circuit on a transparent flexible film, so that the signal conduction circuit is connected with the microneedle array electrodes to construct a complete electrochemical three-electrode sensor.
  • a transparent flexible polymer film with a thickness of 50 ⁇ m is selected as the transparent flexible film in the embodiment of the present invention, including but not limited to polyethylene terephthalate (PET), polyethylene naphthalate (PEN) And transparent polyimide (PI) and other materials; and the transparent film material can transmit most of the visible light to ensure the normal photosynthesis of plant stems and leaves.
  • the signal conduction circuit on the transparent flexible film can be prepared by the following methods:
  • the patterned signal conduction circuit is deposited on the transparent flexible film by the magnetron sputtering process, the deposited metal is gold or platinum, and the deposited thickness is about 100nm, and the patterning can be realized by a pre-customized mask;
  • conductive silver glue (which is located in the opening 122 of the transparent flexible substrate as shown in Figures 1 and 5) to connect the patterned signal conduction circuit and the corresponding microneedle array electrodes;
  • dimethylsiloxane is used to bond and seal the transparent flexible film and the transparent flexible substrate to protect the internal signal conduction circuit from rain and the like.
  • the electrochemical sensor using the microneedle array as the electrode is mainly due to:
  • Electrochemical sensors are suitable for the development of in-situ, real-time, and living plant-transmitted devices.
  • existing plant electrochemical sensors such as silicon-based electrochemical probes, have problems such as complex processing, easy breakage and damage; Sensing electrodes can only detect biological signals on the surface of plants, but cannot detect various biomarkers in plants.
  • the microneedle array prepared based on polymer material is used as an electrochemical sensor electrode. First, it can efficiently penetrate the plant epidermis in a minimally invasive way to detect the biomarker molecules inside the plant. Secondly, after the microneedle array penetrates the plant epidermis It can play the role of a fixed sensor, which is convenient for in-situ and continuous biosensing, and also avoids the use of adhesion and fixing devices to damage the plant. Again, the microneedle array electrode based on polymer materials has good biocompatibility. , high environmental tolerance, suitable for long-term continuous sensing.
  • the sensor of the embodiment of the present invention is transparent, flexible and can be applied, and the reasons for this feature are as follows:
  • the microneedle array electrode in the embodiment of the present invention is integrated on the substrate of flexible PDMS material (transparent flexible substrate), which can better stick to the stem and leaf surface of the plant; at the same time, the transparent flexible substrate and the transparent flexible film are both flexible
  • the transparent material ensures that most of the visible light can pass through the sensor to the plant surface.
  • the porous structure adopted by the microneedle array electrode in the embodiment of the present invention can ensure long-term sensing stability, which is mainly reflected in:
  • a porous structure is prepared on the surface and inside of the microneedle array electrode (the structure is prepared by acid etching zinc oxide microspheres), on the one hand, the sensing modification structure and material can be attached to the inner surface of the porous structure, which is relatively To a large extent, the damage of the sensing structure caused by the stress caused by the contact with the plant tissue is avoided, and on the other hand, the specific surface area of the sensing electrode is increased, thereby increasing the attachment area of the sensing response material, and correspondingly improving the sensor. Detection sensitivity.
  • this embodiment may include the following contents:
  • the present invention also provides a sensing system 20 for detecting plant active small molecules, comprising:
  • the above-mentioned sensor 10 is fixed at the stem and leaf of the plant;
  • the electrochemical workstation 21 is connected to the sensor 10 through a signal transmission cable, so as to realize the in-situ and continuous monitoring of plant active small molecule signals.
  • the electrochemical workstation 21 can be a portable device, and further, the electrochemical workstation can also be replaced with a wireless transmission module, such as Bluetooth, 5G, etc., for wirelessly transmitting the collected sensor signals to a remote data terminal for processing, This enables remote sensing management.
  • a wireless transmission module such as Bluetooth, 5G, etc.
  • this embodiment may include the following contents:
  • the working electrode is functionalized to obtain the ability to detect specific active small molecules (such as reactive oxygen species ROS).
  • the three-electrode sensing system composed of microneedle array electrodes works by electrochemical measurement.
  • the sensing electrodes microneedle array electrodes
  • subcutaneous ROS molecules such as hydrogen peroxide, superoxide, etc.
  • An electrochemical reaction occurs on the working electrode and the counter electrode, and a current signal is generated, which is conducted to the electrochemical workstation along the loop of the microneedle array electrode-signal conduction circuit-electrochemical workstation.
  • the electrochemical workstation measures the magnitude of the current, and then according to the current -Concentration standard curve to convert the concentration value of ROS molecules.
  • this embodiment may include the following contents:
  • a soybean plant at the flowering stage is selected, and a microneedle array sensor for glucose detection is applied to its main stem and the upper, middle and lower compound leaves of the plant, and a thin wire is used to connect the sensor signal conduction circuit to the Portable electrochemical workstation. Electrochemical signals were collected every 6 hours to determine the glucose level of the plant, and the duration of each collection was 10 minutes; the collection was continuous for 2 to 3 weeks, and the nutritional status of soybean plants was evaluated according to the monitored glucose level change trend, and the corresponding water was taken. , fertilizer control measures, and then continue to monitor 1-2 weeks to the pod setting stage.

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Abstract

一种检测植物活性小分子的传感器(10)和制备方法，该传感器(10)包括：微针阵列电极(11)，其用于检测植物活性小分子浓度；透明柔性衬底(12)，其一侧具有固定微针阵列电极(11)的凹槽(121)；透明柔性薄膜(13)，其固定于透明柔性衬底(12)远离凹槽(121)的另一侧；多个信号传导电路(14)，其布置于透明柔性衬底(12)和透明柔性薄膜(13)间，并且每一信号传导电路(14)穿过透明柔性衬底(12)与微针阵列电极(11)连接。可以贴附于植物的叶片、茎等表面，起到传感和固定的双重功能，并且可以使得可见光透过传感器(10)照射至植物表面。

Description

一种检测植物活性小分子的传感器和制备方法 技术领域
本发明属于植物传感器领域，尤其涉及一种检测植物活性小分子的传感器和制备方法。
背景技术
根际微生物在植物生长过程中意义重大，且相当种类的根际微生物对于植物生长有益。如豆类蔬菜能与对应的根瘤菌进行共生固氮作用，从而减少氮肥的投入，使得豆类蔬菜不但在蔬菜供应中占据重要地位，为农民带来巨大的经济价值，而且能为减少农业面源污染做出实质性的贡献。
根际微生物对植物促生和胁迫的不同响应机制，会在激素等水平上反馈出来。然而目前的标准检测方法都是破坏植物样本的离体检测，仅能反应某一时间点的静态浓度或累积效应。并且大部分的研究集中在构建比表面积大的纳米结构、合成新的催化材料和进行离体检测，而对植物小分子的动态、在体检测较少涉及，难以为相关研究和精准农业的实践提供实时动态的信息支撑。可以预见，未来植物传感器和传感系统会朝着微型化、智能化、在体实时检测等方向发展，这是一个崭新的研究领域，也是精准农业必由之路。
传统的植物激素等活性小分子的检测方法属于离体检测，需要昂贵的仪器和复杂的样品制备过程，采集往往造成了植物体的损伤甚至死亡，而且得到的数据只能反映的是静态结果和累积效应。植物激素等小分子在植物体内含量低而且容易被分解，如何对它们进行简便、快速和准确的测定一直是学界寻求重大创新与突破的课题。
近年来，随着生物电子工程技术的飞速发展，尤其是借助微纳技术的进展，为新型植物传感器件的开发提供了巨大的潜能。目前，基于微纳技术开发的智能传感器件已在植物健康状态监测、植物响应调制等应用领域进行了一些探索性研究。然而现有的传感器件多为贴敷于植物表皮，仅能检测表皮温湿度等植株-环境界面处的信息，而难以检测植株茎叶等器官的内部生物信号，如活性小分子浓度水平等。另外，现有的传感器具有可穿透植物表皮的功能，其直接同组织液接触实现生物传感，然而其多数基于动物透皮传感需求开发，直接套用至植物传感时仍存在诸多待解决问题，如电极阵列由于非柔性的特点，导致其不适合植物较为轻薄的叶和弯曲的茎，又如动物透皮传感对传感电极生物安全性要求较高，而植物透皮传感则对可靠性如耐高温/高湿环境、耐阳光直射以及传感器透明度(不阻碍茎叶的光合作用)要求较高。因此，现阶段的植物传感器仍存在较大缺陷。综上，开发一种实时、在体的用于植物透皮传感的传感器，支持植物体内激素时空分布和变化的基础研究和精准农业的发展，具有重要的科研和产业价值。
发明内容
针对上述现有技术中存在的缺陷，本发明的目的在于提供一种针对植物茎、叶使用的柔性微针阵列电极传感器，可以贴附于植物的叶片、茎等表面，起到传感和固定的双重功能，并且可以使得可见光透过传感器照射至植物表面。
第一方面，本发明提供一种检测植物活性小分子的传感器，包括：
微针阵列电极，其用于检测植物活性小分子浓度；
透明柔性衬底，其一侧具有固定所述微针阵列电极的凹槽；
透明柔性薄膜，其固定于所述透明柔性衬底远离所述凹槽的另一侧；
多个信号传导电路，其布置于所述透明柔性衬底和透明柔性薄膜间，并且每一所述信号传导电路穿过所述透明柔性衬底与所述微针阵列电极连接。
其中，所述微针阵列电极包括构成电化学三电极传感体系的工作电极、对电极和参比电极。
第二方面，本发明还提供一种制备如权利要求1所述传感器的方法，所述微针阵列电极采用以下方法制备：
制备氧化锌微球溶液；
制备微针阵列电极的模具；
将氧化锌微球溶液与制备微针阵列电极的第一混合溶液混合，制备成第二混合溶液；
将第二混合溶液填充于所述微针阵列电极的模具内固化；
在第二混合溶液固化后，酸蚀去除氧化锌微球，以得到多孔的微针阵列电极；
将多孔的微针阵列电极进行修饰得到包括工作电极、对电极和参比电极的微针阵列电极。
其中，所述氧化锌微球采用以下方法制备：
配制浓度为20～80mmol/L六水合硝酸锌溶液和20～80mmol/L乌洛托品溶液并以体积比1:2～2:1混合；
将混合溶液与浓度20～60mmol/L的柠檬酸钠溶液以体积比1:1～3:1的比例混合获得氧化锌微球。
其中，所述微针阵列电极具体采用以下方法制备：
制备微针阵列电极母模，该微针阵列电极母模与微针阵列电极外形相同；
将二甲基硅氧烷与固化剂按9:1～11:1的体积比混合，并浇铸于微针阵列电极母模上，固化后脱模得到微针阵列电极模具；
将聚乙二醇二丙烯酸酯和光交联剂按体积比8:1～10:1混合得到第一混合溶液；
将含有氧化锌微球的溶液与第一混合溶液按体积比1:10～2:10混合得到第二混合溶液；
将第二混合溶液填充至微针阵列电极模具中，固化后脱模获得微针阵列电极；
将微针阵列电极放入浓度0.5～1.5mol/L的盐酸中，以去除氧化锌微球后得到多孔的微针阵列电极。
其中，所述工作电极采用以下方法制备：
将质量分数为0.5～1.5％的牛血清蛋白溶液与质量分数为0.2～0.8％的戊二醛溶液或L-赖氨酸溶液，按照体积比8:1～10:1混合；
在混合溶液中加入质量分数为0.05～0.15％的单壁碳管或单层石墨烯；
加入质量分数为0.5～1.5％的传感所需的酶或传感受体分子；
将最后的混合溶液滴涂至微针阵列电极，以完成微针阵列电极的修饰得到工作电 极。
其中，所述传感所需的酶或传感受体分子为检测葡萄糖所需的葡萄糖氧化酶，检测水杨酸所需的二茂铁衍生物类，以及检测活性氧所需的超氧化物歧化酶中的一种或多种。
其中，所述对电极和参比电极采用以下方法制备：
在微针阵列电极表面溅射铂层，以完成微针阵列电极的修饰得到对电极；
将微针阵列电极浸入Ag/AgCl浆料并捞出干燥，以完成微针阵列电极的修饰得到参比电极。
其中，所述透明柔性衬底采用以下方法制备：
制备浇筑聚二甲基硅氧烷衬底的模具；
将聚二甲基硅氧烷与固化剂按9:1～10:1的体积比混合后，浇铸于模具上，固化后脱模获得厚度在0.5mm～0.8mm的柔性衬底。
其中，所述信号传导电路采用以下方法制备：
选取厚度在30～70μm的透明柔性薄膜；
在透明柔性薄膜上沉积厚度为90～110nm的信号传导电路；
通过导电银胶连接信号传导电路和对应的微针阵列电极；
采用聚二甲基硅氧烷将透明柔性薄膜和透明柔性衬底固定。
与现有技术相比，本发明通过微针阵列电极刺破植物表皮插入植物组织，使得传感器可以贴附于植物的叶片、茎等表面，从而起到传感和固定的双重功能；另外，通过透明柔性衬底统合微针阵列电极和信号传导电路，可以使得传感器固定于植物上时，可见光仍可以透过传感器照射至植物表面，从而可以达到不影响植物的光合作用，进一步，柔性的透明柔性衬底和透明柔性薄膜，可以适应植物较为轻薄的叶和弯曲的茎等部位。
附图说明
通过参考附图阅读下文的详细描述，本公开示例性实施方式的上述以及其他目的、特征和优点将变得易于理解。在附图中，以示例性而非限制性的方式示出了本公开的若干实施方式，并且相同或对应的标号表示相同或对应的部分，其中：
图1是示出根据本发明实施例的一种检测植物活性小分子的传感器的结构示意图；
图2是示出根据本发明某一实施例的传感器的立体结构示意图；
图3是示出根据本发明某一实施例的传感器的爆炸结构示意图；
图4是示出根据本发明某一实施例的传感器的立体结构示意图；
图5是示出根据本发明某一实施例的传感器的爆炸结构示意图；
图6是示出根据本发明某一实施例的制备微针阵列电极的示意图；以及
图7是示出根据本发明实施例的一种检测植物活性小分子的传感系统的示意图。
具体实施方式
在本发明实施例中使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本发明。在本发明实施例和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该” 也旨在包括多数形式，除非上下文清楚地表示其他含义，“多种”一般包含至少两种。
应当理解，尽管在本发明实施例中可能采用术语第一、第二、第三等来描述…，但这些…不应限于这些术语。这些术语仅用来将…区分开。例如，在不脱离本发明实施例范围的情况下，第一…也可以被称为第二…，类似地，第二…也可以被称为第一…。
本发明是基于生物相容性聚合物材料的多孔微针阵列电极，获得工作电极、对电极和参比电极，随后整合至透明柔性衬底并集成为三电极电化学传感器，贴附于植物叶片、茎等表面，使微针电极刺破植物表皮插入植物组织，可起到传感和固定的双重功能；传感器通过导线与电化学测量仪器相连，可以实现原位、实时、微创且持续的植物活性小分子浓度检测。本发明可进行检测的活性小分子指标可大致分为三大类：植物营养成分(如葡萄糖)，植物激素(如脱落酸ABA、水杨酸SA、生长素IAA)以及植物逆境胁迫应答指标(如活性氧ROS)。通过对这些植物活性小分子的原位、在体监测，能够及时地反映出不同根际微生物对植物的促生或胁迫效果，为完善植株的健康生长评估体系提供技术基础，也有助于精准农业技术的推广应用，还能为研究微生物多样性与植物促生及抗逆机理等领域提供理论依据。
下面结合附图详细说明本发明的可选实施例。
实施例一
参见图1所示，本发明实施例提供一种检测植物活性小分子的传感器10，包括：
微针阵列电极11，其用于检测植物活性小分子浓度；
透明柔性衬底12，其一侧具有固定所述微针阵列电极11的凹槽121；
透明柔性薄膜13，其固定于所述透明柔性衬底12远离所述凹槽121的另一侧；
多个信号传导电路14，其布置于所述透明柔性衬底12和透明柔性薄膜13间，并且每一所述信号传导电路14穿过所述透明柔性衬底12与所述微针阵列电极11连接。
实施例二
在实施例一的基础上，本实施还可以包括以下内容：
参见图1至图5所示，所述微针阵列电极11包括固定于植物上的微针阵列和固定有所述微针阵列的基底，该微针阵列由多个微针排列形成。与之对应地，透明柔性衬底12的凹槽121具有固定该基底的结构设置。在一个应用场景中，透明柔性衬底12的凹槽121截面为T型，基底一侧面的中部位置设有所述微针阵列。在另一个应用场景中，透明柔性衬底12的凹槽121底部具有一个通孔，从而使得透明柔性衬底12的两侧通过通孔连通，对应该结构的基底截面为工字型，由于透明柔性衬底12为柔性的，因此该微针阵列电极11可以直接通过透明柔性衬底12的柔性特性进行安装固定。另外，为了避免固定于透明柔性衬底12上的微针阵列电极11发生转动的情况，可以对透明柔性衬底12和微针阵列电极11设置对应的结构进行限制。在一个应用场景中，微针阵列电极11的基底为方形、三角形或其他不规则形状，透明柔性衬底12对应的凹槽121为与之匹配的方形、三角形或其他不规则形状。在另一个应用场景中，微针阵列电极11的微针阵列的微针排列方式为方形、三角形或其他不规则形状(例如，整个微针阵列由9根微针排列成3×3的正方形)，透明柔性衬底12对应的凹槽121靠近槽口的侧壁的截面为方形、三角形或其他不规则形状。
进一步地，微针阵列的单根微针可以为四棱锥形状或者圆锥形状，其长度为650μm，底部(微针与基底连接的部位)边长或直径为200μm，相邻的微针的中心距约为500μm。另 外，本发明实施例的所述微针阵列电极11包括构成电化学三电极传感体系的工作电极、对电极和参比电极。
实施例三
在实施例一的基础上，本实施例还可以包括以下内容：
本发明实施例提供一种制备上述传感器的方法，所述微针阵列电极11采用以下方法制备：
制备氧化锌(ZnO)微球溶液；
制备微针阵列电极的模具；
将氧化锌微球溶液与制备微针阵列电极的第一混合溶液混合，制备成第二混合溶液；
将第二混合溶液填充于所述微针阵列电极的模具内固化；
在第二混合溶液固化后，酸蚀去除氧化锌微球，以得到多孔的微针阵列电极；
将多孔的微针阵列电极进行修饰得到包括工作电极、对电极和参比电极的微针阵列电极。
实施例四
在实施例三的基础上，本实施例还可以包括以下内容：
所述氧化锌微球采用以下方法制备：
配制浓度为20～80mmol/L六水合硝酸锌溶液和20～80mmol/L乌洛托品溶液并以体积比1:2～2:1混合；
将混合溶液与浓度20～60mmol/L的柠檬酸钠溶液以体积比1:1～3:1的比例混合获得氧化锌微球。
在一个应用场景中，可以配制浓度均为50mmol/L的六水合硝酸锌溶液[Zn(NO 3) 2·6H 2O]和乌洛托品(HTMA)溶液并以体积比1:1混合混合；
将混合溶液与浓度为38mmol/L的柠檬酸钠(Na 3C 6H 5O 7)溶液以体积比2:1的比例混合，并于90℃反应一定时间(90min)获得氧化锌微球，并将溶液浓缩后备用。其中，该氧化锌微球的直径可通过溶液混合比和反应时长进行调整，从而使得最终得到的微针阵列电极满足不同的需求，提高其适应性。
实施例五
在上述实施例的基础上，本实施例可以包括以下内容：
所述微针阵列电极具体采用以下方法制备：
制备微针阵列电极母模，该微针阵列电极母模与微针阵列电极外形相同；
将二甲基硅氧烷与固化剂按9:1～11:1的体积比混合，并浇铸于微针阵列电极母模上，固化后脱模得到微针阵列电极模具；
将聚乙二醇二丙烯酸酯和光交联剂按体积比8:1～10:1混合得到第一混合溶液；
将含有氧化锌微球的溶液与第一混合溶液按体积比1:10～2:10混合得到第二混合溶液；
将第二混合溶液填充至微针阵列电极模具中，固化后脱模获得微针阵列电极；
将微针阵列电极放入浓度0.5～1.5mol/L的盐酸中，以去除氧化锌微球后得到多孔的微针阵列电极。
其中，基于聚乙二醇二丙烯酸酯材料所制得微针阵列电极刚性较高，可以以此保证穿刺植物茎、叶表皮的效率。
参见图6所示，在一个应用场景中，所述微针阵列电极可以采用以下方法制备：
利用光刻技术制备出SU-8光刻胶材质的微针阵列母模(如图6左上角所示结构)；
将二甲基硅氧烷(PDMS)与固化剂按10:1的体积比混合，待均匀搅拌后进行离心去气，以避免最终形成的模具存在气泡而影响微针阵列电极的成型质量；
将离心去气后的混合溶液浇铸到SU-8光刻胶材质的微针阵列母模上(如图6中的步骤①，其中，混合溶液和微针阵列母模位于容器内)，并置于40℃加热装置(烘箱)内，在固化一定时间(24小时)后进行脱模(如图6中的步骤②)，制得微针阵列模具；
将小分子量聚乙二醇二丙烯酸酯(PEGDA，Mn～700)和光交联剂按体积比9:1进行混合，在搅拌均匀后超声去气；
将氧化锌微球的浓缩液与混合光交联剂后的聚乙二醇二丙烯酸酯溶液按体积比1:8混合，在超声一定时间(30min)使其充分混合，从而使得作为模板的氧化锌微球均匀地分布在聚乙二醇二丙烯酸酯溶液中；其中，可以通过调整氧化锌微球的比例实现微针阵列电极不同的孔隙率。
超声停止后(即充分混合后)，立即将最后制得的混合溶液填充至二甲基硅氧烷通过固化剂形成的模具中(如图6中的步骤③)，每个模具(或模具单次)加入量约为100μL，再对其进行离心(如图6中的步骤④)，待离心充模后立即进行紫外光固化(如图6中的步骤⑤)，然后脱模获得微针阵列电极(如图6中的步骤⑥)。
将脱模后的微针阵列电极放入浓度为1mol/L的盐酸中并超声一定时间(60min)，以将微针阵列电极中的氧化锌微球腐蚀，在超声后取出微针阵列电极用去离子水进行充分清洗后放入真空罐进行充分干燥，最终获得多孔微针阵列(如图6中的步骤⑦)。
其中，本发明实施例采用氧化锌微球作为牺牲材料和结构，同制备微针阵列电极的母溶液进行预先混合，随后在微针阵列电极固化脱模后，通过酸蚀将该氧化锌微球溶解，从而获得微针阵列电极结构(包括微针和基底)中连续的微米级孔洞形成的通道。形成的这些微米级孔洞和通道提供了丰富的传感修饰材料(不同的电极具有不同的传感修饰材料)的附着位点，同时增加了微针阵列电极的比表面积，并且通道本身的毛细作用能够使植物组织液充分浸润微针传感电极。而这三方面的共同作用能够提升传感器的检测灵敏度以及传感修饰材料附着的稳定性，保障传感器能够灵敏、持续地原位监测植物体内活性小分子的浓度水平。
实施例六
在上述实施例的基础上，本实施例可以包括以下内容：
所述微针阵列电极通过对应方法制备得到构成电化学三电极传感体系的工作电极、对电极和参比电极。
进一步地，所述工作电极采用以下方法制备：
将质量分数为0.5～1.5％的牛血清蛋白溶液与质量分数为0.2～0.8％的戊二醛溶液或L-赖氨酸溶液，按照体积比8:1～10:1混合；
在混合溶液中加入质量分数为0.05～0.15％的单壁碳管或单层石墨烯；
加入质量分数为0.5～1.5％的传感所需的酶或传感受体分子；
将最后的混合溶液滴涂至微针阵列电极，以完成微针阵列电极的修饰得到工作电极。
更进一步地，所述对电极和参比电极采用以下方法制备：
在微针阵列电极表面溅射铂层，以完成微针阵列电极的修饰得到对电极；
将微针阵列电极浸入Ag/AgCl浆料并捞出干燥，以完成微针阵列电极的修饰得到参比电极，其中，Ag/AgCl表示浆料中含有Ag和AgCl。
在一个应用场景中，工作电极可以采用以下方法制备：
将质量分数为1％的牛血清蛋白溶液与质量分数为0.5％的戊二醛或L-赖氨酸溶液，按照体积比9:1进行混合；
随后在混合溶液中加入质量分数为0.1％的单壁碳管或单层石墨烯，超声以进行充分混合；
再加入质量分数在1％左右的传感所需的酶或其他类型传感受体分子(传感修饰材料)，例如，检测葡萄糖所需的葡萄糖氧化酶，检测水杨酸所需的二茂铁衍生物类氧化还原微针阵列电极，以及检测活性氧所需的超氧化物歧化酶等材料中的一种或多种；
在最后的混合溶液充分混合后，将其滴涂至多孔微针阵列电极，并在低温(4℃)的干燥环境下静置一定时间(24小时)，从而完成传感工作电极的修饰。
对电极可以采用以下方法制备：
可以采用射频磁控溅射工艺进行制备对电极。具体地，溅射材料选用铂，溅射功率为80W，溅射时长为10分钟，按照该溅射参数在微针阵列表面均匀溅射一层铂，从而获得经过修饰的对电极；
参比电极可以采用以下方法制备：
可以采用浸泡法制备参比电极。具体地，可以将微针阵列电极浸入商用Ag/AgCl浆料中30分钟，随后捞出并置于40℃的加热装置(烘箱)内一定时间(12小时)，使得浆料干燥并固定在微针阵列电极上，从而获得经过修饰的参比电极。
实施例七
在上述实施例的基础上，本实施例可以包括以下内容：
所述透明柔性衬底12采用以下方法制备：
制备浇筑聚二甲基硅氧烷衬底的模具；
将聚二甲基硅氧烷与固化剂按9:1～10:1的体积比混合后，浇铸于模具上，固化后脱模获得厚度在0.5mm～0.8mm的柔性衬底。
在一个应用场景中，为获得柔性的传感器件，本发明实施例使用柔性的二甲基硅氧烷材料制作微针阵列电极的透明柔性衬底，以起到支持和固定的作用。所述透明柔性衬底可以采用以下方法制备：
采用3D打印技术制备浇筑二甲基硅氧烷衬底的模具；
随后按前述步骤向模具内浇筑二甲基硅氧烷和固化剂的混合物，该固化后获得的透明柔性衬底整体厚度为0.7mm。另外，该透明柔性衬底上包含三个用于固定微针阵列电极的凹槽121，并利用二甲基硅氧烷的弹性(或者其他固定结构)将微针阵列电极卡持(或固定)于凹槽121内。进一步，透明柔性衬底另一面(远离凹槽121的一面)留有开孔(可以为方形开孔或圆形开孔等)，该开孔用于透明柔性薄膜上的信号传导电路与微针阵列电极的背 部(基底远离微针的一侧)进行连接。
实施例八
在上述实施例的基础上，本实施例可以包括以下内容：
所述信号传导电路采用以下方法制备：
选取厚度在30～70μm的透明柔性薄膜；
在透明柔性薄膜上沉积厚度为90～110nm的信号传导电路；
通过导电银胶连接信号传导电路和对应的微针阵列电极；
采用聚二甲基硅氧烷将透明柔性薄膜和透明柔性衬底固定。
在一个应用场景中，本发明实施例在透明柔性薄膜上制备图案化信号传导电路，使得信号传导电路同微针阵列电极连接，以构建成完整的电化学三电极传感器。其中，选取厚度为50μm的透明柔性聚合物薄膜作为本发明实施例的透明柔性薄膜，包括但不限于聚对苯二甲酸乙二醇酯(PET)、聚萘二甲酸乙二醇酯(PEN)和透明聚酰亚胺(PI)等材料；而该透明的薄膜材料能够透过大部分可见光，保障植物茎叶的正常光合作用的进行。另外，透明柔性薄膜上的所述信号传导电路可以采用以下方法制备：
采用磁控溅射工艺在透明柔性薄膜上沉积图案化信号传导电路，沉积的金属为金或铂，沉积的厚度约为100nm，图案化可以通过预先定制的掩模版实现；
制得图案化信号传导电路后，使用导电银胶(其位于如图1和5中透明柔性衬底的开孔122内)连接图案化信号传导电路和相对应的微针阵列电极；
待导电银胶固化后，采用二甲基硅氧烷将透明柔性薄膜和透明柔性衬底粘合、密封，以保护内部信号传导电路不受雨水等影响。
本发明实施例以微针阵列作为电极的电化学传感器，其主要原因在于：
电化学传感器适合开发为实现原位、实时、活体植物传的器件，但现有的植物电化学类传感器如硅基的电化学探针，存在加工复杂、易破碎损坏等问题；又如平面的传感电极，则仅能检测植物表面的生物信号，而无法探测植物体内的各类生物标志物。
而基于聚合物材料制备的微针阵列作为电化学传感器电极，首先其能以微创的方式高效地穿透植物表皮，对植物内部生物标志物分子进行检测，其次微针阵列穿透植物表皮后可起到固定传感器的作用，便于开展原位、持续的生物传感，同时也避免了使用粘连、固定装置对植株的损伤，再次基于聚合物材料的微针阵列电极，其生物相容性好，环境耐受度高，适合开展长时间的持续传感。
本发明实施例的传感器透明且柔性可贴敷，使其具有该特点的原因为：
现有植物传感器、传感探头多为金属、硅等刚性材料制成，无法贴敷于植株表面且不透明。
而本发明实施例的微针阵列电极集成在柔性PDMS材质(透明柔性衬底)的衬底上，能够较好贴敷植株的茎、叶表面；同时透明柔性衬底以及透明柔性薄膜均为柔性透明材质，保障了大部分可见光可透过传感器照射至植物表面。这两方面的共同作用使得本发明的传感器对植物造成的影响控制在较低的水平。
本发明实施例中微针阵列电极采用的多孔结构可以保证长期传感稳定性，其主要体现于：
现有的基于各类微纳传感结构和材料的植物传感器，其电极表面均需要进行精细 的结构修饰。而这些精细的传感核心结构直接同植物表面或植物组织接触，容易在机械摩擦、挤压等作用下被破坏，使得传感器无法在较长时间持续监测过程中保持稳定的性能。
而本发明实施例通过在微针阵列电极表面和内部制备多孔结构(通过酸蚀氧化锌微球制得该结构)，一方面使得传感修饰结构和材料能够附着于该多孔结构内表面，较大程度地避免了同植物组织接触带来的应力造成的传感结构破坏，另一方面还增加了传感电极的比表面积，从而增加了传感响应材料的附着面积，相应地提高了传感器的检测灵敏度。
实施例九
在上述实施例的基础上，本实施例可以包括以下内容：
参见图7所示，本发明还提供一种检测植物活性小分子的传感系统20，其包括
上述的传感器10，其固定于植株茎叶处；
电化学工作站21，其通过信号传输线缆与所述传感器10连接，以实现植物活性小分子信号的原位、连续监测。
其中，电化学工作站21可以为便携式设备，进一步地，该电化学工作站还可以替换为无线传输模块，如蓝牙、5G等，用于将采集的传感信号通过无线传送至远程数据终端进行处理，从而实现远程传感管理。
实施例十
在上述实施例的基础上，本实施例可以包括以下内容：
本发明实例的电极传感器，其工作电极通过功能化修饰获得检测特定活性小分子(如活性氧ROS)的能力。微针阵列电极构成的三电极传感体系以电化学测量的方式工作，当传感电极(微针阵列电极)检测到植物皮下ROS分子(如过氧化氢，超氧化物等)时，会在工作电极和对电极上发生电化学反应并产生电流信号，沿微针阵列电极-信号传导电路-电化学工作站的回路传导至该电化学工作站，由电化学工作站测量出电流的大小，再根据电流-浓度标准曲线换算出ROS分子的浓度值。
实施例十一
在上述实施例的基础上，本实施例可以包括以下内容：
在一个实际应用场景中，选取开花期的大豆植株，在其主茎和植物上、中和下部的复叶上分别施加一片检测葡萄糖的微针阵列传感器，并用细导线将传感器信号传导电路连接至便携式电化学工作站。每6小时采集一次电化学信号测定植株的葡糖糖水平，每次采集持续时间10分钟；连续采集2～3周，根据监测到的葡萄糖水平变化趋势评估大豆植株营养情况，并采取对应的水、肥调控措施，再继续监测1-2周至结荚期。
以上介绍了本发明的较佳实施方式，旨在使得本发明的精神更加清楚和便于理解，并不是为了限制本发明，凡在本发明的精神和原则之内，所做的修改、替换、改进，均应包含在本发明所附的权利要求概括的保护范围之内。

Claims (10)

  1. 一种检测植物活性小分子的传感器，其特征在于，包括：
    微针阵列电极，其用于检测植物活性小分子浓度；
    透明柔性衬底，其一侧具有固定所述微针阵列电极的凹槽；
    透明柔性薄膜，其固定于所述透明柔性衬底远离所述凹槽的另一侧；
    多个信号传导电路，其布置于所述透明柔性衬底和透明柔性薄膜间，并且每一所述信号传导电路穿过所述透明柔性衬底与所述微针阵列电极连接。
  2. 如权利要求1所述传感器，其特征在于，所述微针阵列电极包括构成电化学三电极传感体系的工作电极、对电极和参比电极。
  3. 一种制备如权利要求1所述传感器的方法，其特征在于，所述微针阵列电极采用以下方法制备：
    制备氧化锌微球溶液；
    制备微针阵列电极的模具；
    将氧化锌微球溶液与制备微针阵列电极的第一混合溶液混合，制备成第二混合溶液；
    将第二混合溶液填充于所述微针阵列电极的模具内固化；
    在第二混合溶液固化后，酸蚀去除氧化锌微球，以得到多孔的微针阵列电极；
    将多孔的微针阵列电极进行修饰得到包括工作电极、对电极和参比电极的微针阵列电极。
  4. 如权利要求3所述的方法，其特征在于，所述氧化锌微球采用以下方法制备：
    配制浓度为20～80mmol/L六水合硝酸锌溶液和20～80mmol/L乌洛托品溶液并以体积比1:2～2:1混合；
    将混合溶液与浓度20～60mmol/L的柠檬酸钠溶液以体积比1:1～3:1的比例混合获得氧化锌微球。
  5. 如权利要求3所述方法，其特征在于，所述微针阵列电极具体采用以下方法制备：
    制备微针阵列电极母模，该微针阵列电极母模与微针阵列电极外形相同；
    将二甲基硅氧烷与固化剂按9:1～11:1的体积比混合，并浇铸于微针阵列电极母模上，固化后脱模得到微针阵列电极模具；
    将聚乙二醇二丙烯酸酯和光交联剂按体积比8:1～10:1混合得到第一混合溶液；
    将含有氧化锌微球的溶液与第一混合溶液按体积比1:10～2:10混合得到第二混合溶液；
    将第二混合溶液填充至微针阵列电极模具中，固化后脱模获得微针阵列电极；
    将微针阵列电极放入浓度0.5～1.5mol/L的盐酸中，以去除氧化锌微球后得到多孔的微针阵列电极。
  6. 如权利要求3所述的方法，其特征在于，所述工作电极采用以下方法制备：
    将质量分数为0.5～1.5％的牛血清蛋白溶液与质量分数为0.2～0.8％的戊二醛溶液或L-赖氨酸溶液，按照体积比8:1～10:1混合；
    在混合溶液中加入质量分数为0.05～0.15％的单壁碳管或单层石墨烯；
    加入质量分数为0.5～1.5％的传感所需的酶或传感受体分子；
    将最后的混合溶液滴涂至微针阵列电极，以完成微针阵列电极的修饰得到工作电极。
  7. 如权利要求6所述的方法，其特征在于，所述传感所需的酶或传感受体分子为检测葡 萄糖所需的葡萄糖氧化酶，检测水杨酸所需的二茂铁衍生物类，以及检测活性氧所需的超氧化物歧化酶中的一种或多种。
  8. 如权利要求3所述的方法，其特征在于，所述对电极和参比电极采用以下方法制备：
    在微针阵列电极表面溅射铂层，以完成微针阵列电极的修饰得到对电极；
    将微针阵列电极浸入Ag/AgCl浆料并捞出干燥，以完成微针阵列电极的修饰得到参比电极。
  9. 如权利要求3所述的方法，其特征在于，所述透明柔性衬底采用以下方法制备：
    制备浇筑聚二甲基硅氧烷衬底的模具；
    将聚二甲基硅氧烷与固化剂按9:1～10:1的体积比混合后，浇铸于模具上，固化后脱模获得厚度在0.5mm～0.8mm的柔性衬底。
  10. 如权利要求3所述的方法，其特征在于，所述信号传导电路采用以下方法制备：
    选取厚度在30～70μm的透明柔性薄膜；
    在透明柔性薄膜上沉积厚度为90～110nm的信号传导电路；
    通过导电银胶连接信号传导电路和对应的微针阵列电极；
    采用聚二甲基硅氧烷将透明柔性薄膜和透明柔性衬底固定。
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