Preparation method of electrochemical sensor for starch detection, electrochemical sensor and application

Abstract

The invention relates to a preparation method of an electrochemical sensor for starch detection, the electrochemical sensor and application, and the method comprises the following steps: patterning the surface of a polyimide film through a laser system to prepare three graphene electrodes; transferring the graphene three-electrode to the surface of the polycaprolactone to obtain a formed polycaprolactone-graphene three-electrode; coating Ag / AgCl silver paste on a reference electrode area in the polycaprolactone-graphene three-electrode, heating and curing, then insulating and packaging outside the three-electrode area, and activating the electrode; the electrochemical sensor for starch detection is obtained by modifying a carbon nanocage, platinum-palladium nanoparticles and glucoamylase layer by layer in a working electrode area. The prepared polycaprolactone degradable electrode can realize environment-friendly detection of starch, the degradable electrode material is soft and bendable, the starch content of various tissue parts of a plant can be detected, and the problems that an existing sensor is difficult to be effectively attached to the plant to be detected and environmental pollution is caused are effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a preparation method of an electrochemical sensor for starch detection, an electrochemical sensor and applications thereof. Background Art

[0002] Starch, a carbohydrate found widely in plants, is stored as polysaccharides in fruits, seeds, and roots, serving as a crucial energy storage and supply. In industrial applications, it not only serves as a food processing additive to improve product taste, but also as a filler in composite materials for degradation and modification of synthetic polymers. Therefore, accurate determination of starch content is crucial for quality control in the brewing, food, and pharmaceutical industries.

[0003] Currently, common methods for determining starch content include titration, colorimetry, enzymatic hydrolysis, infrared spectroscopy, etc. Although these methods have their own characteristics in terms of sensitivity and stability, they generally have the following technical defects: the detection instruments are expensive, the professional skills of the operators are strictly required, and the sample pretreatment process is complicated and time-consuming, making it difficult to meet the needs of rapid detection and large-scale application.

[0004] Compared to the aforementioned methods, electrochemical methods offer advantages such as ease of operation and fast response time, making them suitable for rapid plant starch detection. However, traditional rigid sensors (such as gold electrodes) struggle to adhere effectively to the plant surface, impose significant limitations on the material being tested, and can easily cause environmental pollution after long-term use or disposal. Summary of the Invention

[0005] In light of this, the main purpose of the present invention is to provide a method for preparing an electrochemical sensor for starch detection, an electrochemical sensor, and its applications. This method utilizes laser-induced graphene to create an electrode pattern, which is then transferred to a polycaprolactone-based biodegradable substrate. This method achieves environmentally friendly starch detection. The flexible and bendable nature of the biodegradable electrode material enables detection of starch content in various plant tissues, effectively addressing the challenges of existing sensors, which struggle to effectively adhere to the plant being tested and can easily cause environmental pollution.

[0006] In a first aspect, the present invention provides a method for preparing an electrochemical sensor for starch detection, comprising the following steps: The surface of the polyimide film is patterned by a laser system to prepare a graphene three-electrode; Transferring the graphene three-electrode to the surface of polycaprolactone to obtain a formed polycaprolactone-graphene three-electrode; Applying Ag / AgCl silver paste to the reference electrode region of the polycaprolactone-graphene three-electrode, heating and curing, insulating and encapsulating the three-electrode region, and activating the encapsulated polycaprolactone-graphene three-electrode; The working electrode region of the activated polycaprolactone-graphene triple electrode is modified layer by layer with carbon nanocages, platinum-palladium nanoparticles and glucoamylase to obtain an electrochemical sensor for starch detection.

[0007] In some embodiments, patterning the surface of the polyimide film by a laser system to prepare the graphene three-electrode includes: After the polyimide film is attached to the mold, the polyimide film is cleaned with distilled water and ethanol; The laser system is controlled by a computer to pattern the cleaned polyimide film to obtain a graphene three-electrode.

[0008] In some embodiments, transferring the graphene three-electrode to a polycaprolactone surface to obtain a formed polycaprolactone-graphene three-electrode comprises: The polycaprolactone melt is evenly coated on the polyimide film with the pattern at 80-100 rpm for 60-90 s; The polyimide film coated with the polycaprolactone melt is heated at 80-100° C. for 10-15 hours, cooled to room temperature for curing, and the formed polycaprolactone-graphene three-electrode is mechanically peeled off from the polyimide film.

[0009] In some embodiments, the step of coating the reference electrode region of the polycaprolactone-graphene three-electrode with Ag / AgCl silver paste, insulating and encapsulating the three-electrode region, heating and curing the three-electrode, and activating the encapsulated polycaprolactone-graphene three-electrode comprises: The reference electrode region of the polycaprolactone-graphene three-electrode is coated with Ag / AgCl silver paste, the region outside the three-electrode is insulated and encapsulated with polydimethylsiloxane, and the encapsulated polycaprolactone-graphene three-electrode is obtained after heating and curing; The encapsulated polycaprolactone-graphene three-electrode was placed in a 0.01 M phosphate buffer solution and subjected to chronoamperometry scanning to perform activation treatment.

[0010] In some embodiments, the working electrode region of the activated polycaprolactone-graphene triple electrode is modified layer by layer with carbon nanocages, platinum-palladium nanoparticles, and glucoamylase to obtain an electrochemical sensor for starch detection, comprising: drop-coating a carbon nanocage dispersion on the working electrode region of the activated polycaprolactone-graphene triple electrode to obtain a CNCs / LIG working electrode, followed by drying; The dried CNCs / LIG working electrode is immersed in a mixture of chloroauric acid and palladium chloride, and Pt-Pd nanoparticles are prepared by electroplating the electrode at room temperature. After drying, a Pt-Pd / CNCs / LIG working electrode is obtained; The chitosan solution and the glucoamylase mixture were dropped onto the Pt-Pd / CNCs / LIG working electrode to obtain an electrochemical sensor with GA / Pt-Pd / CNCs / LIG as the working electrode.

[0011] In some embodiments, the concentration of the carbon nanocage dispersion is 0.5-2 mg / mL; The concentration of the mixed solution of chloroauric acid and palladium chloride is 0.5-2 mg / mL; The mass fraction of the chitosan solution is 0.5-2%, the enzymatic activity of the glucoamylase is 110-120 U / mg, and the concentration is 80-200 mg / mL.

[0012] In a second aspect, an embodiment of the present application provides an electrochemical sensor prepared according to any of the above-mentioned methods for preparing an electrochemical sensor for starch detection.

[0013] In a third aspect, an embodiment of the present application provides an application of the electrochemical sensor described above in starch detection, comprising the following steps: Prepare starch-phosphate buffer solutions of different concentrations, perform amperometric detection using the electrochemical sensor, and draw a standard curve; Obtaining a sample to be tested, and pre-treating the sample to be tested to obtain a test solution; The electrochemical sensor is inserted into the liquid to be tested, connected to an electrochemical workstation, and the starch current is recorded by chronoamperometry. The actual starch concentration is calculated based on the standard curve.

[0014] In some embodiments, obtaining a sample to be tested and pre-treating the sample to be tested to obtain a test solution includes: Obtaining a sample to be tested and drying it; wherein the sample to be tested includes plant material; The dried sample to be tested is crushed, and the powder is repeatedly washed with anhydrous ethanol, and the corresponding precipitate is collected and dried after centrifugation to obtain a sugar-free powder; The sugar-removed powder is mixed with a phosphate buffer solution and heated in a boiling water bath to obtain a gelatinized solution; After the gelatinized liquid is precipitated with n-butanol, the corresponding precipitate is redissolved with the phosphate buffer to obtain a test solution.

[0015] In a fourth aspect, an embodiment of the present application provides another application of the electrochemical sensor described above in starch detection, comprising the following steps: Prepare starch-phosphate buffer solutions of different concentrations, perform chronoamperometry detection using the electrochemical sensor, and draw a standard curve; After a small number of small holes were punched on the leaf to be tested, the electrochemical sensor was fixed to the lower surface of the leaf to be tested and connected to an electrochemical workstation. The starch current was recorded by chronoamperometry, and the actual starch concentration was calculated based on the standard curve. During starch detection, the working electrode of the electrochemical starch detection sensor was facing the small hole, and 5-20 μL PBS was dripped on the small hole.

[0016] Technical effects of the present invention: This application uses laser printing on a polyimide film to pattern it and prepare a graphene three-electrode. The pattern is then transferred to the polycaprolactone surface by mechanical stripping to obtain a polycaprolactone-graphene three-electrode, and Ag / AgCl is coated in the reference electrode area to prepare a reference electrode. In addition, carbon nanocages CNCs, platinum palladium nanoparticles Pt-Pd and glucoamylase GA are modified layer by layer in the working electrode area to construct a working electrode, thereby obtaining a degradable electrochemical sensor with Ag / AgCl as the reference electrode and GA / Pt-Pd / CNCs / LIG as the working electrode. The sensor of this application can not only achieve highly sensitive detection of plant starch, but also the degradable electrode material can achieve environmentally friendly detection of starch. At the same time, its flexible and bendable characteristics enable it to detect starch in leaves and processed grain solutions, meeting more detection needs, and also have the advantages of excellent electrical performance and high integration.

[0017] Further features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the disclosure and, together with the description, serve to explain the principles of the disclosure.

[0019] Figure 1 Schematic diagram showing a process of preparing an electrochemical sensor for starch detection according to an embodiment of the present application; Figure 2 Shown is a schematic structural diagram of an electrochemical sensor for starch detection according to an embodiment of the present application; Figure 3Shown is a diagram of the working electrode modification process of an electrochemical sensor for starch detection according to an embodiment of the present application; Figure 4 A diagram showing an application process of the electrochemical sensor according to an embodiment of the present application in starch detection; Figure 5 Shown is a standard relationship curve diagram of the electrochemical sensor of the embodiment of the present application in starch detection application; Figure 6 Another process diagram of the application of the electrochemical sensor in starch detection according to an embodiment of the present application is shown; Figure 7 Shown is a graph showing the current difference results corresponding to Example 1 and Comparative Examples 2 and 3 in starch detection applications of the electrochemical sensor according to the embodiment of the present application. DETAILED DESCRIPTION

[0020] Various exemplary embodiments, features, and aspects of the present disclosure will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise indicated.

[0021] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0022] In addition, numerous specific details are provided in the following detailed description to better illustrate the present disclosure. Those skilled in the art will appreciate that the present disclosure can be practiced without certain specific details. In some instances, methods, means, components, and circuits well known to those skilled in the art are not described in detail in order to highlight the main points of the present disclosure.

[0023] like Figure 1 As shown, in a first aspect, the embodiments of the present application provide a method for preparing an electrochemical sensor for starch detection, comprising the following steps: S100, patterning the surface of the polyimide film using a laser system to prepare a graphene three-electrode substrate; In this step, a degradable triangular electrode is first prepared using laser-induced graphene technology. Specifically, a laser is controlled to scan and pattern a polyimide film to produce a three-dimensional porous graphene LIG, or graphene trielectrode. The graphene trielectrode substrate has a side length of 25-35 mm and is printed on polyimide tape using a computer-controlled laser system. The resulting graphene trielectrode exhibits excellent conductivity, and laser-induced graphene technology is suitable for mass production and the production process is convenient and fast.

[0024] S200, transferring the graphene three-electrode to the surface of polycaprolactone to obtain a formed polycaprolactone-graphene three-electrode; In this step, the graphene trielectrode is transferred to the polycaprolactone surface via mechanical exfoliation, resulting in a flexible polycaprolactone-graphene trielectrode, or LIG / PCL electrode, comprising a reference electrode region, a working electrode region, and a counter electrode region. Polycaprolactone ensures the complete transfer of the pattern on the polyimide film, and the resulting polycaprolactone-graphene trielectrode exhibits high conductivity and biocompatibility, while also possessing flexibility. Furthermore, due to its biodegradable nature, using polycaprolactone as a substrate effectively meets the environmental requirements of current starch detection applications.

[0025] S300, coating the reference electrode region of the polycaprolactone-graphene three-electrode with Ag / AgCl silver paste, heating and curing, insulating and encapsulating the three-electrode region, and activating the encapsulated polycaprolactone-graphene three-electrode; In this step, Ag / AgCl silver paste is applied to the exposed reference electrode area and heat-cured to produce a polycaprolactone-graphene trielectrode with Ag / AgCl as the reference electrode. Following this heat-curing process, the areas outside the reference, working, and counter electrode areas are insulated and encapsulated. The encapsulated trielectrode is then activated in a buffer solution to remove surface impurities.

[0026] It should be noted that Ag / AgCl is used as a reference electrode, which is soft and flexible. Therefore, when analyzing the starch content in plant tissues, it is not only possible to detect starch in leaves, but also in treated seed solutions, etc., that is, there are fewer requirements and restrictions on the test materials, and it is suitable for different periods and environments of plant growth. The test site is not limited to tissues such as roots, stems, leaves, and fruits of plants, and the physical adaptability is stronger. At the same time, the soft feature can also avoid affecting plant growth during testing and prevent the induction of plant stress responses.

[0027] S400: The working electrode region of the activated polycaprolactone-graphene triple electrode is modified layer by layer with carbon nanocages, platinum-palladium nanoparticles and glucoamylase to obtain an electrochemical sensor for starch detection.

[0028] In this step, the working electrode is obtained by drop-coating carbon nanocages (CNCs), platinum-palladium nanoparticles (Pt-Pd), and glucoamylase (GA) onto LIG. Specifically, the working electrode region of an activated polycaprolactone-graphene triple electrode is layered with carbon nanocages (CNCs), platinum-palladium nanoparticles (Pt-Pd), and glucoamylase (GA), resulting in a biodegradable electrochemical sensor using GA / Pt-Pd / CNCs / LIG as the working electrode. This method not only enables highly sensitive detection of plant starch and rapid and accurate acquisition of starch information, providing a theoretical basis for plant breeding, but also makes electrochemical sensors based on biodegradable electrode materials more environmentally friendly and safer, meeting the growing demand for environmental protection.

[0029] In some embodiments, patterning is performed on the surface of a polyimide film using a laser system to prepare a graphene three-electrode, including: After attaching the polyimide film to the mold, the polyimide film was cleaned with distilled water and ethanol; The cleaned polyimide film is patterned using a computer-controlled laser system to obtain a graphene three-electrode.

[0030] In this example, the preparation of the graphene trielectrode involves first attaching a polyimide film, specifically polyimide tape, to a mold. The polyimide tape (PI tape) is then cleaned with distilled water and then ethanol. The PI tape is then patterned using a computer-controlled laser system to obtain a three-dimensional porous LIG, or graphene trielectrode. It should be noted that laser printing of polyimide to graphene is more convenient, enabling carbonization at room temperature and pressure. The resulting graphene trielectrode substrate has a three-dimensional porous structure, imparting a certain degree of flexibility while maintaining the integrity of the conductive network, enabling it to be attached to plant leaves for in-situ testing.

[0031] In some embodiments, the graphene three-electrode is transferred to the polycaprolactone surface to obtain a formed polycaprolactone-graphene three-electrode, comprising: The polycaprolactone melt is evenly coated on the patterned polyimide film at 80-100 rpm for 60-90 s; The polyimide film coated with the polycaprolactone melt is heated at 80-100° C. for 10-15 hours, cooled to room temperature for solidification, and the formed polycaprolactone-graphene three-electrode is mechanically peeled off from the polyimide film.

[0032] In this embodiment, a spin coater is used to evenly cover the polycaprolactone melt PCL on a patterned polyimide tape so that the PCL melt can be immersed in the three-dimensional porous LIG structure. Subsequently, the heat treatment is carried out in a vacuum drying oven, and after cooling to room temperature, the formed polycaprolactone-graphene three-electrode is mechanically peeled off from the polyimide film, that is, the transfer and fixation of the graphene three-electrode are achieved, and a flexible and stretchable LIG / PCL electrode is obtained. It should be noted that the transfer of the pattern to the polycaprolactone surface by mechanical stripping can make the graphene pattern clear and complete, the transfer effect is better, and the formed polycaprolactone-graphene three-electrode is degradable, which can reduce environmental pollution and is suitable for eco-friendly agriculture. At the same time, the flexibility of polycaprolactone allows the electrode to maintain the integrity of the conductive network under deformation, and it can fit more closely with the plant in the plant starch detection process, reduce the interface resistance, and improve the stability of the electrochemical signal.

[0033] Specifically, the present embodiment utilizes a spin coater to evenly spin-coat the polycaprolactone melt at a rotation speed of 80-100rpm for 60-90s. Here, it should be noted that the rotation speed of 80-100rpm is relatively low and the centrifugal force is weak. Under this condition, it is rotated for 60-90s, which can effectively avoid the situation where the solution is thick at the edge and thin in the middle due to high-speed throwing out, thereby forming a smooth and uniform film, a good adaptation to graphene transfer, and providing a stable foundation for subsequent device integration. In addition, the temperature of the vacuum drying oven is set to 80-100°C, and heated under this condition for 10-15h. The residual solvent after spin coating is removed by the synergistic effect of thermal drive and vacuum, while improving the surface flatness of the film, making it more suitable for graphene transfer, and the resulting polycaprolactone-graphene three-electrode structure more reliable.

[0034] In some embodiments, Ag / AgCl silver paste is coated on the reference electrode region of a polycaprolactone-graphene three-electrode, insulated and encapsulated outside the three-electrode region, heated and cured, and the encapsulated polycaprolactone-graphene three-electrode is activated, including: The reference electrode region of the polycaprolactone-graphene three-electrode is coated with Ag / AgCl silver paste, and the region outside the three-electrode region is insulated and encapsulated with polydimethylsiloxane, which is then heated and cured to obtain the encapsulated polycaprolactone-graphene three-electrode; The encapsulated polycaprolactone-graphene three-electrode was placed in a 0.01 M phosphate buffer solution and subjected to current scanning for activation treatment.

[0035] In this example, an Ag / AgCl silver paste was applied to the exposed reference electrode area and cured by heat. Polydimethylsiloxane was then used to insulate and encapsulate the area outside the three-electrode region. Specifically, after constructing the Ag / AgCl reference electrode, the areas outside the reference, working, and counter electrode regions of the polycaprolactone-graphene trielectrode were insulated and encapsulated with polydimethylsiloxane (PDMS). Subsequently, the entire electrode was placed in 0.01M phosphate buffer and subjected to a chronoamperometric scan (1.7V) for activation and removal of impurities on the electrode surface.

[0036] In some embodiments, the working electrode region of the activated polycaprolactone-graphene triple electrode is modified layer by layer with carbon nanocages, platinum-palladium nanoparticles, and glucoamylase to obtain an electrochemical starch detection sensor, comprising: The carbon nanocage dispersion is drop-coated on the working electrode region of the activated polycaprolactone-graphene triple electrode to obtain a CNCs / LIG working electrode, which is then dried. The dried CNCs / LIG working electrode was immersed in a mixture of chloroauric acid and palladium chloride, and Pt-Pd nanoparticles were prepared by electroplating the electrode at room temperature. After drying, a Pt-Pd / CNCs / LIG working electrode was obtained. The chitosan solution and glucoamylase mixture were dropped onto the Pt-Pd / CNCs / LIG working electrode to obtain an electrochemical starch detection sensor with GA / Pt-Pd / CNCs / LIG as the working electrode.

[0037] In this embodiment, the working electrode is constructed by layer-by-layer coating of carbon nanocages, platinum-palladium nanoparticles, and glucoamylase on a LIG basis, so that the resulting biosensor can achieve highly sensitive detection of plant starch.

[0038] Specifically, in the process of constructing the working electrode, 5 to 10 μL, preferably 5 μL, of a CNCs dispersion (specifically, a concentration of 0.5 to 2 mg / mL, preferably 1 mg / mL) is drop-coated onto the working electrode region of the polycaprolactone-graphene trielectrode to form a CNCs / LIG working electrode, which is then dried. Modifying the working electrode region with carbon nanocages enables efficient starch adsorption during starch detection, enhancing the detection signal intensity.

[0039] Subsequently, the dried CNCs / LIG working electrode is immersed in a mixture of chloroauric acid (HPtCl6) and palladium chloride (PdCl2) and subjected to chronoamperometric electroplating at -0.1V for 800-1500s, preferably 1000s, to prepare a Pt-Pd / CNCs / LIG working electrode. The concentration of the mixture of chloroauric acid and palladium chloride is preferably 0.5 to 2 mg / mL, preferably 1 mg / mL. It should be noted that the structural characteristics of CNCs enable uniform dispersion of Pt-Pd particles during the electroplating process and prevent particle shedding. Furthermore, Pt-Pd particles have a stronger ability to adsorb and dissociate glucose in starch detection. At the same time, the conductive network of LIG shortens the electron transmission path, thereby enhancing the current signal of starch detection and achieving higher sensitivity.

[0040] Finally, 5 to 10 μL, preferably 5 μL, of the chitosan solution and glucoamylase mixture is dropped onto the Pt-Pd / CNCs / LIG working electrode to obtain a degradable biosensor with GA / Pt-Pd / CNCs / LIG as the working electrode. The chitosan solution has a mass fraction of 0.5-2%, preferably 0.3%, and the enzyme activity in the glucoamylase mixture is preferably 110-120 U / mg, with a concentration of 80-200 mg / mL, preferably 100 mg / mL. Specifically, the chitosan solution and glucoamylase mixture is prepared by dissolving glucoamylase in the chitosan solution and ultrasonically mixing the mixture to form a uniform mixture. It should be noted that glucoamylase can hydrolyze starch into glucose. The glucose generated under the action of chitosan can quickly diffuse to the surface of the Pt-Pd nanoparticles and be catalytically oxidized, thereby improving detection efficiency. At the same time, the biocompatibility of chitosan can reduce plant tissue damage, making it suitable for in situ detection of plants.

[0041] It can be seen that the present application uses laser printing on a polyimide film to pattern it and prepare a graphene three-electrode substrate. The pattern is then transferred to the polycaprolactone surface by mechanical stripping to obtain a polycaprolactone-graphene three-electrode, and Ag / AgCl is coated in the reference electrode area to prepare a reference electrode. In addition, carbon nanocages CNCs, platinum palladium nanoparticles Pt-Pd and glucoamylase GA are modified layer by layer in the working electrode area to construct a working electrode, thereby obtaining a degradable electrochemical sensor with Ag / AgCl as a reference electrode and GA / Pt-Pd / CNCs / LIG as a working electrode. The biosensor of the present application can not only achieve highly sensitive detection of plant starch, but also the degradable electrode material can achieve environmentally friendly detection of starch. At the same time, its flexible and bendable characteristics enable it to detect both starch in leaves and treated grain solutions, meeting more detection needs, and also have the advantages of excellent electrical performance and high integration.

[0042] like Figure 2 and 3 As shown, in a second aspect, an embodiment of the present application provides an electrochemical sensor prepared by any of the above-mentioned methods for preparing an electrochemical sensor for starch detection.

[0043] It should be noted that the electrochemical sensor in this embodiment is a degradable electrode starch sensor with a triangular structure. It comprises three graphene electrodes, one covered with a polycaprolactone membrane and the other with a GA / Pt-Pd / CNCs / LIG working electrode, an Ag / AgCl reference electrode, and a LIG counter electrode. The polycaprolactone substrate in this electrochemical starch detection sensor is biodegradable and flexible, meeting environmental requirements while also enabling measurement of multiple plant tissues, making it more versatile.

[0044] like Figure 4 As shown, in a third aspect, the present application provides an application of the above electrochemical sensor in starch detection, comprising the following steps: S10, preparing starch-phosphate buffer solutions of different concentrations, performing chronoamperometry detection using an electrochemical sensor, and drawing a standard curve; In this step, starch-phosphate buffer solutions of 0, 1, 5, 10, 15, 20, and 25% (w / w) were prepared, and the pH was 4.5. Subsequently, the electrochemical sensor prepared by the above method was used for chronoamperometry detection. The specific potential was 0.4V and the reaction time was 50s. The current corresponding to different starch concentrations was obtained to make a concentration-current electrode relationship curve. Among them, the linear range can reach 0.05-25%. The obtained standard relationship curve is as follows Figure 5 The values ​​shown are used for subsequent calculation of actual sample concentrations.

[0045] S20, obtaining a sample to be tested, and pre-treating the sample to be tested to obtain a test solution; In this step, the test object needs to be pre-processed after acquisition to obtain a test solution suitable for detection. The test samples include plant materials, specifically crops, flowers, vegetables, and fruits, and the test sites can be plant tissues such as roots, stems, leaves, and fruits. This allows for highly sensitive starch detection in a variety of plants and tissues.

[0046] S30, inserting the electrochemical starch detection sensor into the test solution, connecting it to an electrochemical workstation, recording the starch current by chronoamperometry, and calculating the actual starch concentration based on the standard curve.

[0047] In this step, after the electrode of the degradable electrochemical starch detection sensor is inserted into the test liquid, it is connected to the electrochemical workstation, wherein the specific electrochemical workstation is a CHI 760e electrochemical workstation, and then the starch current is recorded by chronoamperometry, and the starch concentration of the actual sample is calculated according to the current-concentration standard relationship curve obtained in step S10, thereby realizing the measurement of plant starch.

[0048] In some embodiments, obtaining a sample to be tested and pre-treating the sample to be tested to obtain a test solution includes: Obtaining a sample to be tested and drying it; wherein the sample to be tested includes plant material; The dried sample to be tested is crushed, and the powder is repeatedly washed with anhydrous ethanol. After centrifugation, the corresponding precipitate is collected and dried to obtain a sugar-free powder; The sugar-removed powder is mixed with a phosphate buffer solution and heated in a boiling water bath to obtain a gelatinized solution; After the gelatinized liquid was precipitated with n-butanol, the corresponding precipitate was redissolved with phosphate buffer to obtain a test solution.

[0049] In the present embodiment, after obtaining the sample to be tested, it needs to be pretreated. Specifically, before the formal measurement, the sample to be tested needs to be dried and the dried sample is crushed to 80-100 mesh fine powder to ensure that the starch is fully exposed. Subsequently, the powder is repeatedly washed with anhydrous ethanol to remove the interference of soluble sugars, the precipitate is collected after centrifugation, and dried at 75-80 ° C to remove ethanol to obtain a powder after desugaring. Further, the desugared powder is mixed with a phosphate buffer and heated in a boiling water bath for 10-30 minutes to completely gelatinize and dissolve the starch to obtain a gelatinized liquid. Finally, the gelatinized liquid is precipitated with n-butanol, the precipitate is dried, and redissolved with a hot buffer to obtain a final liquid to be tested. The electrochemical sensor prepared by the above preparation method is inserted into the liquid to be tested, connected to the CHI 760e electrochemical workstation, and the current is recorded by chronoamperometry. Finally, the actual starch concentration of the liquid to be tested is calculated using the standard curve to achieve the measurement of starch.

[0050] like Figure 6 As shown, in a fourth aspect, an embodiment of the present application provides another application of the electrochemical starch detection sensor in starch detection, comprising the following steps: S1. Prepare starch-phosphate buffer solutions of different concentrations, perform chronoamperometry detection using an electrochemical sensor, and draw a standard curve; S2. After making a small number of small holes on the leaf to be tested, fix the electrochemical sensor on the lower surface of the leaf to be tested, connect it to the electrochemical workstation, record the starch current by chronoamperometry, and calculate the actual starch concentration based on the standard curve; wherein, during starch detection, the working electrode of the electrochemical starch detection sensor is facing the small holes, and 5-20 μL PBS is dripped on the small holes.

[0051] In this embodiment, the standard curve is the same as the standard curve obtained in step S10, and the repeated parts are not repeated. After obtaining the above-mentioned standard relationship curve, several small holes are punched on the leaf to be tested to release starch, wherein the number of small holes can be freely selected according to actual needs and is not limited to this. The electrochemical starch sensor is then fixed to the lower surface of the leaf to be tested, and 5-20 μL of PBS is dripped at the small holes to promote the chemical connection between the electrode and the leaf. Among them, the amount of PBS is preferably 5 μL. Further, the CHI 760e electrochemical workstation is connected, the current is recorded by the chronoamperometry, and finally the actual starch concentration of the test liquid is calculated using the standard curve to achieve the measurement of starch on the leaves. The electrochemical starch sensor in this embodiment is a degradable electrode material with soft and flexible characteristics. Therefore, it can detect the treated test liquid and directly detect starch on the leaf. It can not only achieve environmentally friendly detection of starch, but also meet the use requirements of on-site and real-time analysis of starch concentration.

[0052] The present invention is further illustrated below by examples and comparative examples. The experimental methods in the following examples and comparative examples, unless otherwise specified, are conventional methods, performed according to the techniques or conditions described in literature in the art or according to product specifications. The materials and reagents used in the following examples, unless otherwise specified, are commercially available.

[0053] It should be noted that the research subjects of Example 1 and Comparative Example 1 were both rice in the latex stage.

[0054] Example 1 Rice grains of two different varieties, "Lianjing No. 7" and "Wuyunjing No. 23," from the same period were selected and treated according to the above pretreatment method. The starch content was then detected by electrochemical method using the biosensor prepared using the above preparation method.

[0055] Comparative Example 1 Rice grains of the same varieties as in Example 1, Lianjing No. 7 and Wuyunjing No. 23, were pretreated as described above and then tested for starch content using spectrophotometry according to GB / T 15683-2008. The relative error between the two methods was within ±15%, indicating that the electrochemical sensor results were reliable.

[0056] The test results of Example 1 and Comparative Example 1 are shown in Table 1.

[0057]

[0058] Table 1 Schematic table of starch measurement results of Example 1 and Comparative Example 1 Comparative Example 2 Comparative Example 2 provides an electrochemical sensor for starch detection, wherein its preparation method is different from that of the above-mentioned Example 1 in that: in Comparative Example 2, Pt-Pd is omitted when preparing the working electrode, and the sensor GA / CNCs / LIG is prepared.

[0059] Subsequently, the sensor performance was tested by cyclic voltammetry (CV), and the CV scan was performed in 5 mM potassium ferrocyanide and potassium ferrocyanide solution (containing 0.1 M potassium chloride). The CV scan curve of GA / CNCs / LIG can be found in Figure 7 It can be seen that the peak current of the sensor prepared in Comparative Example 2 is relatively small, and the detection effect is not as good as that of Experimental Example 1.

[0060] Comparative Example 3 Comparative Example 3 provides an electrochemical sensor for starch detection, wherein its preparation method is different from that of Example 1 in that: in Comparative Example 3, when preparing the working electrode, Pt-Pd / CNCs is omitted, and a sensor GA / LIG is prepared.

[0061] Subsequently, the sensor performance was tested by cyclic voltammetry (C), and the CV scan was performed in 5 mM potassium ferrocyanide and potassium ferrocyanide solution (containing 0.1 M potassium chloride). The CV scan curve of GA / LIG can be found in Figure 7 As shown, the peak current is too small, which shows that the detection effect of comparative example 3 is not as good as that of embodiment 1.

[0062] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0063] Unless otherwise defined, the technical terms or scientific terms involved in this application should have the usual meanings understood by people with ordinary skills in the technical field to which this application belongs. The words "one", "the" and similar words involved in this application do not indicate quantity restrictions and can indicate the singular or plural. The terms "include", "comprise", "have" and any variations thereof involved in this application are intended to cover non-exclusive inclusions. The words "connect", "connected", "coupled" and similar words involved in this application are not limited to physical or mechanical connections, but include electrical connections, whether direct or indirect. The "multiple" involved in this application refers to two or more, and "and / or" describes the association relationship of associated objects, indicating that three relationships can exist. The character " / " generally indicates that the previous and next associated objects are in an "or" relationship. The terms "first", "second", "third" and the like involved in this application are merely to distinguish similar objects and do not represent a specific ordering of objects.

[0064] The above embodiments merely illustrate several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make several modifications or improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A method for preparing an electrochemical sensor for starch detection, characterized in that: The steps include: The surface of the polyimide film is patterned by a laser system to prepare a graphene three-electrode; Transferring the graphene three-electrode to the surface of polycaprolactone to obtain a formed polycaprolactone-graphene three-electrode; Applying Ag / AgCl silver paste to the reference electrode region of the polycaprolactone-graphene three-electrode, heating and curing, insulating and encapsulating the three-electrode region, and activating the encapsulated polycaprolactone-graphene three-electrode; The working electrode region of the activated polycaprolactone-graphene triple electrode is modified layer by layer with carbon nanocages, platinum-palladium nanoparticles and glucoamylase to obtain an electrochemical sensor for starch detection.

2. The method for preparing an electrochemical sensor for starch detection according to claim 1, wherein The method of patterning the surface of the polyimide film by a laser system to prepare the graphene three-electrode comprises: After the polyimide film is attached to the mold, the polyimide film is cleaned with distilled water and ethanol; The laser system is controlled by a computer to pattern the cleaned polyimide film to obtain a graphene three-electrode.

3. The method for preparing an electrochemical sensor for starch detection according to claim 1, wherein: The step of transferring the graphene three electrodes to the surface of polycaprolactone to obtain a formed polycaprolactone-graphene three electrodes comprises: The polycaprolactone melt is evenly coated on the polyimide film with the pattern at 80-100 rpm for 60-90 s; The polyimide film coated with the polycaprolactone melt is heated at 80-100° C. for 10-15 hours, cooled to room temperature for curing, and the formed polycaprolactone-graphene three-electrode is mechanically peeled off from the polyimide film.

4. The method for preparing an electrochemical sensor for starch detection according to claim 1, wherein The process of coating the reference electrode region of the polycaprolactone-graphene three-electrode with Ag / AgCl silver paste, insulating and encapsulating the three-electrode region, heating and curing the three-electrode, and activating the encapsulated polycaprolactone-graphene three-electrode comprises: The reference electrode region of the polycaprolactone-graphene three-electrode is coated with Ag / AgCl silver paste, the region outside the three-electrode region is insulated and encapsulated with polydimethylsiloxane, and the encapsulated polycaprolactone-graphene three-electrode is obtained after heating and curing; The encapsulated polycaprolactone-graphene three-electrode was placed in a 0.01 M phosphate buffer solution and subjected to chronoamperometry scanning to perform activation treatment.

5. The method for preparing an electrochemical sensor for starch detection according to any one of claims 1 to 4, characterized in that: The method comprises modifying the working electrode region of the activated polycaprolactone-graphene triple electrode with carbon nanocages, platinum-palladium nanoparticles and glucoamylase layer by layer to obtain an electrochemical sensor for starch detection, comprising: drop-coating a carbon nanocage dispersion on the working electrode region of the activated polycaprolactone-graphene triple electrode to obtain a CNCs / LIG working electrode, followed by drying; The dried CNCs / LIG working electrode is immersed in a mixture of chloroauric acid and palladium chloride, and Pt-Pd nanoparticles are prepared by electroplating the electrode at room temperature. After drying, a Pt-Pd / CNCs / LIG working electrode is obtained; The chitosan solution and the glucoamylase mixture were dropped onto the Pt-Pd / CNCs / LIG working electrode to obtain an electrochemical sensor with GA / Pt-Pd / CNCs / LIG as the working electrode.

6. The method for preparing an electrochemical sensor for starch detection according to claim 5, wherein The concentration of the carbon nanocage dispersion is 0.5-2 mg / mL; The concentration of the mixed solution of chloroauric acid and palladium chloride is 0.5-2 mg / mL; The mass fraction of the chitosan solution is 0.5-2%, the enzymatic activity of the glucoamylase is 110-120 U / mg, and the concentration is 80-200 mg / mL.

7. An electrochemical sensor prepared according to the method for preparing an electrochemical sensor for starch detection according to any one of claims 1 to 6.

8. Use of the electrochemical sensor according to claim 7 in starch detection, characterized in that: The steps include: Prepare starch-phosphate buffer solutions of different concentrations, perform chronoamperometry detection using the electrochemical sensor, and draw a standard curve; Obtaining a sample to be tested, and pre-treating the sample to be tested to obtain a test solution; The electrochemical sensor is inserted into the liquid to be tested, connected to an electrochemical workstation, and the starch current is recorded by chronoamperometry. The actual starch concentration is calculated based on the standard curve.

9. The use according to claim 8, characterized in that The method of obtaining a sample to be tested and pre-treating the sample to be tested to obtain a liquid to be tested comprises: Obtaining a sample to be tested and drying it; wherein the sample to be tested includes plant material; The dried sample to be tested is crushed, and the powder is repeatedly washed with anhydrous ethanol, and the corresponding precipitate is collected and dried after centrifugation to obtain a sugar-free powder; The sugar-removed powder is mixed with a phosphate buffer solution and heated in a boiling water bath to obtain a gelatinized solution; After the gelatinized liquid is precipitated with n-butanol, the corresponding precipitate is redissolved with the phosphate buffer to obtain a test solution.

10. Use of the electrochemical sensor according to claim 7 in starch detection, characterized in that: The steps include: Prepare starch-phosphate buffer solutions of different concentrations, perform chronoamperometry detection using the electrochemical sensor, and draw a standard curve; After a small number of small holes were punched on the leaf to be tested, the electrochemical sensor was fixed to the lower surface of the leaf to be tested and connected to an electrochemical workstation. The starch current was recorded by chronoamperometry and the actual starch concentration was calculated based on the standard curve. During starch detection, the working electrode of the electrochemical sensor was facing the small hole, and 5-20 μL of PBS was dripped on the small hole.

Images