Preparation method of proline electrochemical sensor and method for detecting proline concentration

Abstract

This invention provides a method for preparing a proline electrochemical sensor and a method for detecting proline concentration, comprising: electrodepositing a metal selenide on the surface of a carbon cloth working electrode to obtain a modified electrode; applying a composite material comprising porous carbon material, a conductive metal-organic framework, and a redox probe to the surface of the modified electrode; polymerizing proline as a template molecule on the surface of the modified electrode to form a molecularly imprinted polymer; and eluting the template molecule from the molecularly imprinted polymer to obtain the proline electrochemical sensor. This invention enables the preparation of a proline electrochemical sensor with both high sensitivity and high selectivity.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensor technology, and in particular to a method for preparing a proline electrochemical sensor and a method for detecting proline concentration. Background Technology

[0002] Proline is an important physiologically active substance in plants, and changes in its content are a crucial physiological indicator reflecting plant stress resistance. Accurate and rapid monitoring of proline content in plants is of great significance in agricultural production and plant science research. Currently, conventional methods for proline detection mainly include colorimetry, high-performance liquid chromatography (HPLC), and gas chromatography (GC). However, these traditional methods have significant drawbacks, such as expensive instruments, complex and time-consuming sample pretreatment processes, and the potential for damage to the plant. They also fail to achieve in vivo, in-situ, and non-destructive detection of proline content in plants.

[0003] To address the problems of traditional detection methods, electrochemical sensing technology has been applied to the field of substance detection. Electrochemical methods have attracted considerable attention due to their advantages such as simple operation, rapid response, high sensitivity, and low cost. Among these, flexible carbon cloth as the electrode substrate, with its good conductivity, large specific surface area, and excellent flexibility, is suitable for modifying and loading functional materials and can adhere well to plant tissue surfaces, offering the possibility of developing portable electrochemical sensors for in vivo detection. However, when using this technology for proline detection, the sensor performance largely depends on the modification materials and structural design of the electrode surface.

[0004] Despite the immense potential of electrochemical sensors in detection, the sensitivity and selectivity of conventional electrochemical sensors still need improvement when faced with complex sample environments such as plant fluids. Various amino acids, ions, and other small molecule compounds present in biological samples may interfere with the detection signal of the target analyte, proline, affecting the accuracy of the results. Furthermore, to achieve accurate determination of trace amounts of proline, the sensor's signal response intensity needs further amplification. Therefore, developing a proline electrochemical sensor that combines high sensitivity and high selectivity has become a pressing problem in this field. Summary of the Invention

[0005] This invention provides a method for preparing a proline electrochemical sensor and a method for detecting proline concentration, in order to solve the technical problem of how to prepare a proline electrochemical sensor with both high sensitivity and high selectivity.

[0006] This invention provides a method for preparing a proline electrochemical sensor, comprising: A modified electrode is obtained by electrodepositing a metal selenide on the surface of a carbon cloth working electrode. A composite material comprising porous carbon material, a conductive metal-organic framework, and a redox probe is applied to the surface of the modified electrode; Using proline as a template molecule, a molecularly imprinted polymer is formed by polymerization on the surface of the modified electrode. The template molecule in the molecularly imprinted polymer is eluted to obtain a proline electrochemical sensor.

[0007] According to the preparation method of a proline electrochemical sensor provided by the present invention, the metal selenide is nickel tetraselenide.

[0008] According to a method for preparing a proline electrochemical sensor provided by the present invention, the method involves electrodepositing a metal selenide on the surface of a carbon cloth working electrode to obtain a modified electrode, comprising: The carbon cloth working electrode, reference electrode, and counter electrode are immersed in a mixed solution containing ammonium chloride, selenite, and nickel sulfate. The modified electrode is obtained by forming nickel tetraselenide on the surface of the carbon cloth working electrode using a constant potential deposition method.

[0009] According to the preparation method of a proline electrochemical sensor provided by the present invention, the porous carbon material is hollow carbon sphere CMK-3, the conductive metal-organic framework is Ni3(HITP)2, and the redox probe is thionine.

[0010] According to a method for preparing a proline electrochemical sensor provided by the present invention, the step of applying a composite material comprising porous carbon material, a conductive metal-organic framework, and a redox probe to the surface of the modified electrode includes: The hollow carbon spheres CMK-3, Ni3(HITP)2, and thionine were mixed in a chitosan solution and subjected to ultrasonic treatment to prepare the composite material. The composite material is applied to the surface of the modified electrode and then dried.

[0011] According to a method for preparing a proline electrochemical sensor provided by the present invention, wherein proline is used as a template molecule to polymerize on the surface of the modified electrode to form a molecularly imprinted polymer, comprising: A molecularly imprinted polymerization solution was prepared by mixing a template molecule of proline with β-cyclodextrin, which is a functional monomer. The modified electrode is immersed in the molecularly imprinted polymer solution for electropolymerization, and the molecularly imprinted polymer is formed on the surface of the modified electrode.

[0012] According to the method for preparing a proline electrochemical sensor provided by the present invention, the electropolymerization is carried out by cyclic voltammetry.

[0013] According to a method for preparing a proline electrochemical sensor provided by the present invention, the template molecule in the molecularly imprinted polymer is eluted to obtain the proline electrochemical sensor, comprising: The electrode bearing the molecularly imprinted polymer is immersed in an alkaline solution; The template molecule in the molecularly imprinted polymer was eluted using cyclic voltammetry to obtain the proline electrochemical sensor.

[0014] This invention also provides a method for detecting proline concentration, comprising: After placing the working electrode, reference electrode, and counter electrode of the proline electrochemical sensor into the test solution, the response oxidation peak current generated by the redox probe in the proline electrochemical sensor is obtained during the differential pulse voltammetry scan. The concentration of proline in the test solution is determined based on the oxidation peak current. The proline electrochemical sensor is prepared according to any of the preparation methods of the proline electrochemical sensor.

[0015] According to a method for detecting proline concentration provided by the present invention, the step of determining the proline concentration in the test solution based on the oxidation peak current includes: The oxidation peak currents corresponding to a series of proline standard solutions with different known concentrations were obtained, resulting in multiple sets of concentration-current data. By fitting multiple sets of concentration-current data, a standard curve between the proline concentration and the oxidation peak current is obtained. The concentration of proline in the test solution is calculated by substituting the oxidation peak current corresponding to the test solution into the standard curve.

[0016] The present invention provides a method for preparing a proline electrochemical sensor and a method for detecting proline concentration. By electrodepositing metal selenides and applying a composite material consisting of porous carbon materials and a conductive metal-organic framework, the specific surface area and conductivity of the electrode are greatly increased, significantly amplifying the electrochemical signal of the redox probe and thus improving the sensor's sensitivity. Furthermore, by constructing a molecularly imprinted polymer layer on the electrode surface and eluting it to form a specific recognition cavity, the sensor can accurately identify proline molecules, effectively avoiding interference from other molecules in complex biological samples, thereby improving the sensor's selectivity. In summary, the present invention can prepare a proline electrochemical sensor with both high sensitivity and high selectivity. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a schematic flowchart of the preparation method of the proline electrochemical sensor provided by the present invention.

[0019] Figure 2 This is a schematic flowchart of the proline concentration detection method provided by the present invention.

[0020] Figure 3 This is a schematic diagram of the electrode modification process provided by the present invention.

[0021] Figure 4 This is a schematic diagram of the in vivo test results of cucumber seedling leaves provided by the present invention.

[0022] Figure 5 This is a schematic diagram of the proline concentration detection device provided by the present invention.

[0023] Figure 6 This is a schematic diagram of the structure of the electronic device provided by the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0025] The following is combined Figures 1 to 6 The preparation method of the proline electrochemical sensor and the detection method of proline concentration of the present invention are described.

[0026] Figure 1 This is a schematic flowchart of the preparation method of the proline electrochemical sensor provided by the present invention, as shown below. Figure 1 As shown, the method includes, but is not limited to, steps S1, S2, S3 and S4.

[0027] Step S1: Electrodeposit a metal selenide on the surface of the carbon cloth working electrode to obtain a modified electrode.

[0028] First, cut the carbon cloth and sonicate it for 2 minutes each in acetone, ethanol, and pure water. Then, rinse the electrode with plenty of pure water to clean the carbon cloth. Next, fix the carbon cloth to the PDMS flexible support substrate with PVC insulating tape. Wrap conductive aluminum tape around the edge of the carbon cloth 0.5 cm from the edge for connecting the electrode to the electrode clamp of the potentiostat. Leave a 1 cm margin. A 1cm carbon cloth working area was prepared. The electrode was placed in a 120℃ oven and heated for 30 minutes to melt the polyethylene insulating tape, thus sealing the non-working area of ​​the electrode and preventing capillary action during the measurement process. The resulting carbon cloth working electrode was then obtained.

[0029] A carbon cloth working electrode is an electrode with a substrate made of a cloth-like material woven from carbon fibers. Due to its excellent conductivity, chemical stability, flexibility, and three-dimensional porous network structure, carbon cloth is ideally suited as a substrate for electrochemical sensors. Its porous structure and rough surface increase the effective surface area of ​​the electrode, providing more active sites for subsequent loading of functional materials.

[0030] Electrodeposition is a method of depositing a thin film of a specific material (i.e., metal selenide) on the surface of a conductive substrate (i.e., a carbon cloth working electrode) through electrolysis. Specific implementation methods can include constant potential deposition, constant current deposition, or pulse deposition.

[0031] By introducing a metal selenide layer on the surface of carbon cloth, the conductivity and electrocatalytic activity of the electrode can be significantly improved, providing preliminary amplification for subsequent electrochemical signals and thus improving the sensitivity of the sensor.

[0032] Step S2 involves applying a composite material comprising porous carbon material, a conductive metal-organic framework, and a redox probe to the surface of the modified electrode.

[0033] Composite materials are multiphase materials composed of two or more materials with different properties. Porous carbon materials are carbon materials with abundant pore structure and huge specific surface area, specifically including activated carbon, mesoporous carbon, graphene, carbon nanotubes, or hollow carbon spheres. The role of porous carbon materials is to provide a huge surface area for loading more conductive metal-organic frameworks and redox probes, thereby further amplifying the signal response of sensors.

[0034] Conductive metal-organic frameworks (MOFs) are crystalline porous materials with a periodic network structure formed by the self-assembly of metal ions or clusters with organic ligands through coordination bonds. Here, "conductive MOF" specifically refers to conductive metal-organic frameworks, which not only possess the large specific surface area and regular pores of traditional MOFs but also exhibit excellent electronic conductivity. Their function is to act as a conductive network and catalytic center, promoting rapid electron transfer at the electrode surface.

[0035] Redox probes are molecules capable of undergoing reversible redox reactions and generating stable electrochemical signals. Specific examples include thionine, methylene blue, ferrocene, and their derivatives. Their function is as signal reporter molecules; their oxidation or reduction peak current changes as the analyte (proline) binds to the sensor. Quantitative analysis of the analyte can be achieved by detecting this current change.

[0036] Composite materials can be applied to the surface of the modified electrode using methods such as drop coating, spin coating, dip coating, or screen printing.

[0037] Step S2 constructs a highly efficient signal conversion and amplification platform. Porous carbon materials and conductive metal-organic frameworks jointly construct a three-dimensional network structure with high specific surface area and high conductivity, while the redox probe is immobilized within this network. The synergistic effect of these three components greatly enhances the electrochemical response signal of the sensor, laying the foundation for achieving high-sensitivity detection.

[0038] Step S3: Using proline as a template molecule, a molecularly imprinted polymer is formed on the surface of the modified electrode through polymerization.

[0039] This invention utilizes molecular imprinting technology (MIT), a biomimetic technology that mimics the antigen-antibody or enzyme-substrate specific recognition mechanism found in nature.

[0040] The template molecule is the target molecule that is intended to be specifically recognized by the sensor; in this invention, it is proline. Molecularly imprinted polymers (MIPs) are high-molecular polymers formed by polymerizing functional monomers in the presence of a template molecule. The functional monomers interact with the template molecule through non-covalent bonds (such as hydrogen bonds or electrostatic interactions) or covalent bonds. After polymerization, a crosslinking agent immobilizes the functional monomers around the template molecule, forming a polymer network with a specific three-dimensional spatial structure and recognition sites.

[0041] Molecularly imprinted polymers can be formed on the surface of modified electrodes through electrochemical polymerization, photopolymerization, or thermal polymerization.

[0042] Step S3 can impart selectivity to the sensor by constructing a layer of artificial antibody on the electrode surface that has a specific recognition ability for proline molecules, thereby effectively eliminating the influence of other interfering substances in the sample.

[0043] Step S4: Elute the template molecules from the molecularly imprinted polymer to obtain the proline electrochemical sensor.

[0044] Elution refers to the treatment of an electrode with a molecularly imprinted polymer using a specific eluent to disrupt the interaction between the template molecule and the functional monomer, thereby removing the template molecule from the polymer network. After elution, memory cavities remain in the polymer that perfectly match the template molecule in size, shape, and functional group arrangement. These cavities can then specifically bind proline molecules in solution again.

[0045] Step S4 is a crucial step in molecular imprinting technology, its significance lying in creating recognition sites for the specific recognition of proline. These cavities can only be used for subsequent detection processes after the template molecule is removed. After this step, a complete, detection-capable electrochemical sensor for proline is obtained.

[0046] As described above, this invention significantly increases the specific surface area and conductivity of the electrode by electrodepositing metal selenides and applying a composite material composed of porous carbon materials and conductive metal-organic frameworks, thereby amplifying the electrochemical signal of the redox probe and improving the sensor's sensitivity. Furthermore, by constructing a molecularly imprinted polymer layer on the electrode surface and eluting it to form a specific recognition cavity, the sensor can accurately recognize proline molecules, effectively avoiding interference from other molecules in complex biological samples, thus improving the sensor's selectivity. In summary, this invention can prepare a proline electrochemical sensor that combines high sensitivity and high selectivity.

[0047] In one embodiment, the metal selenide of the present invention can be nickel tetraselenide.

[0048] Nickel tetraselenide (Ni3Se4) is a novel transition metal chalcogenide electrode material that has attracted attention due to its excellent redox catalytic ability. Ni3Se4 not only improves the conductivity and increases the specific surface area of ​​carbon cloth electrodes, but also acts as a highly efficient electrocatalyst, effectively catalyzing the electrochemical reactions of redox probes and further accelerating the electron transfer rate. This, in addition to achieving high sensitivity, further enhances the signal response intensity of the sensor, thus improving its overall sensitivity.

[0049] In one embodiment, step S1 may further include: The carbon cloth working electrode, reference electrode, and counter electrode are immersed in a mixed solution containing ammonium chloride, selenite, and nickel sulfate. Nickel tetraselenide was formed on the surface of the carbon cloth working electrode by constant potential deposition to obtain a modified electrode.

[0050] First, an electrodeposition solution is prepared, which is a mixed solution containing ammonium chloride, selenite, and nickel sulfate. Nickel sulfate (NiSO4) serves as the nickel source, providing Ni... 2+Ions; selenite (H2SeO3) serves as a selenium source, providing SeO3. 2- Ions; ammonium chloride (NH4Cl) serves as a supporting electrolyte to increase the conductivity of the solution and ensure the stability of the electrodeposition process. The specific concentrations of these reagents can be: 0.1M NH4Cl, 0.05M H2SeO3, and 0.05M nickel sulfate.

[0051] The working area of ​​the carbon cloth working electrode was then immersed in the electrodeposition solution, and electrodeposition was performed using a potentiostatic deposition method. Potentiostatic deposition is an electrochemical synthesis method in which a constant potential is applied to the carbon cloth working electrode immersed in the electrodeposition solution. At this potential, Ni in the solution... 2+ and SeO3 2- The ions undergo a reduction reaction on the electrode surface, forming and depositing an in-situ nickel tetraselenide (Ni3Se4) thin film. This constant potential can be specifically set to -0.95V.

[0052] This invention employs a one-step constant potential electrodeposition method to directly grow nickel tetraselenide in situ on the surface of a carbon cloth electrode. This method is extremely simple to operate, has mild reaction conditions, short processing time, low cost, and makes it easy to control the morphology and thickness of the deposited layer.

[0053] In one embodiment, the porous carbon material of the present invention can be hollow carbon spheres CMK-3, the conductive metal-organic framework can be Ni3(HITP)2, and the redox probe can be thionine.

[0054] Hollow carbon spheres CMK-3 are a typical mesoporous carbon material, which can be regarded as a porous carbon material in a broad sense. They have a hollow core and a porous shell, and possess extremely high specific surface area, good electrical conductivity and high chemical stability. They can provide a high-capacity loading platform for other functional materials.

[0055] The conductive metal-organic framework Ni3(HITP)2 not only possesses a porous structure but also exhibits high conductivity comparable to graphite. Its role is to construct efficient electron conduction pathways and accelerate electron transfer. The mesoporous structure of the hollow carbon spheres CMK-3 also provides excellent support and dispersion for Ni3(HITP)2, preventing its aggregation and detachment during electrochemical processes, thereby improving the stability of the sensor.

[0056] Thionium exhibits good electrochemical activity and a stable redox signal. Its oxidation peak current is highly sensitive to the surrounding chemical environment. When proline binds to the sensor, it hinders electron transfer, leading to a decrease in the oxidation peak current of thionium, thus enabling the detection of proline.

[0057] This invention combines the high specific surface area of ​​hollow carbon spheres CMK-3 with the high conductivity of Ni3(HITP)2 to construct a high-performance signal amplification platform. More importantly, the dispersing and stabilizing effect of the hollow carbon spheres CMK-3 on Ni3(HITP)2 solves the problem of easy aggregation and failure of conductive metal-organic framework materials in applications, significantly improving the long-term stability and repeatability of the sensor. Therefore, this specific material combination not only achieves extremely high sensitivity but also ensures the stability and reliability of the sensor.

[0058] In one embodiment, step S2 may further include: A composite material was prepared by mixing hollow carbon spheres CMK-3, Ni3(HITP)2, and thionine in a chitosan solution and then subjecting the mixture to ultrasonic treatment. The composite material was applied to the surface of the modified electrode and then dried.

[0059] Hollow carbon spheres CMK-3, Ni3(HITP)2, and thionine (Thi) solid powders can be added to a chitosan (CS) solution and sonicated for 30 minutes to obtain a Thi-CMK-3-Ni3(HITP)2-CS composite material.

[0060] Chitosan is a natural high-molecular polymer with good biocompatibility, film-forming properties, and adhesiveness. Here, it mainly acts as a dispersant and binder. Through ultrasonic treatment, utilizing the high energy generated by the cavitation effect of ultrasound, various materials can be uniformly dispersed in the chitosan solution, preventing them from agglomerating and forming a stable suspension, i.e., the dispersion of the composite material.

[0061] For example, 10 mg of CMK-3, 2 mg of Ni3(HITP)2 and 5 mM thionine can be dissolved in 1 mL of 1% acetic acid solution containing 2 mg / mL chitosan and sonicated at room temperature for 2 hours to fully disperse them, thus obtaining Thi-CMK-3-Ni3(HITP)2-CS composite material.

[0062] The prepared composite dispersion is then applied to the surface of the modified electrode (i.e., the Ni3Se4 / CC electrode). The application method can be drop-coating, where 10 μL is dropped onto the modified electrode surface. Afterward, the solvent (such as water and acetic acid) is evaporated by drying, for example, by air drying at room temperature. Once the solvent has evaporated, the chitosan solidifies into a thin film, firmly fixing the hollow carbon spheres CMK-3, Ni3(HITP)2, and thionine to the electrode surface, forming a stable functional layer.

[0063] This invention uses chitosan as a binder and combines ultrasonic dispersion and drop-coating drying methods, which has two advantages: First, the adhesive effect of chitosan ensures a strong bond between the composite material functional layer and the electrode substrate, preventing the functional material from falling off during use, thereby improving the service life and stability of the sensor; Second, the method is simple to operate, has mild conditions, is easy to implement, and provides an economical and efficient way to prepare uniform and stable composite film.

[0064] In one embodiment, step S3 may further include: A molecularly imprinted polymerization solution was prepared by mixing a template molecule of proline with β-cyclodextrin, which is a functional monomer. The modified electrode is immersed in a molecularly imprinted polymer solution for electropolymerization, forming a molecularly imprinted polymer on the surface of the modified electrode.

[0065] First, a molecularly imprinted polymerization solution is prepared. This polymerization solution contains the template molecule proline and the functional monomer β-cyclodextrin. β-cyclodextrin is a cyclic oligosaccharide composed of 7 glucose units, and its molecular structure exhibits a cone-shaped hollow barrel structure that is hydrophilic on the outside and hydrophobic on the inside. Its cavity can encapsulate guest molecules of matching size, and the external hydroxyl functional groups can form hydrogen bonds with other molecules. The proline molecule (template) will self-assemble with the β-cyclodextrin molecule (functional monomer) through non-covalent interactions such as hydrogen bonds to form a supramolecular complex. For example, 1 mM proline and 2 mM β-cyclodextrin can be mixed and sonicated for 10 min, with the concentration ratio of template molecule to functional monomer being 1:4 to 2:1, preferably 1:2.

[0066] Electropolymerization then proceeds. The modified electrode, with the pre-modified composite material, is used as the working electrode and immersed in the aforementioned molecularly imprinted polymerization solution. By applying a specific electrochemical procedure (such as cyclic voltammetry), the polymerization reaction of β-cyclodextrin molecules is initiated. The β-cyclodextrin molecules interconnect on the electrode surface, forming an insoluble polymer film (poly-β-cyclodextrin), while simultaneously immobilizing the proline-β-cyclodextrin complex within the polymer network.

[0067] This invention employs β-cyclodextrin as a functional monomer and utilizes electropolymerization to form a molecularly imprinted polymer film in situ on the electrode surface. The advantages of this method are: First, β-cyclodextrin is widely available, inexpensive, and biocompatible, and its abundant hydroxyl groups facilitate electropolymerization, making it an ideal functional monomer. Second, electropolymerization is a highly controllable film preparation technique. By precisely controlling electrochemical parameters (such as potential, time, and cycle number), the thickness, density, and morphology of the imprinted polymer film can be easily adjusted, thereby optimizing sensor performance.

[0068] In one embodiment, the electropolymerization of the present invention can be performed using cyclic voltammetry.

[0069] Cyclic voltammetry (CV) is a commonly used electrochemical research method. The process involves scanning the potential back and forth at a constant rate within a defined potential range. When the potential reaches a value sufficient to initiate the polymerization of the functional monomer (β-cyclodextrin), the polymerization reaction occurs at the electrode surface. By repeating multiple potential scans (i.e., multiple cycles), the polymer film gradually thickens.

[0070] The specific parameters for cyclic voltammetry can be set as follows: In a three-electrode system, the modified electrode is used as the working electrode, the silver / silver chloride electrode as the reference electrode, and the platinum wire as the counter electrode, all immersed together in a molecularly imprinted polymer solution. The potential scan range is set to -0.4V to 1.0V, the scan rate is 100mV / s, and 10 cycles are performed. Through these 10 cycles, a molecularly imprinted polymer film of suitable thickness can be grown on the electrode surface.

[0071] Cyclic voltammetry can monitor current changes simultaneously during polymerization, allowing for real-time monitoring of polymer film growth. By observing changes in the CV spectrum, the success of polymerization and the film growth status can be determined. This method offers good repeatability and precise control, ensuring that the imprinted layer on the sensor surface has similar thickness and properties in each fabrication, thereby improving batch-to-batch reproducibility of sensor fabrication.

[0072] In one embodiment, step S4 may further include: The electrode with the molecularly imprinted polymer is immersed in an alkaline solution; The template molecules in the molecularly imprinted polymer were eluted using cyclic voltammetry to obtain a proline electrochemical sensor.

[0073] First, an alkaline solution is chosen as the eluent. The interaction between proline and β-cyclodextrin is mainly hydrogen bonding. Under alkaline conditions (high pH), the carboxyl group (-COOH) of proline deprotonates to form a carboxylate group (-COO). - Furthermore, the hydroxyl groups (-OH) of β-cyclodextrin may also be partially deprotonated. This change in charge state disrupts the original hydrogen bonding forces between them, making it easier for proline molecules to detach from the polymer cavity. For example, the alkaline solution could be a PBS buffer solution (pH 7.0) containing 50 mM sodium hydroxide (NaOH).

[0074] Elution was then performed using cyclic voltammetry. The electrode with the imprinted polymer was immersed in an alkaline eluent, and cyclic voltammetry scans were performed. The periodic changes in potential caused minute swelling and contraction of the polymer chains, which may have altered the intermolecular electrostatic forces, accelerating the diffusion and detachment of template molecules from the imprinted pores. The CV parameters for elution were set as follows: potential range -0.8V to 1.1V, scan rate 100mV / s, and 10 cycles. Continuous scanning continued until the redox peak representing the template molecules disappeared on the CV curve, indicating that the template molecules had been completely eluted.

[0075] This invention employs an alkaline solution combined with cyclic voltammetry for elution. The alkaline environment chemically weakens the binding force between the template and the monomer, while cyclic voltammetry physically promotes the detachment of the template. The synergistic effect of these two methods can significantly shorten the elution time and ensure that the template molecules are removed more thoroughly, thereby obtaining a greater number and higher quality recognition sites, ultimately improving the sensitivity and response range of the sensor.

[0076] Figure 2 This is a flowchart illustrating the method for detecting proline concentration provided by the present invention, as shown in Figure 2. The method includes, but is not limited to, steps S5 and S6.

[0077] Step S5: After placing the working electrode, reference electrode, and counter electrode of the proline electrochemical sensor into the test solution, the response oxidation peak current generated by the redox probe in the proline electrochemical sensor is acquired during the differential pulse voltammetry scan. The proline electrochemical sensor is prepared according to any of the preparation methods for proline electrochemical sensors.

[0078] The test solution can be a sample solution containing an unknown concentration of proline, such as plant extracts or buffer solutions.

[0079] The detection system of the present invention adopts a standard three-electrode system, including: a working electrode as the sensing core (i.e., the prepared proline electrochemical sensor), a reference electrode (such as an Ag / AgCl electrode) that provides a stable potential reference, and a counter electrode (such as a platinum wire electrode) that constitutes a current loop.

[0080] Differential pulse voltammetry (DPV) is a highly sensitive electrochemical analysis technique. It superimposes a series of small-amplitude voltage pulses onto a linearly increasing voltage baseline. By measuring the current difference between two time points before and after each pulse and plotting the voltage, background interference such as non-Radaic current (charging current) can be effectively eliminated, resulting in a signal with a higher signal-to-noise ratio.

[0081] The oxidation peak current refers to the peak current value of the peak signal that appears on the DPV spectrum when a redox probe (such as thionine) undergoes an oxidation reaction. When the sensor binds to proline molecules in the test solution, the proline molecules occupy the imprinted cavity, hindering electron transfer between the redox probe and the electrode surface, resulting in a decrease in the oxidation peak current. The more proline molecules bind, the more significant the current decrease.

[0082] The oxidation peak current can be automatically acquired by connecting to an electrochemical workstation or portable detection device connected to a three-electrode system. The device performs a DPV scan and records the current-voltage curve, and then automatically or manually extracts the peak current data from the curve.

[0083] Step S6: Determine the proline concentration in the test solution based on the oxidation peak current.

[0084] The oxidation peak current value of this invention has a certain functional relationship with the proline concentration in the test solution. When the proline concentration varies within a certain range, the decrease in oxidation peak current (or the current value itself) is usually linearly related to the logarithm of the proline concentration. Therefore, the corresponding proline concentration can be calculated by substituting the measured oxidation peak current value into a pre-established standard curve.

[0085] This invention employs highly sensitive differential pulse voltammetry (DPV) for proline concentration detection. Compared to other voltammetric techniques such as cyclic voltammetry (CV), DPV effectively suppresses background current interference, significantly improving the signal-to-noise ratio and sensitivity, enabling the detection of even lower proline concentrations. Combined with the aforementioned highly selective sensor, this method achieves accurate, rapid, and highly sensitive quantitative analysis of proline in complex sample environments.

[0086] In one embodiment, step S6 may further include: The oxidation peak currents corresponding to a series of proline standard solutions with different known concentrations were obtained, resulting in multiple sets of concentration-current data. By fitting multiple sets of concentration-current data, a standard curve between proline concentration and oxidation peak current was obtained. The concentration of proline in the test solution was calculated by substituting the oxidation peak current corresponding to the test solution into the standard curve.

[0087] First, prepare a series of proline standard solutions with known concentrations, such as 0 mol / L, 10 mol / L, etc. -9 moL / L, 10 -8 moL / L, 10 -7 moL / L, 10 -6 mol / L, 10 -5 moL / L, 10 -4A mol / L proline standard solution was prepared. Then, using the same proline electrochemical sensor, DPV was sequentially performed on these standard solutions, and the oxidation peak current value corresponding to each concentration was recorded. This yielded a series of paired proline concentration-oxidation peak current data points.

[0088] The relevant detection parameters can be set as follows: voltage range -0.5~0.05V, pulse period 0.5s, pulse width 0.05s, amplitude potential 0.025V, and the detection process is carried out at room temperature (25℃). The proline molecularly imprinted working electrode, reference electrode, and counter electrode are connected to the DPV detection device. The three electrodes are immersed in an electrolyte containing the lowest concentration of proline. After incubation for 2 minutes, solution calibration begins, detection is performed, and the detection signal is wirelessly transmitted to the computer. The computer's data processing function acquires the DPV peak current data and automatically fills in the current corresponding to that concentration. The above steps are repeated to obtain the peak current values ​​corresponding to different concentrations of proline standard solutions.

[0089] Then, multiple sets of concentration-current data were fitted to obtain a standard curve between proline concentration and oxidation peak current. This standard curve best represents the variation trend of these data points and represents the quantitative relationship between the current signal and proline concentration.

[0090] Finally, by substituting the oxidation peak current of the test solution into the standard curve equation, the concentration of proline in the test sample can be calculated.

[0091] This invention establishes a standard curve to precisely correlate complex electrochemical signals with the concentration of proline to be measured, providing a scientific basis and specific operating procedures for the quantitative application of the sensor, and ensuring the accuracy and reliability of the detection results.

[0092] Based on the preceding text, we can conclude that... Figure 3The illustrated technical solution first involves growing a nanostructured nickel tetraselenide (Ni3Se4) on the surface of carbon cloth (CC) fibers. Next, a composite material, Thi-CMK-3-Ni3(HITP)2-CS, is applied to the surface of the modified electrode via drop-casting. This composite material contains thionine (Thi), hollow carbon spheres (CMK-3), a conductive metal-organic framework Ni3(HITP)2, and chitosan (CS). Then, a molecularly imprinted polymer film is polymerized on the modified electrode surface using MIP electropolymerization, where template molecules (i.e., proline) can be seen encapsulated within the polymer. Subsequently, the template molecules are removed from the polymer through an elution process, leaving specific recognition cavities or imprinted sites on the polymer surface that match the shape, size, and functional group arrangement of proline molecules. The sensor fabrication is thus complete.

[0093] In applications, proline molecules in the test solution can be specifically captured by the recognition cavity on the sensor surface through an incubation process. This binding causes a change in the electrochemical signal, which is ultimately reflected in the DPV (Current-Potential) curve below, enabling quantitative detection.

[0094] This invention selects cucumber seedling leaves as the test object. A proline molecularly imprinted carbon cloth electrode is attached to the leaf and fixed with a clip. A small hole is punched on the leaf surface using a puncher. Because there is very little sap in plant leaves, and the three-electrode system requires sufficient electrolyte to ensure reliable signal, PBS buffer is added to the hole. Then, a portable DPV detection device is used to collect the DPV peak current of the sensor and wirelessly transmit it to the data processing module of a tablet computer. The data processing module compares and analyzes the detection data with a standard curve to obtain the concentration of free proline in the plant leaves, which is immediately displayed on the tablet computer screen and can be saved.

[0095] The experimental results of cucumber seedling leaves are as follows: Figure 4As shown in the figure, the red curve represents the baseline signal of the sensor, indicating the current response obtained when the sensor is placed in a phosphate-buffered saline (PBS) solution without proline. In this solution, electron transfer of the redox probe (thionine) is unimpeded, resulting in a high oxidation peak current. The black curve represents the sample signal of the sensor, indicating the current response obtained when the sensor is attached to a cucumber seedling leaf and endogenous proline is detected in the leaf. Because proline molecules in the leaf bind to the imprinted sites on the sensor surface, electron transfer is hindered, leading to a significant decrease in the oxidation peak current. The difference between the peak currents of the two curves is directly related to the concentration of proline in the leaf; a larger difference indicates a higher proline concentration. Figure 4 This strongly demonstrates that the sensor can be successfully used for the detection of proline in living plants.

[0096] To compare the performance of the proline molecularly imprinted electrochemical sensor in the in vivo detection of proline in cucumber seedlings, this invention compares the results with those obtained by gas chromatography, as shown in Table 1.

[0097] Table 1

[0098] This invention also provides a portable detection system for proline, comprising the aforementioned proline electrochemical sensor, a portable DPV detection device, and a tablet computer. The portable DPV detection device includes a power module, a detection module, and a Bluetooth module. The detection module includes a control unit (with an ADuCM355 chip as the core processor), a potentiostat module, an analog-to-digital converter circuit, a current-to-voltage converter circuit, and a digital-to-analog converter circuit. The proline electrochemical sensor is directly inserted into the interface of the portable detection device. The portable detection device acquires the signal via the potentiostat and transmits the detection signal to the tablet computer via the Bluetooth module.

[0099] The tablet PC's main functions include communication, detection control, and data processing. Communication refers to establishing effective communication with the portable detection system via Bluetooth, including sending control commands and receiving detection data. Detection control involves the tablet PC controlling the detection process of the substance to be tested, ensuring the user can intuitively control the experiment and receive real-time feedback. Data processing involves the tablet PC further analyzing and processing the collected detection data, displaying the analysis results intuitively on the user interface, and saving the raw data for subsequent detailed analysis. For example, the tablet PC's data processing function can perform baseline calibration and calculate the concentration value of the sample based on a standard working curve.

[0100] The portable detection system of the present invention can be used to monitor the proline content in living plants. The system is low in cost, sensitive and fast in detection, and can be applied to different scenarios and on-site detection.

[0101] The device for detecting proline concentration provided by the present invention will be described below. The device for detecting proline concentration described below can be referred to in correspondence with the method for detecting proline concentration described above.

[0102] like Figure 5 As shown, the proline concentration detection device provided by the present invention includes: The acquisition module is used to acquire the response oxidation peak current generated by the redox probe in the proline electrochemical sensor during the differential pulse voltammetry scan after the working electrode, reference electrode and counter electrode of the proline electrochemical sensor are placed in the test solution. The determination module is used to determine the concentration of proline in the test solution based on the oxidation peak current. The proline electrochemical sensor was prepared according to any of the preparation methods for proline electrochemical sensors.

[0103] Figure 6 A schematic diagram of the physical structure of an electronic device is provided. This electronic device may include a processor, a communications interface, memory, and a communication bus, wherein the processor, communications interface, and memory communicate with each other via the communication bus. The processor can invoke logical instructions stored in the memory to execute a method for detecting proline concentration.

[0104] Furthermore, the logical instructions in the aforementioned memory can be implemented as software functional units and sold or used as independent products, and can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, or the part that contributes to the prior art, or a part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of the present invention. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0105] On the other hand, the present invention also provides a computer program product, which includes a computer program that can be stored on a non-transitory computer-readable storage medium. When the computer program is executed by a processor, the computer is able to perform the proline concentration detection method provided by the above methods.

[0106] In another aspect, the present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, is implemented to perform the proline concentration detection method provided by the methods described above.

[0107] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this embodiment according to actual needs. Those skilled in the art can understand and implement this without any creative effort.

[0108] Through the above description of the embodiments, those skilled in the art can clearly understand that each embodiment can be implemented by means of software plus necessary general-purpose hardware platforms, and of course, it can also be implemented by hardware. Based on this understanding, the above technical solutions, in essence or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product can be stored in a computer-readable storage medium, such as ROM / RAM, magnetic disk, optical disk, etc., and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute the methods described in the various embodiments or some parts of the embodiments.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a proline electrochemical sensor, characterized in that, include: A modified electrode is obtained by electrodepositing a metal selenide on the surface of a carbon cloth working electrode. A composite material comprising porous carbon material, a conductive metal-organic framework, and a redox probe is applied to the surface of the modified electrode; Using proline as a template molecule, a molecularly imprinted polymer is formed by polymerization on the surface of the modified electrode. The template molecule in the molecularly imprinted polymer is eluted to obtain a proline electrochemical sensor.

2. The method for preparing the proline electrochemical sensor according to claim 1, characterized in that, The metal selenide is nickel tetraselenide.

3. The method for preparing the proline electrochemical sensor according to claim 2, characterized in that, The modified electrode is obtained by electrodepositing a metal selenide on the surface of the carbon cloth working electrode, including: The carbon cloth working electrode, reference electrode, and counter electrode are immersed in a mixed solution containing ammonium chloride, selenite, and nickel sulfate. The modified electrode is obtained by forming nickel tetraselenide on the surface of the carbon cloth working electrode using a constant potential deposition method.

4. The method for preparing the proline electrochemical sensor according to claim 1, characterized in that, The porous carbon material is hollow carbon sphere CMK-3, the conductive metal-organic framework is Ni3(HITP)2, and the redox probe is thionine.

5. The method for preparing the proline electrochemical sensor according to claim 4, characterized in that, The process of applying a composite material comprising porous carbon material, a conductive metal-organic framework, and a redox probe to the surface of the modified electrode includes: The hollow carbon spheres CMK-3, Ni3(HITP)2, and thionine were mixed in a chitosan solution and subjected to ultrasonic treatment to prepare the composite material. The composite material is applied to the surface of the modified electrode and then dried.

6. The method for preparing the proline electrochemical sensor according to claim 1, characterized in that, The polymer formed by polymerizing proline-templated molecules on the surface of the modified electrode to form a molecularly imprinted polymer includes: A molecularly imprinted polymerization solution was prepared by mixing a template molecule of proline with β-cyclodextrin, which is a functional monomer. The modified electrode is immersed in the molecularly imprinted polymer solution for electropolymerization, and the molecularly imprinted polymer is formed on the surface of the modified electrode.

7. The method for preparing the proline electrochemical sensor according to claim 6, characterized in that, The electropolymerization is performed using the cyclic voltammetry method.

8. The method for preparing the proline electrochemical sensor according to claim 1, characterized in that, The template molecule is eluted from the molecularly imprinted polymer to obtain a proline electrochemical sensor, comprising: The electrode bearing the molecularly imprinted polymer is immersed in an alkaline solution; The template molecule in the molecularly imprinted polymer was eluted using cyclic voltammetry to obtain the proline electrochemical sensor.

9. A method for detecting proline concentration, characterized in that, include: After placing the working electrode, reference electrode, and counter electrode of the proline electrochemical sensor into the test solution, the response oxidation peak current generated by the redox probe in the proline electrochemical sensor is obtained during the differential pulse voltammetry scan. The concentration of proline in the test solution is determined based on the oxidation peak current. The proline electrochemical sensor is prepared according to the preparation method of any one of the proline electrochemical sensors described in claims 1-8.

10. The method for detecting proline concentration according to claim 9, characterized in that, The step of determining the proline concentration in the test solution based on the oxidation peak current includes: The oxidation peak currents corresponding to a series of proline standard solutions with different known concentrations were obtained, resulting in multiple sets of concentration-current data. By fitting multiple sets of concentration-current data, a standard curve between the proline concentration and the oxidation peak current is obtained. The concentration of proline in the test solution is calculated by substituting the oxidation peak current corresponding to the test solution into the standard curve.

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