Electrochemical sensor based on metal cluster composite and preparation method and application thereof

Abstract

To provide an electrochemical sensor based on a metal cluster composite material, and its manufacturing method.SOLUTION: The Au25 graphene composite solutions, the kidney bean estrase-chitosan solutions, and the Nafion solutions were dropped on glassy carbon electrodes and dried to obtain electrodes for electrochemical biosensors. The metal clusters consist of 25 gold atoms and a defined number of organic ligands and have a precise structure and a stable geometry. The Au25 graphene composite has excellent conductivity and a cooperative electrocatalytic effect, and can effectively promote the transfer of electrons, thereby improving the response of the sensor. The electrochemical sensor can measure the content of profenofos by using an enzyme inhibition method using kidney bean esterase as an enzyme source.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to the technical field of biosensors, in particular to electrochemical sensors based on metal cluster composites, and their fabrication methods and applications. [Background technology]

[0002] Profenofos is a representative organophosphate pesticide widely used in modern agricultural production. It is an asymmetric organophosphate insecticide with contact lethality and toxic effects, a broad insecticidal spectrum, and long-lasting efficacy, enabling it to control harmful insects and mites on crops such as cotton, vegetables, and fruit trees. However, the widespread use of organophosphate pesticides has led to the problem of pesticide residues, which not only seriously pollute the ecological environment but also pose serious risks to human health. For example, highly toxic profenofos is easily absorbed by the human body, causing serious damage to vital organs such as the liver and kidneys, and can even cause epilepsy and toxic shock. It can damage the central nervous system, gastrointestinal perforation or bleeding, respiratory failure, liver and kidney damage, and ultimately harm human health. Therefore, accurate detection and analysis of pesticide residues in the environment and agricultural products is essential for environmental monitoring and management and food safety.

[0003] Currently, traditional detection methods mainly include gas chromatography, high-performance liquid chromatography, and colorimetry. Gas chromatography is widely used, but it has poor stability and is not suitable for detecting substances that are not easily volatile. High-performance liquid chromatography is suitable for detecting complex samples, but it is expensive and prone to contamination. Colorimetry is easy to operate, but it requires the absorption characteristics of the test substance.

[0004] Compared with conventional detection methods, a new type of electrochemical biosensor can be constructed to measure the inhibition rate of profenofos in the enzyme-catalyzed reaction, thereby quickly analyzing the residual amount of profenofos in the sample. Currently, the manufacturing materials for electrochemical biosensors mainly include carbon material supports and metal nanomaterials, and commonly used carbon materials include amorphous carbon and graphene, but there are certain deficiencies in response sensitivity. Summary of the Invention [Means for solving the problem]

[0005] The object of the present invention is to provide an electrochemical sensor based on a metal cluster composite material to solve the problems proposed in the background art.

[0006] In order to achieve the above object, the present invention provides the following technical means: 1. An electrochemical sensor based on a metal cluster composite, comprising: The electrochemical sensor is composed of a glassy carbon electrode, metal clusters@graphene, kidney bean esterase-chitosan, and Nafion. TM (Nafion TM ) membrane.

[0007] 1. A method for fabricating an electrochemical sensor based on a metal cluster composite, comprising: Weigh out 0.1 to 10 mmol of metal precursor and surfactant, add them to 5 to 500 mL of organic solvent, and thoroughly stir and mix them to obtain a suspension. Add 1 to 10 times the molar amount of the metal precursor to the suspension and stir thoroughly to completely mix and react. Stir further in an ice bath. Then, add 0.1 to 2 mol / L of sodium borohydride (NaBH4) solution dropwise to the solution and continue stirring for 2 to 12 hours. After evaporating and concentrating the reacted mixture, wash with a large amount of ice water, then wash again with an organic solvent, and repeat the washing process 2 to 3 times. Extract the resulting product with an organic solvent and grow a single crystal to obtain pure Au. 25 S1, which obtains the cluster and synthesizes the metal cluster; By uniformly dispersing graphene and an organic solvent with ultrasonic waves, adding the metal cluster solution prepared in advance, and then subjecting it to ultrasonic treatment again, Au 25 After obtaining the G solution, kidney bean powder is added to ultrapure water and stirred uniformly, and then the mixture is placed in a refrigerator for infusion. The refrigerator is set at 4°C, and after centrifugation, the supernatant (crude enzyme solution, KbE) is sucked up. Chitosan (CS) is added and the mixture is stirred uniformly to obtain a KbE-CS solution, which is then used to produce an electrode composite material. S2 This Au 25 The G composite solution, KbE-CS solution, and Nafion solution (NF) were dropped onto a clean bare electrode (GCE). After modification, the electrode was dried under an infrared lamp, and then eluted with ultrapure water. The electrode was then dried in an earwashing bowl to obtain NF / KbE-CS / Au. 25 By assembling the @G / GCE electrochemical biosensor, NF / KbE-CS / Au 25 S3, which obtained the @G / GCE electrochemical biosensor.

[0008] Preferably, the metal precursors described in S1 include, but are not limited to, chloroauric acid, (triphenylphosphine)gold(I) chloride.

[0009] Preferably, the surfactants described in S1 include, but are not limited to, tetra-n-octylammonium bromide, tetrabutylammonium chloride, and trimethylstearylammonium chloride.

[0010] Preferably, the organic solvents described in S1 and S2 include, but are not limited to, methanol, ethanol, acetonitrile, dichloromethane, toluene, acetone, tetrahydrofuran, isopropyl alcohol, n-butanol, and N,N-dimethylformamide.

[0011] Preferably, the organic solvent described in S1 is a mixture of toluene and methanol, and the ratio of toluene to methanol includes, but is not limited to, 1 / 30, 1 / 15, 1 / 10, 1 / 6, 1 / 3, and 1 / 2.

[0012] Preferably, the organic ligands described in S1 include, but are not limited to, 2-phenylethanethiol, 2,4-dimethylbenzenethiol, 4-tert-butylbenzenethiol, cyclohexanethiol, and benzyl mercaptan.

[0013] Preferably, the solvent of the NaBH4 solution described in S1 includes, but is not limited to, water, methanol, ethanol, acetonitrile, dichloromethane, toluene, acetone, tetrahydrofuran, isopropyl alcohol, and n-butanol.

[0014] Preferably, the electrochemical biosensor manufactured by the method for manufacturing an electrochemical sensor based on a metal cluster composite material is applied to the detection of profenofos. [Effects of the Invention]

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention relates to NF / KbE-CS / Au 25 A new type of electrochemical sensor based on @G / GCE was constructed, in which the core structure of the sensor was Au 25 The fabrication method includes the steps of synthesizing metal clusters by size aggregation, compounding the metal clusters with carbon materials, compounding enzymes with chitosan, and layer-by-layer assembly of an electrochemical biosensor electrode. The clusters are composed of gold atoms and organic ligands, with 25 gold atoms, and have a precise and stable three-dimensional structure. At the same time, the metal cluster / carbon material composite has excellent conductivity and cooperative electrocatalytic effect, which can effectively promote electron transfer, improving the response sensitivity of the sensor of the present invention and enabling more efficient detection of residual profenofos.

[0016] At the same time, this invention uses kidney bean esterase as an enzyme source and uses enzyme inhibition method to measure the content of profenofos, which can be applied to the detection of actual samples, and NF / KbE-CS / Au 25 @G / GCE has excellent repeatability, stability and interference resistance during testing, and Au 25Under optimal experimental conditions, with a G loading of 5 μL, a PBS buffer solution at pH 7.0, and a profenofos inhibition time of 15 minutes, the electrochemical biosensor's linear range for detecting profenofos was 1-2000 μg / L, with the linear equation Y=19.8626lgc+13.3459 and correlation coefficient R2=0.97316. The electrochemical biosensor of the present invention exhibited excellent response sensitivity and showed great potential for practical applications, being convenient, efficient, and efficient. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a UV-visible spectrogram of Au25(PET)18 of the present invention. [Figure 2] 1 is a line graph of the peak current I versus different loading amounts of Au25(PET) of the present invention. [Figure 3] 1 is a line graph of different inhibition times and inhibition rates of profenofos of the present invention. [Figure 4] 1 is a linear relationship graph between different mass concentrations of profenofos of the present invention and the inhibition rate. [Figure 5] FIG. 10 is a diagram showing the stability of repeated SWV detection using the same electrode of the present invention. [Figure 6] FIG. 1 is a comparative diagram of the influence of each interfering substance of the present invention on the response signal of the NF / KbE-CS / Au25@G / GCE biosensor. DETAILED DESCRIPTION OF THE INVENTION

[0018] Hereinafter, the technical aspects of the embodiments of the present invention will be clearly and completely described in connection with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and are not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present invention.

[0019] Example 1 675 mg of tetra-n-octylammonium bromide (TOAB) was weighed and 30 mL of tetrahydrofuran (THF) was added to dissolve the TOAB. A HAuCl4 solution (496 mg dissolved in 5 mL of THF) was then added dropwise, causing the solution to turn reddish-brown. After stirring for 30 minutes, 2-phenylethanethiol (1020 μL) was added and the mixture was stirred until colorless. The mixture was then stirred for another 30 minutes in an ice bath. Next, 536 mg of NaBH4 was dispersed in 8 mL of ice water, and the NaBH4 was quickly dissolved and added to the flask. The solution immediately turned black. The reaction was continued for 12 hours in an ice bath. After the reaction was complete, the concentrated reaction solution was slowly evaporated under vacuum. The concentrated reaction solution was then washed with a large amount of ice water 3-5 times, and then the same procedure was repeated with methanol 3-5 times. Finally, the resulting product was placed in a toluene / methanol mixture to grow crystals. Needle-shaped Au 25 Crystals gradually precipitate within 1-2 days.

[0020] Weighed 4 mg of multi-layer graphene was added to a 10 mL centrifuge tube, and then 4 mL of N,N-dimethylformamide was immediately added. The multi-layer graphene was uniformly dispersed in the solution by ultrasonic treatment, and then the previously prepared Au 25 A solution of the clusters in N,N-dimethylformamide (concentration: 1 mg / mL) was added to the centrifuge tube and sonicated again for 5 minutes to obtain Au clusters. 25 A KbE solution was obtained. Kidney bean powder was mixed with 5 mL of ultrapure water per 1 g of powder, stirred uniformly, and then soaked overnight in a refrigerator at 4°C for extraction. After centrifugation, the supernatant (crude enzyme solution) was aspirated and stored refrigerated. 1 mg of chitosan was accurately weighed using an analytical balance, dissolved in 5 mL of the KbE crude enzyme supernatant, and shaken to obtain a 0.2 g / L KbE-CS solution.

[0021] Pipette 5 μL of Au sequentially. 25The G composite solution, 10 μL of 1 g / L KbE-CS solution, and 4 μL of 0.5% Nafion solution were applied dropwise to a clean bare electrode (GCE). After modification, the electrode was dried under an infrared lamp, eluted with ultrapure water, and dried in an earwashing bowl to obtain NF / KbE-CS / Au. 25 A @G / GCE electrochemical biosensor was obtained.

[0022] NF / KbE-CS / Au 25 After incubating the @G / GCE electrode in the prepared profenophos solutions with different mass concentrations for 15 minutes, each electrode was transferred to a PBS buffer solution containing 0.8 mmol / L 1-NA at pH 7.0 and SWV scans were performed. The SWV curves corresponding to this series of concentrations were recorded and analyzed. A good linear relationship between the profenophos mass concentration (lgC) and the inhibition rate (Y) was observed in the range of 1 to 2000 μg / L. The linear equation was Y = 13.459 lgC + 19.8626, with a correlation coefficient R. 2 =0.97316.

[0023] First, a PBS test solution of purple cabbage juice was obtained by polishing, extraction, centrifugation, and dilution. 25 The @G / GCE modified electrode was detected in 0.1 mol / L PBS containing 0.8 mmol / L NA at pH 7.0, and a square wave pulse volt-ampere characteristic curve was obtained, followed by I0. Then, the NF / KbE-CS / Au 25 The @G / GCE modified electrode was incubated in a PBS mixed solution containing purple cabbage juice for 15 minutes, and then further incubated in a PBS mixed solution containing 0.8 mmol / L 1-NA at pH 7.0 (0.1 mol / L) to measure the SWV. I1 was obtained. The inhibition rate Y was expressed by the following equation:

[0024]

number

[0025] By inputting the standard curve, it was found that the concentration of profenofos was below the residual amount specified in the Chinese national standard.

[0026] Example 2 675 mg of tetra-n-octylammonium bromide (TOAB) was weighed and 30 mL of tetrahydrofuran (THF) was added to dissolve the TOAB. A HAuCl4 solution (496 mg dissolved in 5 mL of THF) was then added dropwise, causing the solution to turn reddish-brown. After stirring for 30 minutes, 2-phenylethanethiol (1020 μL) was added and the mixture was stirred until colorless. The mixture was then stirred for another 30 minutes in an ice bath. Next, 536 mg of NaBH4 was dispersed in 8 mL of ice water, and the NaBH4 was quickly dissolved and added to the flask. The solution immediately turned black. The reaction was continued for 12 hours in an ice bath. After the reaction was complete, the concentrated reaction solution was slowly evaporated under vacuum. The concentrated reaction solution was then washed with a large amount of ice water 3-5 times, and then the same procedure was repeated with methanol 3-5 times. Finally, the resulting product was placed in a toluene / methanol mixture to grow crystals. Needle-shaped Au 25 Crystals gradually precipitate within 1-2 days.

[0027] Weighed 4 mg of multi-layer graphene was added to a 10 mL centrifuge tube, and then 4 mL of N,N-dimethylformamide was immediately added. The multi-layer graphene was uniformly dispersed in the solution by ultrasonic treatment, and then the previously prepared Au 25 A solution of the clusters in N,N-dimethylformamide (concentration: 1 mg / mL) was added to the centrifuge tube and sonicated again for 5 minutes to obtain Au clusters. 25 A KbE solution was obtained. Kidney bean powder was mixed with 5 mL of ultrapure water per 1 g of powder, stirred uniformly, and then soaked overnight in a refrigerator at 4°C for extraction. After centrifugation, the supernatant (crude enzyme solution) was aspirated and stored refrigerated. 1 mg of chitosan was accurately weighed using an analytical balance, dissolved in 5 mL of the KbE crude enzyme supernatant, and shaken to obtain a 0.2 g / L KbE-CS solution.

[0028] Pipette 5 μL of Au sequentially. 25The G composite solution, 10 μL of 1 g / L KbE-CS solution, and 4 μL of 0.5% Nafion solution were applied dropwise to a clean bare electrode (GCE). After modification, the electrode was dried under an infrared lamp, eluted with ultrapure water, and dried in an earwashing bowl to obtain NF / KbE-CS / Au. 25 A @G / GCE electrochemical biosensor was obtained.

[0029] NF / KbE-CS / Au 25 After incubating the @G / GCE electrode in the prepared profenophos solutions with different mass concentrations for 15 minutes, each electrode was transferred to a PBS buffer solution containing 0.8 mmol / L 1-NA at pH 7.0 and SWV scans were performed. The SWV curves corresponding to this series of concentrations were recorded and analyzed. A good linear relationship between the profenophos mass concentration (lgC) and the inhibition rate (Y) was observed in the range of 1 to 2000 μg / L. The linear equation was Y = 13.459 lgC + 19.8626, with a correlation coefficient R. 2 =0.97316.

[0030] First, the chive juice PBS test solution was obtained by polishing, extraction, centrifugation, and dilution. 25 The @G / GCE modified electrode was detected in 0.1 mol / L PBS containing 0.8 mmol / L NA at pH 7.0, and a square wave pulse volt-ampere characteristic curve was obtained, followed by I0. Then, the NF / KbE-CS / Au 25 After incubating the @G / GCE modified electrode in the PBS mixed solution containing Chinese chive juice for 15 minutes, SWV was measured in the PBS mixed solution containing 0.8 mmol / L 1-NA at pH 7.0 (0.1 mol / L) to obtain I1. The inhibition rate Y is expressed by the following equation 2:

[0031]

number

[0032] By inputting the standard curve, it was found that the concentration of profenofos was below the residual amount specified in the Chinese national standard.

[0033] Example 3 675 mg of tetra-n-octylammonium bromide (TOAB) was weighed and 30 mL of tetrahydrofuran (THF) was added to dissolve the TOAB. A HAuCl4 solution (496 mg dissolved in 5 mL of THF) was then added dropwise, causing the solution to turn reddish-brown. After stirring for 30 minutes, 2-phenylethanethiol (1020 μL) was added and the mixture was stirred until colorless. The mixture was then stirred for another 30 minutes in an ice bath. Next, 536 mg of NaBH4 was dispersed in 8 mL of ice water, and the NaBH4 was quickly dissolved and added to the flask. The solution immediately turned black. The reaction was continued for 12 hours in an ice bath. After the reaction was complete, the concentrated reaction solution was slowly evaporated under vacuum. The concentrated reaction solution was then washed with a large amount of ice water 3-5 times, and then the same procedure was repeated with methanol 3-5 times. Finally, the resulting product was placed in a toluene / methanol mixture to grow crystals. Needle-shaped Au 25 Crystals gradually precipitate within 1-2 days.

[0034] Weighed 4 mg of multi-layer graphene was added to a 10 mL centrifuge tube, and then 4 mL of N,N-dimethylformamide was immediately added. The multi-layer graphene was uniformly dispersed in the solution by ultrasonic treatment, and then the previously prepared Au 25 A solution of the clusters in N,N-dimethylformamide (concentration: 1 mg / mL) was added to the centrifuge tube and sonicated again for 5 minutes to obtain Au clusters. 25 A KbE solution was obtained. Kidney bean powder was mixed with 5 mL of ultrapure water per 1 g of powder, stirred uniformly, and then soaked overnight in a refrigerator at 4°C for extraction. After centrifugation, the supernatant (crude enzyme solution) was aspirated and stored refrigerated. 1 mg of chitosan was accurately weighed using an analytical balance, dissolved in 5 mL of the KbE crude enzyme supernatant, and shaken to obtain a 0.2 g / L KbE-CS solution.

[0035] Pipette 5 μL of Au sequentially. 25The G composite solution, 10 μL of 1 g / L KbE-CS solution, and 4 μL of 0.5% Nafion solution were applied dropwise to a clean bare electrode (GCE). After modification, the electrode was dried under an infrared lamp, eluted with ultrapure water, and dried in an earwashing bowl to obtain NF / KbE-CS / Au. 25 A @G / GCE electrochemical biosensor was obtained.

[0036] NF / KbE-CS / Au 25 After incubating the @G / GCE electrode in the prepared profenophos solutions with different mass concentrations for 15 minutes, each electrode was transferred to a PBS buffer solution containing 0.8 mmol / L 1-NA at pH 7.0 and SWV scans were performed. The SWV curves corresponding to this series of concentrations were recorded and analyzed. A good linear relationship between the profenophos mass concentration (lgC) and the inhibition rate (Y) was observed in the range of 1 to 2000 μg / L. The linear equation was Y = 13.459 lgC + 19.8626, with a correlation coefficient R. 2 =0.97316.

[0037] First, a PBS test solution of millet extract was obtained by polishing, extraction, centrifugation, and dilution. 25 The @G / GCE modified electrode was detected in 0.1 mol / L PBS containing 0.8 mmol / L NA at pH 7.0, and a square wave pulse volt-ampere characteristic curve was obtained, followed by I0. Then, the NF / KbE-CS / Au 25 After incubating the @G / GCE modified electrode in the PBS mixed solution containing the millet extract for 15 minutes, the SWV was measured in the PBS mixed solution containing 0.8 mmol / L 1-NA at pH 7.0 (0.1 mol / L) to obtain I1, and the inhibition rate Y was calculated by the following equation:

[0038]

number

[0039] By inputting the standard curve, it was found that the concentration of profenofos was below the residual amount specified in the Chinese national standard.

[0040] Example 4 675 mg of tetra-n-octylammonium bromide (TOAB) was weighed and 30 mL of tetrahydrofuran (THF) was added to dissolve the TOAB. A HAuCl4 solution (496 mg dissolved in 5 mL of THF) was then added dropwise, causing the solution to turn reddish-brown. After stirring for 30 minutes, 2-phenylethanethiol (1020 μL) was added and the mixture was stirred until colorless. The mixture was then stirred for another 30 minutes in an ice bath. Next, 536 mg of NaBH4 was dispersed in 8 mL of ice water, and the NaBH4 was quickly dissolved and added to the flask. The solution immediately turned black. The reaction was continued for 12 hours in an ice bath. After the reaction was complete, the concentrated reaction solution was slowly evaporated under vacuum. The concentrated reaction solution was then washed with a large amount of ice water 3-5 times, and then the same procedure was repeated with methanol 3-5 times. Finally, the resulting product was placed in a toluene / methanol mixture to grow crystals. Needle-shaped Au 25 Crystals gradually precipitate within 1-2 days.

[0041] Weighed 4 mg of multi-layer graphene was added to a 10 mL centrifuge tube, and then 4 mL of N,N-dimethylformamide was immediately added. The multi-layer graphene was uniformly dispersed in the solution by ultrasonic treatment, and then the previously prepared Au 25 A solution of the cluster in N,N-dimethylformamide (1 mg / mL) was added to the centrifuge tube and sonicated again for 5 minutes to obtain the Au25@G solution. Kidney bean powder was mixed with 5 mL of ultrapure water per 1 g of powder, stirred uniformly, and then soaked overnight in a refrigerator at 4°C for extraction. After centrifugation, the supernatant (crude enzyme solution) was aspirated and stored refrigerated. 1 mg of chitosan was accurately weighed using an analytical balance, dissolved in 5 mL of the KbE crude enzyme supernatant, and then shaken to obtain a 0.2 g / L KbE-CS solution.

[0042] Pipette 5 μL of Au sequentially. 25The G composite solution, 10 μL of 1 g / L KbE-CS solution, and 4 μL of 0.5% Nafion solution were applied dropwise to a clean bare electrode (GCE). After modification, the electrode was dried under an infrared lamp, eluted with ultrapure water, and dried in an earwashing bowl to obtain NF / KbE-CS / Au. 25 A @G / GCE electrochemical biosensor was obtained.

[0043] NF / KbE-CS / Au 25 After incubating the @G / GCE electrode in the prepared profenophos solutions with different mass concentrations for 15 minutes, each electrode was transferred to a PBS buffer solution containing 0.8 mmol / L 1-NA at pH 7.0 and SWV scans were performed. The SWV curves corresponding to this series of concentrations were recorded and analyzed. A good linear relationship between the profenophos mass concentration (lgC) and the inhibition rate (Y) was observed in the range of 1 to 2000 μg / L. The linear equation was Y = 13.459 lgC + 19.8626, with a correlation coefficient R. 2 =0.97316.

[0044] First, a PBS test solution of cabbage juice was obtained by polishing, extraction, centrifugation, and dilution. 25 The @G / GCE modified electrode was detected in 0.1 mol / L PBS containing 0.8 mmol / L NA at pH 7.0, and a square wave pulse volt-ampere characteristic curve was obtained, followed by I0. Then, the NF / KbE-CS / Au 25 The @G / GCE modified electrode was incubated in the PBS mixed solution containing cabbage juice for 15 minutes, and then further incubated in the PBS mixed solution containing 0.8 mmol / L 1-NA at pH 7.0 (0.1 mol / L) to measure the SWV. I1 was obtained. The inhibition rate Y was expressed by the following equation:

[0045]

number

[0046] By inputting the standard curve, it was found that the concentration of profenofos was below the residual amount specified in the Chinese national standard.

[0047] The present invention relates to NF / KbE-CS / Au 25 @G / GCE electrochemical biosensor was fabricated and applied to the detection of profenofos.

[0048] The electrochemical biosensor was constructed using a glassy carbon electrode, metal clusters@graphene, kidney bean esterase-chitosan, and Nafion. TM (Nafion TM ) film, and has 25 metal atoms.

[0049] The response of the electrochemical biosensor of the present invention to the residual amount of profenofos was investigated, and a method was proposed in which the inhibition rate was used as the premise for detecting the concentration. The specific test procedure is as follows: (1) Construct a standard curve and measure NF / KbE-CS / Au 25 The @G / GCE electrode was incubated in the prepared profenophos solutions with different mass concentrations for a while, and then the electrode was transferred to a PBS buffer solution containing a fixed concentration of 1-NA, and square-wave pulse SWV scans were performed.

[0050] With increasing profenofos concentration, the peak current value decreased and the inhibition rate increased. Within a certain concentration range, the mass concentration of profenofos (lgC) and the inhibition rate (Y) showed a good linear relationship, and the linear equation and standard curve were obtained.

[0051] The expressions applied to agricultural crops are: (2) Measuring the actual sample: the sample is accurately weighed and thoroughly polished, then added to the PBS solution, the mixture is centrifuged, and the supernatant is taken and tested.

[0052] NF / KbE-CS / Au using square wave pulse method 25 The @G / GCE modified electrode was probed with PBS containing a fixed concentration of 1-NA to obtain a square wave pulse volt-ampere characteristic curve, and then I0 was obtained. 25After incubating the @G / GCE modified electrode in a PBS mixed solution containing the sample solution for a certain period of time, the SWV is measured in a PBS mixed solution containing a certain concentration of 1-NA to obtain I1, and the inhibition rate is expressed by the following equation 1, which is input into the standard curve to calculate the profenofos concentration.

[0053] The electrochemical detection method used in this invention is an enzyme inhibition method, and it has excellent repeatability, stability, and interference resistance, and can maintain its operation under various conditions. 25 @G / GCE electrochemical biosensor is used.

[0054] The mass concentration of the profenofos solution described in step (1) is 1 to 2000 μg / L, the incubation time is 5 to 25 min, the 1-NA concentration is 0.1 to 2.0 mmol / L, the pH of the PBS buffer is 5.0 to 8.0, and the inhibition rate Y is represented by the number 5.

[0055]

number

[0056] The test conditions described in (2) are the same as the preferred test conditions described in (1).

[0057] This invention uses kidney bean esterase as an enzyme source and uses enzyme inhibition method to measure the content of profenofos, which is then applied to the detection of actual samples, and the NF / KbE-CS / Au 25 @G / GCE has excellent repeatability, stability and interference resistance during testing, and Au 25 Under optimal experimental conditions, with a G loading of 5 μL, a PBS buffer solution at pH 7.0, and a profenofos inhibition time of 15 minutes, the electrochemical biosensor's linear range for detecting profenofos was 1-2000 μg / L, with the linear equation Y=19.8626lgc+13.3459 and correlation coefficient R2=0.97316. The electrochemical biosensor of the present invention exhibited excellent response sensitivity and showed great potential for practical applications, being convenient, efficient, and efficient.

[0058] While embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that changes, modifications, substitutions and variations can be made without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. 1. An electrochemical sensor based on a metal cluster composite, comprising: The electrochemical sensor is composed of a glassy carbon electrode, metal clusters @ graphene, kidney bean esterase-chitosan, and Nafion. TM (Nafion TM ) An electrochemical sensor based on a metal cluster composite material, characterized in that it comprises a film.

2. 1. A method for fabricating an electrochemical sensor based on a metal cluster composite, comprising: Weigh out 0.1 to 10 mmol of metal precursor and surfactant, add them to 5 to 500 mL of organic solvent, and thoroughly stir and mix them to obtain a suspension; Add 1 to 10 times the molar number of the organic ligand to the metal precursor, stir thoroughly, and allow to react completely. Stir further in an ice bath, and then add 0.1 to 2 mol / L of sodium borohydride (NaBH ４ ) solution, add it dropwise to the solution, and continue stirring for 2 to 12 hours. After evaporating and concentrating the reacted mixed solution, wash it with a large amount of ice water, then wash it again with an organic solvent, and wash it 2 to 3 times. The resulting product is extracted with an organic solvent and single crystal grown to obtain pure Au. ２５ S1, which obtains the cluster and synthesizes the metal cluster; Graphene and an organic solvent are uniformly dispersed by ultrasonic waves, and the previously prepared metal cluster solution is added. Then, ultrasonic treatment is performed again to obtain Au ２５ S2: obtaining the G solution, adding kidney bean powder to ultrapure water and stirring uniformly, then placing in a refrigerator for infusion, setting the refrigerator at 4°C, centrifuging, and then sucking up the supernatant (crude enzyme solution, KbE), adding chitosan (CS), and shaking uniformly to obtain a KbE-CS solution, thereby producing an electrode composite material; This Au ２５ The G composite solution, KbE-CS solution, and Nafion solution (NF) were applied dropwise onto a clean bare electrode (GCE). After modification, the electrode was dried with an infrared lamp, and then eluted with ultrapure water and dried with an earwashing bowl to obtain NF / KbE-CS / Au. ２５ By assembling the NF / KbE-CS / Au@G / GCE electrochemical biosensor, ２５ S3, which obtained the @G / GCE electrochemical biosensor; 1. A method for producing an electrochemical sensor based on a metal cluster composite material, comprising:

3. 3. The method for fabricating an electrochemical sensor based on a metal cluster composite material according to claim 2, wherein the metal precursors described in S1 include, but are not limited to, chloroauric acid, (triphenylphosphine)gold(I) chloride.

4. The method for manufacturing an electrochemical sensor based on a metal cluster composite material according to claim 2, characterized in that the surfactants described in S1 include, but are not limited to, tetra-n-octylammonium bromide, tetrabutylammonium chloride, and trimethylstearylammonium chloride.

5. The method for manufacturing an electrochemical sensor based on a metal cluster composite material according to claim 2, characterized in that the organic solvents described in S1 and S2 include, but are not limited to, methanol, ethanol, acetonitrile, dichloromethane, toluene, acetone, tetrahydrofuran, isopropyl alcohol, n-butanol, and N,N-dimethylformamide.

6. The method for manufacturing an electrochemical sensor based on a metal cluster composite material as described in claim 2, characterized in that the organic solvent described in S1 is a mixture of toluene and methanol, and the ratio of toluene to methanol includes, but is not limited to, 1 / 30, 1 / 15, 1 / 10, 1 / 6, 1 / 3, and 1 / 2.

7. The method for manufacturing an electrochemical sensor based on a metal cluster composite material according to claim 2, characterized in that the organic ligands described in S1 include, but are not limited to, 2-phenylethanethiol, 2,4-dimethylbenzenethiol, 4-tert-butylbenzenethiol, cyclohexanethiol, and benzyl mercaptan.

8. NaBH ４ 3. The method for fabricating an electrochemical sensor based on a metal cluster composite material according to claim 2, characterized in that the solvent of the solution includes, but is not limited to, water, methanol, ethanol, acetonitrile, dichloromethane, toluene, acetone, tetrahydrofuran, isopropyl alcohol, and n-butanol.

9. A method for using an electrochemical biosensor manufactured by the method for manufacturing an electrochemical sensor based on a metal cluster composite material according to claim 2 in detecting profenofos.

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