Electrochemical sensors based on metal cluster composites, their fabrication methods and applications

Abstract

Electrochemical sensors based on metal cluster composites and methods of manufacture are provided. [Solution] Au 25 The graphene composite solution, the bean esterase chitosan solution, and the Nafion solution are dropped onto a glassy carbon electrode and then dried to obtain an electrochemical biosensor electrode. The metal cluster is composed of 25 gold atoms and a well-defined number of organic ligands, and has a precise composition and stable geometric structure. Au 25 The graphene composite has excellent electrical conductivity and cooperative electrocatalytic effect, which can effectively promote the transfer of electrons and improve the response sensitivity of the sensor. The electrochemical sensor can measure the content of profenofos by enzyme inhibition method using kidney bean esterase as the enzyme source.

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 organophosphorus pesticide that has been widely used in modern agricultural production. It is an asymmetric organophosphorus insecticide with contact lethality and toxic effects, a wide insecticidal spectrum, and long-lasting efficacy, which can prevent harmful insects and mites in cotton, vegetables, fruit and other crops. However, the problem of pesticide residues caused by the widespread use of organophosphorus pesticides not only seriously pollutes the ecological environment, but also causes serious damage to human health. For example, highly toxic profenofos is easily absorbed by the human body, and not only seriously damages important organs such as the liver and kidneys, but can also cause epilepsy, toxic shock, etc. It can damage the central nervous system, gastrointestinal perforation or bleeding, respiratory failure, liver and kidneys, etc., and ultimately harm human health. Therefore, for environmental monitoring and management and food safety, it is necessary to accurately detect and analyze pesticide residues in the environment and agricultural products.

[0003] At present, 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 pollution. Colorimetry is easy to operate, but has requirements on the absorption characteristics of the test substance.

[0004] Compared with traditional detection methods, a new type of electrochemical biosensor can be constructed and the amount of profenofos remaining in a sample can be rapidly analyzed by measuring the inhibition rate of profenofos in the enzyme-catalyzed reaction. At present, the manufacturing materials for electrochemical biosensors mainly include carbon material carriers 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 producing an electrochemical sensor based on a metal cluster composite, comprising the steps of: Weigh out 0.1 to 10 mmol of metal precursor and surfactant, add them to 5 to 500 mL of organic solvent, and thoroughly mix and stir to obtain a suspension. Add 1 to 10 times the molar amount of the metal precursor to the suspension, stir thoroughly, and allow the mixture to react completely. Stir further under ice bath. Then, add 0.1 to 2 mol / L of sodium borohydride (NaBH4) solution dropwise to the solution. Stir 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 wash 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 a pre-prepared metal cluster solution, and then subjecting the mixture to ultrasonic treatment again, Au 25 S2: Obtain the G solution, add kidney bean powder to ultrapure water and stir evenly, then put it in a refrigerator to infuse, set the refrigerator temperature at 4°C, and after centrifugation, suck up the supernatant (crude enzyme solution, KbE), add chitosan (CS) and shake evenly to obtain KbE-CS solution to produce an electrode composite material; This Au 25 The @G composite solution, KbE-CS solution, and Nafion solution (NF) were dropped onto a clean bare electrode (GCE), and after modification was completed, 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 The @G / GCE electrochemical biosensor was constructed using NF / KbE-CS / Au 25 S3, which obtained the @G / GCE electrochemical biosensor.

[0008] Preferably, the metal precursors described in S1 above 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 prepared by the method for preparing an electrochemical sensor based on a metal cluster composite material is applied to detect profenofos. Effect 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 electrochemical sensor of @G / GCE was constructed, in which the core composition of the sensor is Au 25 @G, the preparation method includes the steps of synthesizing metal clusters by size aggregation, compounding metal clusters with carbon materials, compounding enzymes with chitosan, and assembling electrochemical biosensor electrodes layer by layer. The clusters are composed of gold atoms and organic ligands, the number of gold atoms is 25, and have a precise and stable three-dimensional structure. At the same time, the metal cluster-carbon material composite has good electrical conductivity and cooperative electrocatalytic effect, which can effectively promote the transfer of electrons, improve the response sensitivity of the sensor of the present invention, and more efficiently detect the residual amount of profenofos.

[0016] At the same time, the present invention uses kidney bean esterase as an enzyme source, and uses the 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 25Under the optimal experimental conditions of @G loading amount 5μL, PBS buffer pH 7.0, and profenofos inhibition time 15min, the linear range of the electrochemical biosensor to detect profenofos is 1-2000μg / L, the linear equation is Y=19.8626lgc+13.3459, and the correlation coefficient R2=0.97316. The electrochemical biosensor of the present invention shows excellent response sensitivity, and shows broad potential for practical application, being convenient and efficient. [Brief description of the drawings]

[0017] [Figure 1] 1 is a UV-visible spectrogram of Au25(PET)18 of the present invention. [Diagram 2] 1 is a line graph of different Au25(PET) loadings and peak current I of the present invention. [Diagram 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 inhibition rate. [Diagram 5] FIG. 13 shows the stability of the present invention when SWV detection is performed repeatedly using the same electrode. [Figure 6] This is a comparative diagram of the effects 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 PREFERRED EMBODIMENTS

[0018] Hereinafter, the technical aspects of the embodiments of the present invention will be described clearly and completely in conjunction 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 in the present invention, all other embodiments obtained by those skilled in the art without performing creative labor belong to the scope of protection of the present invention.

[0019] Example 1 Weigh out 675 mg of tetra-n-octylammonium bromide (TOAB), add 30 mL of tetrahydrofuran (THF) to dissolve TOAB, and add HAuCl4 solution (496 mg dissolved in 5 mL THF) dropwise, and the solution turned reddish brown. After stirring for 30 minutes, add 2-phenylethanethiol (1020 μL) ligand, continue stirring until it becomes colorless, and stir for another 30 minutes under ice bath. Next, take 536 mg of NaBH4 and disperse it in 8 mL of ice water, quickly dissolve NaBH4, and add it to the flask, and the solution immediately turns black. Continue reacting in an ice bath environment for 12 hours, and after the reaction is completed, gradually evaporate the concentrated reaction solution under a vacuum environment. After that, wash the concentrated reaction solution with a large amount of ice water, repeat 3 to 5 times, then perform the same operation using methanol, and wash again 3 to 5 times. Finally, place the obtained product in a toluene / methanol mixed system to grow crystals. Acicular 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 Au 25 The N,N-dimethylformamide solution of the clusters (concentration 1 mg / mL) was added to the centrifuge tube and sonicated again for 5 min to obtain Au 25 A KbE-CS solution was obtained. The kidney bean powder was mixed with 5 mL of ultrapure water per 1 g of powder, and after stirring uniformly, the refrigerator was set to 4°C and the mixture was soaked overnight for extraction. After centrifugation, the supernatant (crude enzyme solution) was sucked off and stored in a refrigerator. 1 mg of chitosan was accurately weighed using an analytical balance, and dissolved in 5 mL of the KbE crude enzyme solution supernatant. The mixture was then shaken uniformly to obtain a 0.2 g / L KbE-CS solution.

[0021] Pipette 5 μL of Au 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 was completed, the electrode was dried with an infrared lamp, eluted with ultrapure water, and dried with an earwashing ball 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 profenophos solutions with different mass concentrations for 15 min, each electrode was transferred to a PBS buffer solution containing 0.8 mmol / L 1-NA at pH 7.0 for SWV scanning. By recording and analyzing the SWV curves corresponding to this series of concentrations, it was found that the profenophos mass concentration (lgC) and the inhibition rate (Y) showed a good linear relationship in the range of 1 to 2000 μg / L, and 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 grinding, extraction, centrifugation and dilution. 25 The @G / GCE modified electrode was detected in 0.1 mol / L PBS containing 0.8 mmol / L 1-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 purple cabbage juice for 15 minutes, and then the SWV was measured in the PBS mixed solution of pH=7.0 0.1mol / L containing 0.8mmol / L 1-NA to obtain I1, and the inhibition rate Y was expressed by the following formula 1:

[0024]

number

[0025] By inputting it into 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 Weigh out 675 mg of tetra-n-octylammonium bromide (TOAB), add 30 mL of tetrahydrofuran (THF) to dissolve TOAB, and add HAuCl4 solution (496 mg dissolved in 5 mL THF) dropwise, and the solution turned reddish brown. After stirring for 30 minutes, add 2-phenylethanethiol (1020 μL) ligand, continue stirring until it becomes colorless, and stir for another 30 minutes under ice bath. Next, take 536 mg of NaBH4 and disperse it in 8 mL of ice water, quickly dissolve NaBH4, and add it to the flask, and the solution immediately turns black. Continue reacting in an ice bath environment for 12 hours, and after the reaction is completed, gradually evaporate the concentrated reaction solution under a vacuum environment. After that, wash the concentrated reaction solution with a large amount of ice water, repeat 3 to 5 times, then perform the same operation using methanol, and wash again 3 to 5 times. Finally, place the obtained product in a toluene / methanol mixed system to grow crystals. Acicular 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 Au 25 The N,N-dimethylformamide solution of the clusters (concentration 1 mg / mL) was added to the centrifuge tube and sonicated again for 5 min to obtain Au 25 A KbE-CS solution was obtained. The kidney bean powder was mixed with 5 mL of ultrapure water per 1 g of powder, and after stirring uniformly, the refrigerator was set to 4°C and the mixture was soaked overnight for extraction. After centrifugation, the supernatant (crude enzyme solution) was sucked off and stored in a refrigerator. 1 mg of chitosan was accurately weighed using an analytical balance, and dissolved in 5 mL of the KbE crude enzyme solution supernatant. The mixture was then shaken uniformly to obtain a 0.2 g / L KbE-CS solution.

[0028] Pipette 5 μL of Au 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 was completed, the electrode was dried with an infrared lamp, eluted with ultrapure water, and dried with an earwashing ball 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 profenophos solutions with different mass concentrations for 15 min, each electrode was transferred to a PBS buffer solution containing 0.8 mmol / L 1-NA at pH 7.0 for SWV scanning. By recording and analyzing the SWV curves corresponding to this series of concentrations, it was found that the profenophos mass concentration (lgC) and the inhibition rate (Y) showed a good linear relationship in the range of 1 to 2000 μg / L, and 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 grinding, extraction, centrifugation and dilution. 25 The @G / GCE modified electrode was detected in 0.1 mol / L PBS containing 0.8 mmol / L 1-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, the SWV was measured in the PBS mixed solution of pH=7.0 containing 0.1mol / L of 0.8mmol / L 1-NA to obtain I1, and the inhibition rate Y was expressed by the following formula 2:

[0031]

number

[0032] By inputting it into 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 Weigh out 675 mg of tetra-n-octylammonium bromide (TOAB), add 30 mL of tetrahydrofuran (THF) to dissolve TOAB, and add HAuCl4 solution (496 mg dissolved in 5 mL THF) dropwise, and the solution turned reddish brown. After stirring for 30 minutes, add 2-phenylethanethiol (1020 μL) ligand, continue stirring until it becomes colorless, and stir for another 30 minutes under ice bath. Next, take 536 mg of NaBH4 and disperse it in 8 mL of ice water, quickly dissolve NaBH4, and add it to the flask, and the solution immediately turns black. Continue reacting in an ice bath environment for 12 hours, and after the reaction is completed, gradually evaporate the concentrated reaction solution under a vacuum environment. After that, wash the concentrated reaction solution with a large amount of ice water, repeat 3 to 5 times, then perform the same operation using methanol, and wash again 3 to 5 times. Finally, place the obtained product in a toluene / methanol mixed system to grow crystals. Acicular 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 Au 25 The N,N-dimethylformamide solution of the clusters (concentration 1 mg / mL) was added to the centrifuge tube and sonicated again for 5 min to obtain Au 25 A KbE-CS solution was obtained. The kidney bean powder was mixed with 5 mL of ultrapure water per 1 g of powder, and after stirring uniformly, the refrigerator was set to 4°C and the mixture was soaked overnight for extraction. After centrifugation, the supernatant (crude enzyme solution) was sucked off and stored in a refrigerator. 1 mg of chitosan was accurately weighed using an analytical balance, and dissolved in 5 mL of the KbE crude enzyme solution supernatant. The mixture was then shaken uniformly to obtain a 0.2 g / L KbE-CS solution.

[0035] Pipette 5 μL of Au 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 was completed, the electrode was dried with an infrared lamp, eluted with ultrapure water, and dried with an earwashing ball 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 profenophos solutions with different mass concentrations for 15 min, each electrode was transferred to a PBS buffer solution containing 0.8 mmol / L 1-NA at pH 7.0 for SWV scanning. By recording and analyzing the SWV curves corresponding to this series of concentrations, it was found that the profenophos mass concentration (lgC) and the inhibition rate (Y) showed a good linear relationship in the range of 1 to 2000 μg / L, and the linear equation was Y = 13.459 lgc + 19.8626, with a correlation coefficient R 2 =0.97316.

[0037] First, the PBS test solution of millet extract was obtained by polishing, extraction, centrifugation and dilution. 25 The @G / GCE modified electrode was detected in 0.1mol / L PBS containing 0.8 mmol / L 1-NA at pH=7.0, and a square wave pulse volt-ampere characteristic curve was obtained, and then the I0 was obtained. Then, the NF / KbE-CS / Au 25 After incubating the @G / GCE modified electrode in the PBS mixed solution containing millet extract for 15 minutes, the SWV was measured in the PBS mixed solution of pH=7.0 0.1mol / L containing 0.8mmol / L 1-NA to obtain I1, and the inhibition rate Y was expressed by the following formula 3:

[0038]

number

[0039] By inputting it into 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 Weigh out 675 mg of tetra-n-octylammonium bromide (TOAB), add 30 mL of tetrahydrofuran (THF) to dissolve TOAB, and add HAuCl4 solution (496 mg dissolved in 5 mL THF) dropwise, and the solution turned reddish brown. After stirring for 30 minutes, add 2-phenylethanethiol (1020 μL) ligand, continue stirring until it becomes colorless, and stir for another 30 minutes under ice bath. Next, take 536 mg of NaBH4 and disperse it in 8 mL of ice water, quickly dissolve NaBH4, and add it to the flask, and the solution immediately turns black. Continue reacting in an ice bath environment for 12 hours, and after the reaction is completed, gradually evaporate the concentrated reaction solution under a vacuum environment. After that, wash the concentrated reaction solution with a large amount of ice water, repeat 3 to 5 times, then perform the same operation using methanol, and wash again 3 to 5 times. Finally, place the obtained product in a toluene / methanol mixed system to grow crystals. Acicular 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 Au 25 The cluster solution in N,N-dimethylformamide (concentration 1 mg / mL) was added to the centrifuge tube and ultrasonicated again for 5 minutes to obtain the Au25@G solution. The kidney bean powder was mixed with 5 mL of ultrapure water per 1 g of powder, and after uniform stirring, the refrigerator temperature was set to 4°C and the mixture was soaked overnight for extraction. After centrifugation, the supernatant (crude enzyme solution) was sucked off and stored in a refrigerator. 1 mg of chitosan was accurately weighed using an analytical balance, dissolved in 5 mL of the KbE crude enzyme solution supernatant, and shaken uniformly to obtain a 0.2 g / L KbE-CS solution.

[0042] Pipette 5 μL of Au 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 was completed, the electrode was dried with an infrared lamp, eluted with ultrapure water, and dried with an earwashing ball 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 profenophos solutions with different mass concentrations for 15 min, each electrode was transferred to a PBS buffer solution containing 0.8 mmol / L 1-NA at pH 7.0 for SWV scanning. By recording and analyzing the SWV curves corresponding to this series of concentrations, it was found that the profenophos mass concentration (lgC) and the inhibition rate (Y) showed a good linear relationship in the range of 1 to 2000 μg / L, and the linear equation was Y = 13.459 lgc + 19.8626, with a correlation coefficient R 2 =0.97316.

[0044] First, the PBS test solution of cabbage juice was obtained by polishing, extraction, centrifugation and dilution. 25 The @G / GCE modified electrode was detected in 0.1mol / L PBS containing 0.8 mmol / L 1-NA at pH=7.0, and a square wave pulse volt-ampere characteristic curve was obtained, and then the I0 was obtained. 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 to obtain I1. The inhibition rate Y is given by the following equation 4:

[0045]

number

[0046] By inputting it into 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 A @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, bean esterase-chitosan, and Nafion. TM (Nafion TM ) film, with 25 metal atoms.

[0049] The response of the electrochemical biosensor of the present invention to the residual amount of profenofos was studied, 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 into a PBS buffer solution containing a constant concentration of 1-NA to perform square-wave pulse SWV scans.

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

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

[0052] NF / KbE-CS / Au using the 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. Then, the NF / KbE-CS / Au 25After incubating the @G / GCE modified electrode in the PBS mixed solution containing the sample solution for a certain period of time, the SWV is measured in the PBS mixed solution containing a certain concentration of 1-NA to obtain I1, and the inhibition rate is shown by the following equation 1, and the inhibition rate is input into the standard curve to calculate the profenofos concentration.

[0053] The electrochemical detection method used in the present invention is an enzyme inhibition method, which has excellent repeatability, stability and interference resistance, and can maintain its operating ability 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 in (1).

[0057] This invention uses kidney bean esterase as an enzyme source, and uses the enzyme inhibition method to determine 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 the optimal experimental conditions of @G loading amount 5μL, PBS buffer pH 7.0, and profenofos inhibition time 15min, the linear range of the electrochemical biosensor to detect profenofos is 1-2000μg / L, the linear equation is Y=19.8626lgc+13.3459, and the correlation coefficient R2=0.97316. The electrochemical biosensor of the present invention shows excellent response sensitivity, and shows broad potential for practical application, being convenient and efficient.

[0058] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that the same may be modified, altered, substituted, and changed without departing from the principles and spirit of the present invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for producing an electrochemical sensor based on a metal cluster composite, comprising the steps of: 0.1 to 10 mmol of metal precursor and surfactant are weighed out, added to 5 to 500 mL of organic solvent, and thoroughly mixed and stirred to obtain a suspension; Add 1 to 10 times the molar amount of the organic ligand to the metal precursor, thoroughly stir, and completely mix and react with each other. Stir further in an ice bath. Then, add 0.1 to 2 mol / L of sodium borohydride (NaBH ４ ) solution, dropwise, and continue stirring for 2 to 12 hours. The reacted mixed solution is evaporated and concentrated, then washed with a large amount of ice water, and then washed again with an organic solvent. After washing 2 to 3 times, the resulting product is extracted with an organic solvent, and single crystal growth is performed to obtain pure Au. ２５ S1, which obtains a cluster and synthesizes a metal cluster; Graphene and an organic solvent are uniformly dispersed by ultrasonic waves, and the previously prepared metal cluster solution is added and ultrasonically treated 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, and centrifugally treating the mixture to remove 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 each applied dropwise onto a clean glassy carbon electrode (GCE). After modification, the electrode was dried under an infrared lamp, eluted with ultrapure water, and dried with an earwashing bowl to obtain NF / KbE-CS / Au. ２５ The NF / KbE-CS / Au@G / GCE electrochemical biosensor was assembled. ２５ S3 to obtain the @G / GCE electrochemical biosensor; 2. A method for producing an electrochemical sensor based on a metal cluster composite material, comprising:

2. The method for producing an electrochemical sensor based on a metal cluster composite material according to claim 1, characterized in that the metal precursors described in S1 include chloroauric acid, (triphenylphosphine)gold(I) chloride.

3. The method for manufacturing an electrochemical sensor based on a metal cluster composite material according to claim 1, characterized in that the surfactants described in S1 include tetra-n-octylammonium bromide, tetrabutylammonium chloride, and trimethylstearylammonium chloride.

4. The method for producing an electrochemical sensor based on a metal cluster composite material according to claim 1, characterized in that the organic solvents described in S1 and S2 include methanol, ethanol, acetonitrile, dichloromethane, toluene, acetone, tetrahydrofuran, isopropyl alcohol, n-butanol, and N,N-dimethylformamide.

5. The method for manufacturing an electrochemical sensor based on a metal cluster composite material as described in claim 1, characterized in that the organic solvent described in S1 is a mixture of toluene and methanol, and the ratio of toluene to methanol includes 1 / 30, 1 / 15, 1 / 10, 1 / 6, 1 / 3, and 1 / 2.

6. The method for producing an electrochemical sensor based on a metal cluster composite material according to claim 1, characterized in that the organic ligands described in S1 include 2-phenylethanethiol, 2,4-dimethylbenzenethiol, 4-tert-butylbenzenethiol, cyclohexanethiol, and benzyl mercaptan.

7. NaBH ４ The method for preparing an electrochemical sensor based on a metal cluster composite material according to claim 1, characterized in that the solvent of the solution includes water, methanol, ethanol, acetonitrile, dichloromethane, toluene, acetone, tetrahydrofuran, isopropyl alcohol, and n-butanol.

8. 2. 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 1 in detecting profenofos.

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