A method for detecting benzocaine based on in-situ growth of prussian blue nanoszyme

Abstract

The application discloses a method for detecting benzocaine based on in-situ growth of Prussian blue nanoszyme, which comprises the following steps: S1, growing Prussian blue nanoszyme in-situ; S2, preparing a nano probe; S3, preparing a colorimetric immunosensor: preparing a colorimetric immunosensor based on the nano probe obtained in step S2; S4, constructing a standard curve; and S5, determining benzocaine in a sample. In the application, polydopamine containing iron ions is added to potassium ferrocyanide under acidic conditions to grow Prussian blue nanoszyme in-situ. The Prussian blue nanoszyme can generate -OH by catalyzing H2O2, so that the color developing substrate tetramethyl benzidine changes from colorless to blue. The benzocaine antibody is labeled by an electrostatic adsorption method, the specific recognition of the antigen and the antibody is utilized, the target object is added, the target object and the antigen compete, and then an indirect competitive enzyme-linked immunization colorimetric sensing analysis method is constructed. The method is used for trace detection of benzocaine, and the detection limit is 0.042 ng / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a method for detecting benzocaine based on in-situ growth of Prussian blue nanoszyme. BACKGROUND

[0002] In fishery production, fish are prone to struggle and injury, infection of fish disease or death due to lack of oxygen due to long-distance and high-density transportation. In order to increase the survival rate of fish and reduce economic losses, benzocaine is widely used as a fish anesthetic in the circulation link. It can forcibly change the physiological state of fish during transportation, reduce the metabolic intensity in fish, and make fish move slowly and enter a state similar to hibernation, thereby preventing fish from moving violently in the container and causing injury. With the increasing concern and demand of people for food safety, the residue of food and drugs has become a social hot issue.

[0003] At present, the detection of benzocaine mainly relies on instrumental analysis methods such as liquid chromatography, liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry. For example, the Chinese invention patent CN112526027A discloses a detection method of benzocaine related substances RT-HPLC, which comprises the following steps: ① preparing an analysis solution: dissolving the sample with a mobile phase (methyl alcohol-ice acetic acid aqueous solution) to prepare an analysis solution, wherein the volume percentage of the organic phase in the methyl alcohol-ice acetic acid aqueous solution is greater than 50%; ② chromatographic conditions: the chromatographic column is a reversed-phase chromatographic column, the mobile phase is an acid or phosphate aqueous solution, the water phase solution is weakly acidic, the organic phase is methyl alcohol, and the elution mode adopts isocratic elution; the high-performance liquid flow rate is set to 0.8-1.2 ml / min; the column temperature is 25-40 DEG C, and the ultraviolet detector is used with a detection wavelength of 220-290 nm; ③ on-machine detection: 20 μL of the analysis solution prepared in step ① is injected into the high-performance liquid chromatograph for chromatographic analysis, and the chromatogram is recorded. For another example, the Chinese invention patent CN110596272A discloses a method for detecting local anesthetic components of adult external preparations. In the embodiments of the present application, high-performance liquid chromatography is used for detection, which comprises the following steps: a, preparing a test solution; b, detecting the test solution and the control solution by high-performance liquid chromatography; the preparation method of the test solution: taking adult external preparations as samples and adding them to a methyl alcohol solution containing hydrochloric acid, ultrasonicating, cooling to room temperature, diluting, oscillating and mixing uniformly, centrifuging if necessary, filtering through a membrane to obtain the test solution; the preparation method of the control solution: preparing a series of mixed standard solutions with different concentrations by using methyl alcohol containing hydrochloric acid, and obtaining a series of control solutions with different concentrations according to the preparation method of the test solution. The local anesthetic components include ester local anesthetics, amide local anesthetics and effective active ingredients of Syzygium aromaticum; the ester local anesthetics include procainamide, procaine, chloroprocaine, benzocaine, bupivacaine, oxybuprocaine, tetracaine and dibucaine; the amide local anesthetics include lidocaine, prilocaine, ropivacaine, bupivacaine and pramoxine; and the effective active ingredients of Syzygium aromaticum include eugenol, eugenol formaldehyde and isoeugenol formaldehyde.

[0004] However, although these methods have accurate and reliable detection results, they excessively rely on expensive instruments, have high operation conditions, are time-consuming and laborious, and are difficult to perform rapid screening in large quantities, which need to be solved urgently. SUMMARY

[0005] In order to make up for the defects of the prior art, the present application provides a method for detecting benzocaine based on in-situ growth of Prussian blue nanoszyme.

[0006] The technical problems to be solved by the present application are solved by the following technical solutions:

[0007] A method for detecting benzocaine based on in-situ growth of Prussian blue nanoszyme, comprising the following steps:

[0008] S1. In-situ growth of Prussian blue nanoszyme: dissolve dopamine and ferric chloride completely in deionized water, react, and obtain a mixed solution; add Tris solution to the mixed solution, continue to react, centrifuge, then wash with ultrapure water, and freeze-dry to obtain iron-based polydopamine nanospheres; re-disperse the iron-based polydopamine nanospheres in ionized water, add potassium ferrocyanide aqueous solution and hydrochloric acid, react, centrifuge, wash with ultrapure water, and freeze-dry to obtain Prussian blue nanoszyme;

[0009] S2. Preparation of nanoprobes: re-disperse the in-situ growth Prussian blue nanoszyme prepared according to step S1 in ultrapure water, add benzocaine antibodies, stir at room temperature, then add bovine serum albumin for blocking, centrifuge to remove the supernatant, and finally disperse with a probe reconstitution solution for standby;

[0010] S3. Preparation of a colorimetric immunosensor: prepare a colorimetric immunosensor based on the nanoprobes obtained in step S2; specifically comprising the following steps:

[0011] (1) Antigen coating: dilute benzocaine coating stock solution to a working solution concentration with carbonate buffer, coat on a 96-well plate, and incubate overnight at 4°C; after incubation, spin dry the enzyme-labeled plate, wash the plate twice with PBS containing 0.05% Tween-20, and dry by tapping for standby;

[0012] (2) Blocking: use blocking buffer to block the enzyme-labeled plate coated in step (1), incubate at 37°C for 1-3 hours, spin dry the enzyme-labeled plate after incubation, and dry in an oven at 37°C for standby;

[0013] (3) Sample addition: dilute the target to a working concentration with 0.01M PBS, take 50μL and add to the enzyme-labeled plate blocked in step (2), then add 50μL of the diluted nanoprobes obtained in step S2; incubate at 37°C, and wash the plate 5 times with phosphate buffer containing Tween-20;

[0014] S4. Construction of a standard curve: configure benzocaine standard solution of different concentrations, add to the enzyme-labeled plate coated with benzocaine coating stock solution, add an equal volume of Prussian blue nanoprobes, react in a water bath until complete, then add color developing solution, detect the absorbance value after color development with an enzyme-labeled instrument, and obtain a standard curve of absorbance value versus antigen concentration;

[0015] S5. Determination of benzocaine in a sample: replace the benzocaine standard solution with an equal volume of treated sample, determine the sample according to the method of the standard curve, and substitute into the standard curve obtained in step S4 to obtain the concentration of benzocaine in the sample.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The present application utilizes polydopamine containing iron ions to grow Prussian blue nanoszyme in situ under acidic conditions, and the Prussian blue nanoszyme can generate -OH by catalyzing H2O2, so that the colorless substrate tetramethyl benzidine changes to blue. By electrostatic adsorption method, benzocaine antibody is labeled, and by the specific recognition of antigen and antibody, the target substance is added, and by the competition of the target substance and the antigen, an indirect competitive enzyme-linked immunosorbent colorimetric sensing analysis method is constructed. The detection limit of the method for trace detection of benzocaine is 0.042 ng / mL.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The method for detecting benzocaine based on the in-situ growth of Prussian blue nanoszyme provided by the present application has the advantages of high sensitivity, wide linear range, simple and fast operation, strong stability, good accuracy, low cost, etc., and can be used for detecting benzocaine in environmental samples, with an addition recovery rate of 82.38% to 109.07% and a coefficient of variation of less than 14.93%, which indicates that the method for detecting benzocaine in samples has good accuracy and stability, and can directly trace the amount of benzocaine drug residues in food. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the optimization of potassium ferrocyanide in the preparation of in-situ growth Prussian blue nanoszyme in S1 step of the present application;

[0021] Figure 2 is the optimization of hydrochloric acid in the preparation of in-situ growth Prussian blue nanoszyme in S1 step of the present application;

[0022] Figure 3The reaction time optimization of in-situ growth of Prussian blue nanoszyme prepared in S1 step of the application;

[0023] Figure 4 The enzyme activity determination of in-situ growth of Prussian blue nanoszyme prepared in S1 step of the application;

[0024] Figure 5 The optimization of in-situ growth of Prussian blue nanoszyme probe prepared in S2 step of the application;

[0025] Figure 6 The UV absorption spectrum of in-situ growth of Prussian blue nanoszyme and its probe prepared in S1 and S2 steps of the application;

[0026] Figure 7 The ZETA potential diagram of in-situ growth of Prussian blue nanoszyme and its probe prepared in S1 and S2 steps of the application;

[0027] Figure 8 The standard curve of detecting benzocaine in S4 step of the application. DETAILED DESCRIPTION

[0028] The raw materials and equipment used in the application are conventional raw materials and equipment in the art unless otherwise specified; the methods used in the application are conventional methods in the art unless otherwise specified.

[0029] Unless otherwise defined, all terms used in the present description, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Terms of approximation include terms such as "approximately", "substantially", and "about" and are used as terms of approximation meaning nearly or nearly the same as a stated value or reference quantity.

[0030] As used herein, the terms "comprise", "comprising", "include", "including", "have", "has", "contain", "containing", "characterized by", "characterized" or variants thereof are open-ended, non-limiting terms that also mean "consisting essentially of and "consisting of". The compositions and methods / processes of the present application comprise, consist essentially of and consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps or limitations described herein.

[0031] All numerical values or expressions representing elements of a quantity, a process condition, or the like, used in the specification and claims are to be understood as "about" the value or condition per se. All ranges recited are inclusive of the endpoints, which are combinable. Since these ranges are continuous, they include every value within that range. It is also understood that any numerical range recited in this application is intended to include all sub-ranges of the same.

[0032] As described in the above technical background, the detection of benzocaine mainly relies on instrumental analysis methods such as liquid chromatography, liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry. Although these methods have accurate and reliable detection results, they excessively rely on expensive instruments, have high operation conditions, are time-consuming and laborious, and are difficult to perform rapid screening in large quantities. Moreover, there is no reported colorimetric sensing analysis method for detecting benzocaine based on in-situ growth of Prussian blue nanoscale enzyme probes.

[0033] To this end, the present inventors have conducted a large number of experiments and developed a method for detecting benzocaine based on in-situ growth of Prussian blue nanoscale enzyme. The detection line of this method for benzocaine can be as low as 0.042 ng / mL, and has a wide detection range of 0.411-1021.6 ng / mL. Compared with existing instrumental detection, this method has the advantages of high sensitivity, simple and fast operation, strong stability, good accuracy, low cost, etc., and can be used for the detection of benzocaine in samples, and has a wide application prospect.

[0034] Specifically, the present application provides a method for detecting benzocaine based on in-situ growth of Prussian blue nanoscale enzyme, which comprises the following steps:

[0035] S1. In-situ growth of Prussian blue nanoscale enzyme: dissolve dopamine and ferric chloride completely in deionized water, react to obtain a mixed solution; add Tris solution to the above mixed solution, continue to react, centrifuge, then wash with ultrapure water, freeze-dry to obtain iron-based polydopamine nanospheres; redisperse the iron-based polydopamine nanospheres in ionized water, add potassium ferrocyanide aqueous solution and hydrochloric acid, react, centrifuge, wash with ultrapure water, and freeze-dry to obtain Prussian blue nanoscale enzyme;

[0036] S2. Preparation of nanoprobes: redisperse the in-situ grown Prussian blue nanoscale enzyme prepared according to step S1 in ultrapure water, add benzocaine antibody, stir at room temperature, then add bovine serum albumin for blocking, centrifuge to remove the supernatant, and finally disperse with probe redissolving solution for standby;

[0037] S3. Preparation of colorimetric immunosensor: prepare a colorimetric immunosensor based on the nanoprobes obtained in step S2; specifically comprising the following steps:

[0038] (1) Antigen coating: dilute the benzocaine coating stock solution to the working solution concentration with carbonate buffer, coat on a 96-well plate, and incubate overnight at 4℃; after incubation, spin dry the enzyme-labeled plate, wash the plate twice with PBS containing 0.05% Tween-20, and dry for standby;

[0039] (2) Blocking: block the enzyme-labeled plate coated in step (1) with blocking buffer, incubate at 37℃ for 1-3 h, spin dry the enzyme-labeled plate after incubation, and dry in an oven at 37℃ for standby;

[0040] (3) Add sample: dilute the target to the working concentration with 0.01M PBS, take 50 μL and add to the enzyme-labeled plate after blocking in step (2), then add 50 μL of the diluted nanoprobes obtained in step S2; incubate at 37°C, and wash the plate 5 times with phosphate buffer containing Tween-20;

[0041] S4. Construct a standard curve: configure benzocaine standard solution of different concentrations, add to the enzyme-labeled plate coated with benzocaine coating agent, add an equal volume of Prussian blue nanoprobes, after complete reaction in a water bath, add color developing solution, after color development, detect the absorbance value with an enzyme-labeled instrument to obtain a standard curve of the absorbance value and the antigen concentration;

[0042] S5. Determine benzocaine in a sample: replace the benzocaine standard solution with an equal volume of the treated sample, determine the sample according to the method of the standard curve, and substitute the standard curve obtained in step S4 to obtain the benzocaine concentration in the sample.

[0043] The application will be described in detail below with reference to examples, which are only preferred embodiments of the application and are not a limitation of the application. Unless otherwise specified, the solution concentration herein is volume concentration.

[0044] Example 1

[0045] The embodiment provides a method for detecting benzocaine based on in-situ growth of Prussian blue nanoscale enzymes, specifically comprising the following steps:

[0046] S1. In-situ growth of Prussian blue nanoscale enzymes: weigh 56.9 mg of dopamine and 2.03 mg of ferric chloride into a beaker, add 15 mL of deionized water to completely dissolve, and react at room temperature for 1 hour; add 20 mL of Tris solution with a concentration of 25 mg / mL to the above mixture, continue to stir for 2 hours, centrifuge at 10000 rp for 10 min with a high-speed centrifuge, then wash with ultrapure water, and freeze-dry to obtain iron-based polydopamine nanospheres. According to the obtained iron-based polydopamine nanospheres, redispersion is performed in 15 mL of ultrapure water, 15 mL of potassium ferrocyanide aqueous solution with a concentration of 2 mM and 7 mL of hydrochloric acid with a concentration of 0.6 M are added, stirring is performed at room temperature for 20 min, centrifugation is performed, ultrapure water is used for washing, and freeze-drying is performed to obtain the final product Prussian blue nanoscale enzyme, which is redispersed in ultrapure water before use and diluted 5 times for use. The concentration of potassium ferrocyanide and the concentration of hydrochloric acid and the reaction time are the best reaction conditions after optimization, as shown in Table 1. Figures 1-3

[0047] ​The enzyme activity of the in-situ grown Prussian blue nanoszyme was determined by measuring the UV absorption spectra of TMB+H2O2 (TMB is tetramethylbenzidine), PDA@PB (PDA is polydopamine and PB is Prussian blue), PDA@PB+TMB, PDA@PB+H2O2 and PDA@PB+TMB+H2O2, respectively, as shown in Figure 4 In the presence of H2O2, PDA@PB can catalyze TMB, and the color of TMB changes from colorless to blue, and a characteristic peak is generated at about 650 nm, proving that the in-situ grown Prussian blue nanoszyme prepared by the method has peroxidase-like activity.

[0048] S2. Preparation of nanoprobes: The in-situ grown Prussian blue nanoszyme prepared according to step S1 was re-dispersed in ultrapure water, 4 μg / ml benzocaine antibody was added, and it was slowly shaken at room temperature for 1 h, then 10% bovine serum albumin was added for blocking for 1 h, and it was centrifuged at 7000 rp for 5 min, the supernatant was removed, and finally it was dispersed with 1 mL probe redissolution solution, and it was diluted 4 times to the working concentration with 0.01M PBS. The concentration of benzocaine antibody is the optimal concentration after optimization, as shown in Figure 5 .

[0049] As shown in Figure 6 , the UV scanning determination (200-800 nm) of the in-situ grown Prussian blue nanoszyme and its probe found that the characteristic absorption peak of the Prussian blue nanoprobes shifted to the characteristic absorption peak position of the Prussian blue nanoszyme, and a new characteristic peak was generated, indicating that the benzocaine antibody was successfully labeled on the Prussian blue nanoszyme. Combined with Figure 7 , the ZETA potential of the Prussian blue nanoprobes is higher than that of the Prussian blue nanoszyme, indicating that the Prussian blue nanoprobes are successfully prepared.

[0050] S3. Preparation of colorimetric immunosensor and establishment of indirect enzyme-linked immunosorbent colorimetric sensor analysis method:

[0051] (1) Antigen coating: The benzocaine coating agent was diluted to the working solution concentration with carbonate buffer solution, coated on a 96-well plate, and incubated at 4℃ overnight; after incubation, the enzyme-labeled plate was shaken dry, washed with PBS solution containing 0.05% Tween-20 twice, and dried for use;

[0052] (2) Blocking: The enzyme-labeled plate coated in step (1) was blocked with blocking buffer, incubated at 37℃ for 1-3 h, the enzyme-labeled plate was shaken dry after incubation, and dried in an oven at 37℃ for standby;

[0053] (3) Sample addition: Dilute the target substance to the working concentration with 0.01M PBS, take 50μL and add it to the enzyme-labeled plate blocked in step (2), and then add 50μL of the diluted in situ-grown Prussian blue nanoprobe; incubate at 37℃, and wash the plate 5 times with phosphate buffer containing Tween-20;

[0054] (4) Color development: Add 100 μL of color development reagent AB solution to each well, wherein solution A (mainly contains sodium acetate, sodium citrate and hydrogen peroxide) and solution B (mainly contains EDTC, sodium citrate, glycerol and TMB) each contain 50 μL;

[0055] (5) Termination: Add 50 μL of termination solution to each well;

[0056] (6) Detection: The absorbance at 450 nm was measured using an ELISA reader (A). 450 ).

[0057] S4. Construct the standard curve:

[0058] Benzocaine standard solutions of different concentrations were prepared using 0.01M PBS (pH 6.4). 50 μL of each solution was added to the ELISA plate prepared in step S3 above, along with an equal volume of Prussian blue nanoprobes. The plate was incubated at 37°C. After incubation, the plate was washed five times with phosphate buffer containing Tween-20. The plate was then patted dry, and 100 μL of chromogenic solution was added. After 20 min of development, 50 μL of stop solution was added. The absorbance at 450 nm was measured using an ELISA reader. 450 Plotting benzocaine concentration on the x-axis and absorbance on the y-axis, with the logarithmic value of the corresponding standard concentration on the x-axis, a four-parameter curve was fitted using Origin 8.5 software: y = (AD) / [1 + (X / C)B] + D, where A and D represent the minimum and maximum absorbance values ​​of the drug concentration, respectively, and C is the midpoint concentration. The absorbance value when the standard concentration equals C is (A + D) / 2, which is at the inflection point of the curve, and the half-maximal inhibitory concentration (IC50) is 1 / 2. 50 B represents the steepness of the curve, called the slope factor, expressed as IC. 10 The detection limit is defined as IC20 to IC10. 80 This refers to the detection range.

[0059] A standard curve was established using benzocaine as a standard, and the results are as follows: Figure 8 As shown, the standard curve established using benzocaine as a standard exhibits a typical S-shaped curve, indicating good detection sensitivity. The detection limit for benzocaine using this method is as low as 0.042 ng / mL, with an IC50 value of [missing value]. 50 The concentration was 20.5 ng / mL, and the detection range was 0.411–1021.6 ng / mL.

[0060] S5. Determine the benzocaine content in the sample:

[0061] The same volume of the treated sample is used instead of the benzocaine standard solution, and the A 450 The standard curve obtained in step S4 is substituted to obtain the benzocaine concentration in the sample.

[0062] Detection of actual samples of fish, shrimp and water: fish, shrimp and water samples added at levels of 50 ng / g, 250 ng / g, 500 ng / g and 1000 ng / g were determined for benzocaine according to the steps in step S4. The recovery rate was calculated according to the formula: recovery rate (%) = (determined value after addition - blank value) / addition amount x 100%. The results are shown in Table 1. The recovery rate in this addition was between 82.38% and 109.07%, and the coefficient of variation was less than 14.93%, indicating that the method for determining benzocaine in samples has good accuracy and stability, and can directly trace the amount of benzocaine drug residues in food.

[0063] Table 1.

[0064]

[0065] Example 2

[0066] The embodiment provides a method for detecting benzocaine based on in-situ growth of Prussian blue nanoszyme, and specifically comprises the following steps:

[0067] S1. In-situ growth of Prussian blue nanoszyme: 56.9 mg of dopamine and 2.03 mg of ferric chloride are weighed and placed in a beaker, 15 mL of deionized water is added to completely dissolve, and the reaction is carried out at room temperature for 1 hour; 20 mL of Tris solution with a concentration of 25 mg / mL is added to the above mixture, and stirring is continued for 2 hours; then, an ultra-high-speed centrifuge is used for centrifugation at 10000 rp for 10 min, and then the product is washed with ultrapure water and freeze-dried to obtain iron-based polydopamine nanospheres. The obtained iron-based polydopamine nanospheres are redispersed in 15 mL of ultrapure water, 15 mL of potassium ferrocyanide aqueous solution with a concentration of 2 mM and 7 mL of hydrochloric acid with a concentration of 0.6 M are added, stirring is carried out at room temperature for 20 min, centrifugation is carried out, the product is washed with ultrapure water and freeze-dried to obtain the final product Prussian blue nanoszyme, which is redispersed in ultrapure water before use and diluted by 8 times for use.

[0068] S2. Preparation of nanoprobes: the in-situ growth Prussian blue nanoszyme prepared according to step S1 is redispersed in ultrapure water, 4 μg / ml of benzocaine antibody is added, and slow oscillation is carried out at room temperature for 1 h; then, 10% bovine serum albumin is added for blocking for 1 h, centrifugation is carried out at 7000 rp for 5 min, the supernatant is removed, and finally, 1 mL of probe redissolving solution is used for dispersion, which is diluted by 4 times to the working concentration with 0.01M PBS when used.

[0069] S3. Preparation of colorimetric immunosensor and establishment of indirect enzyme-linked immunosorbent colorimetric sensor analysis method:

[0070] (1) Antigen coating: Dilute the benzocaine coating agent to the working solution concentration with carbonate buffer, coat it on a 96-well plate, and incubate overnight at 4°C; after incubation, shake off the enzyme-labeled plate, wash the plate twice with PBS containing 0.05% Tween-20, and dry it for use;

[0071] (2) Blocking: Use blocking buffer to block the enzyme-labeled plate coated in step (1), incubate at 37°C for 1-3 hours, shake off the enzyme-labeled plate after incubation, and dry it in an oven at 37°C for standby;

[0072] (3) Sample addition: Dilute the target to the working concentration with 0.01M PBS, take 50μL and add it to the enzyme-labeled plate after blocking in step (2), and then add 50μL of diluted in-situ growth Prussian blue nanoprobes; incubate at 37°C and wash the plate 5 times with phosphate buffer containing Tween-20;

[0073] (4) Color development: Add 100μL of color developing agent A liquid (mainly containing sodium acetate, sodium citrate and hydrogen peroxide) and 50μL of color developing agent B liquid (mainly containing EDTC, sodium citrate, glycerol and TMB) to each well;

[0074] (5) Termination: Add 50μL of termination liquid to each well;

[0075] (6) Detection: Detect the 450nm absorbance (A 450 ) with an enzyme-labeled instrument.

[0076] S4. Construction of standard curve:

[0077] Prepare benzocaine standard solution of different concentrations with 0.01M PBS (PH 6.4), take 50μL and add it to the enzyme-labeled plate prepared in step S3 above, add an equal volume of Prussian blue nanoprobes, and react in a 37°C water bath; after incubation, wash the plate 5 times with phosphate buffer containing Tween-20; dry it, add 100μL of color developing liquid, develop color for 20 minutes, add 50μL of termination liquid, and detect the 450nm absorbance (A 450 ) with an enzyme-labeled instrument.

[0078] S5. Determination of benzocaine in samples:

[0079] Replace the benzocaine standard solution with an equal volume of treated sample, follow the steps in step S4 above, and substitute the measured A 450 into the standard curve obtained in step S4 to obtain the concentration of benzocaine in the sample.

[0080] Example 3

[0081] The embodiment provides a method for detecting benzocaine based on in-situ growth of Prussian blue nanoszyme, and specifically comprises the following steps.

[0082] S1. In-situ growth of Prussian blue nanoszyme: 56.9 mg of dopamine and 2.03 mg of ferric chloride are weighed and placed in a beaker, 15 mL of deionized water is added to completely dissolve, and reaction is carried out at room temperature for 1 hour; 20 mL of Tris solution with a concentration of 25 mg / mL is added to the above mixture, and stirring is continued for 2 hours; then, an ultrahigh-speed centrifuge is used for centrifugation at 10000 rp for 10 min, and then washing is performed with ultrapure water; after freeze-drying, iron-based polydopamine nanospheres are obtained. The iron-based polydopamine nanospheres obtained according to the above are redispersed in 15 mL of ultrapure water, 15 mL of potassium ferrocyanide aqueous solution with a concentration of 2 mM and 7 mL of hydrochloric acid with a concentration of 0.6 M are added, stirring is performed at room temperature for 20 min, centrifugation is performed, washing is performed with ultrapure water, freeze-drying is performed to obtain the final product Prussian blue nanoszyme; when used, the Prussian blue nanoszyme is redispersed in ultrapure water and diluted by 10 times for use.

[0083] S2. Preparation of a nanoprobe: the in-situ growth Prussian blue nanoszyme prepared according to step S1 is redispersed in ultrapure water, 4 μg / ml of benzocaine antibody is added, and slow oscillation is performed at room temperature for 1 h; then, 10% bovine serum albumin is added for blocking for 1 h, centrifugation is performed at 7000 rp for 5 min, the supernatant is removed, and finally, 1 mL of probe redissolving solution is used for dispersion for standby; when used, the probe is diluted by 4 times to a working concentration with 0.01M PBS.

[0084] S3. Preparation of a colorimetric immunosensor and establishment of an indirect enzyme-linked immunocolorimetric sensor analysis method:

[0085] (1) Antigen coating: the benzocaine coating agent is diluted to a working solution concentration with a carbonate buffer solution, coated on a 96-well plate, and incubated overnight at 4 ℃; after incubation, the enzyme-labeled plate is shaken dry, washed with a PBS solution containing 0.05% Tween-20 for 2 times, and then taken out for standby after being dried in an oven at 37 ℃;

[0086] (2) Blocking: the enzyme-labeled plate coated in step (1) is blocked with a blocking buffer solution, incubated at 37 ℃ for 1-3 h, shaken dry after incubation, and taken out for standby after being dried in an oven at 37 ℃;

[0087] (3) Sample addition: the target object is diluted to a working concentration with 0.01M PBS, 50 μL of which is added to the enzyme-labeled plate after blocking in step (2), and then 50 μL of the in-situ growth Prussian blue nanoprobe after dilution is added; incubation is performed at 37 ℃, and the plate is washed with a phosphate buffer solution containing Tween-20 for 5 times;

[0088] (4) Color development: add 100 μL of color developing agent AB liquid to each well, wherein A liquid (mainly containing sodium acetate, sodium citrate and hydrogen peroxide) and B liquid (mainly containing EDTC, sodium citrate, glycerol and TMB) each contains 50 μL;

[0089] (5) Termination: add 50 μL of termination liquid to each well;

[0090] (6) Detection: detect the absorbance value (A450) at 450 nm by using an enzyme label instrument. 450 ).

[0091] S4. Construct a standard curve:

[0092] Prepare benzocaine standard solution with different concentrations by using 0.01 M PBS (PH 6.4), take 50 μL and add to the enzyme label plate prepared in the above step S3, add an equal volume of Prussian blue nanoprobes, react in a 37°C water bath, after incubation, wash the plate 5 times with phosphate buffer containing Tween-20; dry, add 100 μL of color developing liquid, color develop for 20 min, add 50 μL of termination liquid, and detect the absorbance value (A450) at 450 nm by using an enzyme label instrument. 450 ).

[0093] S5. Determine benzocaine in a sample:

[0094] Replace the benzocaine standard solution with an equal volume of treated sample, and operate according to the steps of the above step S4, and substitute the measured A 450 into the standard curve obtained in the step S4 to obtain the concentration of benzocaine in the sample.

[0095] Example 4

[0096] The present embodiment provides a method for detecting benzocaine based on in-situ growth of Prussian blue nanoscale enzymes, which specifically comprises the following steps:

[0097] S1. In-situ growth of Prussian blue nanoscale enzymes: weigh 56.9 mg of dopamine and 2.03 mg of ferric chloride into a beaker, add 15 mL of deionized water to completely dissolve, react at room temperature for 1 hour; add 20 mL of Tris solution with a concentration of 25 mg / mL to the above mixture, continue to stir for 2 hours, centrifuge at 10000 rp for 10 min by using a high-speed centrifuge, then wash with ultrapure water, freeze-dry to obtain iron-based polydopamine nanospheres. According to the obtained iron-based polydopamine nanospheres, redispersed in 15 mL of ultrapure water, add 15 mL of potassium ferrocyanide aqueous solution with a concentration of 2 mM and 7 mL of hydrochloric acid with a concentration of 0.6 M, stir at room temperature for 20 min, centrifuge, wash with ultrapure water, freeze-dry to obtain the final product Prussian blue nanoscale enzyme, which is redispersed in ultrapure water before use and diluted 5 times for use.

[0098] S2. Preparation of nanoprobes: The in-situ grown Prussian blue nanoscale enzyme prepared according to step S1 is re-dispersed in ultrapure water, 4 μg / ml benzocaine antibody is added, and slowly shaken at room temperature for 1 h, 10% bovine serum albumin is added for blocking for 1 h, centrifuged at 7000 rp for 5 min, the supernatant is removed, and finally dispersed with 1 mL of probe redissolution solution, and diluted 5 times with 0.01M PBS to the working concentration when used.

[0099] S3. Preparation of colorimetric immunosensor and establishment of indirect enzyme-linked immunocolorimetric sensor analysis method:

[0100] (1) Antigen coating: The benzocaine coating agent is diluted to the working solution concentration with carbonate buffer, coated on a 96-well plate, and incubated at 4°C overnight; after incubation, the enzyme-labeled plate is shaken dry, washed with PBS containing 0.05% Tween-20 twice, and dried for use;

[0101] (2) Blocking: The enzyme-labeled plate coated in step (1) is blocked with blocking buffer, incubated at 37°C for 1-3 h, the enzyme-labeled plate is shaken dry after incubation, and placed in an oven at 37°C for drying;

[0102] (3) Sample addition: The target is diluted to the working concentration with 0.01M PBS, 50 μL is taken and added to the enzyme-labeled plate after blocking in step (2), and 50 μL of diluted in-situ grown Prussian blue nanoprobes is added; incubate at 37°C, and wash the plate with phosphate buffer containing Tween-20 for 5 times;

[0103] (4) Color development: 100 μL of color developing agent AB liquid is added to each well, wherein A liquid (mainly containing sodium acetate, sodium citrate and hydrogen peroxide) and B liquid (mainly containing EDTC, sodium citrate, glycerol and TMB) each contain 50 μL;

[0104] (5) Termination: 50 μL of termination liquid is added to each well;

[0105] (6) Detection: The absorbance (A 450 ) at 450 nm is detected by an enzyme-labeled instrument.

[0106] S4. Construction of standard curve:

[0107] Different concentrations of benzocaine standard solution are prepared with 0.01M PBS (PH 6.4), 50 μL is taken and added to the enzyme-labeled plate prepared in step S3 above, and an equal volume of Prussian blue nanoprobes is added, and reacted in a 37°C water bath, after incubation, the plate is washed with phosphate buffer containing Tween-20 for 5 times; dry, add 100 μL of color developing liquid, color develop for 20 min, add 50 μL of termination liquid, and detect the absorbance (A 450 ) at 450 nm by an enzyme-labeled instrument.

[0108] S5. Determining the benzocaine in the sample:

[0109] Instead of benzocaine standard solution, an equal volume of the treated sample was used to operate according to the steps of step S4 above, and the measured OD 450 was substituted into the standard curve obtained in step S4 to obtain the concentration of benzocaine in the sample.

[0110] Example 5

[0111] The present embodiment provides a method for detecting benzocaine based on in-situ growth of Prussian blue nanoszyme, which specifically comprises the following steps:

[0112] S1. In-situ growth of Prussian blue nanoszyme: 56.9 mg of dopamine and 2.03 mg of ferric chloride were weighed into a beaker, 15 mL of deionized water was added to completely dissolve, and the reaction was carried out at room temperature for 1 hour; 20 mL of Tris solution with a concentration of 25 mg / mL was added to the above mixture, and stirring was continued for 2 hours; then it was centrifuged at 10000 rp for 10 min using an ultra-high speed centrifuge, and then washed with ultrapure water, and after freeze-drying, iron-based polydopamine nanospheres were obtained. The iron-based polydopamine nanospheres obtained according to the above were redispersed in 15 mL of ultrapure water, 15 mL of potassium ferrocyanide aqueous solution with a concentration of 2 mM and 7 mL of hydrochloric acid with a concentration of 0.6 M were added, and stirring was carried out at room temperature for 20 min, and then it was centrifuged and washed with ultrapure water, and freeze-dried to obtain the final product Prussian blue nanoszyme, which was redispersed in ultrapure water before use, and diluted 5 times for use.

[0113] S2. Preparation of nanoprobes: The in-situ growth Prussian blue nanoszyme prepared according to step S1 was redispersed in ultrapure water, 4 μg / ml of benzocaine antibody was added, and slow shaking was carried out at room temperature for 1 h, then 10% bovine serum albumin was added for blocking for 1 h, and then it was centrifuged at 7000 rp for 5 min, and the supernatant was removed, and finally it was dispersed with 1 mL of probe redissolving solution for standby, and when used, it was diluted 8 times to the working concentration with 0.01 M PBS.

[0114] S3. Preparation of colorimetric immunosensor and establishment of indirect enzyme-linked immunocolorimetric sensor analysis method:

[0115] (1) Antigen coating: The benzocaine coating agent was diluted to the working solution concentration with carbonate buffer solution, and coated on a 96-well plate, and incubated at 4℃ overnight; after incubation, the enzyme-labeled plate was shaken dry, and then washed with PBS solution containing 0.05% Tween-20 twice, and then shaken dry for standby;

[0116] (2) Blocking: The enzyme-labeled plate coated in step (1) was blocked with blocking buffer, and incubated at 37℃ for 1-3 h, and then the enzyme-labeled plate was shaken dry, and then placed in an oven at 37℃ for drying standby;

[0117] (3) Add sample: dilute the target to working concentration with 0.01M PBS, take 50 μL and add to the enzyme-labeled plate after blocking in step (2), then add 50 μL of the diluted in-situ growth Prussian blue nanoprobes; incubate at 37°C, and wash the plate 5 times with phosphate buffer containing Tween-20;

[0118] (4) Color development: add 100 μL of color developing agent AB liquid to each well, wherein A liquid (sodium acetate, sodium citrate, hydrogen peroxide) and B liquid (EDTC, sodium citrate, glycerol, TMB) each contain 50 μL;

[0119] (5) Termination: add 50 μL of termination liquid to each well;

[0120] (6) Detection: detect the absorbance (A450) at 450 nm with an enzyme-labeled instrument. 450 )。

[0121] S4. Construct a standard curve:

[0122] Dilute the benzocaine standard solution to different concentrations with 0.01M PBS (PH 6.4), take 50 μL and add to the enzyme-labeled plate prepared in step S3 above, add an equal volume of Prussian blue nanoprobes, and react in a 37°C water bath; after incubation, wash the plate 5 times with phosphate buffer containing Tween-20; pat dry, add 100 μL of color developing liquid, color develop for 20 min, add 50 μL of termination liquid, and detect the absorbance (A450) at 450 nm with an enzyme-labeled instrument. 450 )。

[0123] S5. Determine benzocaine in a sample:

[0124] Replace the benzocaine standard solution with an equal volume of treated sample, follow the steps in step S4 above, and substitute the measured A 450 into the standard curve obtained in step S4 to obtain the concentration of benzocaine in the sample.

[0125] Example 6

[0126] This example provides a method for detecting benzocaine based on in-situ growth of Prussian blue nanoscale enzymes, which specifically includes the following steps:

[0127] S1. In-situ growth of Prussian blue nanoszyme: 56.9 mg of dopamine and 2.03 mg of ferric chloride were weighed into a beaker, 15 mL of deionized water was added to completely dissolve, and reacted at room temperature for 1 hour; 20 mL of Tris solution with a concentration of 25 mg / mL was added to the above mixture, and stirring was continued for 2 hours, then centrifuged at 10000 rp for 10 min with an ultra-high speed centrifuge, then washed with ultrapure water, and after freeze-drying, iron-based polydopamine nanospheres were obtained. The iron-based polydopamine nanospheres obtained according to the above were redispersed in 15 mL of ultrapure water, 15 mL of 2 mM aqueous potassium ferrocyanide solution and 7 mL of 0.6 M hydrochloric acid were added, stirred at room temperature for 20 min, centrifuged, washed with ultrapure water, and freeze-dried to obtain the final product Prussian blue nanoszyme, which was redispersed in ultrapure water for use, and diluted 5 times for use.

[0128] S2. Preparation of nanoprobes: The in-situ growth Prussian blue nanoszyme prepared according to step S1 was redispersed in ultrapure water, 4 μg / ml benzocaine antibody was added, and slowly oscillated at room temperature for 1 h, then 10% bovine serum albumin was added for blocking for 1 h, centrifuged at 7000 rp for 5 min, the supernatant was removed, and finally dispersed with 1 mL of probe redissolution solution for standby, which was diluted 10 times to working concentration with 0.01M PBS when used.

[0129] S3. Preparation of colorimetric immunosensor and establishment of indirect enzyme-linked immunocolorimetric sensor analysis method:

[0130] (1) Antigen coating: The benzocaine coating agent was diluted to the working solution concentration with carbonate buffer solution, coated on a 96-well plate, and incubated at 4°C overnight; after incubation, the enzyme-labeled plate was shaken dry, washed with PBS solution containing 0.05% Tween-20 twice, and dried for standby;

[0131] (2) Blocking: The enzyme-labeled plate coated in step (1) was blocked with blocking buffer, incubated at 37°C for 1-3 h, the enzyme-labeled plate was shaken dry after incubation, and dried in an oven at 37°C for standby;

[0132] (3) Sample addition: The target was diluted to the working concentration with 0.01M PBS, 50 μL was taken and added to the enzyme-labeled plate after blocking in step (2), and 50 μL of diluted in-situ growth Prussian blue nanoprobes was added; incubated at 37°C, and washed the plate 5 times with phosphate buffer solution containing Tween-20;

[0133] (4) Color development: 100 μL of color developing agent AB solution was added to each well, wherein A solution (mainly containing sodium acetate, sodium citrate and hydrogen peroxide) and B solution (mainly containing EDTC, sodium citrate, glycerol and TMB) each contained 50 μL;

[0134] (5) Termination: 50 μL of termination solution was added to each well;

[0135] (6) Detection: detect 450nm absorbance (A 450 ) with a microplate reader.

[0136] S4. Construct a standard curve:

[0137] Prepare benzocaine standard solution with different concentrations using 0.01M PBS (PH 6.4), take 50μL and add to the enzyme-labeled plate prepared in step S3 above, add an equal volume of Prussian blue nanoprobes, react in a 37℃ water bath, after incubation, wash the plate 5 times with phosphate buffer containing Tween-20; pat dry, add 100μL color developing solution, color develop for 20min, add 50μL stop solution, detect 450nm absorbance (A 450 ) with a microplate reader.

[0138] S5. Determine benzocaine in a sample:

[0139] Replace the benzocaine standard solution with an equal volume of the treated sample, follow the steps in step S4 above, and substitute the measured A 450 into the standard curve obtained in step S4 to obtain the benzocaine concentration in the sample.

[0140] The above examples only express the embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent of the present application, and any technical solution obtained by equivalent substitution or equivalent transformation should fall within the protection scope of the present application.

Claims

1. A method for detecting benzocaine based on in-situ grown Prussian blue nanozymes, characterized in that, It includes the following steps: S1. In-situ growth of Prussian blue nanozyme: Dopamine and ferric chloride were completely dissolved in deionized water and reacted to obtain a mixture; Tris solution was added to the mixture and the reaction was continued. After centrifugation, the mixture was washed with ultrapure water and freeze-dried to obtain iron-based polydopamine nanospheres; the iron-based polydopamine nanospheres were redispersed in deionized water, potassium ferrocyanide aqueous solution and hydrochloric acid were added, the mixture was reacted, centrifuged, washed with ultrapure water, and freeze-dried to obtain Prussian blue nanozyme; S2. Preparation of nanoprobes: The in-situ grown Prussian blue nanozyme prepared in step S1 is redispersed in ultrapure water, benzocaine antibody is added, stirred at room temperature, bovine serum albumin is added for blocking, the supernatant is removed by centrifugation, and finally dispersed with probe reconstitution solution for later use. S3. Preparation of colorimetric immunosensor: A colorimetric immunosensor is prepared based on the nanoprobe obtained in step S2; S4. Constructing a standard curve: Prepare benzocaine standard solutions of different concentrations, add them to an ELISA plate coated with benzocaine, add an equal volume of Prussian blue nanoprobe, and after the reaction is complete in a water bath, add the colorimetric solution. After the colorimetric reaction is complete, use an ELISA reader to detect the absorbance value to obtain a standard curve of absorbance value versus antigen concentration. S5. Determine the benzocaine concentration in the sample: Replace the benzocaine standard solution with an equal volume of the treated sample and determine the sample according to the standard curve method. Substitute the sample into the standard curve obtained in step S4 to obtain the concentration of benzocaine in the sample. In step S1, the concentration of dopamine is 20 mM, the concentration of ferric chloride is 0.5 mM, the concentration of potassium ferrocyanide is 1-5 mM, the concentration of hydrochloric acid is 0.4-0.8 M, and the reaction time for in-situ growth of Prussian blue nanozyme is 10-50 min.

2. The method for detecting benzocaine based on in-situ grown Prussian blue nanozyme according to claim 1, characterized in that, In step S2, the final concentration of benzocaine antibody is 3–5 μg / mL.

3. The method for detecting benzocaine based on in-situ grown Prussian blue nanozyme according to claim 1, characterized in that, In step S2, the probe reconstitution solution is a 0.02M borate buffer solution.

4. The method for detecting benzocaine based on in-situ grown Prussian blue nanozyme according to claim 1, characterized in that, Step S3 specifically includes the following steps: (1) Antigen coating: The benzocaine coating agent was diluted with carbonate buffer to the working solution concentration and coated on a 96-well plate. It was incubated overnight at 4°C. After incubation, the plate was shaken dry, washed twice with PBS solution containing 0.05% Tween-20, and patted dry for later use. (2) Blocking: Block the microplate coated in step (1) with blocking buffer, incubate at 37°C for 1-3 hours, spin dry the microplate after incubation, and dry it in an oven at 37°C for later use. (3) Sample addition: Dilute the target substance to the working concentration with 0.01M PBS, add 50μL to the enzyme-labeled plate after blocking in step (2), and then add 50μL of the diluted nanoprobe obtained in step S2; incubate at 37℃, and wash the plate 5 times with phosphate buffer containing Tween-20.

5. The method for detecting benzocaine based on in-situ grown Prussian blue nanozyme according to claim 4, characterized in that, In step S3, the blocking buffer is a phosphate buffer containing 0.2% gelatin and 2% casein.

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