A ratio fluorescence immunoassay method for benzocaine

Abstract

The present invention discloses a ratio fluorescence immunoassay method for benzocaine, which comprises the following steps: S1. Synthesis of blue fluorescent carbon dots; S2. Coating of microplates; S3. Establishment of standard curve; S4. Determination of benzocaine concentration in samples. The present invention performs immunoassay based on the ratio change of the fluorescence intensity of the emission peaks of two fluorescent materials. Since the two fluorescent materials are subjected to the same external interference, the fluorescence intensity ratio of the two offsets the interference of external factors such as temperature, polarity, and fluorophore concentration to a certain extent, and has a self-contained internal standard effect, so it has higher precision, signal-to-noise ratio and sensitivity. The method has a detection limit of 1fg / mL for benzocaine, which is significantly lower than the existing detection method, and can be used for rapid detection of benzocaine, with broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescence analysis, and in particular to a ratio fluorescence immunoassay method for detecting benzocaine based on blue fluorescent carbon dots and dopamine. Background Art

[0002] Benzocaine is a widely used local anesthetic in medicine. It is mainly used for surface anesthesia, such as oral anesthesia, soothing sore throats, and treating skin ulcers. In recent years, benzocaine has also been used for fish anesthesia because of its low price and faster anesthesia and resuscitation of fish, so as to reduce damage to the fish during operation and transportation. However, ingestion of aquatic products containing benzocaine residues can be harmful to human health. Benzocaine can cause adverse reactions such as dermatitis, urticaria, burning, stinging, and angioedema. In addition, ingestion of benzocaine can also lead to a fatal complication - methemoglobinemia. Methemoglobinemia refers to the abnormal presence of excessive amounts of iron hemoglobin in the blood that cannot carry oxygen, resulting in dyspnea, cyanosis, hypoxia and damage to multiple organs, and even death. It is estimated that the incidence of methemoglobinemia after benzocaine ingestion is as high as 0.1%-0.15%. In addition, benzocaine residues may spread in water and soil, posing a serious threat to the environment. Therefore, the evaluation and monitoring of trace anesthetics is necessary to protect human health and control environmental pollution.

[0003] At present, the main methods for detecting benzocaine are high performance liquid chromatography (A new validated method for the simultaneous determination of benzocaine, propylparaben and benzyl alcohol in a bioadhesive gel by HPLC / A high performance liquid chromatography method for the simultaneous determination of benzocaine, propylparaben and benzyl alcohol in a bioadhesive gel by HPLC / Perez-Lozano et al. Journal of pharmaceutical and biomedical analysis 2005, 39, 920-927), spectrophotometry (Simultaneous determination of benzocaine and cetylpiridinium chloride in tablets by first-derivative spectrophotometric method / First-order derivative spectrophotometric method for simultaneous determination of benzocaine and cetylpiridinium chloride in tablets, Paschoal, LR; Ferreira, WA Farmaco (Societa chimica italiana: 1989) 2000, 55, 687-693), and electrochemical sensor (Highly sensitive determination of anesthetic drug benzocaine Based on hydroxypropyl-beta-cyclodextrin-carbonblack nanohybrids / Highly sensitive determination of the anesthetic benzocaine based on hydroxypropyl-beta-cyclodextrin-carbon black nanohybrids, Wang et al. Anal Methods 2022, 14, 900-906) and other analytical methods, they can detect benzocaine residues in biological and food samples. However, these analytical methods still have some defects. For example, chromatographic analysis requires expensive instruments, spectrophotometry has low accuracy and sensitivity, and electrochemical sensors have low detection flux.

[0004] The ELISA method based on antigen-antibody specific binding reaction has the advantages of low cost, high throughput, simple operation, good accuracy, etc., and has attracted much attention in the rapid screening of food contaminants. For example, Chinese invention patent CN110938011A discloses a benzocaine hapten, artificial antigen, antibody and its preparation and application, wherein a method for detecting benzocaine by immunoassay comprises the following steps: (1) immunizing an animal with an artificial benzocaine antigen shown in formula (II) to prepare a polyclonal antibody; (2) using the artificial benzocaine antigen shown in formula (III) as a coating source, coating it to a microwell, adding the polyclonal antibody prepared in step (1) to the microwell, then adding a sample to be tested, and using a competitive ELISA method to determine the content of benzocaine in the sample to be tested. However, the traditional ELISA has the disadvantage of insufficient sensitivity for samples with a relatively complex matrix such as aquatic products, and there is no report on a ratio fluorescence immunoassay method for benzocaine. Summary of the invention

[0005] In order to make up for the defects of the existing technology, the present invention provides a ratio fluorescence immunoassay method for benzocaine. The method has the advantages of convenience, simple operation, ultra-high sensitivity (the detection limit can be as low as 1 fg / mL), and a wide detection range. It can be used for the rapid detection of benzocaine and has broad application prospects.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0007] A ratio fluorescence immunoassay method for benzocaine comprises the following steps:

[0008] S1. Synthesis of blue fluorescent carbon dots: Add diammonium citrate and urea to a beaker and add ultrapure water to dissolve them. After dissolution, transfer them to a high-pressure reactor and heat them for reaction. After the reaction is completed, cool them to room temperature and take them out. After filtering, dialyze the filtrate with ultrapure water to obtain a blue fluorescent carbon dot aqueous solution.

[0009] S2. Coating of microplate: Add benzocaine coating agent to the microplate, place in refrigerator for a period of time, then wash off the coating solution; pat the microplate dry, add blocking solution, incubate for a period of time, pat the microplate dry, dry and set aside;

[0010] S3. Establishment of standard curve: adding different concentrations of benzocaine standard solution and antibody to the microplate obtained in step S2 at the same time, incubating for a certain period of time, washing the plate, adding alkaline phosphatase-labeled goat anti-mouse secondary antibody, incubating for a period of time, washing the plate, adding L-ascorbic acid-2-phosphate trisodium salt solution for incubation, and then adding the blue fluorescent carbon dot aqueous solution and dopamine solution prepared in step S1, incubating; under 350nm excitation, recording the fluorescence emission spectrum in the range of 380-600nm, and then under 430nm excitation, recording the fluorescence emission spectrum in the range of 460-700nm, and calculating the fluorescence intensity ratio F 460 / F 510 , with benzocaine concentration as the horizontal axis, F 460 / F 510 Plot a standard curve for the ordinate;

[0011] S4. Determination of benzocaine concentration in the sample: Replace the benzocaine standard solution with an equal volume of the processed sample and follow the steps in step S3. 460 / F 510 Substitute into the standard curve obtained in step S3 to obtain the concentration of benzocaine in the sample.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention is to coat a benzocaine coating source in a microplate, use a benzocaine monoclonal antibody (Ab-BZC) as a recognition element, apply the specific binding reaction of the antigen and antibody, construct a competitive reaction mode, then wash away the excess drugs and antibodies, and then use an alkaline phosphatase-labeled goat anti-mouse secondary antibody (Ab2-ALP) as a signal amplification element to bind to the antibody, then wash away the excess secondary antibody, and the ALP connected to the microplate reacts with the subsequently added AAP (L-ascorbic acid-2-phosphate trisodium salt) to generate AA (ascorbic acid), and AA reacts with the subsequently added fluorescent carbon dots (B-CDs) and dopamine (DA). DA will spontaneously oxidize in an alkaline environment to generate a fluorescent substance polydopamine (PDA) and quench the fluorescence of B-CDs. The emission wavelength of B-CDs is 460nm, and the emission wavelength of PDA is 510nm. Therefore, due to different drug concentrations, the competitive reaction can make the subsequent enzyme-catalyzed AA concentration different, and finally the unquenched B-CDs and the produced PDA content are different. Finally, the fluorescence intensity ratio at wavelengths of 460nm and 510nm is calculated to achieve the purpose of ultrasensitive detection of benzocaine.

[0014] The present invention performs immunoassay based on the ratio change of the fluorescence intensity of the emission peaks of two fluorescent materials. Since the two fluorescent materials are subject to the same external interference, the fluorescence intensity ratio of the two offsets the interference of external factors such as temperature, polarity, and fluorophore concentration to a certain extent, and has a self-contained internal standard effect, so it has higher precision, signal-to-noise ratio and sensitivity. The detection limit of benzocaine in this method is as low as 1fg / mL, which is significantly lower than the existing detection method, and can be used for rapid detection of benzocaine, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the ratiometric fluorescence immunoassay method for ultrasensitive detection of benzocaine based on blue fluorescent carbon dots and dopamine.

[0016] Figure 2 This is a transmission electron microscope image of the blue fluorescent carbon dots prepared in step S11 of the present invention.

[0017] Figure 3 This is a fluorescence spectrum of the blue fluorescent carbon dots prepared in step S11 of the present invention.

[0018] Figure 4 This is a transmission electron microscopy image of the solution after adding DA and B-CDs in step S13 of the present invention.

[0019] Figure 5 The standard curve for the detection of benzocaine. DETAILED DESCRIPTION

[0020] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0021] Unless otherwise specified, the meanings of the terms in this specification are the same as those generally understood by those skilled in the art. However, if there is any conflict, the definitions in this specification shall prevail.

[0022] As used herein, "includes," "comprising," "including," "containing," "having," or other variations are intended to encompass non-exclusive inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the end result may be added. The term "comprising" also includes the terms "consisting of" and "consisting essentially of." The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0023] All numerical values ​​or expressions used in the specification and claims relating to component amounts, process conditions, etc. should be understood to be modified by "about" in all cases. All ranges relating to the same component or property include endpoints, which can be independently combined. Since these ranges are continuous, they include every numerical value between the minimum and maximum values. It should also be understood that any numerical range cited in the application is expected to include all subranges within the range.

[0024] As described in the above technical background, the traditional ELISA has the disadvantage of insufficient sensitivity for samples with a relatively complex matrix such as aquatic products, and there is no report on the ratio fluorescence immunoassay method for benzocaine.

[0025] To this end, the inventors conducted a large number of experiments to develop a ratio fluorescence immunoassay method for benzocaine, specifically, it comprises the following steps:

[0026] S1. Synthesis of blue fluorescent carbon dots: Add diammonium citrate and urea in a mass ratio of 3:4 into a beaker and add ultrapure water to dissolve. After dissolution, transfer to a high-pressure reactor and heat for reaction at 180°C for 6 to 8 hours. After the reaction is completed, cool to room temperature and take out. After filtering, dialyze the filtrate with ultrapure water to obtain a blue fluorescent carbon dot aqueous solution, which is diluted 50 to 100 times when used.

[0027] S2. Coating of microplate: 100 μL of benzocaine coating agent is added to the microplate, and the plate is placed in a refrigerator for a period of time, preferably 12 to 15 hours, and then the coating solution is washed off; the microplate is patted dry, 100 to 200 μL of blocking solution is added, and after incubation for a period of time, preferably 1 to 3 hours, the microplate is patted dry, dried, and set aside;

[0028] S3. Establishment of standard curve: Benzocaine standard solution of different concentrations was mixed with 1-5 μg L -1 The antibodies are simultaneously added to the microplate obtained in step S2, and incubated for a certain period of time, preferably 30 to 40 minutes; the plate is washed, and a sheep anti-mouse secondary antibody labeled with alkaline phosphatase is added. After incubation for a period of time, the plate is washed, and a 0.1 to 1 mM L-ascorbic acid-2-phosphate trisodium salt solution is added and incubated for 30 to 40 minutes, and then the blue fluorescent carbon dot aqueous solution prepared in step S1 and 5 to 10 mM dopamine solution are added and incubated for 30 to 40 minutes; under 350 nm excitation, the fluorescence emission spectrum within the range of 380 to 600 nm is recorded, and then under 430 nm excitation, the fluorescence emission spectrum within the range of 460 to 700 nm is recorded, and the fluorescence intensity ratio F is calculated. 460 / F 510 , with benzocaine concentration as the horizontal axis, F 460 / F 510 Plot a standard curve for the ordinate;

[0029] S4. Determination of benzocaine concentration in the sample: Replace the benzocaine standard solution with an equal volume of the processed sample and follow the steps in step S3. 460 / F 510 Substitute into the standard curve obtained in step S3 to obtain the concentration of benzocaine in the sample.

[0030] The present invention is described in detail below in conjunction with examples, which are only preferred embodiments of the present invention and are not limitations of the present invention. Unless otherwise specified, the reagents, methods and instruments used in the present invention are conventional reagents, methods and instruments in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] The unit "M" involved in the present invention represents the molar concentration of a substance, and can also be expressed as mol / L. Common units include nM (nmol / L), μM (umol / L), mM (mMol / L), and M (mol / L).

[0032] Example 1

[0033] This embodiment provides a ratiometric fluorescence immunoassay method for ultrasensitive detection of benzocaine, which specifically comprises the following steps:

[0034] Step S11. Synthesis of blue fluorescent carbon dots: weigh 75 mg of diammonium citrate and 100 mg of urea in a beaker, add 3 mL of ultrapure water to dissolve, transfer to a high-pressure reactor after dissolution, and place in an electrochemical oven at 180°C for 7 hours. After the reaction is completed, cool to room temperature and take out, filter and dialyze the filtrate with ultrapure water at 4°C for 6 hours to obtain a blue fluorescent carbon dot aqueous solution (B-CDs), store in a refrigerator at 4°C away from light, and dilute 100 times when used;

[0035] from Figure 2 It can be seen that the prepared blue fluorescent carbon dots are round particles with a particle size of about 2.5 nm. The optimal excitation wavelength of the blue fluorescent carbon dots is 350 nm, and the maximum emission wavelength is 460 nm (e.g. Figure 3 ).

[0036] Step S12. Coating of microplate: Pipette 100 μL of benzocaine coating agent into the microplate, place in a 4°C refrigerator for 12 h, remove and wash the coating solution; add 120 μL of blocking solution for 3 h, spin dry the blocking solution, and place in 37°C for drying;

[0037] Step S13. Establishment of standard curve: Add 50 μL of benzocaine standard solution of different concentrations and 50 μL of antibody solution to the microplate, mix, incubate for 40 minutes, and then wash the plate with washing solution; add 100 μL of 500-fold diluted alkaline phosphatase-labeled goat anti-mouse secondary antibody solution to each well, incubate for 30 minutes, and then wash the plate with washing solution; add 100 μL of 0.5 mM AAP solution to each well, incubate for 40 minutes; add 50 μL of B-CDs solution and 50 μL of 10 mM DA solution, incubate for 40 minutes, record the fluorescence emission spectrum in the range of 380 to 600 nm under 350 nm excitation, and then record the fluorescence emission spectrum in the range of 460 to 700 nm under 430 nm excitation. Calculate the fluorescence intensity ratio F 460 / F 510 , with benzocaine concentration as the horizontal axis, F 460 / F 510 Plot a standard curve for the ordinate;

[0038] from Figure 4 It can be seen that after the reaction, dopamine polymerizes to form polydopamine and attaches to the surface of carbon dots to aggregate the carbon dots. Figure 5 As shown in the figure, with the increase of benzocaine concentration, the antibody competes with the drug, the amount of monoclonal antibody connected to the microplate decreases, and then the amount of alkaline phosphatase-labeled goat anti-mouse decreases, the AA content produced by catalysis decreases, and after adding B-CDs and DA to react, the amount of PDA produced increases, and the fluorescence of B-CDs is quenched, which is specifically manifested as F 460 / F 510 The value decreases, F 460 / F 510 It is negatively correlated with the concentration of benzocaine, and the detection limit of benzocaine is 1fg / mL, which is significantly lower than the method for detecting benzocaine in the prior art.

[0039] Step S14. Determination of benzocaine concentration in the sample: Replace the benzocaine standard solution with an equal volume of the processed sample, and follow the steps of step S13 to measure the F 460 / F 510 Substitute into the standard curve to obtain the benzocaine concentration in the sample.

[0040] For actual samples of fish and shrimp: fish and shrimp samples with 100 μg / kg, 500 μg / kg, and 1000 μg / kg levels of benzocaine were taken, and benzocaine was determined according to the steps described in step S13. The recovery rate was calculated according to the formula: recovery rate (%) = (measured value after addition-blank value) / addition amount × 100%. The results are shown in Table 1. The recovery rate of this addition is between 94.1% and 102.5%, and the coefficient of variation is less than 2.1%, indicating that the method for determining benzocaine has good accuracy and stability.

[0041]

[0042] Example 2

[0043] This embodiment provides a ratiometric fluorescence immunoassay method for ultrasensitive detection of benzocaine, which specifically comprises the following steps:

[0044] Step S21. Synthesis of blue fluorescent carbon dots: Weigh 75 mg of diammonium citrate and 100 mg of urea in a beaker, add 3 mL of ultrapure water to dissolve, transfer to a high-pressure reactor after dissolution, and place in an electrochemical oven at 180°C for 6 hours. After the reaction is completed, cool to room temperature and take out, filter and dialyze the filtrate with ultrapure water at 4°C for 6 hours to obtain a blue fluorescent carbon dot aqueous solution (B-CDs), store in a refrigerator at 4°C away from light, and dilute 100 times when used;

[0045] Step S22. Coating of microplate: Pipette 100 μL of benzocaine coating agent into the microplate, place in a 4°C refrigerator for 12 h, remove and wash the coating solution; add 120 μL of blocking solution for 3 h, spin dry the blocking solution, and place in 37°C for drying;

[0046] Step S23. Establishment of standard curve: Add 50 μL of benzocaine standard solution of different concentrations and 50 μL of antibody solution to the microplate, mix, incubate for 40 minutes, and then wash the plate with washing solution; add 100 μL of 500-fold diluted alkaline phosphatase-labeled goat anti-mouse secondary antibody solution to each well, incubate for 30 minutes, and then wash the plate with washing solution; add 100 μL of 0.5 mM AAP solution to each well, incubate for 40 minutes; add 50 μL of B-CDs solution and 50 μL of 10 mM DA solution, incubate for 40 minutes, record the fluorescence emission spectrum in the range of 380 to 600 nm under 350 nm excitation, and then record the fluorescence emission spectrum in the range of 460 to 700 nm under 430 nm excitation. Calculate the fluorescence intensity ratio F 460 / F 510 , with benzocaine concentration as the horizontal axis, F 460 / F 510 Plot a standard curve for the ordinate;

[0047] Step S24. Determination of benzocaine concentration in the sample: Replace the benzocaine standard solution with an equal volume of the processed sample, and follow the steps of step S23 to measure the F 460 / F 510 Substitute into the standard curve to obtain the benzocaine concentration in the sample.

[0048] Example 3

[0049] This embodiment provides a ratiometric fluorescence immunoassay method for ultrasensitive detection of benzocaine, which specifically comprises the following steps:

[0050] Step S31. Synthesis of blue fluorescent carbon dots: Weigh 75 mg of diammonium citrate and 100 mg of urea in a beaker, add 3 mL of ultrapure water to dissolve, transfer to a high-pressure reactor after dissolution, and place in an electrochemical oven at 180°C for 8 hours. After the reaction is completed, cool to room temperature and take out, filter and dialyze the filtrate with ultrapure water at 4°C for 6 hours to obtain a blue fluorescent carbon dot aqueous solution (B-CDs), store in a refrigerator at 4°C away from light, and dilute 100 times when used;

[0051] Step S32. Coating of microplate: Pipette 100 μL of benzocaine coating agent into the microplate, place in a 4°C refrigerator for 12 hours, remove and wash the coating solution; add 120 μL of blocking solution for 3 hours, spin dry the blocking solution, and place in 37°C for drying;

[0052] Step S33. Establishment of standard curve: Add 50 μL of benzocaine standard solution of different concentrations and 50 μL of antibody solution to the microplate, mix, incubate for 40 minutes, and then wash the plate with washing solution; add 100 μL of 500-fold diluted alkaline phosphatase-labeled goat anti-mouse secondary antibody solution to each well, incubate for 30 minutes, and then wash the plate with washing solution; add 100 μL of 0.5 mM AAP solution to each well, incubate for 40 minutes; add 50 μL of B-CDs solution and 50 μL of 10 mM DA solution, incubate for 40 minutes, record the fluorescence emission spectrum in the range of 380 to 600 nm under 350 nm excitation, and then record the fluorescence emission spectrum in the range of 460 to 700 nm under 430 nm excitation. Calculate the fluorescence intensity ratio F 460 / F 510 , with benzocaine concentration as the horizontal axis, F 460 / F 510 Plot a standard curve for the ordinate;

[0053] Step S34. Determination of benzocaine concentration in the sample: Replace the benzocaine standard solution with an equal volume of the processed sample, and follow the steps of step S33 to measure the F 460 / F 510 Substitute into the standard curve to obtain the benzocaine concentration in the sample.

[0054] The above-mentioned embodiments merely express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. Any technical solution obtained in the form of equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. A ratio fluorescence immunoassay method for benzocaine, characterized in that: It includes the following steps: S1. Synthesis of blue fluorescent carbon dots: Add diammonium citrate and urea to a beaker and add ultrapure water to dissolve them. After dissolution, transfer them to a high-pressure reactor and heat them for reaction. After the reaction is completed, cool them to room temperature and take them out. After filtering, dialyze the filtrate with ultrapure water to obtain a blue fluorescent carbon dot aqueous solution. S2. Coating of microplate: Add benzocaine coating agent to the microplate, place in refrigerator for a period of time, then wash off the coating solution; pat the microplate dry, add blocking solution, incubate for a period of time, pat the microplate dry, dry and set aside; S3. Establishment of standard curve: adding different concentrations of benzocaine standard solution and antibody to the microplate obtained in step S2 at the same time, incubating for a certain period of time, washing the plate, adding alkaline phosphatase-labeled goat anti-mouse secondary antibody, incubating for a period of time, washing the plate, adding L-ascorbic acid-2-phosphate trisodium salt solution for incubation, and then adding the blue fluorescent carbon dot aqueous solution and dopamine solution prepared in step S1, incubating; under 350nm excitation, recording the fluorescence emission spectrum in the range of 380-600nm, and then under 430nm excitation, recording the fluorescence emission spectrum in the range of 460-700nm, and calculating the fluorescence intensity ratio F 460 / F 510 , with benzocaine concentration as the horizontal axis, F 460 / F 510 Plot a standard curve for the ordinate; S4. Determination of benzocaine concentration in the sample: Replace the benzocaine standard solution with an equal volume of the processed sample and follow the steps in step S3. 460 / F 510 Substituting the standard curve obtained in step S3 into the standard curve to obtain the concentration of benzocaine in the sample; The concentration of the antibody in step S3 is 1-5 μg L -1 , time is 30 to 40 minutes; In step S3, the concentration of L-ascorbic acid-2-phosphate trisodium salt is 0.1-1 mM, and the time is 30-40 min; In step S3, the dopamine concentration is 5-10 mM; In the step S3, the blue fluorescent carbon dot aqueous solution and dopamine solution prepared in step S1 are added and incubated for 30 to 40 minutes.

2. The ratio fluorescence immunoassay method for benzocaine according to claim 1, characterized in that: In step S1, the mass ratio of diammonium citrate to urea is 3:

4.

3. The ratio fluorescence immunoassay method for benzocaine according to claim 1, characterized in that: The heating temperature of the heating reaction in step S1 is 180° C. and the temperature is kept for 6 to 8 hours.

4. The ratio fluorescence immunoassay method for benzocaine according to claim 1, characterized in that: In the step S1, the blue fluorescent carbon dots are diluted 50 to 100 times when used.

5. The ratio fluorescence immunoassay method for benzocaine according to claim 1, characterized in that: In step S2, the original volume of benzocaine coating is 100 μL, and the coating is allowed to stand for 12 to 15 hours.

6. The ratio fluorescence immunoassay method for benzocaine according to claim 1, characterized in that: In step S2, the amount of blocking solution added is 100-200 μL, and the blocking time is 1-3 hours.

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