阅读说明：本技术 一种诺氟沙星的检测方法 (Norfloxacin detection method ) 是由 胡高爽 高山 郝建雄 韩雪 赵丹丹 饶欢 李娜 于 2020-07-24 设计创作，主要内容包括：本发明涉及荧光免疫检测技术领域,具体公开一种诺氟沙星的检测方法。包括以下步骤：将诺氟沙星单克隆抗体与生物素偶联,得免疫配基；将所述免疫配基与羧基化纳米Fe<Sub>3</Sub>O<Sub>4</Sub>微球偶联,得免疫磁珠；将链霉亲和素与羧基化水溶性发绿光上转化纳米粒子偶联,得荧光信号探针；将所述免疫磁珠加入待测样品中,加入磁场分离富集样品,再加入荧光信号探针,充分反应后磁分离富集样品,得样品溶液；将所述样品溶液滴加至含有诺氟沙星人工抗原的免疫层析试纸条上,观察结果。本发明提供的方法实现了快速分离目标物、双重信号放大提高检测灵敏度和增加检测系统稳定性的目的,能够实现对大量未知样品的简单快速筛选,检测成本低,有利于在实践中推广应用。(The invention relates to the technical field of fluorescence immunoassay, and particularly discloses a norfloxacin detection method. The method comprises the following steps: coupling the norfloxacin monoclonal antibody with biotin to obtain an immune ligand; the immune ligand is reacted with carboxylated nano Fe 3 O 4 Coupling the microspheres to obtain immunomagnetic beads; coupling streptavidin with the converted nanoparticles on the carboxylated water-soluble green light to obtain a fluorescent signal probe; adding the immunomagnetic beads into a sample to be detected, adding a magnetic field to separate and enrich the sample, adding a fluorescent signal probe, and carrying out magnetic separation and enrichment on the sample after full reaction to obtain a sample solution; and (3) dropwise adding the sample solution onto an immunochromatographic test strip containing the artificial norfloxacin antigen, and observing the result. The present invention providesThe method realizes the purposes of quickly separating the target object, improving the detection sensitivity by dual signal amplification and increasing the stability of the detection system, can realize simple and quick screening of a large number of unknown samples, has low detection cost, and is beneficial to popularization and application in practice.)

1. The norfloxacin detection method is characterized by comprising the following steps of:

step a, coupling norfloxacin monoclonal antibody with biotin to obtain immune ligand;

step b, the immune ligand and the carboxylated nano Fe₃O₄Coupling the microspheres to obtain immunomagnetic beads;

coupling streptavidin and the converted nanoparticles on the carboxylated water-soluble green light to obtain a fluorescent signal probe;

d, adding the immunomagnetic beads into a sample to be detected, adding a magnetic field to separate and enrich the sample, adding a fluorescent signal probe, and after full reaction, magnetically separating and enriching the sample to obtain a sample solution;

and e, dropwise adding the sample solution to an immunochromatographic test strip containing the artificial norfloxacin antigen, and observing the result.

2. The method for detecting norfloxacin according to claim 1, wherein in step a, 0.13 to 0.15mmol of biotin is coupled to 1mg of norfloxacin monoclonal antibody.

3. The method for detecting norfloxacin of claim 1, wherein in step b, the carboxylated nano-Fe is₃O₄The mass ratio of the microspheres to the norfloxacin monoclonal antibody is 1: 0.18-0.22.

4. The method for detecting norfloxacin of claim 1, wherein in step b, the carboxylated nano-Fe is₃O₄The preparation of the microspheres specifically comprises the following steps:

step 1, taking ferric chloride and ferrous chloride as raw materials, and preparing Fe by adopting a coprecipitation method₃O₄Nanoparticles;

step 2, mixing the Fe₃O₄Mixing nano particles with oleic acid, and reactionSeparating to obtain Fe coated with oleic acid₃O₄Nanoparticles;

step 3, coating the oleic acid with Fe₃O₄Adding potassium permanganate solution into the nano particles, reacting and separating to obtain the carboxylated nano Fe₃O₄And (3) microspheres.

5. The method for detecting norfloxacin according to claim 4, wherein in the step 1, the molar ratio of the ferric chloride to the ferrous chloride is 1.7-2.0:1, the reaction temperature is 75-85 ℃, and the reaction time is 2.5-3.5 h; and/or

In step 2, the Fe₃O₄The molar ratio of the nano particles to the oleic acid is 1:0.8-1.2, the reaction temperature is 65-75 ℃, and the reaction time is 50-70 min; and/or

In the step 3, the concentration of the potassium permanganate solution is 8-12mg/mL, the molar ratio of potassium permanganate to oleic acid is 1:1.5-1.7, the reaction temperature is 20-30 ℃, and the reaction time is 7-9 h.

6. The method for detecting norfloxacin according to claim 1, wherein: in the step c, the mass ratio of the streptavidin to the carboxylated water-soluble green light-emitting conversion nanoparticles is 1: 4-7.

7. The method for detecting norfloxacin according to claim 1, wherein: in the step c, the preparation method of the carboxylated water-soluble green light-emitting up-conversion nano particle comprises the following steps: yttrium acetate, ytterbium acetate and erbium acetate are used as raw materials, hydrophobic up-conversion nanoparticles are prepared by a thermal decomposition method, and then the surfaces of the hydrophobic up-conversion nanoparticles are subjected to carboxylation modification by a ligand exchange method to obtain carboxylated water-soluble green light up-conversion nanoparticles.

8. The method for detecting norfloxacin of claim 7, wherein: the molar ratio of yttrium acetate, ytterbium acetate and erbium acetate is 78-80:18-22: 2.

9. The method for detecting norfloxacin according to claim 1, wherein: the immunochromatographic test strip comprises a bottom plate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the bottom plate, the water absorption pad and the combination pad are respectively overlapped and pressed on the nitrocellulose membrane and are positioned on two opposite sides of the bottom plate, and the sample pad is overlapped and pressed on the combination pad; the cellulose nitrate membrane is provided with a detection line coated with norfloxacin artificial antigen and a quality control line coated with goat anti-mouse polyclonal antibody.

10. The method for detecting norfloxacin of claim 9, wherein: the coating amount of the norfloxacin artificial antigen is 0.05-0.1 mu g/strip, and the coating amount of the goat anti-mouse polyclonal antibody is 0.1-0.3 mu g/strip.

Technical Field

The invention relates to the technical field of fluorescence immunoassay, in particular to a norfloxacin detection method.

Background

Norfloxacin (NOR) is a novel third-generation fluoroquinolone spectrum antibacterial drug, has the characteristics of wide antibacterial spectrum and strong antibacterial activity, is an important synthetic drug for treating various infectious diseases, and is widely applied clinically. In recent years, norfloxacin veterinary drug residues appear in animal-derived foods due to indiscriminate use and abuse of norfloxacin veterinary drugs, and great health risks are brought to consumers, such as allergic reactions, canceration reactions, enhancement of bacterial drug resistance and the like. Therefore, research on norfloxacin detection methods is imminent.

At present, many methods have been reported for detecting norfloxacin in aquatic products, including microbiological method, thin layer chromatography, spectrophotometry, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry, and the like. Although these methods can be used for detecting norfloxacin, the methods require complicated sample pretreatment methods before detection, special detection instruments and professional detection personnel for operation, and are expensive in detection cost, long in detection time and not suitable for rapid detection on site. Therefore, the development of the norfloxacin detection method which has high sensitivity, rapid detection and simple and convenient operation has great significance.

The Immunoassay (IA) is a technique for specifically measuring and analyzing an antibody (or antigen) based on an antigen (or antibody) as a selective chemical reagent, can overcome the defects of the methods such as the microbiological method, the thin-layer chromatography and the instrumental analysis method, and has the advantages of simple operation, strong specificity, high sensitivity and the like. The immunochromatographic test strip is a novel rapid immunoassay technology developed on the basis of an enzyme-linked immunoassay method in the eighties and ninety years of the twentieth century, and has the advantages of simplicity and rapidness in operation, easiness in result judgment, safety, no pollution and the like, so that the immunochromatographic test strip is widely applied to various countries in the world and becomes a trend method developed in the field of in-vitro rapid diagnosis. However, this method uses colloidal gold as a signal marker, and requires relatively complicated pretreatment steps (repeated centrifugation and washing steps for many times) for actual sample detection to eliminate the influence of the substrate on the marker (colloidal gold, etc.), thereby eliminating false positive results, and thus limiting further improvement in the sensitivity of the detection method.

Disclosure of Invention

The invention provides a norfloxacin detection method, aiming at the problems that the existing colloidal gold immunochromatographic test paper for detecting norfloxacin needs complicated pretreatment and the detection sensitivity needs to be further improved.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

a method for detecting norfloxacin comprises the following steps:

step a, coupling norfloxacin monoclonal antibody with biotin to obtain immune ligand;

step b, the immune ligand and the carboxylated nano Fe₃O₄Coupling the microspheres to obtain immunomagnetic beads;

coupling streptavidin and the converted nanoparticles on the carboxylated water-soluble green light to obtain a fluorescent signal probe;

d, adding the immunomagnetic beads into a sample to be detected, adding a magnetic field to separate and enrich the sample, adding a fluorescent signal probe, and after full reaction, magnetically separating and enriching the sample to obtain a sample solution;

and e, dropwise adding the sample solution to an immunochromatographic test strip containing the artificial norfloxacin antigen, and observing the result.

Compared with the prior art, the norfloxacin monoclonal antibody on the immunomagnetic beads is specifically combined with norfloxacin in a sample to be detected, and the specific capture and enrichment of a target in a complex matrix sample can be realized under the action of an external magnetic field, so that the defects of low sensitivity, incapability of realizing field detection and the like caused by centrifugation and dilution required in sample pretreatment in the traditional immunochromatography method are overcome, and a large amount of unknown samples can be simply and rapidly screened; meanwhile, the enriched sample is combined with the fluorescent signal probe through coupling of biotin-chain and avidin, so that the combined detection of immunoassay, magnetic separation and fluorescence detection is realized. The invention utilizes the advantages of large specific surface area of immunomagnetic beads, stable fluorescence performance of fluorescent signal probes, strong matrix interference resistance and the like, increases the bearing capacity of a target object to be detected and the stability of a detection system, breaks through the limitation of the traditional immunochromatography technology, realizes the purposes of quickly separating the target object, amplifying double signals and improving the detection sensitivity and the system stability, can realize the quick qualitative or semi-quantitative detection of norfloxacin under the naked eye condition through the weak fluorescence of a detection line and a quality control line, is suitable for the field quick detection, and has wide application prospect.

The detection mechanism of the invention is as follows: if the sample is positive, the compound formed by the detection sample, the immunomagnetic beads and the fluorescent probe is dripped on the immunochromatography test strip, the sample can move towards the direction of the absorbent paper by the acting force of the capillary, and when the compound passes through the detection line, if the target detection object and the detection antibody completely react, no redundant detection antibody-Fe exists₃O₄The nanoparticle-up-conversion nanoparticles and the capture antigen at the detection line are subjected to specific reaction, so that a fluorescence signal strip cannot be formed on the detection line, and when the sample continues to move to the quality control line, the detection antibody and the second antibody at the detection line are subjected to specific reaction to form a fluorescence signal; if the sample is negative, the sample is free of the target detection substance and Fe₃O₄The detection antibody marked on the nano-particles has a specific reaction, so that fluorescence signal bands can be formed at the detection line and the quality control line; the test strip was proved to be ineffective as long as no fluorescence signal band was observed at the control line.

Preferably, in step a, 0.13-0.15mmol of biotin is coupled to 1mg of norfloxacin monoclonal antibody.

Further preferably, the specific process of step a is as follows: 1mg of norfloxacin monoclonal antibody and 0.13-0.15mmol of biotin were mixed uniformly, 0.12mg of N-hydroxysuccinimide (NHS) was added, the mixture was incubated at room temperature (15-35 ℃) for 50-70min, and dialyzed against 0.1mol/L phosphate buffered saline (PBS solution) having pH of 7.4 at room temperature, and unreacted biotin was removed by filtration, and the resulting norfloxacin monoclonal antibody-biotin complex was redissolved in 0.18-0.22mL of PBS solution (0.1mol/L, pH of 7.4) to obtain an immunophilin.

Preferably, in step b, the carboxylated nano Fe₃O₄The mass ratio of the microspheres to the norfloxacin monoclonal antibody is 1: 0.18-0.22.

Preferably, in step b, the carboxylated nano Fe₃O₄The preparation of the microspheres specifically comprises the following steps:

step 1, taking ferric chloride and ferrous chloride as raw materials, and preparing Fe by adopting a coprecipitation method₃O₄Nanoparticles;

step 2, mixing the Fe₃O₄Uniformly mixing the nano particles and the oleic acid, reacting and separating to obtain Fe coated with the oleic acid₃O₄Nanoparticles;

step 3, coating the oleic acid with Fe₃O₄Adding potassium permanganate solution into the nano particles, reacting and separating to obtain the carboxylated nano Fe₃O₄And (3) microspheres.

Preferably, in the step 1, the molar ratio of the ferric chloride to the ferrous chloride is 1.7-2.0:1, the reaction temperature is 75-85 ℃, and the reaction time is 2.5-3.5 h.

Preferably, in step 2, said Fe₃O₄The molar ratio of the nano particles to the oleic acid is 1:0.8-1.2, the reaction temperature is 65-75 ℃, and the reaction time is 50-70 min.

Preferably, in the step 3, the concentration of the potassium permanganate solution is 8-12mg/mL, the molar ratio of potassium permanganate to oleic acid is 1:1.5-1.7, the reaction temperature is 20-30 ℃, and the reaction time is 7-9 h.

Further preferably, the step b specifically comprises the following steps:

FeCl is weighed according to the molar ratio of 1.7-2.0:1₃·6H₂O and FeCl₂·4H₂And O, adding the mixture into water to prepare a ferric salt mixed solution with ferric chloride concentration of 0.2-0.3g/mL and ferrous chloride concentration of 0.08-0.1 g/mL. Dropwise adding the mixed solution of the iron salt into a sodium hydroxide solution (0.05-0.07g/mL) at 75-85 ℃ in an inert gas atmosphere, wherein the sodium hydroxide and FeCl₃The molar ratio of the Fe to the Fe is 16-18:1, and the reflux reaction is carried out for 2.5-3.5h to obtain the Fe₃O₄Magnetic nanoparticles.

Oleic acid and the Fe are weighed according to the molar ratio of 0.8-1.2:1 under the condition of vigorous stirring₃O₄Magnetic nanoparticles are uniformly mixed, reacted in water bath at 65-75 ℃ for 50-70min under inert atmosphere, and separated by an external magnetic field to obtain Fe coated with oleic acid₃O₄Washing magnetic nanoparticles with absolute ethyl alcohol for 3-4 times, then washing with water until the pH value is 7, adding a potassium permanganate solution (8-12mg/mL) according to the molar ratio of potassium permanganate to oleic acid of 1:1.5-1.7, reacting for 7.5-8.5h under the ultrasonic condition, separating by an external magnetic field, washing, carrying out vacuum freeze drying for 39-41h, adding a PBS (0.1M, pH7.4) solution for dissolving to obtain carboxylated nano Fe with the mass concentration of 4.8-5.2mg/mL₃O₄A microsphere solution.

According to carboxylated nano Fe₃O₄The mass ratio of the microspheres to the norfloxacin monoclonal antibody is 1:0.18-0.22, and the carboxylated nano Fe is transferred₃O₄Adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) into the microsphere solution, and adding EDC and NHS and carboxylated nano Fe₃O₄Reacting at room temperature for 2.5-3.5h, washing with PBS solution, adding 4.5-5.2 wt% OVA solution, sealing for 2h, and washing to obtain immunomagnetic beads.

Preferably, in the step c, the mass ratio of the streptavidin to the carboxylated water-soluble green light-emitting converted nanoparticles is 1: 4-7.

Preferably, in step c, the preparation method of the carboxylated water-soluble green light-emitting upconversion nanoparticle comprises the following steps: yttrium acetate, ytterbium acetate and erbium acetate are used as raw materials, hydrophobic up-conversion nanoparticles are prepared by a thermal decomposition method, and then the surfaces of the hydrophobic up-conversion nanoparticles are subjected to carboxylation modification by a ligand exchange method to obtain carboxylated water-soluble green light up-conversion nanoparticles.

Preferably, the molar ratio of yttrium acetate, ytterbium acetate and erbium acetate is 78-80:18-22: 2.

The carboxylated water-soluble green light up-conversion nano particle prepared by the invention has a characteristic emission peak at 542nm and no other interference peak under the excitation wavelength of 980nm, has the advantages of stable optical signal, small background interference and strong matrix interference resistance, and improves the sensitivity and accuracy of detection.

Further preferably, step c specifically comprises the following steps:

weighing yttrium acetate, ytterbium acetate and erbium acetate according to a molar ratio of 78-80:18-22:2, adding oleic acid and octadecene, wherein the molar ratio of oleic acid to yttrium acetate is 23-25:1, and the molar ratio of octadecene to yttrium acetate is 67-69: 1, heating to 95-105 ℃, vacuumizing for 8-12min, heating to 155-165 ℃, reacting for 25-35min, and naturally cooling to room temperature to obtain a reaction solution. Then, weighing sodium hydroxide and ammonium fluoride according to a molar ratio of 2-3:3-5, adding methanol to prepare a solution with the concentration of the sodium hydroxide of 0.2-0.3mol/L, wherein the molar ratio of the sodium hydroxide to the yttrium acetate is 3-4:1, dropwise adding the solution into the reaction solution, stirring at room temperature for 25-35min, heating to remove the methanol, heating to 95-105 ℃, vacuumizing for 8-12min, heating to 295-305 ℃ under the protection of argon, reacting for 50-70min, naturally cooling, and washing to obtain the hydrophobic up-conversion nanoparticles (OA-UCNPs).

Dispersing OA-UCNPs in toluene to obtain OA-UCNPs dispersion liquid with the concentration of 14-16mg/mL, weighing polyacrylic acid (PAA) according to the mass ratio of 15-17:1, adding the polyacrylic acid (PAA) into the dispersion liquid, uniformly mixing, stirring and reacting at the temperature of 105-115 ℃ for 50-70min, then heating to the temperature of 230-250 ℃ for reacting for 50-70min, and centrifuging to obtain carboxylated water-soluble greenish light-emitting up-conversion nanoparticles (PAA-UCNPs).

Weighing streptavidin and PAA-UCNPs according to the mass ratio of 1:4-7, carrying out low-speed oscillation incubation reaction for 1.5-2.5h at the temperature of 25-35 ℃ by using activated ester reaction, and washing by using HEPES buffer solution to obtain the fluorescent signal probe.

The adding proportion of the immunomagnetic beads, the fluorescent signal probes and the sample to be detected in the invention is the conventional dosage in the field, and can be obtained by conventional adjustment.

Preferably, the immunochromatographic test strip comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the bottom plate, wherein the water absorption pad and the combination pad are respectively overlapped and pressed on the nitrocellulose membrane and are positioned on two opposite sides of the bottom plate, and the sample pad is overlapped and pressed on the combination pad; the cellulose nitrate membrane is provided with a detection line coated with norfloxacin artificial antigen and a quality control line coated with goat anti-mouse polyclonal antibody.

Preferably, the coating amount of the norfloxacin artificial antigen is 0.05-0.1 mu g of coating on each immunochromatographic test strip, and the coating amount of the goat anti-mouse polyclonal antibody is 0.1-0.3 mu g of coating on each immunochromatographic test strip.

More preferably, the coating amount of the norfloxacin artificial antigen is 0.07 mu g of coating amount on each immunochromatographic strip, and the coating amount of the goat anti-mouse polyclonal antibody is 0.2 mu g of coating amount on each immunochromatographic strip.

The norfloxacin detection method provided by the invention can be used for directly dripping the sample of which the immunomagnetic beads and the fluorescent signal probes specifically react with the sample to be detected onto the immunochromatographic test strip, judging whether the sample is a positive sample or not through the fluorescent signals of the detection line on the immunochromatographic test strip, and is quick and intuitive in reading result, high in detection result accuracy and suitable for field detection of the norfloxacin-containing sample, and professional operation is not required.

Drawings

FIG. 1 is a transmission electron micrograph of hydrophobically converted nanoparticles prepared in example 1;

FIG. 2 is a schematic diagram of magnetic separation, enrichment and detection based on an immunochromatographic test strip in example 1 of the present invention;

FIG. 3 is a graph of fluorescence signals determined by the limits of detection in example 2, wherein 1 is norfloxacin standard concentration of 0ng/mL, 2 is norfloxacin standard concentration of 0.05ng/mL, 3 is norfloxacin standard concentration of 0.1ng/mL, and 4 is norfloxacin standard concentration of 0.5 ng/mL;

FIG. 4 is a graph of the fluorescence signals of example 2 after 4 weeks of storage in the stability test item, wherein 1 is a norfloxacin standard concentration of 0ng/mL, 2 is a norfloxacin standard concentration of 0.05ng/mL, and 3 is a norfloxacin standard concentration of 0.1 ng/mL;

FIG. 5 is a graph of fluorescence signals measured after 8 weeks of storage under the stability test item of example 2, wherein 1 is norfloxacin standard concentration of 0ng/mL, 2 is norfloxacin standard concentration of 0.05ng/mL, and 3 is norfloxacin standard concentration of 0.1 ng/mL;

FIG. 6 is a graph of the fluorescence signals of example 2 after 12 weeks of storage in the stability test item, wherein 1 is a norfloxacin standard concentration of 0ng/mL, 2 is a norfloxacin standard concentration of 0.05ng/mL, and 3 is a norfloxacin standard concentration of 0.1 ng/mL;

FIG. 7 is a graph of fluorescence signals measured after 16 weeks of storage under the stability test of example 2, wherein 1 is norfloxacin standard concentration of 0ng/mL, 2 is norfloxacin standard concentration of 0.05ng/mL, and 3 is norfloxacin standard concentration of 0.1 ng/mL;

FIG. 8 is an electron scanning electron micrograph of hydrophobically converted nanoparticles prepared in example 3;

FIG. 9 is a graph of fluorescence intensity of hydrophobically converted nanoparticles prepared in examples 1 and 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The following examples are provided to better illustrate the embodiments of the present invention.