CN111830255B - Detection method of norfloxacin - Google Patents
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Abstract
The invention relates to the technical field of fluorescence immunoassay, and particularly discloses a detection method of norfloxacin. The method comprises the following steps: coupling the norfloxacin monoclonal antibody with biotin to obtain an immune ligand; the immune ligand and carboxylated nano Fe 3 O 4 Coupling the microspheres to obtain immunomagnetic beads; coupling streptavidin with carboxylated water-soluble green light-emitting upper conversion nano particles 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 performing magnetic separation and enrichment on the sample after full reaction to obtain a sample solution; the sample solution was drop-added to an immunochromatographic test strip containing norfloxacin artificial antigen, and the result was observed. The method provided by the invention realizes the purposes of quickly separating the target object, improving the detection sensitivity by double 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 favorable for popularization and application in practice.
Description
Technical Field
The invention relates to the technical field of fluorescence immunoassay, in particular to a detection method of norfloxacin.
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 the misuse and abuse of norfloxacin veterinary drugs, which brings great health risks to consumers, such as allergic reactions, cancerous reactions, and enhancement of bacterial drug resistance. Thus, research on a norfloxacin detection method is urgent.
At present, a number of methods have been reported for detecting norfloxacin in aquatic products, including microbiological methods, 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 norfloxacin detection, complicated sample pretreatment methods are required before detection, specific detection instruments and professional detection personnel are required for operation, the detection cost is high, the detection time is long, and the method is not suitable for on-site rapid detection. Therefore, the development of the norfloxacin detection method with high sensitivity, rapid detection and simple operation has very important significance.
The Immunoassay (IA) is a technology for specifically measuring and analyzing an antibody (or antigen) based on the antigen (or antibody) as a selective chemical reagent, can overcome the defects of the methods such as a microbiological method, a thin-layer chromatography method, an instrumental analysis method and the like, 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 based on an enzyme-linked immunoassay method in the eighth ninety of the twentieth century, and is widely applied to various countries of the world due to the advantages of simplicity and rapidness in operation, easiness in judgment of results, safety, no pollution and the like, so that the immunochromatographic test strip becomes a trend method for developing in vitro rapid diagnosis fields. However, this method uses colloidal gold as a signal marker, and requires relatively complicated pretreatment steps (repeated centrifugation, washing steps are required several times) for actual sample detection to eliminate the influence of the matrix on the marker (colloidal gold, etc.), thereby eliminating false positive results, thus limiting further improvement in the sensitivity of the detection method.
Disclosure of Invention
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, the invention provides a norfloxacin detection method.
In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme:
the norfloxacin detection method comprises the following steps of:
step a, coupling a norfloxacin monoclonal antibody with biotin to obtain an immune ligand;
step b, the immune ligand and carboxylated nano Fe 3 O 4 Coupling the microspheres to obtain immunomagnetic beads;
step c, coupling the streptavidin with carboxylated water-soluble green-emitting upper conversion nano particles to obtain a fluorescent signal probe;
step 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, dripping the sample solution on an immunochromatographic test strip containing the norfloxacin artificial antigen, and observing the result.
Compared with the prior art, the invention can realize specific capture and enrichment of the target in the complex matrix sample under the action of an external magnetic field by the specific combination of the norfloxacin monoclonal antibody on the immunomagnetic beads and the norfloxacin in the sample to be detected, thereby overcoming the defects of low sensitivity, incapability of realizing on-site detection and the like caused by the need of centrifugation and dilution in the pretreatment of the sample in the traditional immunochromatography method, and realizing simple and rapid screening of a large number of unknown samples; meanwhile, through coupling of biotin-chain and avidin, the enriched sample is combined with a fluorescent signal probe, so that the combined detection of immunoassay, magnetic separation and fluorescent detection is realized. The invention utilizes the advantages of large specific surface area of the immunomagnetic beads, stable fluorescence performance of the fluorescent signal probe, strong matrix interference resistance and the like, increases the bearing capacity of the target object to be detected and the stability of a detection system, breaks through the limitations 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 the norfloxacin under the naked eye condition through the fluorescence intensity of the detection line and the quality control line, is suitable for on-site quick detection, and has wide application prospect.
The invention is characterized in thatThe detection mechanism is as follows: if the sample is positive, the complex formed by the detection sample, the immunomagnetic beads and the fluorescent probe is dripped on the immunochromatographic test strip, the sample moves towards the direction of the absorbent paper under the action of the capillary force, and when the complex passes through the detection line, if the target detection object and the detection antibody completely react, no redundant detection antibody-Fe exists 3 O 4 The nanoparticle-up-conversion nanoparticle and the capture antigen at the detection line are subjected to specific reaction, so that a fluorescent signal strip is not formed on the detection line, and when a sample continues to move to the quality control line, the detection antibody and the secondary antibody at the detection line are subjected to specific reaction to form a fluorescent signal; if the sample is negative, no target detection object and Fe are in the sample 3 O 4 The detection antibody marked on the nano-particles has a specific reaction, so that fluorescent signal strips can be formed at the detection line and the quality control line; as long as no fluorescent signal band is observed at the quality control line, the test strip is proved to be invalid.
Preferably, in step a, every 1mg of norfloxacin monoclonal antibody is conjugated with 0.13-0.15mmol of biotin.
Further preferably, the specific process of step a is: uniformly mixing 1mg of norfloxacin monoclonal antibody with 0.13-0.15mmol of biotin, adding 0.12mg of N-hydroxysuccinimide (NHS), incubating for 50-70min at room temperature (15-35 ℃), dialyzing with 0.1mol/L of phosphate buffer salt solution (PBS solution) with pH=7.4 at room temperature, filtering to remove unreacted biotin, and redissolving the obtained norfloxacin monoclonal antibody-biotin complex with 0.18-0.22mL of PBS solution (0.1 mol/L, pH=7.4) to obtain the immune ligand.
Preferably, in step b, the carboxylated nano Fe 3 O 4 The mass ratio of the microsphere to the norfloxacin monoclonal antibody is 1:0.18-0.22.
Preferably, in step b, the carboxylated nano Fe 3 O 4 The preparation of the microsphere specifically comprises the following steps:
step 1, ferric chloride and ferrous chloride are used as raw materials, and a coprecipitation method is adopted to prepare Fe 3 O 4 A nanoparticle;
step 2, the Fe is treated 3 O 4 The nano particles and oleic acid are uniformly mixed, reacted and separated to obtain Fe coated by oleic acid 3 O 4 A nanoparticle;
step 3, coating the oleic acid with Fe 3 O 4 Adding potassium permanganate solution into the nano particles, reacting, and separating to obtain the carboxylated nano Fe 3 O 4 And (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 2.5-3.5h.
Preferably, in step 2, the Fe 3 O 4 The 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 50-70min.
Preferably, in the step 3, the concentration of the potassium permanganate solution is 8-12mg/mL, the molar ratio of the potassium permanganate to the oleic acid is 1:1.5-1.7, the reaction temperature is 20-30 ℃, and the reaction time is 7-9h.
Further preferably, the specific step b comprises the following steps:
FeCl is weighed according to the mol ratio of 1.7-2.0:1 3 ·6H 2 O and FeCl 2 ·4H 2 O is added into water to prepare ferric salt mixed solution with the ferric chloride concentration of 0.2-0.3g/mL and the ferrous chloride concentration of 0.08-0.1 g/mL. The ferric salt mixed solution is dripped into sodium hydroxide solution (0.05-0.07 g/mL) under the inert gas atmosphere of 75-85 ℃, and sodium hydroxide and FeCl are added 3 The molar ratio of (2) is 16-18:1, and the reflux reaction is carried out for 2.5-3.5h to obtain Fe 3 O 4 Magnetic nanoparticles.
Under the condition of intense stirring, oleic acid and the Fe are weighed according to the mol ratio of 0.8-1.2:1 3 O 4 The magnetic nano particles are uniformly mixed, reacted in a water bath at 65-75 ℃ for 50-70min under inert atmosphere, and separated by an external magnetic field to obtain Fe coated by oleic acid 3 O 4 Washing magnetic nano particles with absolute ethyl alcohol for 3-4 times, washing with water to pH=7, adding potassium permanganate solution (8-12 mg/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 ultrasonic condition, and separating by external magnetic fieldWashing, vacuum freeze drying for 39-41h, adding PBS (0.1M, pH 7.4) solution for dissolving to obtain carboxylated nanometer Fe with mass concentration of 4.8-5.2mg/mL 3 O 4 Microsphere solution.
According to carboxylated nano Fe 3 O 4 The mass ratio of the microsphere to the norfloxacin monoclonal antibody is 1:0.18-0.22, and carboxylated nano Fe is removed 3 O 4 Microsphere solution, adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS), EDC and NHS and carboxylated nano Fe 3 O 4 The mass ratio of the microspheres is 3.8-4.2:1, the reaction is carried out for 2.5-3.5 hours at room temperature, the microspheres are washed by PBS solution, are blocked by adding 4.5-5.2wt% of OVA solution for 2 hours, and are washed to obtain the immunomagnetic beads.
Preferably, in the step c, the mass ratio of the streptavidin to the carboxylated water-soluble green-emitting upper conversion nanoparticle is 1:4-7.
Preferably, in step c, the preparation method of the carboxylated water-soluble green light emitting on-conversion nanoparticle comprises the following steps: yttrium acetate, ytterbium acetate and erbium acetate are used as raw materials, hydrophobic upper conversion nano particles are prepared through a thermal decomposition method, and then carboxylation modification is carried out on the surfaces of the hydrophobic upper conversion nano particles through a ligand exchange method, so that carboxylated water-soluble green light-emitting upper conversion nano particles are obtained.
Preferably, the molar ratio of yttrium acetate, ytterbium acetate and erbium acetate is 78-80:18-22:2.
The carboxylated water-soluble green light-emitting up-conversion nanoparticle prepared by the invention has characteristic emission peaks at 542nm and no other interference peaks under 980nm excitation wavelength, 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 steps of:
weighing yttrium acetate, ytterbium acetate and erbium acetate according to the 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 the molar ratio of 2-3:3-5, adding methanol to prepare a solution with the sodium hydroxide concentration of 0.2-0.3mol/L and the molar ratio of sodium hydroxide to yttrium acetate of 3-4:1, dropwise adding the solution into the reaction solution, stirring at room temperature to react 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 upconversion nano particles (OA-UCNPs).
Dispersing OA-UCNPs in toluene to obtain an OA-UCNPs dispersion 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, uniformly mixing, stirring and reacting for 50-70min at 105-115 ℃, heating to 230-250 ℃ and reacting for 50-70min, and centrifuging to obtain carboxylated water-soluble green light-emitting conversion nano particles (PAA-UCNPs).
Weighing streptavidin and PAA-UCNPs according to the mass ratio of 1:4-7, performing low-speed oscillation incubation reaction for 1.5-2.5h at 25-35 ℃ by utilizing an activated ester reaction, and washing by using HEPES buffer solution to obtain the fluorescent signal probe.
The addition ratio of the immunomagnetic beads, the fluorescent signaling probes and the sample to be detected is the conventional dosage in the field, and can be obtained by conventional adjustment.
Preferably, the immunochromatographic test strip comprises a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the bottom plate, wherein the water absorption pad and the binding 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 binding pad; the nitrocellulose 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 mug of each immunochromatographic test paper, and the coating amount of the sheep anti-mouse polyclonal antibody is 0.1-0.3 mug of each immunochromatographic test paper.
More preferably, the coating amount of the norfloxacin artificial antigen is 0.07 mug of coating on each immunochromatographic test paper, and the coating amount of the goat anti-mouse polyclonal antibody is 0.2 mug of coating on each immunochromatographic test paper.
According to the norfloxacin detection method provided by the invention, the sample specifically reacting the immunomagnetic beads, the fluorescent signal probe and the sample to be detected can be directly dripped on the immunochromatographic test strip, whether the sample is a positive sample is judged through the fluorescent signal of the detection line on the immunochromatographic test strip, the reading result is quick and visual, the operation of a professional is not needed, the accuracy of the detection result is higher, and the norfloxacin detection method is suitable for the on-site detection of the norfloxacin-containing sample.
Drawings
FIG. 1 is a transmission electron micrograph of the hydrophobic upconversion nanoparticles prepared in example 1;
FIG. 2 is a schematic diagram of magnetic separation enrichment detection based on immunochromatographic test strips in example 1 of the present invention;
FIG. 3 is a graph of fluorescence signals determined by the detection limit 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 0.5ng/mL;
FIG. 4 is a graph of fluorescence signals detected after 4 weeks of placement under the stability test item in example 2, wherein 1 is 0ng/mL of norfloxacin standard, 2 is 0.05ng/mL of norfloxacin standard, and 3 is 0.1ng/mL of norfloxacin standard;
FIG. 5 is a graph of fluorescence signals detected after 8 weeks of placement under the stability test item in example 2, wherein 1 is 0ng/mL of norfloxacin standard, 2 is 0.05ng/mL of norfloxacin standard, and 3 is 0.1ng/mL of norfloxacin standard;
FIG. 6 is a graph of fluorescence signals detected after 12 weeks of placement under the stability test item of example 2, wherein 1 is 0ng/mL of norfloxacin standard, 2 is 0.05ng/mL of norfloxacin standard, and 3 is 0.1ng/mL of norfloxacin standard;
FIG. 7 is a graph of fluorescence signals detected after 16 weeks of placement under the stability test item in example 2, wherein 1 is 0ng/mL of norfloxacin standard, 2 is 0.05ng/mL of norfloxacin standard, and 3 is 0.1ng/mL of norfloxacin standard;
FIG. 8 is an electron scanning electron microscope image of the hydrophobic upconversion nanoparticles prepared in example 3;
FIG. 9 is a graph showing fluorescence intensity of hydrophobic upconversion nanoparticles prepared in example 1 and example 3.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In order to better illustrate the embodiments of the present invention, the following is further illustrated by examples.
Example 1
The embodiment of the invention provides a detection method of norfloxacin, which comprises the following steps:
1. preparation of immunochromatography test strip
(1) Preparation of norfloxacin antigen
11.7mg of norfloxacin was dissolved in 350. Mu. L N, N-dimethylformamide, 21.2mg of NHS and 70.5mg of EDC were added thereto, and the mixture was stirred overnight at room temperature. Centrifuging at 4deg.C and 10000 Xg for 5min, discarding the precipitate, collecting supernatant, slowly adding dropwise the supernatant to chicken Ovalbumin (OVA) solution (33.0 mg chicken ovalbumin in 5mL PBS (0.01M, pH 7.4)), stirring at room temperature for 3 hr, transferring the solution to dialysis bag, and dialyzing with PBS solution (0.01M, pH 7.4) at 4deg.C for three days to obtain norfloxacin antigen (NOR-OVA).
(2) Coating of nitrocellulose membranes
The prepared norfloxacin antigen is coated on a nitrocellulose membrane by a double-dimensional plane membrane drawing instrument of an Shanghai gold mark to be used as a detection line, wherein the coating amount is 0.07 mug/strip.
Sheep anti-mouse lgG is dissolved in 0.01MPBS and 1wt% OVA solution, diluted to a concentration of 1mg/mL, and coated on a nitrocellulose membrane by a film drawing instrument to serve as a quality control line, wherein the coating amount is 0.2 mug/strip.
(3) Assembly of immunochromatographic test strips
The sample pad, the bonding pad, the nitrocellulose membrane and the water absorbing pad are sequentially adhered on the PVC base plate, cut into small strips with the width of 3.7mm, vacuum packaged and stored at normal temperature.
The sample pad, the bonding pad and the water absorbing pad are all conventional commercial products, the sample pad is glass fiber of SB06 sold by Shanghai gold mark biotechnology company, the bonding pad is GFCP203000 glass fiber pad sold by Shanghai gold mark biotechnology company, and the water absorbing pad is CH37M water absorbing paper sold by Shanghai gold mark biotechnology company.
2. Preparation of fluorescent probes
263.7mg (0.78 mmol) of yttrium acetate tetrahydrate, 84.5mg (0.2 mmol) of ytterbium acetate tetrahydrate and 8.3mg (0.02 mmol) of erbium acetate tetrahydrate are added into a 100.0mL three-necked flask, 6.0mL of oleic acid and 17.0mL of octadecene are added, stirring is carried out at 400-650r/min to fully mix, the reaction system is heated to 100 ℃ and vacuumized for 10min, then heated to 160 ℃ and reacted for 30min, and then the reaction solution is naturally cooled to room temperature, thus obtaining the reaction solution.
100mg (2.5 mmol) of sodium hydroxide and 148.2mg (4 mmol) of ammonium fluoride are weighed, 10mL of methanol is added, the mixture is uniformly mixed, the mixture is dripped into the reaction liquid, the mixture is stirred at room temperature for 30min, then the methanol is removed by heating, the system is heated to 100 ℃ and vacuumized for 10min, the mixture is heated to 300 ℃ under the protection of argon, the reaction is carried out for 1h, the mixture is naturally cooled to room temperature, washed by ethanol for three times, and dried, so that hydrophobic upper conversion nano particles (OA-UCNPs) are obtained, and the prepared OA-UCNPs are uniform spheres with the size of about 30nm, and are shown in a figure 1.
Weighing 30mg of the prepared OA-UCNPs, adding 2mL of toluene, uniformly mixing, adding 500mg of polyacrylic acid, stirring at 110 ℃ for reaction for 1h under the protection of argon, heating to 240 ℃ for reaction for 1h, centrifuging, and washing to obtain carboxylated water-soluble green light emitting upper conversion nanoparticles (PAA-UCNPs).
To 5mg of the PAA-UCNPs prepared above, 1mg of chain and a nucleophilic reagent were added, and the reaction was incubated at 30℃under low temperature shaking for 2 hours by using an activated ester reaction, washed three times with HEPES buffer, and the volume was fixed to 1mL with HEPES buffer, thereby obtaining a fluorescent signal probe.
3. Preparation of immunomagnetic beads
Weigh 8.1g FeCl 3 ·6H 2 O and 3.3g FeCl 2 ·4H 2 O in a small beaker, 38mL of distilled water was added to dissolve the sample to obtain a ferric salt mixed solution. Weighing 20.9g of NaOH, dissolving in 375mL of distilled water, transferring to a round-bottom four-neck flask, placing in a water bath at 80 ℃, gradually dripping the ferric salt mixed solution under stirring, introducing nitrogen, deoxidizing, refluxing and condensing for 3h, separating by a magnet after the reaction is finished, and washing by deionized water to obtain Fe 3 O 4 Magnetic nanoparticles.
Under vigorous stirring, 4.66g (16.5 mmol) of oleic acid and 3.82g (16.5 mmol) of prepared Fe 3 O 4 Uniformly mixing magnetic nano particles, reacting for 1h in a water bath at 70 ℃ under the protection of nitrogen, and separating by an external magnetic field to obtain Fe coated with oleic acid 3 O 4 Washing magnetic nano particles with absolute ethyl alcohol for 4 times, then washing the nano particles with water until the pH value is=7, adding a potassium permanganate solution (1.6 g of potassium permanganate is dissolved in 160mL of water), performing ultrasonic vibration for 8 hours, separating by a magnet, washing the nano particles with deionized water for 3 times, performing vacuum freeze drying for 40 hours, adding a PBS (0.1M, pH 7.4) solution for dissolution, and obtaining carboxylated Fe with the mass concentration of 5.0mg/mL 3 O 4 A nanoparticle solution.
1mg of the monoclonal antibody was uniformly mixed with 0.14mmol of biotin, 0.12mg of N-hydroxysuccinimide (NHS) was added, incubated at room temperature for 1 hour, dialyzed against PBS solution of pH 7.4.1M at room temperature, unreacted biotin was removed, and the biotin-labeled monoclonal antibody was reconstituted with 0.2mL of BS solution (0.1M, pH 7.4).
Taking 100 mu L of carboxylated nano Fe 3 O 4 Adding 2mgEDC and 2mgNHS into the microsphere solution, reacting for 3 hours at room temperature, washing 3 times with PBS buffer, adding 20 mu L of the biotin-labeled monoclonal antibody, stirring overnight, washing 3 times with PBS solution, adding 5% OVA solution for sealing for 2 hours, washing 3 times, redissolving with 1mL of 0.1% OVA solution to obtain immunomagnetic beads, and preserving at 4 ℃ for standby.
4. Detection of samples
(1) Pretreatment of samples
Chicken samples purchased locally from a supermarket (negative samples confirmed by HPLC) were selected as detection samples. 1g of the sample is taken, homogenized by a homogenizer, placed in a 50mL centrifuge tube, added with 0.2mL of sodium hydroxide (0.01M) and 1.8mL of methanol, uniformly mixed, vigorously vibrated for 5min, and diluted 5 times by 0.01MPBS solution (pH 7.4), thus obtaining the sample to be detected.
(2) Detection of
Preparing a working solution: 1.21g of Tris was weighed into 100mL of water, the pH was adjusted to 7.0-8.0 with concentrated hydrochloric acid, 5g of sucrose, 0.5g of BSA,0.5g of PVP,0.5g of F68,0.5mL of 10wt% sodium azide, 0.5mL of PEG200,0.5mL of Triton X-100,1.0mL of Tween-20 were added.
100 mu L of the sample to be detected, the immunomagnetic beads and the fluorescent probe are uniformly mixed, an external magnetic field is added, the enriched sample is separated, then 80 mu LPBS solution (0.1M, pH 7.4) and 20 mu L of working solution are added, the reaction product is redissolved, and finally, the reaction product is slowly dripped on the prepared immunochromatographic test strip, and the result is observed after 10 minutes. Fluorescent signals can be observed on both the detection line and the quality control line of the chromatographic test paper. The detection schematic diagram is shown in fig. 2.
Example 2
2.1 method sensitivity test
Norfloxacin standards (0 ng/mL, 0.05ng/mL, 0.1ng/mL, and 0.5 ng/mL) were configured for a series of gradient concentrations. Then, according to the detection method in example 1, as shown in fig. 3, it can be observed that when the concentration of the norfloxacin standard product is 0.1ng/mL, the fluorescence intensity of the chromatographic immunoassay test strip detection line is obviously different from that of the quality control line by visual inspection, and the fluorescence intensity of the detection line almost completely disappears, so that the visual detection limit of the detection method provided by the invention is 0.1ng/mL.
2.2 method stability test
The immunochromatographic test strip prepared in example 1 of the present invention, the immunomagnetic beads and the fluorescent signal probe were stored at 4℃for 16 weeks. The assays of example 1 were performed at weeks 4, 8, 12 and 16, respectively, with norfloxacin standard concentrations of 0ng/mL, 0.05ng/mL and 0.1ng/mL, respectively.
As shown in the figures 4-7, the fluorescence intensity of the detection line of the 0ng/mL sample is still obvious when the detection is carried out after 16 weeks, the fluorescence intensity of the detection line of the 0.1ng/mL sample is obviously different from that of the quality control line by visual inspection, the fluorescence intensity of the detection line almost completely disappears, and the detection method provided by the invention has good stability and the detection result is still accurate and reliable after 16 weeks.
2.3 detection of labeled samples
The norfloxacin standard is added into chicken negative samples, the final addition concentration of the norfloxacin standard in the samples is 0 mug/L, 1.0 mug/L, 5 mug/L and 15 mug/L, and finally the samples are diluted 5 times by PBS buffer solution, and three samples are prepared in parallel for each concentration and are detected according to the detection method of the example 1. And comparing the detection result with a commercial colloidal gold immunochromatographic test strip (Shijia Dai Biotechnology Co., ltd.).
The test method of the colloidal gold immunochromatographic test strip comprises the following steps:
shearing a tissue sample, weighing 0.5g of the tissue sample, placing the tissue sample into a matched tissue extracting solution of a colloidal immunochromatographic test strip, fully and uniformly mixing, then placing a centrifuge tube into a boiling water bath with the temperature of more than 90 ℃ for heating for 10min, taking out the centrifuge tube, placing the centrifuge tube into a centrifuge, centrifuging for 5min at 4000 rpm, taking the supernatant as a liquid to be detected, and dripping the liquid to be detected onto the colloidal gold immunochromatographic test strip.
The test results are shown in Table 1.
TABLE 1
Through testing, the detection limit of the immunochromatographic test strip is 1 mug/L, and the detection limit of the commercial colloidal gold immunochromatographic test strip is 15 mug/L.
Experimental results show that compared with the traditional colloidal gold immunochromatography technology, the method provided by the invention has higher detection sensitivity, is more suitable for rapid detection of norfloxacin in complex matrixes, and can be seen by comparing detection steps of the two methods, the traditional colloidal gold immunochromatography technology requires equipment such as a centrifuge to centrifuge for about 5min in a sample separation step, and the method directly adopts an external magnetic field to separate for a short time (about 2 min) and has good separation effect. In addition, the colloidal gold immunochromatography test strip takes colloidal gold as a signal marker, so that the cost is high, the interference of environmental media is easy, and the detection limit of a sample is high. The prepared up-conversion nano particles are used as signal marking materials, have stable photochemical properties, are not easily influenced by external storage environments, have strong matrix interference resistance, and can realize a dual method for detecting signals by combining immunomagnetic beads, thereby improving detection sensitivity.
The parameters (such as raw material ratio, reaction temperature, time, etc.) in the preparation method of the fluorescent probe and the preparation method of the immunomagnetic beads in example 1 were replaced with parameters in other ranges defined in the present invention, so long as each parameter was within the range defined in the present invention, and the detection effect substantially equivalent to that in example 1 could be achieved.
Example 3
The step of the detection method of norfloxacin is identical to that of the embodiment 1, and only the preparation process of the up-conversion nano-particles is different, in this embodiment, the preparation process of the up-conversion nano-particles is as follows:
0.7g sodium hydroxide was mixed with 8mL oleic acid and 8mL ethanol and stirred rapidly at room temperature to form a white viscous solution. 5mL of water was added and the solution began to clarify while adding aqueous NaF (0.303 g NaF dissolved in 3mL of water). Weigh 0.88mmolY (NO) 3 ) 3 、0.22mmolYb(NO 3 ) 3 And 0.02mmole Er (NO) 3 ) 3 Adding the mixture into the solution, homogenizing, transferring to a reaction kettle, and reacting at 230 ℃ for 12 hours to obtain the hydrophobic up-conversion nano particles. Carboxylated water-soluble green light emitting upconverting nanoparticles were then prepared by ligand exchange method using hydrophobic upconverting nanoparticles in the same manner as in example 1.
An electron scanning electron microscope image of the hydrophobic upconversion nano particle prepared in the embodiment is shown in fig. 8, and the hydrophobic upconversion nano particle prepared by the method can be seen to have a rod-shaped structure, wherein the diameter is 110nm, and the length is 2 μm. The hydrophobic upconverting nanoparticle prepared in example 1 is larger in size and irregular, which is detrimental to migration on nitrocellulose membranes.
The fluorescence intensity pairs of the hydrophobic upconversion nanoparticle prepared by the method and the hydrophobic upconversion nanoparticle prepared in example 1 are shown in fig. 9, and it can be seen from the fig. that the fluorescence intensity of the hydrophobic upconversion nanoparticle prepared in example 1 is higher, which is more beneficial to improving the detection sensitivity.
In summary, the detection method of norfloxacin provided by the invention can realize rapid separation of the target in the complex matrix through magnetic separation, simplifies the pretreatment steps, has shorter pretreatment and detection time consumption, is rapid in detection, can realize simple and rapid screening of a large number of unknown samples, has low detection cost, and is favorable for popularization and application in practice.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (7)
1. The norfloxacin detection method is characterized by comprising the following steps of:
step a, coupling a norfloxacin monoclonal antibody with biotin to obtain an immune ligand; coupling 0.13-0.15mmol of biotin per 1mg of norfloxacin monoclonal antibody;
step b, the immune ligand and carboxylated nano Fe 3 O 4 Coupling the microspheres to obtain immunomagnetic beads; the carboxylated nano Fe 3 O 4 The mass ratio of the microsphere to the norfloxacin monoclonal antibody is 1:0.18-0.22;
step c, coupling the streptavidin with carboxylated water-soluble green-emitting upper conversion nano particles to obtain a fluorescent signal probe; the mass ratio of the streptavidin to the carboxylated water-soluble green light-emitting upper conversion nano particles is 1:4-7;
step 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, dripping the sample solution on an immunochromatographic test strip containing the norfloxacin artificial antigen, and observing the result.
2. The method for detecting norfloxacin according to claim 1, wherein in step b, the carboxylated nano Fe 3 O 4 The preparation of the microsphere specifically comprises the following steps:
step 1, ferric chloride and ferrous chloride are used as raw materials, and a coprecipitation method is adopted to prepare Fe 3 O 4 A nanoparticle;
step 2, the Fe is treated 3 O 4 The nano particles and oleic acid are uniformly mixed, reacted and separated to obtain Fe coated by oleic acid 3 O 4 A nanoparticle;
step 3, coating the oleic acid with Fe 3 O 4 Adding potassium permanganate solution into the nano particles, reacting, and separating to obtain the carboxylated nano Fe 3 O 4 And (3) microspheres.
3. The method for detecting norfloxacin according to claim 2, wherein in step 1, the molar ratio of ferric chloride to ferrous chloride is 1.7-2.0:1, the reaction temperature is 75-85 ℃, and the reaction time is 2.5-3.5h; and/or
In step 2, the Fe 3 O 4 The 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 50-70min; and/or
In the step 3, the concentration of the potassium permanganate solution is 8-12mg/mL, the molar ratio of the potassium permanganate to the oleic acid is 1:1.5-1.7, the reaction temperature is 20-30 ℃, and the reaction time is 7-9h.
4. 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 on-conversion nanoparticle comprises the following steps: yttrium acetate, ytterbium acetate and erbium acetate are used as raw materials, hydrophobic upper conversion nano particles are prepared through a thermal decomposition method, and then carboxylation modification is carried out on the surfaces of the hydrophobic upper conversion nano particles through a ligand exchange method, so that carboxylated water-soluble green light-emitting upper conversion nano particles are obtained.
5. The method for detecting norfloxacin according to claim 4, wherein: the molar ratio of yttrium acetate to ytterbium acetate to erbium acetate is 78-80:18-22:2.
6. The method for detecting norfloxacin according to claim 1, wherein: the immunochromatographic test strip comprises a bottom plate, a sample pad, a bonding pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the bottom plate, wherein the water absorption pad and the bonding 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 bonding pad; the nitrocellulose membrane is provided with a detection line coated with norfloxacin artificial antigen and a quality control line coated with goat anti-mouse polyclonal antibody.
7. The method for detecting norfloxacin according to claim 6, wherein: the coating amount of the norfloxacin artificial antigen is 0.05-0.1 mug/strip, and the coating amount of the sheep anti-mouse polyclonal antibody is 0.1-0.3 mug/strip.
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