1. A kit for detecting norfloxacin, comprising:

(1) norfloxacin-biotin marker;

(2) norfloxacin polyclonal antibodies; the norfloxacin polyclonal antibody is coated on a multi-well plate;

(3) alkaline phosphatase modified with streptavidin;

(4) gold nanoparticles; the gold nanoparticles comprise gold nanoparticles connected with azide groups and gold nanoparticles connected with alkynyl groups, and the gold nanoparticles are used for detecting monovalent copper ions;

(5) ascorbic acid phosphoric acid;

(6) copper sulfate solution;

(7) a copper ion probe; the copper ion probe is used for detecting bivalent copper ions;

the norfloxacin-biotin label is used for being combined with the norfloxacin polyclonal antibody and used for being combined with the alkaline phosphatase modified with streptavidin;

the alkaline phosphatase modified with streptavidin is used for reducing the ascorbyl phosphate into ascorbic acid, and the ascorbic acid is used for reducing bivalent copper ions in the copper sulfate solution into monovalent copper ions.

2. The kit according to claim 1, wherein the gold nanoparticles have a diameter of 13 to 17 nm;

preferably, the molar ratio of the gold nanoparticles with azide groups attached to the gold nanoparticles with alkyne groups attached to the gold nanoparticles is 1: 1;

more preferably, the concentration of the gold nanoparticles is 0.5-2 OD.

3. The kit according to claim 1, wherein the norfloxacin-biotin label is labeled with 0.02-0.06 mg of biotin per 1mg of norfloxacin; preferably 0.04 mg;

preferably, the concentration of the norfloxacin-biotin marker is 0.6-1 mg/mL, and preferably 0.8 mg/mL;

more preferably, the norfloxacin-biotin label is preserved in methanol.

4. The kit according to claim 1, wherein the coating amount of norfloxacin polyclonal antibody on the multi-well plate is 50-200 μ g/well, preferably 100 μ g/well.

5. The kit according to claim 1, wherein the concentration of ascorbyl phosphate is 1 to 3mM, preferably 2 mM.

6. The kit according to claim 1, wherein the concentration of the copper sulfate solution is 1 to 3mM, preferably 2 mM;

preferably, the concentration of the copper ion probe is 10 to 30 μ M, preferably 20 μ M, and the concentration is measured in Tris-HCl at pH 7.2: CH (CH)₃CN volume ratio of 1: 1.

7. The kit of claim 1, further comprising norfloxacin standard;

preferably, the concentration of the norfloxacin standard is 10^-6pg/mL～10⁷pg/mL；

More preferably, the norfloxacin standard is prepared by using a diluent, wherein the diluent is 1X PBS buffer containing 10% by volume of methanol.

8. The kit according to any one of claims 1 to 7, wherein the copper ion probe is used for outputting a fluorescent signal; the gold nanoparticles are used for outputting visual signals.

9. A method for detecting norfloxacin, which comprises the steps of using the kit of any one of claims 1 to 8 for detection, and comprises at least the following steps:

s1, coating the norfloxacin polyclonal antibody on a multi-well plate; the coating amount of the norfloxacin polyclonal antibody on the porous plate is 50-200 mu g/hole, and preferably 100 mu g/hole;

s2, mixing 50 mu L of norfloxacin standard substance and 50 mu L of norfloxacin-biotin marker for each gradient concentration, adding the mixture to a porous plate, and reacting at 36-38 ℃ for 45-75 minutes, preferably 60 minutes;

s3, adding alkaline phosphatase modified with streptavidin in a volume of 100 mu L, and reacting at 36-38 ℃ for 20-40 minutes, preferably for minutes;

s4, adding 100 mu L of ascorbic acid phosphoric acid, and reacting for 45-75 minutes, preferably 60 minutes, at the temperature of 36-38 ℃;

s5, adding 100 mu L of copper sulfate solution, and reacting for 7-15 minutes, preferably 10 minutes, at room temperature;

s6, adding 100 mu L of copper ion probe into 100 mu L of copper ion probe, detecting fluorescence signals, drawing a standard curve, adding 50 mu L of gold nanoparticle mixed solution into 160 mu L of gold nanoparticle mixed solution, detecting visual signals, and preparing a color change diagram;

s7, drawing a standard curve for each detected gradient concentration fluorescence signal, and preparing a color change diagram for each detected gradient concentration visualization signal;

s8, respectively diluting the samples to be tested by 10³Dilution 10⁶Dilution 10⁹Doubling, namely mixing the stock solution of the sample to be detected and the stock solution 10 of the sample to be detected³Multiple dilution solution, original solution of sample to be measured 10⁶Multiple dilution solution, original solution of sample to be measured 10⁹And (3) detecting the multiple dilution liquid by using methods from S2 to S7, substituting the multiple dilution liquid into a standard curve to calculate the concentration of the object to be detected when the fluorescence value of one dilution multiple appears on the standard curve, and comparing the color change with a color change graph.

10. The assay of claim 9, wherein the norfloxacin-biotin label is diluted 4 x 10 with a diluent prior to detection⁴Doubling;

preferably, the alkaline phosphatase modified with streptavidin is diluted 2000 times by PBS before detection;

more preferably, the buffer solution is diluted 10 times with Tri-HCl buffer solution with pH 7.4 before the detection of ascorbyl phosphate.

Background

Norfloxacin (NOR) antibacterial agents are third generation quinolones that act as bacteriostatics by blocking the production of DNA transcriptase by bacteria and inhibiting the replication of bacterial DNA. With the increase of the norfloxacin usage amount, the abuse phenomenon frequently causes serious residue in food. NOR is usually found in beverages, meat products, milk and most dairy products, is a broad-spectrum antibiotic, is difficult to completely degrade in animal bodies, mostly aggregates and remains in animal bodies, and can cause allergic reaction, anaphylactic reaction, immunosuppression, carcinogenesis, teratogenesis, mutagenesis and the like when food containing NOR with an excessive content is eaten.

The currently established NOR detection technology mainly includes high performance liquid chromatography detection technology, microbiological assay technology, immunological detection technology and the like, wherein the immunological detection technology is mainly used and includes immunodiffusion, agglutination test, radioimmunoassay, enzyme-linked immunosorbent assay, immunofluorescence, chemiluminescence enzyme-linked immunoassay and the like. ELISA is widely used in partial areas as a traditional detection technology, but is easily influenced by complex components in food or human blood in the detection process of actual samples (such as dairy products and meat products), so that the technology still has certain defects in the aspect of sensitivity detection.

Therefore, establishing a high-sensitivity detection technology for NOR in food has great significance for the health of consumers and the clinical diagnosis of food poisoning caused by the bacteria.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a kit and a detection method for detecting norfloxacin.

The second invention aims to provide a norfloxacin detection method.

In order to achieve the purpose of the invention, the technical scheme is as follows:

the invention relates to a kit for detecting norfloxacin, which comprises:

(1) norfloxacin-biotin marker;

(2) norfloxacin polyclonal antibodies; the norfloxacin polyclonal antibody is coated on a multi-well plate;

(3) alkaline phosphatase modified with streptavidin;

(4) gold nanoparticles; the gold nanoparticles comprise gold nanoparticles connected with azide groups and gold nanoparticles connected with alkynyl groups, and the gold nanoparticles are used for detecting monovalent copper ions;

(5) ascorbic acid phosphoric acid;

(6) copper sulfate solution;

(7) a copper ion probe; the copper ion probe is used for detecting bivalent copper ions;

the norfloxacin-biotin label is used for being combined with the norfloxacin polyclonal antibody and used for being combined with the alkaline phosphatase modified with streptavidin;

the alkaline phosphatase modified with streptavidin is used for reducing the ascorbyl phosphate into ascorbic acid, and the ascorbic acid is used for reducing bivalent copper ions in the copper sulfate solution into monovalent copper ions.

Optionally, the diameter of the gold nanoparticles is 13-17 nm;

preferably, the molar ratio of the gold nanoparticles with azide groups attached to the gold nanoparticles with alkyne groups attached to the gold nanoparticles is 1: 1;

more preferably, the concentration of the gold nanoparticles is 0.5-2 OD.

Optionally, in the norfloxacin-biotin marker, each 1mg of norfloxacin is marked with 0.02-0.06 mg of biotin; preferably 0.04 mg;

preferably, the concentration of the norfloxacin-biotin marker is 0.6-1 mg/mL, and preferably 0.8 mg/mL;

more preferably, the norfloxacin-biotin label is preserved in methanol.

Optionally, the coating amount of the norfloxacin polyclonal antibody on the multi-well plate is 50-200 mug/well, and preferably 100 mug/well.

Optionally, the concentration of ascorbyl phosphate is 1-3 mM, preferably 2 mM.

Optionally, the concentration of the copper sulfate solution is 1-3 mM, preferably 2 mM;

preferably, the concentration of the copper ion probe is 10 to 30 μ M, preferably 20 μ M, and the concentration is measured in Tris-HCl at pH 7.2: CH (CH)₃CN volume ratio of 1: 1.

Optionally, the kit further comprises norfloxacin standard;

preferably, the concentration of the norfloxacin standard is 10^-6pg/mL～10⁷pg/mL；

More preferably, the norfloxacin standard is prepared by using a diluent, wherein the diluent is 1X PBS buffer containing 10% by volume of methanol.

Optionally, the copper ion probe is used for outputting a fluorescence signal; the gold nanoparticles are used for outputting visual signals.

The invention relates to a norfloxacin detection method, which adopts the kit for detection and at least comprises the following steps:

s1, coating the norfloxacin polyclonal antibody on a multi-well plate; the coating amount of the norfloxacin polyclonal antibody on the porous plate is 50-200 mu g/hole, and preferably 100 mu g/hole;

s2, mixing 50 mu L of norfloxacin standard substance and 50 mu L of norfloxacin-biotin marker for each gradient concentration, adding the mixture to a porous plate, and reacting at 36-38 ℃ for 45-75 minutes, preferably 60 minutes;

s3, adding alkaline phosphatase modified with streptavidin in a volume of 100 mu L, and reacting at 36-38 ℃ for 20-40 minutes, preferably for minutes;

s4, adding 100 mu L of ascorbic acid phosphoric acid, and reacting for 45-75 minutes, preferably 60 minutes, at the temperature of 36-38 ℃;

s5, adding 100 mu L of copper sulfate solution, and reacting for 7-15 minutes, preferably 10 minutes, at room temperature;

s6, adding 100 mu L of copper ion probe into 100 mu L of copper ion probe, detecting fluorescence signals, drawing a standard curve, adding 50 mu L of gold nanoparticle mixed solution into 160 mu L of gold nanoparticle mixed solution, detecting visual signals, and preparing a color change diagram;

s7, drawing a standard curve for each detected gradient concentration fluorescence signal, and preparing a color change diagram for each detected gradient concentration visualization signal;

s8, respectively diluting the samples to be tested by 10³Dilution 10⁶Dilution 10⁹Doubling, namely mixing the stock solution of the sample to be detected and the stock solution 10 of the sample to be detected³Multiple dilution solution, original solution of sample to be measured 10⁶Multiple dilution solution, original solution of sample to be measured 10⁹And (3) detecting the multiple dilution liquid by using methods from S2 to S7, substituting the multiple dilution liquid into a standard curve to calculate the concentration of the object to be detected when the fluorescence value of one dilution multiple appears on the standard curve, and comparing the color change with a color change graph.

Optionally, 4 × 10 dilution of norfloxacin-biotin label with diluent is performed before detection⁴Doubling;

diluting the alkaline phosphatase modified with streptavidin by 2000 times by using PBS before detection;

the test solution was diluted 10-fold with Tri-HCl buffer solution at pH 7.4 before detection of ascorbyl phosphate.

The invention has at least the following beneficial effects:

the technology has the advantages of high sensitivity, wide detection range, simple operation, good specificity and the like, has important practical significance for the ultra-sensitive detection of norfloxacin, and has good guiding significance for realizing the on-site rapid and ultra-sensitive detection technology.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 is a schematic diagram of a dual channel detection of norfloxacin;

FIG. 2 is a diagram of colloidal gold UV characterization;

FIG. 3 is a transmission electron microscope image of colloidal gold;

FIG. 4 is a diagram showing the particle size of colloidal gold;

FIG. 5 is a graph showing UV contrast between gold colloid and gold colloid modified with azido/alkynyl groups;

FIG. 6 is a transmission electron micrograph of colloidal gold @ azido;

FIG. 7 is a graph of the particle size characterization of colloidal gold @ azido;

FIG. 8 is a zeta potential diagram of gold and gold modified with azido/alkynyl groups;

FIG. 9 is a transmission electron micrograph of colloidal gold @ alkynyl;

FIG. 10 is a graph of particle size characterization for colloidal gold @ alkynyl;

FIG. 11 is a graph of fluorescence intensity at different wavelengths for different concentrations of copper sulfate;

FIG. 12 is a line graph of fluorescence intensity at 560nm for different concentrations of copper sulfate;

FIG. 13 is a graph of fluorescence intensity at different wavelengths for different concentrations of AA;

FIG. 14 is a graph of the fluorescence intensity at 560nm under 480nm excitation light for different concentrations of AA;

FIG. 15 is a graph of fluorescence intensity at different wavelengths at different dilution ratios;

FIG. 16 is a graph showing the fold lines of fluorescence intensity at 560nm under 480nm excitation light at different dilution ratios;

FIG. 17 is a graph showing the color change and UV change of gold colloid caused by different AA concentrations (10 μ M-1000 μ M);

FIG. 18 is a graph showing color change of colloidal gold and UV change induced by SA-ALP (1:500-1:32000) at different dilution ratios;

FIG. 19 is a graph of fluorescence detection standard;

FIG. 20 is a line graph of fluorescence detection;

FIG. 21 is a graph of color change criteria for AuNPs;

FIG. 22 is a specificity detection histogram.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention relates to a kit for detecting norfloxacin, which comprises:

(1) norfloxacin-biotin marker;

(2) norfloxacin polyclonal antibodies; coating norfloxacin polyclonal antibody on a multi-well plate;

(3) alkaline phosphatase modified with streptavidin;

(4) gold nanoparticles; the gold nanoparticles comprise gold nanoparticles connected with azide groups and gold nanoparticles connected with alkynyl groups, and the gold nanoparticles are used for detecting monovalent copper ions;

(5) ascorbic acid phosphoric acid;

(6) copper sulfate solution;

(7) a copper ion probe; the copper ion probe is used for detecting bivalent copper ions;

norfloxacin hydrochloride-a biotin label for binding to a polyclonal antibody against norfloxacin and for binding to streptavidin-modified alkaline phosphatase; alkaline phosphatase modified with streptavidin is used for reducing the ascorbyl phosphate into ascorbic acid, and the ascorbic acid is used for reducing bivalent copper ions in the copper sulfate solution into monovalent copper ions. The detection principle of the invention is shown in fig. 1, and specifically comprises the following steps: NOR (norfloxacin) and NOR-Bio (norfloxacin-biotin) were added simultaneously to a 96-well plate coated with Anti-Nor Ab (norfloxacin polyclonal antibody) and incubated. Then, SA-ALP (alkaline phosphatase-streptavidin) was added for incubation. And adding AAP (ascorbic acid phosphoric acid) for incubation to obtain a reaction product. Taking out the reactant and CuSO₄Reacting at room temperature, adding a copper ion probe (Cu) into a brown centrifugal tube²⁺probe), the fluorescence spectrum is recorded with an F97Pro fluorescence spectrophotometer, the fluorescence intensity appears at 650nm, the reaction is added to a reaction mixture containing CuSO₄And 160. mu.L of AuNPs @ Alkynyl/AuNPs @ Azide (colloidal gold/azido) were subjected to CuAAC reaction, and color change of AuNPs was observed. The copper ion probe is used for outputting a fluorescence signal; the gold nanoparticles are used for outputting a visual signal. The invention simultaneously detects norfloxacin micromolecules by two methods, and can detect not only trace amount but also macroscopic detection in special environment. The advantage of using two means to detect the signal is that norfloxacin antibiotic residues can be detected under different conditions. The visual signal can be used for qualitative detection, and then the fluorescent signal is used for continuous quantitative detection. The fluorescent signal of the kit can meet the requirement of low-concentration detection in a laboratory, the visual signal can realize field detection, and direct reading can be realized according to the change of color under the condition without an instrument.

As a specific implementation manner of the embodiment of the invention, the diameter of the gold nanoparticles is 13-17 nm; the diameter can better modify alkynyl and azide, and the diameter is not easy to generate aggregation. The molar ratio of the gold nanoparticles connected with the azide groups to the gold nanoparticles connected with the alkyne groups is 1: 1.

more preferably, the concentration of the gold nanoparticles is 0.5-2 OD, preferably 1 OD. If the concentration of the gold nanoparticles is too high, aggregation of the gold nanoparticles is likely to occur, and if the concentration is too low, the color is lighter and the change is not easily seen.

As a specific implementation manner of the embodiment of the invention, in the norfloxacin-biotin marker, each 1mg of norfloxacin is marked with 0.02-0.06 mg of biotin; preferably 0.04 mg; the technical advantage of selecting the labeling quantity is that enough biotin can be labeled on the norfloxacin small molecules.

Furthermore, the use concentration of the norfloxacin-biotin marker is 0.02-0.06 mu g/mL, preferably 0.04 mu g/mL; the concentration can be selected to reduce the detection limit, and the norfloxacin-biotin marker is stored in methanol.

As a specific implementation manner of the embodiment of the invention, the coating amount of norfloxacin polyclonal antibody on the multi-well plate is 50-200 mug/well, and preferably 100 mug/well. The coating amount can reduce the detection limit and save complete antigen.

As a specific embodiment of the present invention, the concentration of ascorbyl phosphate is 1 to 3mM, preferably 2 mM; the concentration can be selected to reduce the detection limit.

As a specific implementation manner of the embodiment of the invention, the concentration of the copper sulfate solution is 1-3 mM, preferably 2 mM; the concentration is selected to reduce the detection limit.

As a specific implementation manner of the embodiment of the invention, the concentration of the copper ion probe is 10-30 μ M, preferably 20 μ M, and the concentration is selected to reduce the detection limit. Copper ion probe was stored in Tris-HCl at pH 7.2: CH (CH)₃CN volume ratio of 1: 1.

As a specific implementation manner of the embodiment of the invention, the kit further contains norfloxacin standard; the norfloxacin standard has a concentration of 10^-6pg/mL～10⁷pg/mL; in order of 10^-6pg/mL、10^-5pg/mL、10^-4pg/mL、10^-3pg/mL、10^-2pg/mL、10pg/mL、10²pg/mL、10³pg/mL、10⁴pg/mL、10⁵pg/mL、10⁶pg/mL、10⁷pg/mL. Wherein norfloxacin standard substance is adoptedThe diluent is prepared from 1 XPBS buffer solution containing 10% methanol by volume.

As a specific implementation manner of the embodiment of the invention, the preparation method of the NOR polyclonal antibody comprises the following steps: firstly, preparing norfloxacin complete antigen by an EDC (carbodiimide)/NHS (N-hydroxysuccinimide) method, immunizing a big white rabbit, emulsifying adjuvant, the complete antigen and normal saline by an emulsifying instrument, performing subcutaneous injection after emulsification is completed (emulsification completion identification, which is to say that emulsification is completed by sucking a small amount of emulsified solution and dripping the solution into clear water without dispersion and moderate solidification), taking blood, centrifuging, selecting supernatant, and purifying serum to obtain the norfloxacin polyclonal antibody after the titer meets the requirement.

The embodiment of the invention also relates to a detection method of the norfloxacin, which adopts the kit for detection and at least comprises the following steps:

s1, coating the norfloxacin polyclonal antibody on a multi-well plate; coating amount of norfloxacin polyclonal antibody on the porous plate is 50-200 mug/hole, preferably 100 mug/hole;

s2, mixing 50 mu L of norfloxacin standard substance and 50 mu L of norfloxacin-biotin marker for each gradient concentration, adding the mixture to a porous plate, and reacting at 36-38 ℃ for 45-75 minutes, preferably at 37 ℃ for 60 minutes;

s3, adding alkaline phosphatase modified with streptavidin in a volume of 100 mu L, and reacting at 36-38 ℃ for 20-40 minutes, preferably at 37 ℃ for 30 minutes;

s4, adding 100 mu L of ascorbic acid phosphoric acid, and reacting at 36-38 ℃ for 45-75 minutes, preferably at 37 ℃ for 60 minutes;

s5, adding 100 mu L of copper sulfate solution, and reacting for 7-15 minutes, preferably 10 minutes, at room temperature;

s6, adding 100 mu L of copper ion probe into 100 mu L of copper ion probe, detecting fluorescence signals, drawing a standard curve, adding 50 mu L of gold nanoparticle mixed solution into 160 mu L of gold nanoparticle mixed solution, detecting visual signals, and preparing a color change diagram;

s7, drawing a standard curve for each detected gradient concentration fluorescence signal, and preparing a color change diagram for each detected gradient concentration visualization signal;

s8, respectively diluting the samples to be tested by 10³Dilution 10⁶Dilution 10⁹Doubling, namely mixing the stock solution of the sample to be detected and the stock solution 10 of the sample to be detected³Multiple dilution solution, original solution of sample to be measured 10⁶Multiple dilution solution, original solution of sample to be measured 10⁹And detecting the dilution by using the methods of S2-S7, substituting the dilution into a standard curve to calculate the concentration of the object to be detected when the fluorescence value of one dilution appears on the standard curve, and comparing the color change with a color change graph.

Specifically, 4 × 10 dilution of norfloxacin-biotin marker is adopted before detection⁴Doubling; diluting the alkaline phosphatase modified with streptavidin by 2000 times by using PBS before detection; the test solution was diluted 10-fold with Tri-HCl buffer solution at pH 7.4 before detection of ascorbyl phosphate.

Embodiments of the present invention will be described in detail with reference to examples, wherein the specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer, and wherein chloroauric acid is purchased from carbofuran; norfloxacin standards used in the experiments were purchased from Sigma, copper ion probes from herrison biotechnology limited, streptavidin-alkaline phosphatase from beijing beioots biosynthesis biotechnology, and NOR polyclonal antibodies were prepared and purified by self-manufacture (preparation methods: wudi, weidong; preparation of norfloxacin polyclonal antibody [ J ]. beijing agriculture, 2015(12):6.) mercaptoazide/mercaptoalkynyl/mercaptopolyethylene glycol from siensexi biotechnology limited. The synthesis and modification of biotin and nanoparticles are prepared with the knowledge of the open literature, and copper ion probes are obtained commercially.

Example 1 preparation of norfloxacin-Biotin marker (NOR-Bio)

1) Azidonation of norfloxacin haptens

Weighing 5mg of norfloxacin hapten (purchased from Shanghai-derived leaf Biotech Co., Ltd.) (1eq) and dissolving in 1mL of DMF, adding 0.7mg of HATU (1.5eq) and oscillating at room temperature for 30min at 300r/min,

② 0.4mg of DIPEA (2eq) is added, the oscillating reaction is carried out for 1h at the room temperature of 300r/min,

③ then adding 1mg of 2-azidoethylamine (5eq), oscillating and reacting at room temperature for 12h at 300r/min,

extracting, separating and purifying by using Thin Layer Chromatography (TLC), wherein a developing agent is as follows: the chloroform/methanol/ammonia was 15/10/3,

observing the separated silica gel plate under an ultraviolet analyzer, and scraping the target product (NOR-N)₃) Characteristic band of (1), the product was extracted with 900. mu.L of methanol.

2) Synthesis of norfloxacin-biotin marker

Weighing 0.2mg of biotin alkyne (2eq) and dissolving in DMF,

dropping biotin alkyne into the extracted product under the condition of shaking at room temperature of 300 r/min;

③ mixing 0.1mol/L of CuSO₄Dissolving in 0.2mol/L ascorbic acid solution, pre-mixing, adding 100 μ L into the reaction system, and reacting at room temperature under shaking at 300r/min for 8 h.

Example 2 Synthesis and modification of gold nanoparticles

1) Gold nanoparticle synthesis

Firstly, soaking a three-neck flask, a rotor and brown ground glass with aqua regia overnight to remove impurities before preparation, cleaning with double distilled water and drying for later use,

② 30mg of sodium citrate is weighed and dissolved in 3mL of water to prepare 1 percent sodium citrate solution,

thirdly, the reacted three-neck flask is placed in a magnetic stirrer to be fixed and connected with a condenser pipe, 1mL of 1% chloroauric acid solution and 99mL of water are added, a rotor is added, and the bottle mouth of the three-neck flask is covered by a plug. Adjusting the temperature of the magnetic stirrer to 200 ℃, adjusting the rotating speed to 999r/min, heating and stirring until boiling,

fourthly, 2mL of preheated sodium citrate solution is quickly added,

fifthly, the solution turns to wine red after becoming yellow and clear and then turning black and purple, after continuing to heat for 15min, removing the heat source, naturally cooling to room temperature,

filtering with 0.45 micron cellulose acetate filter membrane,

keeping at 4 deg.C in brown glass bottle,

the results are shown in FIGS. 2 to 10.

Wherein: FIG. 2 is a colloidal gold UV characterization diagram, FIG. 3 is a colloidal gold transmission electron microscope diagram, FIG. 4 is a colloidal gold particle size characterization diagram, FIG. 5 is a colloidal gold UV contrast diagram (AuNPs, AuNPs @ Azide, AuNPs @ Alkynyl from top to bottom between 515 nm and 535 nm) for colloidal gold and modified Azide/Alkynyl, FIG. 6 is a colloidal gold @ Azide transmission electron microscope diagram, FIG. 7 is a colloidal gold @ Azide particle size characterization diagram, FIG. 8 is a colloidal gold zeta potential diagram for colloidal gold and modified Azide/Alkynyl, FIG. 9 is a colloidal gold @ Alkynyl transmission electron microscope diagram, and FIG. 10 is a colloidal gold Alkynyl particle size characterization diagram.

The above experimental results show that: and the azide/alkynyl is successfully modified on the colloidal gold.

2) Gold nanoparticle modification technology

Gold nanoparticles with the particle size of about 13nm are prepared by a sodium citrate reduction method, the gold nanoparticles are stirred for 24 hours at room temperature through a conjugation reaction, and a certain volume of gold colloid is mixed with methanol/water solution, wherein the methanol/water solution contains excessive sulfhydryl polyethylene glycol or sulfhydryl azide. Typically, 500. mu.L of gold nanosol (1mM) was diluted to 5mL with deionized water. The solution was then adjusted to pH 9 with sodium hydroxide. Under vigorous stirring, adding sulfhydryl polyethylene glycol (100 mu L, methanol 0.01M) and sulfhydryl azide (200 mu L, methanol 0.01M) into the nano-gold solution at the same time, stirring for 24h, and centrifuging for 20min to obtain azide functionalized gold nanoparticles. H for the obtained azide functionalized gold nanoparticles₂O/tBuOH (3X 5mL), centrifuged, and finally redispersed in H₂O/tBuOH (1.5 mL).

Alkynyl functionalized gold nanoparticles were prepared using the same procedure, substituting mercaptoalkynyl (200 μ L, 0.01M) for mercaptoazide.

Example 3 Condition optimization

1. Verifying the function of copper ions and searching for the optimal concentration:

taking Cu with different concentrations (0.1 mM-500 mM)²⁺mu.L was reacted with 100. mu.L of a copper ion probe (working concentration 20. mu.M) available from Herrison Biotech Ltd.

The results of the experiment are shown in FIGS. 11 and 12; wherein FIG. 11 is a graph showing fluorescence intensities at different wavelengths of copper sulfate at different concentrations (wherein 2mM, 1mM, 0.5mM, 0.2mM, 0.1mM, 10mM, 20mM, 50mM, 100mM, 200mM, 500mM are shown in the order from top to bottom at the peak position), and FIG. 12 is a graph showing fluorescence intensity curves at 560nm at different concentrations of copper sulfate.

The optimum reaction concentration of copper ions was determined to be 2mM based on the experimental results of FIGS. 11 and 12.

2. Validation of optimal working concentration of AA:

mixing Cu²⁺Mixing with AA (0-2000 mu M) with different concentrations, reacting for 10min, taking 100 mu L of the mixture, adding a copper ion probe (100 mu L), measuring a fluorescence signal,

the results of the experiment are shown in fig. 13 and 14; wherein FIG. 13 is a graph showing fluorescence intensities of different concentrations of AA at different wavelengths (wherein the peak positions are, in order from top to bottom, 0, 1. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M, 200. mu.M, 500. mu.M, 1000. mu.M, 2000. mu.M); FIG. 14 is a line graph of fluorescence intensity at 560nm for different concentrations of AA.

From the experimental results of FIGS. 13 and 14, it was found that the fluorescence signal decreased with increasing AA concentration, and finally it was found that the degree of change in fluorescence intensity was decreased at more than 100. mu.M.

3. Validation of the optimal working concentration of SA-ALP:

when AAP (200. mu.M, twice the amount of AAP selected for obtaining a concentration of more than 100. mu.M) was mixed with SA-ALP, SA-ALP (1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000) at different dilution times was reacted with AAP for 10min, 50. mu.L of the mixture was reacted with copper ions (50. mu.L) for 10min, and finally a copper ion probe (100. mu.L) was added to react for 10min and the fluorescence intensity thereof was measured. The experimental results are shown in fig. 15 and 16; wherein, FIG. 15 is a fluorescence intensity diagram under different wavelengths with different dilution multiples (from top to bottom, 1:16000, 1:8000, 1:4000, 1:2000, 1:1000, 1:500 at the peak position); FIG. 16 is a line graph of fluorescence intensity at 560nm at different dilution factors.

Similarly, AuNPs @ Azide/AuNPs @ Alkynyl was used instead of the copper ion probe to verify the presence of Cu +, and the experimental results are shown in FIG. 17 and FIG. 18, wherein FIG. 17 is a graph of the color change of colloidal gold and the change of ultraviolet light (1000 μ M to 10 μ M in sequence from top to bottom at 560 nm) caused by different concentrations of AA (10 μ M to 1000 μ M), and FIG. 18 is a graph of the color change of colloidal gold and the change of ultraviolet light (1:500 to 1:32000 in sequence from top to bottom at 560 nm) caused by SA-ALP (1:500 to 1:32000) at different dilution times.

Fig. 17 and 18 illustrate that the aggregation of colloidal gold causes a color change and a red shift of the uv peak.

Example 4 kit composition

The composition of the kit is shown in table 1:

TABLE 1

Example 5 establishment of detection method

All reactions were performed in 96-well plates.

Norfloxacin standards were purified with methanol: PBS (v: v, 1:9) buffer diluted to 10^-6pg/mL-10⁷pg/mL。

(1)1: the antibody was coated on a 96-well plate at 4000-fold dilution (3.5. mu.g/mL), 100. mu.L/well, 4 ℃ overnight.

(2) PBST was washed away with excess antibody 220. mu.L/well.times.5.

(3) 1% BSA blocked the excess pore sites at 150. mu.L/well, 37 ℃ for 1 h.

(4) mu.L of norfloxacin standard and 0.04. mu.g/mL norfloxacin-biotin marker (NOR-Bio) (50. mu.L) were premixed and added to a 96-well plate at 100. mu.L/well for 1h at 37 ℃.

(5) PBST was washed away with 220. mu.L/well.times.5 of excess mixture.

(6)1: biotin-streptavidin was diluted 2000-fold, 100. mu.L/well, 37 ℃ for 1 h.

(7) PBST was washed away with 220. mu.L/well of biotin-streptavidin 5 times.

(8) To each well was added 100 μ L of AAP (dissolved in Tri-Hcl buffer at pH 7.4).

(9) 50 μ L of the reacted mixture was removed and transferred to a brown centrifuge tube, and 50 μ L of CuSO was added to each well₄(2mM) solution, reaction for ten minutes, 10 minutes later, 100. mu.L of copper ion probe was added to the brown centrifuge tube, and fluorescence spectra were recorded with an F97Pro fluorescence spectrophotometer with fluorescence intensity appearing at 560 nm. The fluorescence detection standard curve is shown in FIG. 19, the fluorescence detection line graph is shown in FIG. 20, and the detection range is 3.18X 10^-2～6.88×10³pg mL^-1。

50 μ L of the mixture was added to a solution containing 20 μ L of CuSO₄CuAAC reactions were performed in wells of (2mM) and 160. mu.L of alkyne/azide-AuNPs (1:1, v: v), and the color change of the AuNPs was observed as shown in FIG. 21.

Example 6

Based on the two-channel detection method specificity experiment: to verify the specificity of the sensor, other interfering proteins were detected. We selected several interfering substances, including several other fluoroquinolones, OVA, E2 and SAL. The samples were tested at 3 concentrations (0.1pg/mL, 10pg/mL, 1000pg/mL) using the method of example 3. The results of the fluorescence-based specificity test are shown in FIG. 22.

As shown in FIG. 22, at a concentration of 1000pg/mL, OVA, E2 and SAL showed no cross reaction, while norfloxacin structural analogues showed cross reaction, with decreasing cross reaction rate and higher specificity at low concentration.

Although the present application has been described with reference to preferred embodiments, it is not intended to limit the scope of the claims, and many possible variations and modifications may be made by one skilled in the art without departing from the spirit of the application.