CN113295861A - Rapid detection method of near-infrared fluorescent microsphere immunochromatographic test strip - Google Patents
Rapid detection method of near-infrared fluorescent microsphere immunochromatographic test strip Download PDFInfo
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Abstract
The invention provides a method for rapidly detecting a near-infrared fluorescent microsphere immunochromatographic test strip of common antibiotics in milk, which comprises the following steps of S1: preparing a PVC base plate, and a sample pad, a nitrocellulose membrane and absorbent paper which are overlapped and stuck on the base plate by 2 mm; the sample pad is treated by sample pad diluent, and the nitrocellulose membrane is coated with artificial antigen of antibiotic as a detection line and is coated with goat anti-rabbit antibody as a quality control line; s2: the method has the advantages that the near-infrared fluorescent microspheres are coupled with the sulfanilamide monoclonal antibody, the quinolone monoclonal antibody, the lincomycin monoclonal antibody and the rabbit anti-goat antibody respectively, an immunochromatography test strip system can be effectively improved, and the detection method which is high in sensitivity, good in stability, low in cost and capable of being used for rapidly detecting sulfanilamide, quinolone and lincomycin in milk on site is obtained.
Description
[ technical field ]
The invention relates to the technical field of food safety immunodetection, and relates to a method for rapidly detecting a near-infrared fluorescent microsphere immunochromatographic test strip with multiple residues of sulfanilamide, quinolone and lincomycin.
[ background art ]
Sulfanilamide is a general name of a class of components taking sulfanilamide as a basic structure, and sulfanilamide drugs are synthetic derivatives of sulfanilamide drugs, are first artificial synthetic antibacterial drugs and are widely used for livestock breeding. More than 20 sulfanilamide medicines are frequently detected in food. The antibacterial effect of sulfonamides is discovered as early as 40 years in the 20 th century, so that the sulfonamide is an effective treatment method for livestock and poultry bacterial infection, has an antibacterial effect mainly by competitively combining dihydrofolate synthetase with para aminobenzoic acid during the synthesis of bacterial nucleoprotein, and has good killing effects on gram-positive bacteria, part of gram-negative bacteria and various pathogenic bacteria and coccidia. Because of the broad spectrum and low price of the antibacterial effect of the sulfonamides, a plurality of sulfonamides are applied in the livestock breeding industry at present. In recent years, the residue phenomenon of sulfonamides is relatively serious. In view of this, the maximum residual limit of livestock and poultry products in China and European Union is 100ng/g, and the limit of the livestock and poultry products in the United states is 10 ng/g.
Quinolone drugs are also known as pyridonic acids or pyridonic acids, and are synthetic antibacterial drugs. The development of quinolone drugs, particularly fluoroquinolone antibiotics, has been in the past for over 50 years and has been widely used for the prevention and treatment of human and animal diseases. Carbostyril is a broad-spectrum synthetic antibacterial agent used for livestock and aquaculture, and the residue of carbostyril drugs attracts wide attention at home and abroad in recent years, on the basis of which, various countries have clear limits on the residue of carbostyril drugs such as difloxacin, enrofloxacin and the like in livestock products. Wherein, the maximum residual limit amount of quinolone drug residues in China and European Union is 10 ng/g.
Lincomycin is effective on gram-positive bacteria such as pneumococcus and gram-negative bacteria, and can inhibit peptide acyltransferase, prevent ribosome synthesis and kill bacteria. Lincomycin has no obvious toxicity, but the residue of lincomycin in food can cause the generation of microbial drug resistance. The residual situation of lincomycin drugs has therefore received attention from many countries. The maximum residual limit of lincomycin in milk prescribed by European Union and China is 150 mug/kg, and the maximum residual limit of lincomycin prescribed by Switzerland, Britain, Belgium and other countries is 10-50 mug/kg.
Drug resistance is an increasingly serious problem, and sulfonamides, quinolones and lincomycin drugs can increase the drug resistance of bacteria such as salmonella and escherichia coli, so that the effectiveness of the drugs is reduced. In addition, quinolone drugs cause allergic reactions that damage the central nervous system and the like. Although the residual of the three antibiotics is limited in quantity standard at home and abroad, the problem of overproof sulfonamide residues in milk and dairy products occurs due to the overlapping of drug abuse phenomena such as overdose, overrange use, non-compliance with withdrawal periods and the like caused by blind pursuit of economic benefits, so that the residual of the three antibiotics in the milk is very important.
The instrumental analysis method is a gold standard for detecting antibiotic drug residues at present, comprises a liquid chromatography-mass spectrometry (LC-MS) method, has high sensitivity, good specificity, strong separation capacity and accurate and reliable measurement results, and can separate and analyze a multi-component mixture. However, this method relies on expensive equipment, complicated pretreatment processes, and specialized operators, and thus it is difficult to achieve rapid on-site detection. In recent years, immunoassay is a technique for detecting based on a specific reaction of an antigen and an antibody, and has been increasingly introduced to field detection due to its good specificity, convenience and rapidity. The immunoassay mainly comprises Enzyme-linked immunosorbent assay (ELISA), lateral flow immunochromatography and the like, and compared with an instrumental analysis method, the ELISA is simpler and more convenient to operate, can perform qualitative and quantitative analysis, and has the advantages of good recovery rate, strong specificity and the like, so that the immunoassay method is widely applied. The immunochromatography lateral flow technology is simple and convenient to operate, low in cost, free of complex instruments and professional personnel, and widely applied to detection of veterinary drug residues in food safety. However, the immunochromatographic test strip reported at present has low detection sensitivity, mainly detects a single antibiotic, and cannot detect multiple antibiotics simultaneously.
[ summary of the invention ]
In order to overcome the problems in the prior art, the invention provides a method for rapidly detecting a near-infrared fluorescent microsphere immunochromatographic test strip with multiple residues of sulfanilamide, quinolone and lincomycin.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention relates to a near-infrared fluorescent microsphere-based immunochromatography test strip for multi-detection of three antibiotic residues in milk, which comprises a PVC (polyvinyl chloride) base plate, a nitrocellulose membrane, a sample pad and absorbent paper, wherein the sample pad is treated by a sample pad diluent, an artificial antigen coated with antibiotics is used as a detection line on the nitrocellulose membrane, and a goat anti-rabbit antibody coated on the nitrocellulose membrane is used as a quality control line, one end of the nitrocellulose membrane is adhered with the sample pad, and the other end of the nitrocellulose membrane is adhered with the absorbent paper.
The test strip realizes detection based on the principle of a competition method, wherein an antibiotic standard substance in a solution to be detected and a whole antigen of an antibiotic fixed on a T line can be specifically combined with a monoclonal antibody of the antibiotic on the near-infrared fluorescent microsphere. When the antibiotic exists in the sample solution, the fluorescent microsphere-antibody is firstly combined with antibiotic molecules, when the liquid flows through the T line, the microsphere-antibody is combined with antigen molecules, so that the microsphere-antibody cannot be combined with the antigen on the T line, and the near infrared fluorescent microsphere-antibody-antigen molecule combination continuously moves forwards; when no antibiotic molecules are present in the sample solution, the fluorescent microsphere-antibody binds to the antigen molecules on the T-line, thereby immobilizing the fluorescent microsphere on the T-line. I.e. the fluorescence intensity on the T-line is inversely proportional to the concentration of antibiotic in the sample, the higher the antibiotic content, the lower the fluorescence intensity on the T-line and vice versa.
The application also provides a preparation method of the near-infrared fluorescent microsphere labeled antibiotic antibody compound, which comprises the following steps:
And 2, preparing 10mg/mL EDC and 10mg/mL sulfo-NHS aqueous solution (which is used as prepared), sequentially adding 25 mu LEDC and 75 mu Lsulfo-NHS solution into the fluorescent microsphere dispersion, uniformly mixing by ultrasonic waves, and activating at room temperature for 10min in a rotating state.
And 3, centrifuging the activated fluorescent microspheres at 14000r/min for 15min at 4 ℃, discarding the supernatant, washing the microspheres for 3 times by using the coupling solution according to the method in the step 1, redissolving the microspheres into 400 mu L of the coupling solution, ultrasonically dispersing the microspheres uniformly, adding a proper amount of antibody, coupling the microspheres for 3h at room temperature in a rotating state, and ultrasonically dispersing the microspheres for several times to avoid microsphere aggregation.
And 4, adding 30 mu L of 10% ethanolamine aqueous solution into the coupled fluorescent microspheres, sealing for 30min at room temperature under a rotating state, centrifuging for 15min at 4 ℃ at 14000r/min, then discarding the supernatant, adding 1mL of 10% BSA solution, dispersing uniformly by ultrasonic, and sealing for 2h at room temperature under a rotating state.
And 5, centrifuging the completely sealed fluorescent microsphere dispersion solution at 14000r/min at 4 ℃ for 15min, then discarding the supernatant, washing for 3 times by using a final washing solution according to the method in the step 1, redissolving into 1mL of the solution, ultrasonically dispersing uniformly, and storing at 4 ℃ in a dark place for later use.
Preferably, the coupling amount of the near-infrared fluorescent microspheres and the antibodies is 60-90 mug of antibodies per mg of microspheres.
Preferably, the pH selected during the coupling is between 6 and 8.
The invention also provides a preparation method of the near-infrared fluorescent microsphere immunochromatography test strip for sulfanilamide, quinolone and lincomycin in milk, which comprises the following steps:
And 2, treating the cellulose nitrate membrane, diluting the sulfanilamide whole antigen, the quinolone whole antigen, the lincomycin antigen and the goat anti-rabbit secondary antibody to the required concentration by using a coating solution, spraying the four solutions onto an NC membrane by using a membrane scribing instrument at the speed of 0.8 mu L/cm in sequence to be respectively used as a detection line (T1 line, T2 line and T3 line) and a quality control line (C line) of the test strip, wherein the distance from T1 to a pad end of the sample is 3mm, the distance from T2 to T13 mm, the distance from T3 to T23 mm and the distance from C line to T33 mm, and drying for 12 hours in an electrothermal blowing drying box at 37 ℃ for later use.
And 3, sticking the nitrocellulose membrane to a fixed area of the PVC base plate, sticking the absorbent paper and the processed sample pad to the upper side and the lower side of the NC membrane in a mode of overlapping 2mm, cutting the assembled PVC back plate into strip test strips of 80mm multiplied by 3.9mm by a cutting machine, placing the test strips into an aluminum foil bag added with a silica gel drying agent, and storing the test strips in a brown drying dish at room temperature for later use.
Preferably, the detection line on the nitrocellulose membrane is coated with sulfanilamide whole antigen with the concentration of 0.6-1.2mg/mL, quinolone whole antigen with the concentration of 0.6-1.2mg/mL and lincomycin whole antigen with the concentration of 0.6-1.2mg/mL, and the quality control line on the nitrocellulose membrane is coated with goat anti-rabbit antibody with the concentration of 0.5 mg/mL.
Preferably, the trehalose concentration in the coating liquid is 0.5% -2.5%.
Preferably, 2 × PBS is selected as the sample pad treatment buffer system.
Preferably, 0.12% to 0.24% EDTA is added to the sample pad treatment solution.
The invention provides a method for rapidly detecting a near-infrared fluorescent microsphere immunochromatographic test strip of sulfanilamide, quinolone and lincomycin in milk, which comprises the following steps:
And 2, accurately sucking 100 mu L of incubated detection liquid by using a pipette, loading the detection liquid into a sample adding hole of a horizontally placed test strip, enabling the near-infrared fluorescent microspheres to flow to the end of the water absorption paper under the siphoning action along with the liquid to be detected, reacting for a certain time, and detecting by using a fluorescence lateral flow chromatography analyzer, so that a result can be obtained within 10 s.
And 3, recording the fluorescence intensity of each point by the detector to draw a scanning curve, wherein the x axis is a position coordinate, the y axis is the fluorescence intensity of the corresponding position, and the integrals of the scanning curves of the T line and the C line and the x axis are respectively recorded as a T value and a C value to represent the fluorescence intensity of the T, C line.
And 5, detecting the milk sample according to the steps 1-3, and substituting the obtained numerical value into the standard curve obtained in the step 4 to obtain the concentration of the substance to be detected in the sample.
Preferably, 1.0-2.0 μ g of the near-infrared fluorescent microsphere-sulfanilamide antibody complex, 1.0-2.0 μ g of the near-infrared fluorescent microsphere-quinolone antibody complex and 1.0-2.0 μ g of the near-infrared fluorescent microsphere-lincomycin antibody complex are added into 100 μ L of the test solution.
Preferably, the concentration of PVP in the milk diluent is between 1% and 5%.
Preferably, the milk is mixed with the diluent according to the ratio of 1: 1-1: 5 was diluted.
Preferably, the sample is loaded for 10-20min before reading.
The immunochromatographic test strip for quantitatively detecting sulfanilamide, quinolone and lincomycin in milk is combined with an immunochromatographic quantitative analyzer, three antibiotics in milk can be rapidly and quantitatively detected, the detection time is shortened, the large-batch samples can be rapidly and accurately screened, the detection sensitivity of sulfanilamide can reach 46.7pg/mL, 27.6pg/mL and 51.4pg/mL, and compared with a common colloidal gold immunochromatographic test strip detection method, the method can obtain a detection result within 10-15min, and has the characteristics of rapidness and sensitivity.
[ description of the drawings ]
FIG. 1 is a schematic structural diagram of a method for rapidly detecting a near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk.
FIG. 2 is a scanning electron microscope image of the near-infrared fluorescent microsphere.
FIG. 3 is a transmission electron microscope image of the near-infrared fluorescent microsphere.
FIG. 4 is a particle size distribution diagram of the near-infrared fluorescent microsphere.
FIG. 5 optimization of near-infrared fluorescent microsphere-antibiotic antibody probe coupling amount.
FIG. 6 optimization of pH for near-infrared fluorescent microsphere-antibiotic antibody probe coupling.
FIG. 7 optimization of milk diluent buffer system.
FIG. 8 shows the immunoreaction kinetics curve of the near-infrared fluorescent microsphere immunochromatographic test strip.
FIG. 9 is a competitive inhibition curve of the rapid detection method of the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk.
FIG. 10 shows the cross-reaction result of the method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk.
[ detailed description of the invention ]
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail below with reference to the accompanying drawings and 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.
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.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When a component is referred to as being "connected" to another component, it can be directly connected to the other component or intervening components may also be present.
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 invention belongs.
The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Some embodiments of the invention are described in detail below with reference to the accompanying drawings. The embodiments described below and the features of the embodiments can be combined with each other without conflict.
Referring to fig. 1 to 10, a method 1 for rapidly detecting a near-infrared fluorescent microsphere immunochromatographic test strip of common antibiotics in milk according to the present invention includes the following steps,
s1: preparing a PVC base plate, and a sample pad, a nitrocellulose membrane and absorbent paper which are overlapped and stuck on the base plate by 2 mm;
the sample pad is treated by sample pad diluent, and the nitrocellulose membrane is coated with an artificial antigen of antibiotic as a detection line and a goat anti-rabbit antibody as a quality control line;
s2: respectively coupling the near-infrared fluorescent microspheres with a sulfanilamide monoclonal antibody, a quinolone monoclonal antibody, a lincomycin monoclonal antibody and a rabbit anti-goat antibody to obtain a near-infrared fluorescent microsphere-sulfanilamide monoclonal antibody labeled compound, a near-infrared fluorescent microsphere-quinolone monoclonal antibody labeled compound, a near-infrared fluorescent microsphere-lincomycin monoclonal antibody labeled compound and a near-infrared fluorescent microsphere-rabbit anti-goat antibody labeled compound, namely the near-infrared fluorescent microsphere probe;
s3: and (3) uniformly mixing the fluorescent microsphere probe and a sample to be detected, incubating, and detecting by using a near-infrared fluorescent microsphere immunochromatography test strip.
The invention aims to solve the problems of overcoming the defects and shortcomings in the prior art, and provides a method for simultaneously and rapidly screening three antibiotics, namely sulfonamide, quinolone and lincomycin in milk with high sensitivity by using a novel near-infrared II-region fluorescent microsphere; meanwhile, an immunochromatography test strip system is improved, and the detection method which is high in sensitivity, good in stability, low in cost and capable of being used for rapidly detecting sulfonamide, quinolone and lincomycin in milk on site is obtained, so that the detection time is greatly shortened, and the detection sensitivity is improved.
In a preferred embodiment, the three detection lines on the nitrocellulose membrane are respectively coated with sulfonamide whole antigen with concentration of 0.9mg/mL, quinolone whole antigen with concentration of 0.9mg/mL and lincomycin whole antigen with concentration of 1.2mg/mL, and the quality control line on the nitrocellulose membrane is coated with goat anti-rabbit antibody with concentration of 0.5 mg/mL.
In a preferred embodiment, a large number of near-infrared region II fluorophores are encapsulated in the near-infrared fluorescent microspheres, and uniformly dispersed polystyrene microspheres with a large number of carboxyl groups are modified on the surfaces of the microspheres; the scanning electron microscope and the transmission electron microscope can be respectively used for representing the surface and the internal forms of the near-infrared fluorescent microspheres; the scanning electron microscope of the near-infrared fluorescent microsphere shows that the fluorescent microsphere is a monodisperse sphere, has consistent size, uniform shape and flat surface, and the transmission electron microscope shows that the fluorescent microsphere has uniform internal texture, good dispersibility and hydrophilicity in a water phase and is not easy to agglomerate; the particle size distribution of the near-infrared fluorescent microspheres is analyzed through Dynamic Light Scattering (DLS), and the diameter of the near-infrared fluorescent microspheres is 359.6 +/-2.335 nm, and the peak shape is symmetrical and sharp.
In a preferred embodiment, the sulfanilamide monoclonal antibody is a sulfanilamide broad-spectrum monoclonal antibody, the quinolone monoclonal antibody is a quinolone broad-spectrum monoclonal antibody, and the lincomycin monoclonal antibody is a lincomycin broad-spectrum monoclonal antibody.
In a preferred embodiment, the coupling method in step S2 includes the following steps:
and 5, centrifuging the completely sealed fluorescent microsphere dispersion solution at 14000r/min at 4 ℃ for 15min, then discarding the supernatant, washing for 3 times by using a final washing solution according to the method in the step 1, redissolving into 1mL of the solution, ultrasonically dispersing uniformly, and storing at 4 ℃ in a dark place for later use.
In a preferred embodiment, the initial washing liquid in the step 1 is MES solution containing 0.1M.
In a preferred embodiment, the coupling solution in step 3 is a borate buffer solution containing 0.14M boric acid and 0.015M sodium tetraborate decahydrate.
In a preferred embodiment, the final washing solution in the step 5 is a boric acid buffer solution containing 0.5% BSA and 0.4% Tween-20; the detection line adopts a competitive immunization mode.
In a preferred embodiment, the method for preparing the assay strip comprises the following steps:
and 3, sticking the nitrocellulose membrane to a fixed area of the PVC base plate, sticking the absorbent paper and the processed sample pad to the upper side and the lower side of the NC membrane in a mode of overlapping 2mm, cutting the assembled PVC back plate into strip test strips of 80mm multiplied by 3.9mm by a cutting machine, placing the test strips into an aluminum foil bag added with a silica gel drying agent, and storing the test strips in a brown drying dish at room temperature for later use.
In a preferred embodiment, the method comprises the steps of:
2, accurately sucking 100 mu L of incubated detection liquid by using a pipettor, loading the detection liquid into a sample adding hole of a horizontally placed test strip, enabling the fluorescent microspheres to flow towards the end of the water absorption paper under the siphoning action along with the liquid to be detected, reacting for a certain time, and detecting by using a fluorescent lateral flow chromatography analyzer, wherein a result can be obtained within 10 s;
The following are specific examples of the present application:
example 1:
the near-infrared fluorescent microsphere is a monodisperse polystyrene microsphere which is coated with a large amount of near-infrared region II fluorophores and is rich in functionalized carboxyl on the surface, and the characterization method comprises the following steps:
(1) the near-infrared fluorescent microspheres were stored in ultrapure water in the form of a 10mg/mL dispersion. In order to characterize the microscopic morphology, a scanning electron microscope is selected to observe the surface morphology, fluorescent microsphere powder is fixed on an electron microscope objective table by using double-layer conductive adhesive, floating particles which are not firmly adhered are blown off by using an ear washing ball, and scanning imaging is carried out by using SEM (scanning Electron microscope) with the amplification voltage of 3.0kV being 70.0k times. The surface topography of the near-infrared fluorescent microspheres is shown in fig. 2.
(2) Selecting a transmission electron microscope to observe the internal state, diluting the fluorescent microsphere dispersion liquid to 30 mu g/mL by ultrapure water, sucking the fluorescent microsphere dispersion liquid by a dropper, dripping the fluorescent microsphere dispersion liquid on a copper net, standing the copper net for 10min at room temperature, sucking water on the copper net by absorbent paper, and putting the copper net in a TEM (transmission electron microscope) to perform imaging by amplifying the copper net by 80.0k times at an acceleration voltage of 8.0 kV. The internal state of the near-infrared fluorescent microspheres is shown in fig. 3.
(3) The fluorescent microsphere dispersion was diluted to 10. mu.g/mL with ultrapure water and its average particle size was determined with a Malvern dynamic light scattering analyzer Nano ZS. The particle size distribution diagram of the near-infrared fluorescent microspheres is shown in fig. 4.
Example 2:
the detection method of the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk comprises the following steps:
(1) preparing a near-infrared fluorescent microsphere probe: dispersing and cleaning the near-infrared fluorescent microspheres for 3 times by using a primary cleaning solution; adding 25 μ L EDC with concentration of 10mg/mL and 75 μ L sulfo-NHS water solution with concentration of 10mg/mL into the solution, and activating at room temperature for 10min under rotation state; washing the activated fluorescent microspheres with a coupling solution, adding a proper amount of antibody, coupling for 3 hours at room temperature in a rotating state, and ultrasonically dispersing for several times to avoid microsphere aggregation; adding 30 mu L of 10% ethanolamine aqueous solution into the coupled fluorescent microspheres, and sealing in a rotating state; centrifuging the completely sealed fluorescent microsphere dispersion at 14000r/min at 4 ℃ for 15min, discarding the supernatant, washing with the final washing solution for 3 times, redissolving into 1mL of the final washing solution, dispersing uniformly by ultrasonic, and storing at 4 ℃ in a dark place for later use.
(2) Preparing an immunochromatography test strip: the nitrocellulose membrane 3 is pasted on the PVC bottom plate 2, the sample pad 1 is pasted at one end of the nitrocellulose membrane 3, and the absorbent paper 8 is pasted at the other end. The detection line 4, the detection line 5, the detection line 6 and the control line 7 are arranged on the nitrocellulose membrane 3, the sulfonamide whole antigen is coated in the detection line 4, the quinolone whole antigen is coated in the detection line 5, the lincomycin whole antigen is coated in the detection line 6, and the goat anti-rabbit antibody is coated in the control line 7. The test strips 4, 5 and 6 are positioned on the left side of the nitrocellulose membrane 3, the control line 7 is positioned on the right side of the nitrocellulose membrane, the distance between the test strip 4 and the test strip 5 is 3 mm-5 mm, the distance between the test strip 5 and the test strip 6 is 3 mm-5 mm, and the distance between the test strip 6 and the control strip 7 is 3 mm-5 mm. In this example, the test strips 4, 5, and 6 were diluted with the coating solution to the desired concentrations. Respectively spraying the test strips 4, 5 and 6 and the control strip 7 coating solution on the positions, corresponding to the test strips 4, 5 and 6 and the control strip 7, of the nitrocellulose membrane 3, wherein the spraying amount is 0.8 mu L/cm; and then the coated nitrocellulose membrane is dried at 37 ℃. And cutting the test paper board into test paper strips, and filling the test paper strips into test paper strip card shells to form the immunochromatographic test paper strips.
(3) And (3) quantitative detection: incubating the near-infrared fluorescent microsphere probe with a sample, standing for reaction for 10min, and sucking 100 mu L of sample pad added with an immunochromatography test strip for detection. The detection principle of the fluorescent test strip is that a sample to be detected flows to one end of the absorbent paper under the siphoning effect, the near-infrared fluorescent microsphere-antibiotic antibody conjugate can be captured by the antibiotic antigen on the T line, and the signal values of the test bands 4, 5 and 6 and the control band 7 are detected by an instrument, so that the concentrations of sulfanilamide, quinolone and lincomycin in milk can be quantitatively detected.
Specifically, a series of standard curves of substances to be detected with different concentrations are configured, then a fluorescence immunochromatography instrument is used for detection, T values corresponding to standard solutions with different concentrations are used as the standard curves, then the unknown samples to be detected are treated in the same way to obtain corresponding T values, and the content of the substances to be detected is obtained according to the standard curves.
Example 3:
an optimization of a near-infrared fluorescent microsphere immunochromatographic test strip for detecting sulfanilamide, quinolone and lincomycin in milk,
(1) optimizing the preparation condition of the near-infrared fluorescent microsphere fluorescent probe, namely optimizing the coupling amount and the coupling pH value;
referring to example 2, the coupling amount and coupling pH of the near-infrared fluorescent microspheres and the antibiotic antibody are changed, the preparation of the near-infrared fluorescent microsphere fluorescent probe is performed under the same other conditions as in example 1, the immunochromatographic test strip is further prepared, the fluorescence intensity of the test strip T line is detected by a fluorescence detector, the optimized coupling amount result is shown in fig. 5, and the optimal coupling amount is 60 μ g/mg; the coupling pH optimization results are shown in FIG. 6, with an optimum pH of 6.
(2) Optimizing a detection buffer solution;
in the experiment, diluted milk is used as a sample loading detection solution, a buffer system of the dilution solution has a large influence on a detection result, the sample loading detection solution is prepared according to example 2, the immunochromatographic test strip is further prepared, the fluorescence intensity of a T line of the test strip is detected by a fluorescence detector, the optimized result of the buffer system is shown in figure 7, and the test strip has good performance when the buffer system is a PBS (phosphate buffer solution) system.
(3) Performing a test strip immunoreaction dynamics experiment;
the determination of the reaction time of the near-infrared fluorescent microsphere immunochromatographic test strip is the premise of quantitative detection of the test strip. And (3) measuring the T-line immunoreaction kinetic curve of the near-infrared fluorescent microsphere immunochromatographic test strip, and referring to example 2, after a sample is dripped into a sample adding hole, reading the T-line value in the test strip by using a fluorescent immunochromatographic reading instrument every five minutes, tracking and recording for 20 minutes, wherein the result is shown in FIG. 8. The change trend of the three antibiotics is the same, and it can be seen that the T line signal value of the negative sample is stable after 10min, so the quantitative detection time is selected to be 10 min.
Example 4:
taking standard sulfamethazine, ciprofloxacin and lincomycin as examples to establish a standard curve of the near-infrared fluorescent microsphere immunochromatographic test strip;
1. experimental Material
The sulfanilamide monoclonal antibody, the quinolone monoclonal antibody and the lincomycin monoclonal antibody are commercialized reagents and are purchased from Beijing Vidervikang biotechnology limited. Goat anti-rabbit and rabbit anti-goat antibodies were purchased from Changsha Boyou Biotech, Inc.
The preparation method of the immunochromatographic test strip is the same as that of example 1.
2. The standard substance is prepared by preparing a series of standard solutions with the concentrations of 0, 0.012, 0.048, 0.1953, 0.781, 3.125, 12.5 and 50ng/mL respectively by using diluted milk for the sulfamethazine, and the standard solutions are used for test strip detection.
3. Testing the test strip;
and (3) repeatedly detecting each concentration for three times, reading the T value of the test strip by using a fluorescence immunochromatography reading instrument after 10min, and performing Logit-log linear regression on the concentration and the T value to fit a standard curve. The detection sensitivity is defined as the mean value of the detection values of 20 blank samples minus 3 times of standard deviation (AV-3SD) corresponding to sulfadiazine concentration, and the lowest point of the linear range is defined as the mean value minus 5 times of standard deviation (AV-5SD) corresponding to sulfadiazine concentration.
4. The result is shown in FIG. 9, the sulfanilamide detection sensitivity of the near-infrared fluorescent microsphere immunochromatography test strip is 46.7pg/mL, the limit of quantitation is 0.119ng/mL, and the detection range is 0.119-50 ng/mL; the quinolone detection sensitivity is 27.6pg/mL through calculation, the limit of quantitation is 0.054ng/mL, and the detection range is 0.054-50 ng/mL; the lincomycin detection sensitivity is 51.4pg/mL, the limit of quantitation is 0.136ng/mL, and the detection range is 0.136-50 ng/mL.
Example 5;
accuracy and precision of near-infrared fluorescent microsphere immunochromatographic test strip
1. The accuracy and precision of the same batch of near-infrared immunochromatographic test strip in milk are evaluated by a standard addition recovery experiment. Under the optimized optimal condition, the sulfamethazine, the ciprofloxacin and the lincomycin are added to milk to be at three concentrations of high, medium and low, each concentration is detected for 6 times in parallel, and the added standard recovery rate and the intra-batch Coefficient of Variation (CV) are calculated. Wherein, the recovery rate of the added standard is the percentage of the detected concentration and the actual concentration, and the closer the recovery rate of the added standard is to 100 percent, the higher the accuracy of the method is.
2. As shown in Table 1, the recovery rate of the sym-sulfamethazine is 102.6 to 112.3 percent; the recovery rate of the ciprofloxacin is 105.6-113.0%; the recovery rate of the lincomycin is 103.0-116.3%, and the recovery rates are all less than 120%, which indicates that the method has good accuracy. In the process of adding the standard, the CV values in the batch are all lower than 10 percent, which indicates that the method has good precision.
TABLE 1 detection accuracy and precision of near infrared fluorescent microsphere immunochromatographic test strip
Example 6;
cross-reactivity rate of common antibiotics in milk
1. And respectively preparing 50ng/mL solutions of aflatoxin B1, tetracycline, erythromycin, melamine, gentamicin, chloramphenicol, sulfamethazine, ciprofloxacin and lincomycin from the diluted milk, respectively detecting the milk samples by using a multiplex detection test strip, comparing whether a significant difference exists between the detection values of the milk samples and the negative sample detection values, and detecting three samples in parallel. And imaging the test strip obtained by the experiment by using a near-infrared fluorescence imaging system, and performing specificity evaluation on the established immunochromatography multi-linked test strip detection method.
2. As a result, as shown in fig. 10, there was no signal in the T-line when the samples containing sulfamethazine, ciprofloxacin, and lincomycin were tested, and there was a signal in the T-line when the samples containing aflatoxin B1, tetracycline, erythromycin, melamine, gentamicin, and chloramphenicol and the blank samples were tested. Therefore, the immunity lateral flow chromatography detection system established based on the near infrared fluorescent microspheres has good specificity.
Compared with the prior art, the method 1 for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip of common antibiotics in milk overcomes the defects and shortcomings in the prior art, and provides a method for rapidly screening three antibiotics, namely sulfonamide, quinolone and lincomycin in milk at the same time by using a novel near-infrared II-region fluorescent microsphere; meanwhile, an immunochromatography test strip system is improved, and the detection method which is high in sensitivity, good in stability, low in cost and capable of being used for rapidly detecting sulfonamide, quinolone and lincomycin in milk on site is obtained, so that the detection time is greatly shortened, and the detection sensitivity is improved.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (10)
1. A method for rapidly detecting a near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk is characterized by comprising the following steps: comprises the following steps of (a) carrying out,
s1: preparing a PVC base plate, and a sample pad, a nitrocellulose membrane and absorbent paper which are overlapped and stuck on the base plate by 2 mm;
the sample pad is treated by sample pad diluent, and the nitrocellulose membrane is coated with an artificial antigen of antibiotic as a detection line and a goat anti-rabbit antibody as a quality control line;
s2: respectively coupling the near-infrared fluorescent microspheres with a sulfanilamide monoclonal antibody, a quinolone monoclonal antibody, a lincomycin monoclonal antibody and a rabbit anti-goat antibody to obtain a near-infrared fluorescent microsphere-sulfanilamide monoclonal antibody labeled compound, a near-infrared fluorescent microsphere-quinolone monoclonal antibody labeled compound, a near-infrared fluorescent microsphere-lincomycin monoclonal antibody labeled compound and a near-infrared fluorescent microsphere-rabbit anti-goat antibody labeled compound, namely the near-infrared fluorescent microsphere probe;
s3: and (3) uniformly mixing the fluorescent microsphere probe and a sample to be detected, incubating, and detecting by using a near-infrared fluorescent microsphere immunochromatography test strip.
2. The method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk according to claim 1, which is characterized in that: three detection lines on the nitrocellulose membrane are respectively coated with sulfanilamide whole antigen with the concentration of 0.9mg/mL, quinolone whole antigen with the concentration of 0.9mg/mL and lincomycin whole antigen with the concentration of 1.2mg/mL, and a quality control line on the nitrocellulose membrane is coated with goat anti-rabbit antibody with the concentration of 0.5 mg/mL.
3. The method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk according to claim 1, which is characterized in that: a large number of near-infrared region II fluorophores are packaged in the near-infrared fluorescent microspheres, and uniformly dispersed polystyrene microspheres with a large number of carboxyl groups are modified on the surfaces of the near-infrared fluorescent microspheres; the scanning electron microscope and the transmission electron microscope can be respectively used for representing the surface and the internal forms of the near-infrared fluorescent microspheres; the scanning electron microscope of the near-infrared fluorescent microsphere shows that the fluorescent microsphere is a monodisperse sphere, has consistent size, uniform shape and flat surface, and the transmission electron microscope shows that the fluorescent microsphere has uniform internal texture, good dispersibility and hydrophilicity in a water phase and is not easy to agglomerate; the particle size distribution of the near-infrared fluorescent microspheres is analyzed through Dynamic Light Scattering (DLS), and the diameter of the near-infrared fluorescent microspheres is 359.6 +/-2.335 nm, and the peak shape is symmetrical and sharp.
4. The method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk according to claim 1, which is characterized in that: the sulfanilamide monoclonal antibody is a sulfanilamide broad-spectrum monoclonal antibody, the quinolone monoclonal antibody is a quinolone broad-spectrum monoclonal antibody, and the lincomycin monoclonal antibody is a lincomycin broad-spectrum monoclonal antibody.
5. The method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk according to claim 1, which is characterized in that: the coupling method in step S2 includes the steps of:
step 1, adding 100 mu L of near-infrared fluorescent microsphere dispersion liquid into 1mL of primary washing liquid, performing ultrasonic dispersion uniformly, centrifuging at 14000r/min at 4 ℃ for 15min, then discarding supernatant, repeating the operation to clean the fluorescent microspheres for 3 times, redissolving the fluorescent microspheres into 1mL of primary washing liquid, and performing ultrasonic dispersion uniformly;
step 2, preparing 10mg/mL EDC and 10mg/mL sulfo-NHS aqueous solution (for preparation), sequentially adding 25 mu LEDC and 75 mu L sulfo-NHS solution into the fluorescent microsphere dispersion, ultrasonically mixing uniformly, and activating at room temperature for 10min in a rotating state;
step 3, centrifuging the activated fluorescent microspheres at 14000r/min for 15min at 4 ℃, discarding the supernatant, washing 3 times by using a coupling solution according to the method in the step 1, redissolving the mixture into 400 mu L of the coupling solution, ultrasonically dispersing the mixture uniformly, adding a proper amount of antibody, coupling the mixture for 3h at room temperature in a rotating state, and ultrasonically dispersing the mixture for several times to avoid microsphere aggregation;
step 4, adding 30 mu L of 10% ethanolamine aqueous solution into the coupled fluorescent microspheres, sealing for 30min at room temperature under a rotating state, centrifuging for 15min at 4 ℃ at 14000r/min, then discarding the supernatant, adding 1mL of 10% BSA solution, ultrasonically dispersing uniformly, and sealing for 2h at room temperature under the rotating state;
and 5, centrifuging the completely sealed fluorescent microsphere dispersion solution at 14000r/min at 4 ℃ for 15min, then discarding the supernatant, washing for 3 times by using a final washing solution according to the method in the step 1, redissolving into 1mL of the solution, ultrasonically dispersing uniformly, and storing at 4 ℃ in a dark place for later use.
6. The method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk according to claim 5, which is characterized in that: the initial washing liquid in the step 1 is MES solution containing 0.1M.
7. The method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk according to claim 5, which is characterized in that: the coupling solution in step 3 is a borate buffer solution containing 0.14M boric acid and 0.015M sodium tetraborate decahydrate.
8. The method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk according to claim 1, which is characterized in that: the final washing solution in the step 5 is a boric acid buffer solution containing 0.5% BSA and 0.4% Tween-20; the detection line adopts a competitive immunization mode.
9. The method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk according to claim 1, which is characterized in that: the preparation method of the assay test strip comprises the following steps:
step 1, preparing a sample pad: the sample pad is made of glass fiber, the sample pad is cut into strips of 30cm multiplied by 28mm, the strips are laid on a grid plate, 7mL of phosphate buffer solution containing 1.2% Tris, 0.4% BSA, 0.4% Tween-20 and 0.12% EDTA is taken to treat the sample pad, the sample pad is fully wetted, and then the sample pad is placed in an electric heating air blast drying box for 12 hours at 37 ℃ until being dried;
step 2, treating the nitrocellulose membrane, diluting sulfanilamide whole antigen, quinolone whole antigen, lincomycin antigen and goat anti-rabbit secondary antibody to required concentration by using a coating solution containing 2% trehalose and 5% methanol, spraying the four solutions onto an NC membrane by using a membrane scribing instrument at the speed of 0.8 mu L/cm in sequence, and respectively serving as a detection line (T1 line, T2 line and T3 line) and a quality control line (C line) of a test strip, wherein the distance from T1 to a pad end of the sample is 3mm, the distance from T2 to T13 mm, the distance from T3 to T23 mm and the distance from C line to T33 mm are respectively, and drying is carried out for 12 hours at the temperature of 37 ℃ in an electrothermal blowing drying box for later use;
and 3, sticking the nitrocellulose membrane to a fixed area of the PVC base plate, sticking the absorbent paper and the processed sample pad to the upper side and the lower side of the NC membrane in a mode of overlapping 2mm, cutting the assembled PVC back plate into strip test strips of 80mm multiplied by 3.9mm by a cutting machine, placing the test strips into an aluminum foil bag added with a silica gel drying agent, and storing the test strips in a brown drying dish at room temperature for later use.
10. The method for rapidly detecting the near-infrared fluorescent microsphere immunochromatographic test strip for common antibiotics in milk according to claim 1, which is characterized in that: the method comprises the following steps:
step 1, performing ultrasonic treatment on a near-infrared fluorescent probe for 1min to change the aggregation state of the near-infrared fluorescent probe, adding the near-infrared fluorescent probe into a solution to be detected, and incubating for 10min to ensure that the monoclonal antibody on the microsphere can be fully combined with antigen molecules;
2, accurately sucking 100 mu L of incubated detection liquid by using a pipettor, loading the detection liquid into a sample adding hole of a horizontally placed test strip, enabling the fluorescent microspheres to flow towards the end of the water absorption paper under the siphoning action along with the liquid to be detected, reacting for a certain time, and detecting by using a fluorescent lateral flow chromatography analyzer, wherein a result can be obtained within 10 s;
step 3, the detector records the fluorescence intensity of each point and draws a scanning curve, wherein the x axis is a position coordinate, the y axis is the fluorescence intensity of the corresponding position, and the integrals of the scanning curves of the T line and the C line and the x axis are respectively marked as a T value and a C value to represent the fluorescence intensity of the T, C line;
step 4, drawing a standard curve: preparing a series of standard solution with gradient concentration, detecting by using a plurality of immunochromatographic test strips in the same batch, and drawing a standard curve by taking a T-line signal value as a vertical coordinate and the concentration of the standard solution as a horizontal coordinate.
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