CN106526166A - Rapid detection of lean meat powder in pork - Google Patents
Rapid detection of lean meat powder in pork Download PDFInfo
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- CN106526166A CN106526166A CN201510584664.1A CN201510584664A CN106526166A CN 106526166 A CN106526166 A CN 106526166A CN 201510584664 A CN201510584664 A CN 201510584664A CN 106526166 A CN106526166 A CN 106526166A
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- ractopamine
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analyzing Materials By The Use Of Magnetic Means (AREA)
Abstract
The invention discloses a gold-magnetic immunochromatographic test strip for rapid quantitative detection of lean meat powder (clenbuterol hydrochloride and ractopamine) in pork. The test strip comprises a base plate, a sample pad, a conjugate release pad, a porous filter membrane and an absorption pad, wherein the sample pad, the conjugate release pad, the porous filter membrane and the absorption pad adhere to the base plate and are connected in sequence. The porous filter membrane is coated with a detection line T1 for a clenbuterol hydrochloride-BSA coupled antigen, a detection line T2 for a ractopamine-BSA coupled antigen and a quality control line C coated with a goat-anti-mouse secondary antibody in sequence, and a monoclonal antibody, marked with gold-magnetic nanoparticles, for clenbuterol hydrochloride and ractopamine is embedded in the sample pad. The test strip combines the advantages of color development and bio-affinity of a colloidal gold test strip, and the advantages of nano-magnetic beads that separation is easy and quantitative detection can be achieved; by means of the prepared test strip, qualitative detection can be conducted through visual inspection, and quantitative detection can also be conducted through magnetic measurement.
Description
Technical field
The invention belongs to biological immune detection field, more particularly in meat productss the quick detection gold magnetic nano immune chromatograph test strip of clenobuterol hydrochloride and Ractopamine preparation and application.
Background technology
Immunochormatography comes across phase early 1980s, it is a kind of new detection technique combined with chromatographic technique by immunolabelling technique, it reacts to form immune complex using antigen and antibody specific, and then a kind of analysis method with high selectivity of detection by quantitative is carried out to analysans, immunoassay can realize the detection to trace substances such as medicine, microorganism, protein matter, cytokine and nucleic acid, have compared to other detection techniques：Quickly, easy, cheap the characteristics of, its specificity is strong, good stability, disclosure satisfy that the characteristics of high flux, the primary dcreening operation of lower bound amount sample are detected.Immunoassay since the advent of the world, quickly grows with the extremely people's favor of its unique advantage, and it has become a technological revolution of Routine Test Lab diagnosis at present, has promoted the development of the detection fields such as environment, food and some other hard-core technology.
Chromatographic technique with gold colloidal as representative has been developed more than 20 years, still extensively apply at present, immuno-gold labeling technology (immunogold labeling technique) mainly make use of colloid gold particle to have the characteristic of high electron density, at gold mark protein binding, visible dark brown coloured particles under the microscope, when these labels are assembled at corresponding part in a large number, naked eyes red color visible or pink speckle, thus for qualitative or semiquantitative tachysynthesises detection method.But as which is only used for qualitative or semiquantitative detection, it is difficult to meet the requirement of Testing index quantification, while testing result is judged by naked eyes, particularly when testing result is in weakly positive, artificial detection leakage phenomenon is easily caused, therefore there is sensitivity, needed further perfect.At present, research novel immune chromatographic technique has become study hotspot with the deficiency for substituting and improving colloidal gold immunochromatographimethod technology, novel immune chromatographic technique does not have the shortcomings that gold colloidal need to be assembled in a large number and could develop the color, which improves detection sensitivity with distinctive photoelectromagnetic signal amplifying system, reduce the background interference of sample, possess the incomparable advantage of conventional tag thing, immunochromatography technique just develops towards directions such as quantitative, high sensitivity, multiple labeling detections.The present invention is therefore.
The content of the invention
Present invention aim at providing a kind of golden magnetic immuno-chromatographic test paper strip of clenbuterol hydrochloride in meat productss for quick detection and the method being used for quickly detecting using the test strips, party's standard measure, high sensitivity, multiple labeling detection, when avoiding testing result in weakly positive, the problems such as easily cause artificial detection leakage phenomenon.
In order to solve these problems of the prior art, the technical scheme that the present invention is provided is：
A kind of golden magnetic lateral flow chromatographic assay device for detecting clenbuterol hydrochloride in Carnis Sus domestica, including the conjugate release pad, porous membrane that arrange along liquid to be detected flow direction, wherein：
Conjugate release pad deposition has the golden clenobuterol hydrochloride of magnetic nano particle labelling and the monoclonal antibody of Ractopamine as conjugate probe to be released；
Porous membrane is stacked on the support pad of solid phase, is in fluid communication with conjugate release pad, and is provided with detection zone；The detection zone is respectively fixed with clenobuterol hydrochloride-BSA coupled antigens and Ractopamine-BSA coupled antigens.
In preferred technical scheme, along the indicating area that the liquid flow direction is additionally provided with and is physically separated with the detection zone, the indicating area is fixed with sheep anti-Mouse two and resists.
In preferred technical scheme, also include the absorption pad arranged along the liquid flow direction, for collecting the liquid for flowing through the detection zone and indicating area.
In preferred technical scheme, wherein golden magnetic nano particle is nucleocapsid structure, and its particle diameter is in the range of 20 ~ 80nm；Preferably, the particle diameter of golden magnetic nano particle is 40nm.
In preferred technical scheme, it is γ-Fe that wherein golden magnetic nano particle is internal layer2O3, outer layer is the core-shell nano composite of Au claddings.
In preferred technical scheme, wherein detection zone includes 2 detection zones, and the first detection zone is fixed with clenobuterol hydrochloride-BSA coupled antigens, and the second detection zone is fixed with Ractopamine-BSA coupled antigens.
Another object of the present invention is to a kind of golden magnetic lateral flow chromatograph test strip for detecting clenbuterol hydrochloride in Carnis Sus domestica is provided, including：
(1) porous membrane being stacked on solid phase support pad, wherein described porous membrane are provided with the detection zone and indicating area being physically separated along liquid flow direction；The detection zone is respectively fixed with clenobuterol hydrochloride-BSA coupled antigens and Ractopamine-BSA coupled antigens；The indicating area is fixed with sheep anti-Mouse two and resists；
(2) conjugate release pad, it is in fluid communication with the porous membrane, and deposition have contact with fluid sample after releasable conjugate probe, wherein described conjugate probe is the monoclonal antibody of the clenobuterol hydrochloride and Ractopamine of golden magnetic nano particle labelling；
(3) absorption pad, for collecting the liquid for flowing through the detection and indicating area.
A further object of the present invention be provide it is a kind of for detect clenbuterol hydrochloride in Carnis Sus domestica whether there is and its content method, comprise the following steps：
I) provide a kind of golden magnetic lateral flow chromatographic assay device, including the porous membrane that conjugate release pad and conjugate release pad are in fluid communication, in the conjugate release pad, deposition has the conjugate probe with test analyte specific binding, and the conjugate probe is the monoclonal antibody of the clenobuterol hydrochloride and Ractopamine of golden magnetic nano particle labelling；Detection zone, the indicating area in detection zone downstream are set on the porous membrane, the detection zone is respectively fixed with clenobuterol hydrochloride-BSA coupled antigens and Ractopamine-BSA coupled antigens with test analyte specific binding, and the indicating area is fixed with the sheep anti-Mouse two combined with the conjugate probe specificity and is resisted；
Ii the test sample is contacted with the conjugate probe, and make test sample drive the conjugate probe to flow to the detection zone and the indicating area；
Iii) make the detection zone and indicating area respectively through magnetic reading apparatus, obtain the intensity of the magnetic signal respectively by magnetic reading apparatus；Wherein the magnetic signal of indicating area is used as indication signal；
Iv) obtain the quantity of the analyte in the test sample according to the intensity of the detection signal, the quantity of the analyte wherein in the test sample is directly proportional to the intensity of the detection signal.
In preferred technical scheme, the wherein golden clenobuterol hydrochloride of magnetic nano particle labelling and the monoclonal antibody of Ractopamine are prepared in accordance with the following steps：
（1）γ-Fe2O3It is prepared by nano-particle：With FeCl3、FeCl2Fe is obtained by chemical coprecipitation for raw material3O4Nano-particle；Fe will be obtained after cleaning3O4Nano-particle is dispersed in HNO3Solution is heated to 90 DEG C and reacts 1 hour, obtains γ-Fe after reaction completely2O3Nano-particle；
（2）It is prepared by the golden magnetic nano particle of nucleocapsid structure：Obtained γ-Fe2O3 nano-particle is dispersed in pure water and is diluted to 1.1mol/L；NH is sequentially added under conditions of sodium citrate is as surfactant2OH HCl, gold chloride are reacted；Repetition sequentially adds NH2OH HCl, gold chloride carry out reaction and obtain gold magnetic nano particle i.e. Au/Fe2O3Colloidal solid；
（3）It is prepared by the monoclonal antibody of the clenobuterol hydrochloride and Ractopamine of golden magnetic nano particle labelling：The golden magnetic nano particle that will be prepared, after the PBS buffer solution rinses of pH=7.4, Magnet isolates golden magnetic nano particle, then disperse in the PBS buffer solution of pH=7.4, it is slowly added to the antibody of clenbuteral hydrochloride and Anti-ractopamine antibody of equimolar ratio, and concussion shakes up, it is slowly added to PEG20000, continue concussion 20min, 10% bovine serum albumen solution is added to final concentration of 1%, Magnetic Isolation, abandon supernatant, precipitation is cleaned with the PBS buffer solution of pH=7.4, Magnetic Isolation, obtained golden magnetic-antibody of clenbuteral hydrochloride and golden magnetic-Anti-ractopamine antibody are scattered in the PBS buffer solution of pH=7.4, 4 DEG C of preservations.
In preferred technical scheme, wherein described test sample needs to carry out pretreatment before being detected, Carnis Sus domestica sample is stirred 1 minute with homogenizer 5000r/min, weighs the 0.5 ± 0.05g of sample after rubbing in 10mL plastic centrifuge tubes, adds 2mL extracting solution in pipe（The PBS buffer solution of pH=7.4）, vortex 1min, ultrasonic 20min are placed in a centrifuge 8000r/min centrifugation 10min.
The present invention adopts nanometer gold-magnetic composite particle, combines the immunochromatography technique of nanometer gold and magnetic bead dual characteristicses, replaces gold colloidal as marker material by the use of nanometer gold-magnetic composite particle, is applied in immunochromatography detection.The advantage of magnetics, optics and immunochromatography is combined based on the immunochromatography technique of nanometer gold magnetic particle, fully demonstrated traditional Gold-immunochromatography assay technical operation it is simple, it is cheap the advantages of, using magnetic it is quantitative the characteristics of compensate for Gold-immunochromatography assay technology and be unable to the defects such as detection by quantitative, nanometer gold magnetic granule has high specific identification, high affine absorption affinity, target molecule can be separated from the living things system of complicated component, the inspissation of sample is played, so as to substantially increase sensitivity and the recall rate of detection.
Clenbuterol hydrochloride in the detection Carnis Sus domestica that the present invention is provided（Clenobuterol hydrochloride and Ractopamine）Golden magnetic nano immune chromatograph test strip, including base plate and it is attached to sample pad, conjugate release pad, porous membrane and the absorption pad being sequentially connected on base plate, scribble the anti-Quality Control C lines of detection T1 lines, the detection T2 lines of Ractopamine-BSA coupled antigens and the coating sheep anti-Mouse two of clenobuterol hydrochloride-BSA coupled antigens on porous membrane successively, in conjugate release pad, be embedded with the monoclonal antibody of the clenobuterol hydrochloride and Ractopamine of golden magnetic nano particle labelling.
Described gold-magnetic nanoparticle is internal layer γ-Fe2O3The core-shell nano composite of outer layer Au claddings, particle diameter are 40nm or so.The detection T1 lines are formed by clenobuterol hydrochloride BSA coupled antigens.The detection T2 lines are formed by Ractopamine BSA coupled antigens.The Quality Control C lines are formed by sheep anti mouse two is anti-.The clenbuterol hydrochloride of the detection is clenobuterol hydrochloride and Ractopamine.
It is a further object to provide a kind of method for preparing above-mentioned test strips.
The concrete grammar of the preparation test strips that the present invention is provided is as follows：
（1）The preparation of golden magnetic nano particle；
（2）It is prepared by golden magnetic-antibody of clenbuteral hydrochloride and golden magnetic-Anti-ractopamine antibody：
（3）Preparation is perfused with the conjugate release pad of golden magnetic-antibody and the porous membrane containing detection T lines and Quality Control C lines：
（4）According to the structure assembling test strips of golden magnetic lateral flow chromatograph test strip.
Above-mentioned steps（1）Described in prepare the golden magnetic concentration of golden magnetic-antibody be 1.0mg/mL.
Above-mentioned steps（2）Described in prepare the antibody of clenbuteral hydrochloride of golden magnetic-antibody and Anti-ractopamine antibody concentration is 200 μ g/mL.
Above-mentioned steps（1）Described in Fe3O4Nano-particle is dispersed in the HNO of 0.01mol/L3It is heated to 90 DEG C to react 1 hour in solution.
Above-mentioned steps（1）Described in Au/Fe2O3Prepared by golden magnetic nano particle, Fe2O3Mass ratio with gold chloride is 1:9.8(w/w).
Above-mentioned steps（1）Described in antibody and Au/Fe2O3The mass ratio of golden magnetic nano particle is 1:100(w/w).
Clenbuterol hydrochloride in the detection Carnis Sus domestica that the present invention is provided（Clenobuterol hydrochloride and Ractopamine）The beneficial effect of golden magnetic nano particle immuno-chromatographic test paper strip be：Gold colloidal is replaced to prepare immuno-chromatographic test paper strip to clenbuterol hydrochloride in Carnis Sus domestica using nanometer gold-magnetic composite particle（Ractopamine and clenobuterol hydrochloride）Detection by quantitative, golden magnetic nano particle combine nanometer gold colour developing, being easily isolated of bioaffinity feature and nanometer magnetic bead, can quantification the characteristics of detect, the test strips of preparation can carry out qualitative detection by estimating, also can be by the measurement of magnetic come further detection by quantitative.The test strips while nanometer gold immuno-chromatographic test paper strip advantage is remained improve which and are difficult to quantitative deficiency, it is expected to further improve traditional nanometer gold immunochromatography technique, immunochromatography technique is made to move towards the application of quantification, multivariate detection and extensiveization, the research is conducive to foods supervision department to carry out site supervision and quantitative primary dcreening operation to Carnis Sus domestica, ensures people's lives safety.
Fig. 1 shows and a kind of is typically stacked in the lateral flow chromatographic assay device constituted on solid phase support pad 11 by porous membrane 10.The support pad may or may not be significantly translucent for ultraviolet and/or visible ray and/or near-infrared or infrared light.The multiple material that 10 optional test sample of porous membrane can be passed through according to capillarity.For example, porous membrane may include but be not limited to natural and synthesis material, such as cellulosic material, paper and cellulose derivative, cellulose acetate and nitrocellulose, polyether sulfone and nylon membrane.A kind of preferred porous membrane is by made by nitrocellulose and/or polyester sulfone materials.It is provided with porous membrane and fixes one or more detection zones 20,20* with the specific binding of conjugate probe 40.It is additionally provided with porous membrane and is fixed with one or more indicating areas 21 with the specific binding of conjugate probe 40.
Lateral flow chromatographic assay device may also comprise absorption pad 12.Absorption pad is used for absorbing the liquid for having migrated through whole porous membrane by capillarity.The absorbing material for being commonly used for manufacturing absorption pad may include but be not limited to natural and synthetic material, such as cellulosic material, paper and cellulose derivative, cellulose acetate and nitrocellulose.
Lateral flow chromatographic assay device may also comprise conjugate release pad 13, here deposition has and one or more conjugate probes 40, the conjugate probe 40 has the monoclonal antibody of clenobuterol hydrochloride and Ractopamine, and the monoclonal antibody connects upper golden magnetic nano particle by way of covalent bond or physical absorption（The conjugate probe 40 of golden magnetic nano particle labelling）, these conjugate probes can discharge and contact with fluid sample with suspending.The conjugate release pad includes but is not limited to fiberglass packing.
Lateral flow chromatographic assay device may also include the sample pad 14 for placing sample.Sample pad can be made up of natural and synthetic material, such as cellulosic material, paper and cellulose derivative, cellulose acetate and nitrocellulose.Sample pad 14, conjugate release pad 13, porous membrane 10 and absorption pad 12 are generally stacked up to certain overlapping degree, so that these parts are in fluid communication each other.
For being measured, sample can be mixed with conjugate probe first, and be subsequently placed on the porous membrane of the lateral chromatography device with detection zone.In this case, lateral chromatography device can be without the need for sample pad and conjugate release pad.Mixture is subsequently passed through detection zone and is interacted with clenobuterol hydrochloride BSA coupled antigens, Ractopamine BSA coupled antigens, and so as to form detection line, the detection line can be measured by magnetic reading plotter.
When lateral chromatography device has sample pad and conjugate release pad, sample can be placed directly within sample pad 14, and is then flowed at conjugate release pad 13, discharged and be suspended in working fluid from the conjugate release pad in this conjugate probe.Sample liquids flow further to detection zone 20 in company with conjugate probe, in 20* and indicating area 21, and eventually flow on absorption pad 12.Although only showing a conjugate release pad 13, it should be appreciated that multiple conjugate release pads can also be used for the present invention.For contributing to accurately detecting specific analyte, the conjugate probe that can connect in the zones of different application of conjugate release pad.Conjugate probe can be respectively used to the detection to analyte and calibration.
When conjugate probe is using golden magnetic nano particle, the big I of golden magnetic nano particle granule changes, such as the type of selected probe particles, filter sizes and filter membrane composition with some factors.The particle diameter of granule can be in the range of 20nm to 100nm, in some embodiments in the range of in the range of 20nm to 80nm, in some embodiments from the range of 30nm to 60nm.In a kind of specific embodiment, the average diameter of granule is in the range of in the range of 30nm to 50nm.Generally, granule is substantially spherical in shape in shape, although including but not limited to tabular, bar-shaped, strip, erose other shapes are also applied for the present invention.As understood by those skilled, can be in the interior composition for changing granule, shape, size and/or density on a large scale.
Modified probe is needed in some cases so as to be easier to specifically be combined with test analyte.Can be using some specific binding portions come the probe that is modified, to form conjugate probe.Specific binding portion typically refer to for specific binding to two different molecules, one of molecule by chemistry and/or physics in the way of combined with second molecule.For example, the specific binding portion of immunoreation includes antigen, hapten, fit, antibody and these complex, and these complex includes those complex formed by recombinant DNA method or peptide symthesis.Antibody can be the mixture or the mixture in fragment and antibody and other specific binding portions of monoclonal or polyclonal antibody, recombinant protein or these materials.Other common specific bindings are to including but is not limited to biotin and Avidin, biotin and streptavidin, antibody binding proteins matter（Such as a-protein or G）With antibody, carbohydrate and lectin, complementary nucleotide sequence（The labelling for detecting Target Nucleotide Sequence being included in used in DNA hybridization assays and capture nucleotide sequence）, complementary amino acid sequence（Including the complementary amino acid sequence that those are formed by recombination method）, effector and acceptor molecule, hormone and hormonebinding protein matter, enzyme co-factor and enzyme, enzyme inhibitor and enzyme, etc..Additionally, specific binding is to the analog also including former specific binding portion.For example, the derivant or fragment of analyte（That is the analog of analyte）As long as can also use with analyte identical epi-position with least one.
Various lateral flow chromatographic assay devices can be built together with magnetic reading plotter.These specific binding portions capture the bound site on the complex combined with analyte by conjugate probe by binding specificity ground.For example, the analyte of such as antibody, antigen has two basic change position.Once reaching detection zone 20, in these bound sites is just occupied by the specific binding portion of Complex Probes.However, the free bound site of analyte is can be coupled in the specific binding portion of these fixations.Conjugate probe is once attached in fixed specific binding portion, just forms new ternary sandwich complex.
Construction regardless of magnetic reading plotter, by the magnetic signal Is of the probe captured in detection zone 20 is associated with the advance standard curve for building, the content of measurable analyte.Based on the signal strength range of detection zone 20, it may be determined that the general concentration range of analyte.So can be in the case of roughly the same while test of being calibrated and be sampled, thus provides reliable quantitatively or semi-quantitatively result, and sensitivity is higher.
Although having been described above the various embodiments of device configuration, it should be appreciated that the device of the present invention could generally have any desired configuration, and need not include all parts mentioned above.
Relative to scheme of the prior art, it is an advantage of the invention that：
Technical solution of the present invention provides clenbuterol hydrochloride in a kind of Quantitative detection Carnis Sus domestica（Clenobuterol hydrochloride and Ractopamine）Golden magnetic immuno-chromatographic test paper strip, the test strips include base plate and are attached to sample pad, conjugate release pad, porous membrane and the absorption pad being sequentially connected on base plate, scribble the anti-Quality Control C lines of detection T1 lines, the detection T2 lines of Ractopamine-BSA coupled antigens and the coating sheep anti-Mouse two of clenobuterol hydrochloride-BSA coupled antigens on porous membrane successively, in sample pad, be embedded with the monoclonal antibody of the clenobuterol hydrochloride and Ractopamine of golden magnetic nano particle labelling.The test strips combine colloidal gold strip colour developing, being easily isolated of bioaffinity feature and nanometer magnetic bead, can quantification the characteristics of detect, the test strips of preparation can carry out qualitative detection by estimating, also can be by the measurement of magnetic come further detection by quantitative.
Description of the drawings
Below in conjunction with the accompanying drawings and embodiment the invention will be further described：
Fig. 1 is present invention gold magnetic lateral flow chromatographic assay device schematic diagram.Wherein 14 is sample pad；13 is conjugate release pad；10 is porous membrane；12 is absorption pad；10 is support pad；20 are detection T1 lines；20* is detection T2 lines；21 is Quality Control C lines.
Fig. 2 is another structural representation of gold magnetic lateral flow chromatograph test strip of the invention；
Fig. 3 is the transmission electron microscope figure of golden magnetic nano particle.
Fig. 4 is positive findingses process decision chart of the present invention, and wherein T1 represents clenobuterol hydrochloride detection line, and T2 represents Ractopamine detection line, and C represents nature controlling line.
Fig. 5 is negative findings process decision chart of the present invention, and wherein C represents nature controlling line.
Specific embodiment
It is noted that described further below is all exemplary, it is intended to provide further instruction to the application.Unless otherwise specified, all technologies used herein and scientific terminology are with the identical meanings being generally understood that with the application person of an ordinary skill in the technical field.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to illustrative embodiments of the restricted root according to the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative is also intended to include plural form, in addition, it is to be further understood that, when in this manual using "comprising" and/or " including " is belonged to, which indicates existing characteristics, step, operation, part or module, component and/or combinations thereof.
It should be noted that the description and claims of this application and the term " first " in above-mentioned accompanying drawing, " second " etc. are for distinguishing similar object, without being used for describing specific order or precedence.It should be appreciated that the data for so using can be exchanged in the appropriate case, so that presently filed embodiment described herein for example can be implemented with the order in addition to those for illustrating here or describing.In addition, term " comprising " and " having " and their any deformation, it is intended to cover non-exclusive including, for example, process, method, system, product or the equipment for containing series of steps or unit is not necessarily limited to those steps clearly listed or unit, but may include other steps clearly do not list or intrinsic for these processes, method, product or equipment or unit.
For the ease of description, space relative terms can be used here, as " ... on ", " in ... top ", " in ... upper surface ", " above " etc., for describing such as a part shown in the figure or module or feature and miscellaneous part or the spatial relation of module or feature.It should be appreciated that space relative terms are intended to comprising the different azimuth in use or operation in addition to orientation described in figure of part or module.For example, if the part in accompanying drawing or module are squeezed, be described as " above miscellaneous part or module or construction " or " on miscellaneous part or module or construction " part or module after will be positioned as " below miscellaneous part or module or construction " or " under miscellaneous part or module or construction ".Thus, exemplary term " in ... top " can include " in ... top " and " in ... lower section " two kinds of orientation.The part or module can also other different modes positioning（It is rotated by 90 ° or in other orientation）, and respective explanations are made to the relative description in space used herein above.
As background technology is introduced, district vehicles turnover control mode of the prior art is single or cannot flexibly be controlled according to the demand of people, in order to solve as above problem, present applicant proposes a kind of function base station service reservation system based on car ring.
It should be noted that in the case where not conflicting, the feature in embodiment and embodiment in the application can be mutually combined.Below with reference to the accompanying drawings and in conjunction with the embodiments describing the present invention in detail.
Embodiment
Referring to shown in Fig. 1 and Fig. 2, the golden magnetic immuno-chromatographic test paper strip of the quick measure clenbuterol hydrochloride, including support pad（Base plate）11 and be attached to be sequentially connected on base plate sample pad 14, with reference to release pad 13, porous membrane 10 and absorption pad 12, the anti-Quality Control C lines 21 of the detection T1 lines 20 for scribbling clenobuterol hydrochloride-BSA coupled antigens on porous membrane successively, the detection T2 lines 20* for scribbling Ractopamine-BSA coupled antigens and coating sheep anti-Mouse two, are embedded with the monoclonal antibody of the clenobuterol hydrochloride and Ractopamine of golden magnetic nano particle labelling in sample conjugate release pad 13.
The monoclonal anti preparation step of the clenobuterol hydrochloride or Ractopamine of the golden magnetic nano particle labelling wherein in conjugate release pad is as follows：
（1）It is prepared by golden magnetic nano particle：
γ-Fe2O3Prepare：2.6gFeCl is sequentially added in 12.5ml aqueous solutions3、1.0gFeCl2And the HCl solution of 0.43mL 12mol/L, the mixed liquor is dropwise dropped to after mix homogeneously in the NaOH solution of 125mL 1.5mol/L, keep being stirred vigorously.Black precipitate Fe of generation3O4Collected with Magnet, discard after supernatant centrifugation, cleaned with pure water 3 times after the precipitate collected twice is merged, black precipitate is added to into 250mL
It is uniformly dispersed in the HCl solution of 0.01mol/L, the colloid solution for obtaining is separated with Magnet again, cleans 2 times with pure water.Completely Fe will be cleaned3O4Nano-particle is dispersed in the HNO of 0.01mol/L3It is heated to 90 DEG C to react 1 hour in solution, after reaction completely, obtains γ-Fe2O3Nano-particle.It is placed in 4 DEG C of refrigerators and preserves.
Au/Fe2O3It is prepared by golden magnetic nano particle：By γ-Fe obtained above2O3Nano-particle pure water is diluted to 1.1mol/L, takes 1mLFe2O3Solution is mixed 10 minutes with the sodium citrate solution of 0. 1mol/L of 1mL, and the mixture of gained adds pure water to 20mL, adds the NH of 1mL 80mmol/L in the solution2OH HCl stir, and in the solution are added dropwise over 1%（Mass percent concentration）Chlorauric acid solution 2mL, stir 50min.Repetition adds above-mentioned same amount of NH2OH HCl and chlorauric acid solution 3 times, ultimately form colloidal solid russet.The golden magnetic grain diameter is 40nm or so, and its transmission electron microscope figure is shown in Fig. 3.
(2) prepared by golden magnetic-antibody of clenbuteral hydrochloride and golden magnetic-Anti-ractopamine antibody：
The golden magnetic granule of above-mentioned cold preservation is taken, with 500 μ LPBS buffer solution（pH=7.4）Rinse 2-3 time, Magnet separating nano-particles, with the golden magnetic granule of 500 μ L of PBS buffer solution dispersions of pH=7.4, are slowly added to antibody of clenbuteral hydrochloride and Anti-ractopamine antibody（1:1）Totally 400 μ L shake up, be put into constant-temperature table concussion（37 DEG C, 180rmp）20min, is slowly added to 1%（Mass percent）PEG20000 to final concentration of 0.1%（Mass percent）, continue concussion 20min, add 10%（Mass percent）Bovine serum albumen solution to final concentration of 1%（Mass percent）, Magnetic Isolation abandons supernatant, and precipitation clean with the PBS buffer solution of pH=7.4, Magnetic Isolation, and obtained golden magnetic-antibody of clenbuteral hydrochloride and gold magnetic-Anti-ractopamine antibody are scattered in the PBS buffer solution of pH=7.4, and 4 DEG C preserve.
Conjugate release pad preparation process is as follows：
Glass fiber filter paper is cut into into the wide slices of 6mm, is put into containing 1%（Mass percent）BSA and 0.5%（Mass percent）30min, 37 DEG C of dry for standby are soaked in the PBS buffer solution of Tween20.Will be above-mentioned（2）Golden magnetic-antibody the granule of preparation is poured on film, and vacuum freeze-drying is standby after 4 hours.
The preparation process of porous membrane is as follows：
Clenobuterol hydrochloride BSA coupled antigens and Ractopamine BSA coupled antigens are diluted to into 200 μ g/mL with the PBS buffer solution of pH=7.4, are sprayed on T1 the and T2 lines of cellulose acetate film with point film instrument, quantity for spray is 1.0 μ L/cm.200 μ g/mL being diluted to the PBS buffer solution of pH=7.4 by sheep anti mouse two anti-, being sprayed on the C lines of cellulose acetate film with point film instrument, quantity for spray is 1.0 μ L/cm.The porous membrane for preparing is placed in 37 DEG C of vacuum constant temperatures, and to be dried more than 4h standby.
The assembling of test strips：
Sample pad, conjugate release pad, porous membrane and absorption pad are pasted on test strips support chip successively.The end of sample pad is connected with the top of conjugate release pad, and the end of conjugate release pad is connected with the top of porous membrane, and the end of porous membrane is connected with the top of absorption pad, all pastes in gripper shoe, obtain quick detection clenbuterol hydrochloride after assembling（Clenobuterol hydrochloride and Ractopamine）Immuno-chromatographic test paper strip.
Application examples
Operating procedure：
The negative Carnis Sus domestica sample of our unit's detection is taken, sample is stirred 1 minute with homogenizer 5000r/min, the 0.5 ± 0.05g of sample after rubbing is weighed in 10mL plastic centrifuge tubes, add 2mL extracting solution in pipe（The PBS buffer solution of pH=7.4）, vortex 1min, ultrasonic 20min are placed in a centrifuge 8000r/min centrifugation 10min.Take the sample application zone that 200 μ L of supernatant vertically drop in test strips, liquid is flowed up by chromatography effect, as liquid flows to above test strips after the nanometer gold magnetic granule redissolution of binding antibody, waits 5-10min, only there is a brownish red band in C lines, be as a result negative.
0.5 ± the 0.05g of sample after above-mentioned rubbing is taken in 10mL plastic centrifuge tubes, 2mL extracting solution is added in pipe（The PBS buffer solution of pH=7.4）, it is 4.0 μ g/kg, vortex 1min, ultrasonic 20min to add clenobuterol hydrochloride standard substance to make the concentration in sample, is placed in a centrifuge 8000r/min centrifugation 10min.Take the sample application zone that 200 μ L of supernatant vertically drop in test strips, liquid is flowed up by chromatography effect, as liquid flows to above test strips after the nanometer gold magnetic granule redissolution of binding antibody, waits 5-10min, there is a brownish red band in T1 lines and C lines in test strips, are as a result positive.
0.5 ± the 0.05g of sample after above-mentioned rubbing is taken in 10mL plastic centrifuge tubes, 2mL extracting solution is added in pipe（The PBS buffer solution of pH=7.4）, it is 4.0 μ g/kg, vortex 1min, ultrasonic 20min to add Ractopamine standard substance to make the concentration in sample, is placed in a centrifuge 8000r/min centrifugation 10min.Take the sample application zone that 200 μ L of supernatant vertically drop in test strips, liquid is flowed up by chromatography effect, as liquid flows to above test strips after the nanometer gold magnetic granule redissolution of binding antibody, waits 5-10min, there is a brownish red band in T2 lines and C lines in test strips, are as a result positive.
As shown in Figure 4,5, measurement result can directly carry out range estimation judgement, and it is in detection T lines that sample is positive（T1 or T2）Two brownish red bands are all shown with Quality Control C lines, when sample is negative, only a brownish red band is shown in Quality Control C lines, detect T lines（T1 and T2）All do not show that brownish red band explanation test strips are invalid with Quality Control C lines；When measured matter concentration is relatively low, T lines are detected（T1 or T2）Colour developing can carry out interpretation with magnetic reading apparatus when unobvious, and ELISA test strip position is inserted into the reading area of magnetic reading apparatus, and magnetic reading apparatus can test the magnetic number of measured object automatically, show the size of T lines region magnetic number by the display screen of instrument.Detection by quantitative can be carried out to the Ractopamine and clenobuterol hydrochloride in sample by means of magnetic reading apparatus, improve detection sensitivity.
Examples detailed above technology design only to illustrate the invention and feature, its object is to allow person skilled in the art to be to will appreciate that present disclosure and implement according to this, can not be limited the scope of the invention with this.All equivalent transformations done according to spirit of the invention or modification, should all be included within the scope of the present invention.
Claims (10)
1. a kind of golden magnetic lateral flow chromatographic assay device for detecting clenbuterol hydrochloride in Carnis Sus domestica, including the conjugate release pad, porous membrane that arrange along liquid to be detected flow direction, wherein：
Conjugate release pad deposition has the golden clenobuterol hydrochloride of magnetic nano particle labelling and the monoclonal antibody of Ractopamine as conjugate probe to be released；
Porous membrane is stacked on the support pad of solid phase, is in fluid communication with conjugate release pad, and is provided with detection zone；The detection zone is respectively fixed with clenobuterol hydrochloride-BSA coupled antigens and Ractopamine-BSA coupled antigens.
2. the golden magnetic lateral flow chromatographic assay device according to claim 1, along the indicating area that the liquid flow direction is additionally provided with and is physically separated with the detection zone, the indicating area is fixed with sheep anti-Mouse two and resists.
3. according to claim
Golden magnetic lateral flow chromatographic assay device described in 2, also includes the absorption pad arranged along the liquid flow direction, for collecting the liquid for flowing through the detection zone and indicating area.
4. the golden magnetic lateral flow chromatographic assay device according to claim 1, wherein golden magnetic nano particle is nucleocapsid structure, and its particle diameter is in the range of 20 ~ 80nm；Preferably, the particle diameter of golden magnetic nano particle is 40nm.
5. according to claim
Golden magnetic lateral flow chromatographic assay device described in 4, it is γ-Fe that wherein golden magnetic nano particle is internal layer2O3, outer layer is the core-shell nano composite of Au claddings.
6. the golden magnetic lateral flow chromatographic assay device according to claim 1, wherein detection zone include 2 detection zones, and the first detection zone is fixed with clenobuterol hydrochloride-BSA coupled antigens, and the second detection zone is fixed with Ractopamine-BSA coupled antigens.
7. a kind of golden magnetic lateral flow chromatograph test strip for detecting clenbuterol hydrochloride in Carnis Sus domestica, including：
(1) porous membrane being stacked on solid phase support pad, wherein described porous membrane are provided with the detection zone and indicating area being physically separated along liquid flow direction；The detection zone is respectively fixed with clenobuterol hydrochloride-BSA coupled antigens and Ractopamine-BSA coupled antigens；The indicating area is fixed with sheep anti-Mouse two and resists；
(2) conjugate release pad, it is in fluid communication with the porous membrane, and deposition have contact with fluid sample after releasable conjugate probe, wherein described conjugate probe is the monoclonal antibody of the clenobuterol hydrochloride and Ractopamine of golden magnetic nano particle labelling；
(3) absorption pad, for collecting the liquid for flowing through the detection and indicating area.
8. it is a kind of for detect clenbuterol hydrochloride in Carnis Sus domestica whether there is and its content method, comprise the following steps：
I) provide a kind of golden magnetic lateral flow chromatographic assay device, including the porous membrane that conjugate release pad and conjugate release pad are in fluid communication, in the conjugate release pad, deposition has the conjugate probe with test analyte specific binding, and the conjugate probe is the monoclonal antibody of the clenobuterol hydrochloride and Ractopamine of golden magnetic nano particle labelling；Detection zone, the indicating area in detection zone downstream are set on the porous membrane, the detection zone is respectively fixed with clenobuterol hydrochloride-BSA coupled antigens and Ractopamine-BSA coupled antigens with test analyte specific binding, and the indicating area is fixed with the sheep anti-Mouse two combined with the conjugate probe specificity and is resisted；
Ii the test sample is contacted with the conjugate probe, and make test sample drive the conjugate probe to flow to the detection zone and the indicating area；
Iii) make the detection zone and indicating area respectively through magnetic reading apparatus, obtain the intensity of the magnetic signal respectively by magnetic reading apparatus；Wherein the magnetic signal of indicating area is used as indication signal；
Iv) obtain the quantity of the analyte in the test sample according to the intensity of the detection signal, the quantity of the analyte wherein in the test sample is directly proportional to the intensity of the detection signal.
9. according to claim
Method described in 8, the wherein golden clenobuterol hydrochloride of magnetic nano particle labelling and the monoclonal antibody of Ractopamine are prepared in accordance with the following steps：
（1）γ-Fe2O3It is prepared by nano-particle：With FeCl3、FeCl2Fe is obtained by chemical coprecipitation for raw material3O4Nano-particle；Fe will be obtained after cleaning3O4Nano-particle is dispersed in HNO3Solution is heated to 90 DEG C and reacts 1 hour, obtains γ-Fe after reaction completely2O3Nano-particle；
（2）It is prepared by the golden magnetic nano particle of nucleocapsid structure：Obtained γ-Fe2O3 nano-particle is dispersed in pure water and is diluted to 1.1mol/L；NH is sequentially added under conditions of sodium citrate is as surfactant2OH HCl, gold chloride are reacted；Repetition sequentially adds NH2OH HCl, gold chloride carry out reaction and obtain gold magnetic nano particle i.e. Au/Fe2O3Colloidal solid；
（3）It is prepared by the monoclonal antibody of the clenobuterol hydrochloride and Ractopamine of golden magnetic nano particle labelling：The golden magnetic nano particle that will be prepared, after the PBS buffer solution rinses of pH=7.4, Magnet isolates golden magnetic nano particle, then disperse in the PBS buffer solution of pH=7.4, it is slowly added to the antibody of clenbuteral hydrochloride and Anti-ractopamine antibody of equimolar ratio, and concussion shakes up, it is slowly added to PEG20000, continue concussion 20min, 10% bovine serum albumen solution is added to final concentration of 1%, Magnetic Isolation, abandon supernatant, precipitation is cleaned with the PBS buffer solution of pH=7.4, Magnetic Isolation, obtained golden magnetic-antibody of clenbuteral hydrochloride and golden magnetic-Anti-ractopamine antibody are scattered in the PBS buffer solution of pH=7.4, 4 DEG C of preservations.
10. according to claim
Method described in 8, wherein described test sample need to carry out pretreatment before being detected, Carnis Sus domestica sample is stirred 1 minute with homogenizer 5000r/min, weigh the 0.5 ± 0.05g of sample after rubbing in 10mL plastic centrifuge tubes, add 2mL extracting solution in pipe（The PBS buffer solution of pH=7.4）, vortex 1min, ultrasonic 20min are placed in a centrifuge 8000r/min centrifugation 10min.
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| CN107101991A (en) * | 2017-05-31 | 2017-08-29 | 东南大学 | A kind of high sensitivity multiplex detection chromatograph test strip |
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| CN116496785B (en) * | 2023-02-06 | 2024-04-26 | 陕西理工大学 | Fe3O4@HAp@Au composite nano fluorescent probe and its preparation method and application |
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Application publication date: 20170322 |