CN102798720B - Immune chromatography test paper for quantitative determination of clenbuterol based on up-conversion fluorescent nanoparticle label and preparation method thereof - Google Patents
Immune chromatography test paper for quantitative determination of clenbuterol based on up-conversion fluorescent nanoparticle label and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an immune chromatography test paper strip for the quantitative determination of clenbuterol based on up-conversion fluorescent nanoparticle label and a preparation method thereof. The test paper strip comprises a supporting layer, an adsorption layer, and a protection layer, wherein the adsorption layer comprises an adsorption fibrous layer, a fluorescent antibody fibrous layer, a cellulose film and an absorbent material layer at the handle end, the cellulose film is provided with detection blots printed by using a CL coupling carrier protein solution and control blots printed by using goat anti-mouse IgG antibodies, and the fluorescent antibody is an NaYF4:Yb:Er nanoparticle labelled CL monoclonal antibody or polyclonal antibody. According to the invention, the application of immune chromatography based on the up-conversion fluorescent nanoparticle label in the quantitative determination of CL residues is realized, so that the detection of the CL residues has no background interference; and the test paper strip has the advantages of strong specificity, high sensitivity, simple, and accurate detection, low cost, wide application scope, and easiness in popularization and application.
Description
Technical field
The present invention relates to a kind of immune chromatography test paper, particularly relate to more than one conversion luminescent materials as biomarker for quantitative immuno-chromatographic test paper strip detecting residual of kelengtelu and preparation method thereof.
background technology
Clenbuterol (Clenbuterol, CL), is commonly called as " clenbuterol hydrochloride ", and clenobuterol hydrochloride is its hydrochloride, and have and promote muscle development and lipolysis effect, Clenbuterol can improve the carcass lean meat percentage of meat animals.Because consumer is to the special preferences of lean meat, the pig that lean meat output capacity is high can be fetched a good price, and adds " clenbuterol hydrochloride " and brings extra returns to raiser.Therefore, ordering about by undue profits, raiser is everlasting cultivating link interpolation " clenbuterol hydrochloride " in animal feed.
But Clenbuterol is a kind of selectivity β
2-adrenoceptor agonists, spinoff is very big, can cause the infringement of cardiovascular system and serious nervous symptoms when intake is larger.After people have eaten the pluck and meat containing residual of kelengtelu, acute or chronic kreotoxism can be caused, the health of harm consumer.Neither one state approval " Clenbuterol " can be used in animal productiong so far, but there is the situation of illegal use for a long time always, causes the event of serious food poisoning not rarely seen all over the world.Also there is " clenbuterol hydrochloride " poisoning in the multiple city of China in recent years, greatly threaten the physical and mental health of consumer.
The method detected for residual of kelengtelu is more, mainly contains (1) physico-chemical analysis method, as high pressure liquid chromatograph, gas chromatography, thin-layer chromatography, electrophoresis etc.; (2) immunoassay, as radioimmunology, enzyme linked immunosorbent assay etc.; (3) bioassay method, as microbiological assay, radioreceptor assay etc.But these methods need expensive instrument and equipment, need skilled professional's operation, operating process is complicated, and detection time is longer, limits its range of application, is difficult to apply in actual production.
Immune test paper method has sxemiquantitative and certain quantitation capabilities, can provide the preliminary information of determinand, and this method is highly sensitive, and analytic process is simple, and the examination being used as " clenbuterol hydrochloride " has unique advantage, is the detection technique needing to first develop.Up-conversion fluorescence technology (UPT), that one has high sensitivity, new bio detection technique based on upper converting phosphor body (UCP), UCP has unique physical arrangement and optical characteristics, it is a kind of phosphor of complete inertia, after finishing with activation, can be used as novel markings thing and apply in field of biological detection.Through retrieval, for detecting Pesticide, the UCP immune chromatography test paper of residue of veterinary drug has no report, although colloid gold test paper is applied wider in this regard, but it can not accomplish quantitative detection, as the research of more responsive, stable, flexible, safe UPT-LF, that the strong of colloid gold immune detection technique supplements, the research and exploitation of this aspect in urgent need of strengthening.
Summary of the invention
The technical problem to be solved in the present invention: the deficiency existed for prior art, provide a kind of special, sensitive, quick, easy, can quantitative immuno-chromatographic test paper strip detecting residual of kelengtelu and preparation method thereof.
Technical scheme of the present invention:
A kind of immuno-chromatographic test paper strip of the detection Clenbuterol based on upper conversion fluorescent nano particle mark, bottom is supporting layer, middle layer is adsorbed layer, protective seam is fixed on adsorbed layer, adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, fluorescence antibody fibrage, cellulose rete and handle end from test lead, stealth cellulose rete is provided with the carrier protein solution of coupling CL is printed detects trace, is also provided with the stealth contrast trace printed with goat anti-mouse igg, rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution; Described fluorescence antibody fibrage adopts the glass fibre cotton of absorption fluorescence antibody to make, and fluorescence antibody adopts NaYF
4: the CL monoclonal antibody of Yb:Er nanoparticle label or polyclonal antibody.
Described NaYF
4: Yb:Er nano particle is with NaYF
4for matrix, Yb
3+for sensitizer, the diameter formed by Yb and Er codope is the nano particle of 50-200nm.
Described NaYF
4: the CL monoclonal antibody of Yb:Er nanoparticle label or polyclonal antibody are prepared by following methods, and concrete steps are:
(1) surface siliconization of fluorescent nano particle
By NaYF
4: it is in the ethanolic solution of 10% that Yb:Er fluorescent nano particle is dispersed in mass concentration, under agitation adds 5ml strong aqua, and reaction 10min, then adds 2ml tetraethyl orthosilicate, room temperature reaction 3h; The centrifugal 5min of 6000rpm, disperses with ethanol after washing;
(2) surface amination of fluorescent nano particle
The fluorescent nano particle of surface siliconization is added in the mixed solution of methyl alcohol that volume ratio is 3:5, acetone, disperse through ultrasound wave, add N-(2-the aminoethyl)-3-aminopropyl trimethoxysilane of 5 μ l, 70 DEG C of water-bath 1h, the centrifugal 5min of 6000rpm, the precipitation ethanol obtained is washed, dry;
(3) mark of CL antibody
By the NaYF that surface is modified through amination
4: Yb:Er nano particle PBS buffer solution disperses, add the glutaraldehyde solution of mass concentration 25% wherein, room temperature reaction 3 hours, centrifugally wash away unnecessary glutaraldehyde, then be dispersed in PBS buffer solution by reactant liquor, add monoclonal antibody or the polyclonal antibody of CL, 4 DEG C are reacted 3 hours, through centrifuge washing, namely obtain NaYF
4: the CL monoclonal antibody of Yb:Er nanoparticle label or polyclonal antibody, be placed in PBS buffer solution, 4 DEG C of preservations.
Described adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film; Absorbent material layer absorbent filter, the supporting layer toughness material do not absorbed water; Cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film;
the carrier protein of coupling CL is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin.
Described stealth detect trace and stealthy contrast trace be arranged in parallel "
︱ ︱" orthoscopic trace, or be " 10 " font arrangement trace, or be " ┬ ┬ " font arrangement trace, or be " ┴ ┴ " font arrangement trace, or be " ├ ├ " font arrangement trace, or be " ┤ ┤ " font arrangement trace.
Described adsorbing fiber layer, fluorescence antibody fibrage and absorbent material layer are coated with diaphragm; the diaphragm that adsorbing fiber layer is corresponding with fluorescence antibody fibrage intersection is printed with sample mark line, this mark line deflection adsorbing fiber layer side 0.3-0.7cm.
Described NaYF4:Yb:Er nano particle adopts coprecipitation preparation, and concrete grammar is as follows:
Get the Y (NO that concentration is 0.2mol/L respectively
3)
3yb (the NO of 20ml, 0.2mol/L
3)
3er (the NO of 2ml, 0.2mol/L
3)
3the EDTA-Na21ml of 0.5ml, 1mol/L, adds in flask, and 50 DEG C of oil bath stirring reaction 1h, obtain reactant liquor A;
Get the NaF solution of 50ml, 1mol/L, add in polytetrafluoroethylcontainer container, under 50 DEG C of stirred in water bath, reactant liquor A is poured in NaF solution, reaction 1min; The centrifugal 5min of 6000rpm, abandoning supernatant must precipitate; Add water in precipitation, ultrasonic washing 3 times, the centrifugal 5min of 7000rpm; Again by precipitation absolute ethyl alcohol ultrasonic washing 2 times, the centrifugal 5min of 10000rpm, is placed in 80 DEG C of baking oven dried overnight by the solid obtained; Dried solid is put into muffle furnace, in 500 DEG C of calcining 5h, namely obtains NaYF
4: Yb:Er fluorescent nano particle.
Described NaYF
4: Yb:Er nano particle also can adopt hydrothermal synthesis method to prepare, and concrete grammar is as follows:
Get the Y (NO that concentration is 0.2mol/L respectively
3)
34ml, Yb (NO
3)
30.5ml, Er (NO
3)
30.1ml, EDTA-Na4ml, mix and fully react, and obtains reactant liquor A; Take NaOH 0.7g, join in the mixed liquor of 8ml oleic acid and 12ml ethanol, mixing, the under agitation slow NaF solution injecting 10mL, 1mol/L in mixed liquor, after filling, continue to be stirred to solution and to be translucent shape; Reactant liquor A is poured in described solution, mixes, ageing 20-30min; Then potpourri is transferred in polytetrafluoroethylcontainer container, 130-150 DEG C of oil bath reaction 12-18h, the product obtained after filtration, washing, dry, namely obtain NaYF
4: Yb:Er fluorescent nano particle.
The preparation method of described immuno-chromatographic test paper strip, is characterized in that: the method comprises the following steps:
(1) preparation of CL monoclonal antibody or polyclonal antibody;
(2) preparation of fluorescence antibody: first prepare NaYF
4: Yb:Er fluorescent material, described fluorescent material is with NaYF
4for matrix, Yb
3+for sensitizer, formed the nano particle of diameter 50-200nm by Yb and Er codope; Then CL monoclonal antibody or polyclonal antibody are carried out fluorescence labeling;
(3) preparation of adsorbing fiber layer: adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film are made;
(4) preparation of cellulose rete: cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film, with point sample instrument diverse location specking detection trace and contrast trace respectively on cellulose membrane, dries;
(5) assembling of test strips: adsorbing fiber layer, fluorescence antibody fibrage, cellulose rete, absorbent material layer are attached to from right to left successively and scribble on the supporting layer of bonding agent, successively supporting layer, adsorbed layer and protective seam are assembled into test strips.
Described fluorescently-labeled concrete steps are as follows:
(1) surface siliconization of fluorescent nano particle
By NaYF
4: it is in the ethanolic solution of 10% that Yb:Er fluorescent nano particle is dispersed in mass concentration, under agitation adds 5ml strong aqua, and reaction 10min, then adds 2ml tetraethyl orthosilicate, room temperature reaction 3h; The centrifugal 5min of 6000rpm, disperses with ethanol after washing;
(2) surface amination of fluorescent nano particle
The fluorescent nano particle of surface siliconization is added in the mixed solution of methyl alcohol that volume ratio is 3:5, acetone, disperse through ultrasound wave, add N-(2-the aminoethyl)-3-aminopropyl trimethoxysilane of 5 μ l, 70 DEG C of water-bath 1h, the centrifugal 5min of 6000rpm, the precipitation ethanol obtained is washed, dry;
(3) mark of CL antibody
By the NaYF that surface is modified through amination
4: Yb:Er nano particle PBS buffer solution disperses, add the glutaraldehyde solution of mass concentration 25% wherein, room temperature reaction 3 hours, centrifugally wash away unnecessary glutaraldehyde, then be dispersed in PBS buffer solution by reactant liquor, add monoclonal antibody or the polyclonal antibody of CL, 4 DEG C are reacted 3 hours, through centrifuge washing, namely obtain NaYF
4: the CL monoclonal antibody of Yb:Er nanoparticle label or polyclonal antibody, be placed in PBS buffer solution, 4 DEG C of preservations.
positive beneficial effect of the present invention:
(1) high specificity, highly sensitive.Test strips of the present invention is with upconversion fluorescence nano material NaYF
4: Yb:Er marks based on CL antibody and makes, can under infrared light direct observed result, due to NaYF
4: the outstanding optical characteristics of Yb:Er nano particle, make the detection of CL eliminate the interference of bias light, sensitivity improves greatly, minimumly a trace residue for gram level detected.
(2) stability is high, and dirigibility is good.In test strips, the luminescence phenomenon of UCP is because the pure physical process of its inside configuration produces, and completely avoid Autonomous test sample etches and the luminous cancellation caused that self decays; What UCP had the diversified characteristic spectrum (absorption spectrum and emission spectrum) of independent assortment can be applicable to multiple analysis.
(3) security is good.Upconversion fluorescence nano material in test strips has the feature of inorganic inert, infrared ray excited, VISIBLE LIGHT EMISSION, makes detection based on UCP for tester, detected product and environment all without any harm.
(4) easy, quick.Test strips can carry out direct-detection to whole blood, urine, saliva, tissue homogenate etc., need not carry out the pre-service of sample.Use test strips of the present invention directly can read value by phosphor reader, realize quantitatively detecting, can execute-in-place.As long as test strips to be inserted test sample 10 ~ 20 second, in 5 minutes, testing result can be judged.
(5) result display is vivid, directly perceived, accurate.Test strips of the present invention with show redness "
︱" and "
︱ ︱" (or " ten ", " ┬ ", " ┴ ", " ├ ", " ┤ ") trace as the positive detected and negative marker, namely show on cellulose membrane redness "
︱" trace, represent in test sample containing CL; Show two redness "
︱ ︱" trace, represent in test sample not containing CL.This result can with the naked eye directly be observed, and it is vivid, directly perceived, accurate, simple and clear to judge, not easily occurs the artificially erroneous judgement such as false positive and false negative.
(6) applied widely, easy to utilize.Present invention achieves the application of immunochromatography technique in quantitative detection CL remains based on upconversion fluorescence nano material mark, the residual detection of CL is disturbed without background; This test strips is easy and simple to handle, can meet the needs of different levels personnel, comprises specialty chemical examination, customs quarantine control, health quarantine, quality monitoring, livestock products processing, raiser and consumer individual etc.The present invention ensuring food safety, Protection of consumer healthy in be extremely important, obvious economic benefit and social benefit will be had.
(7), when prepared by test strips of the present invention, fluorescence antibody adopts NaYF
4: the CL monoclonal antibody of Yb:Er nanoparticle label or polyclonal antibody, labeling process is simple, only need by amidized NaYF
4: Yb:Er nano particle is directly connected with antibody, avoid the tedious steps that collaurum-antibody is connected, and by controlling the Conjugate ratio of volumetric molar concentration adjustment fluorescent nano particle and antibody, thus can effectively improve the sensitivity based on the Sidestream chromatography immune test paper of upconversion fluorescence nano material.
Accompanying drawing explanation
Fig. 1 is the structural representation of immuno-chromatographic test paper strip of the present invention;
Fig. 2 is the plan structure schematic diagram of immuno-chromatographic test paper strip of the present invention;
Fig. 3 is the regression curve of immuno-chromatographic test paper strip of the present invention to CL.
Embodiment
Test strips preparation process of the present invention comprises: the preparation of the preparation of CL monoclonal or polyclonal antibody, the preparation of fluorescence antibody fibrage (pad), adsorbing fiber layer (sample pad), the step such as the preparation of cellulose rete (analyzing film) and the assembling of test strips.
1, the preparation of CL monoclonal antibody or polyclonal antibody
The preparation of monoclonal antibody: comprise the immunogenic preparation of CL, immune animal (mouse), Fusion of Cells, the screening of monoclonal antibody, the preparation of monoclonal antibody; The preparation of polyclonal antibody: comprise the immunogenic preparation of CL, immune animal (rabbit), the screening of polyclonal antibody, the preparation of polyclonal antibody.
(1) preparation of CL artificial antigen:
100 mg CL standard items are dissolved in the hydrochloric acid of 10mL, 1mol/L, slowly drip the NaNO of 30%
2solution, by starch KI detection paper, be added to when KI test paper is brown purple and stop dripping, stirring reaction 45 min, drips the sulfaminic acid ammonium salt solution of 5%, with its reaction end of starch KI detection paper.The CL solution of azo slowly dripped the PBS(pH7.4 in being dissolved with 300 mg BSA with dropper) in, and by the NaOH solution adjust ph to 9.0 of 1 mol/L, after 4 DEG C of lucifuges react 24 h, 4 DEG C of dialysis 48 h, packing ,-20 DEG C save backup.In like manner prepare coating antigen.
(2) preparation of CL monoclonal antibody:
With obtained CL artificial antigen with 50 μ g ~ 100 μ g/ consumption immunity BALB/c mouse in 6 ~ 8 week age only 3 ~ 4 times, each immunization interval 3 ~ 5 weeks, determine antibody titer meet the requirements after superpower immunity, 3 ~ 4 days afterwards by under immune mouse socket of the eye hole blood sampling, be separated positive serum; De-neck is lethal, and the alcohol-pickled mouse 5 ~ 10min with 75% sterilizes body surface, asepticly gets its spleen, is shredded by spleen and grinds, filtering, the centrifugal 10min of 1000rpm through 120 order nylon gauzes, collection splenocyte.By 1 × 10
8splenocyte mix in the ratio of 10:1 with NS0 myeloma cell, the centrifugal 10min of 1000rpm, abandons supernatant, and cell precipitation thing slowly adds the PEG4000 effect 1min of 0.7 ~ 1.0mL 50% in 37 DEG C of water-baths, then serum-free 1640 nutrient culture media 15mL is slowly added, to stop the effect of PEG; 37 DEG C of water-bath 5 ~ 10 min, 1000rpm is centrifugal, and 10min abandons supernatant, is resuspended in HAT Selective agar medium by cell precipitation thing, and adds 96 porocytes cultivation plate hole (100 μ L/ hole, μ L ~ 200), is placed in 37 DEG C, 5%CO
2cultivate in incubator.Cultivate 10 ~ 14 days, carry out positive hole sizer choosing with indirect elisa method, selection strong positive, the hole that inhibiting rate is high, Growth of Cells is vigorous carry out 3 limited dilution clonings, then expand cultivation, set up hybridoma cell strain.The monoclonal antibody of prepared hybridoma secretion can be reacted with CL specifically, and affinity constant reaches 10
10~ 10
12, light chain subtype is κ or λ, and heavy chain subgroup is IgG
1, IgG
2a, IgG
2b, IgG
3, for the monoclonal antibody of CL specific epitope, for the preparation of fluorescence antibody glass fibre cotton.
(3) preparation of CL polyclonal antibody:
With obtained CL artificial antigen immunize New Zealand white rabbits, immunizing dose is 200 μ g ~ 500 μ g/ time, and dorsal sc divides 4 ~ 6 injections.Head exempts from, and dissolves CL artificial antigen, mix with equivalent FCA with aseptic PBS, fully emulsified; Booster immunization, dissolves CL carrier protein couplet thing with aseptic PBS, mix with equivalent FIA, fully emulsified, within 2 ~ 3 weeks, carries out, continuous immunity 4 ~ 5 times, every minor tick 2 ~ 3 weeks after head exempts from, last immune latter 10 ~ 15 days, surveys it surely tire and reach 10 with ELISA method
5time above, blood sampling is separated and collected hyper-immune serum also.IgG antibody is extracted with saturated ammonium sulfate salting out method, namely get 1 portion of hyper-immune serum and add 2 parts of PBS(pH7.2) mixing, add the mixing of equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerator 12h, 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant, again with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration 33%, put 4 DEG C of refrigerator 2h, 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant, with appropriate PBS(pH7.2) dissolution precipitation, put 4 DEG C of interior PBS(pH7.2 of refrigerator) dialyse 48 ~ 72h, liquid is changed for several times in centre, 4 DEG C, the centrifugal 15min of 12000rpm, collect supernatant, obtain the anti-CL polyclonal antibody of purifying,-20 DEG C frozen, for the preparation of fluorescence antibody glass fibre cotton.
2, the preparation of fluorescence antibody fibrage (pad)
The fibrolaminar preparation of fluorescence antibody, first needs to prepare NaYF
4: Yb:Er nano particle, then with the NaYF of preparation
4: Yb:Er nanoparticle label CL antibody.
(1) NaYF
4: the preparation of Yb:Er nano particle, can adopt coprecipitation or hydrothermal synthesis method.
A. coprecipitation
Get the Y (NO that concentration is 0.2mol/L respectively
3)
3yb (the NO of 20ml, 0.2mol/L
3)
3er (the NO of 2ml, 0.2mol/L
3)
3the EDTA-Na21ml of 0.5ml, 1mol/L, adds in 100ml flask, and 50 DEG C of oil bath stirring reaction 1h, obtain reactant liquor A;
Get the NaF aqueous solution of 50ml, 1mol/L, add in polytetrafluoroethylcontainer container, under magnetic agitation, A liquid is poured in NaF aqueous solution in 50 DEG C of water-baths, water-bath 1min; The centrifugal 5min of 6000rpm, abandoning supernatant must precipitate, and adds water in precipitation, ultrasonic washing 3 times, the centrifugal 5min of 7000rpm; With absolute ethyl alcohol ultrasonic washing 2 times, the centrifugal 5min of 10000rpm, is placed in 80 DEG C of baking oven dried overnight by the solid obtained; Dried solid is put into muffle furnace, in 500 DEG C of calcining 5h, namely obtains NaYF
4: Yb:Er fluorescent nano particle.
B. hydrothermal synthesis method
Get the Y (NO that concentration is 0.2mol/L respectively
3)
34ml, Yb (NO
3)
30.5ml, Er (NO
3)
30.1ml, EDTA-Na4ml, mix and after abundant reaction, obtain reactant liquor A;
Take NaOH 0.7g, join in the mixed liquor of 8ml oleic acid and 12ml ethanol and mix, under agitation the slow NaF solution injecting 10mL, 1mol/L in mixed liquor, after filling, continue to stir, to be translucent shape to solution; Reactant liquor A is poured in described solution, mixes, ageing 20min; Then potpourri is transferred in polytetrafluoroethylcontainer container, 130 DEG C of oil baths reaction 12h, the product obtained after filtration, washing, dry, namely obtain NaYF
4: Yb:Er fluorescent nano particle.
(2) NaYF
4: Yb:Er nanoparticle label CL antibody
Labeling process comprises the following steps: NaYF
4: the surface siliconization of Yb:Er nano particle, NaYF
4: the surface amination of Yb:Er nano particle, NaYF
4: the coupling of Yb:Er nano particle and CL antibody.
A. NaYF
4: the surface siliconization of Yb:Er fluorescent nano particle
By NaYF
4: Yb:Er fluorescent nano particle is dispersed in the ethanol water of mass concentration 10%, adds 5ml strong aqua (concentration 28%) under magnetic stirring, and reaction 10min, then adds 2ml tetraethyl orthosilicate, room temperature reaction 3h; The centrifugal 5min of 6000rpm, washes 3 times, finally disperses with ethanol;
B. NaYF
4: the surface amination of Yb:Er fluorescent nano particle
The fluorescent nano particle of surface siliconization is added in the mixed solution of methyl alcohol that volume ratio is 3:5, acetone, disperse through ultrasound wave, add APTSA(N-(2-the aminoethyl)-3-aminopropyl trimethoxysilane of 5ul), 70 DEG C of water-bath 1h, the centrifugal 5min of 6000rpm, the precipitation ethanol obtained is washed 3 times, dry, preserve;
C. the mark (glutaraldehyde method) of CL antibody
By the NaYF that surface is modified through amination
4: the PBS buffer solution dispersion of Yb:Er nano particle pH=7.4, add the glutaraldehyde of mass concentration 25% wherein, room temperature reaction 3 hours, centrifugally wash away unnecessary glutaraldehyde, then be dispersed in the PBS buffer solution of pH=7.4, then add monoclonal antibody or the polyclonal antibody of CL wherein, 4 DEG C are reacted 3 hours, centrifuge washing for several times, namely obtains NaYF again
4: the antibody coupling matter of Yb:Er-CL, with 4 DEG C, the PBS buffer solution preservation of pH=7.4.
3, the preparation of adsorbing fiber layer (sample pad)
Prepared by test lead adsorbing fiber layer glass fibre cotton, nylon membrane, polyvinylidene fluoride pvdf membrane or polyester film, fibrous material is cut into the band of wide 1.5cm specification, puts it in sample pad confining liquid and soak 30min, in 37 DEG C of oven dry, for subsequent use.
4, the preparation of cellulose rete (analyzing film)
Cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film, cut into the band of wide 1.5cm specification, with point sample instrument diverse location specking CL antigen and goat anti-mouse igg antibody (or rabbit anti-mouse IgG, goat anti-rabbit igg antibody) respectively on cellulose membrane, make stealthy detection trace band and contrast trace band, in 37 DEG C of dry for standby.
Wherein goat-anti or rabbit anti-mouse IgG(or goat anti-rabbit igg) preparation method of antibody is as follows:
CL negative mice serum IgG (or negative rabbit anteserum IgG) is extracted with saturated ammonium sulfate, get 1 part of mice serum (or rabbit anteserum) and add 2 parts of PBS(pH7.2) mixing, add the mixing of equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerator 12h, 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant; Again with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration 33%, put 4 DEG C of refrigerator 2h, 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant; With appropriate PBS(pH7.2) dissolution precipitation, put in 4 DEG C of refrigerators, with PBS(pH7.2) dialyse 48h, liquid is changed 3 times in centre, 4 DEG C, the centrifugal 15min of 12000rpm, collects supernatant, measures its protein concentration with ultraviolet spectrophotometer; With mice serum (or rabbit anteserum) IgG of 50 μ g ~ 100 μ g/kg body weight through the healthy goat of subcutaneous and intramuscular injection or rabbit 3 ~ 4 times, final injection is after 10 days, with ELISA measure its serum titer reach more than 1:2000 time, heart or arterial blood drawing, separated and collected hyper-immune serum, goat-anti or rabbit anti-mouse IgG(or goat anti-rabbit igg is extracted with saturated ammonium sulfate) antibody (method is identical with extracting mice serum IgG, no longer repeats), the preparation of trace is contrasted for CL test strip.
5, the assembling of immuno-chromatographic test paper strip
Adsorbing fiber layer (sample pad), fluorescence antibody fibrage (pad), cellulose rete (analyzing film), absorbent material layer are attached to successively from right to left on the supporting layer (base plate) with bonding agent, and are cut into the wide test strips of 3-4cm.
6, the detection reaction principle of immuno-chromatographic test paper strip of the present invention
After test strips test lead inserts testing sample solution, solution to be measured drives the fluorescence antibody in CL to be measured and fluorescence antibody glass fibre cotton to spread to cellulose rete together by syphonic effect, and the final absorbent material layer infiltrating handle end.In diffusion process, CL to be measured can combine with fluorescence antibody, and then the antigen-combining site of CL on closed fluorescence antibody, the detection trace of the CL artificial antigen of fluorescence antibody on cellulose membrane is stoped to be combined, detection trace can not be shown, sheep or rabbit anti-mouse IgG(or goat anti-rabbit igg) antibody then can be combined with fluorescence antibody, under infrared excitation (or using phosphor reader directly to read value) formed red contrast trace band "
︱", namely red zone "
︱" trace is positive expression; Otherwise time in sample solution without CL, then the CL artificial antigen of fluorescence antibody on cellulose membrane can not be stoped to detect trace and to be combined, display red detection trace band "
︱", same goat-anti or rabbit anti-mouse IgG(or goat anti-rabbit igg) antibody is also combined with golden labeling antibody, display red contrast trace band "
︱", formed two red zones "
︱ ︱" be negative expression.If without any red trace band display on cellulose membrane, then show that test strips lost efficacy.
following examples illustrate structure and the detection method of test strips.
Embodiment one: see Fig. 1 and Fig. 2.In figure, 1 is supporting layer, make with plastic slice bar, 2 is adsorbing fiber layer, make with glass fibre cotton, fluorescence antibody fibrage 3 is adsorbed with the fluorescence antibody glass fibre cotton of anti-CL monoclonal antibody, cellulose rete 4 adopts nitrocellulose filter, the absorbent material layer 5 of handle end is made with absorbent filter, adsorbing fiber layer 2, fluorescence antibody fibrage 3, cellulose rete 4, each layer of absorbent material layer 5 are pasted and fixed on successively from right to left on supporting layer 1, intersection fiber crosses one another infiltration each other.Cellulose rete 4 is provided with stealthy detection trace 6, makes with the bovine serum albumin solution (BSA) of coupling CL; Stealthy contrast trace 7 goat anti-mouse igg antibody solution trace on cellulose membrane is made "
︱", two trace bands are arranged in parallel, formation combination trace band "
︱ ︱".
8-1 is the sample end white diaphragm covered above adsorbing fiber layer 2 and fluorescence antibody fibrage 3; 8-2 is other color diaphragm (as yellow) covered above absorbent material layer 5; 9 is sample mark line; this mark line is positioned at the adsorbing fiber layer 2 white diaphragm corresponding with fluorescence antibody fibrage 3 intersection and is partial to adsorbing fiber layer 2 side and is about 0.5cm place, diaphragm is printed on arrow and max printed words on the right side of mark line.
The preparation of testing sample and detecting step:
Detect meat sample: to be shredded by sample, levigate, make the sample suspension of 1:2 ~ 10 with normal saline dilution;
Method of operating: CL test strips sample end inserted in testing sample, insertion depth is no more than mark line, and about 10 ~ 20 seconds took out test strips, observed result under 980nm infrared excitation, or directly read value by phosphor reader.
Result judge: if (a) positive show on cellulose membrane a red trace band "
︱", represent that testing result is positive, illustrate in testing sample containing CL; If (b) feminine gender show on cellulose membrane two red trace bands "
︱ ︱", represent that testing result is negative, illustrate in testing sample not containing CL; On cellulose membrane, do not have red zone to show if c () is lost efficacy, then showed that test strips lost efficacy.
Embodiment two: test strips structure is substantially identical with embodiment one; difference is: fluorescence antibody fibrage 3 is adsorbed with the polyclonal antibody of anti-CL; adsorbing fiber layer 2 is made with nylon membrane; cellulose rete 4 adopts pure cellulose film; the stealthy trace band that detects is " ten " with stealthy contrast trace band, and 8-2 is the blue look diaphragm of handle end covered above absorbent material layer 5.
Detect milk sample: the sample suspension with physiological saline, milk sample dilution being made 1:2 ~ 5.
Result judge: if (a) positive show on cellulose membrane a red trace band "
ten", represent that testing result is positive, illustrate in testing sample containing CL; If (b) feminine gender show on cellulose membrane two red trace bands "
ten", represent that testing result is negative, illustrate in testing sample not containing CL; On cellulose membrane, do not have red zone to show if c () is lost efficacy, then showed that test strips lost efficacy.Method of operating is with embodiment one.
Embodiment three: test strips structure is substantially identical with embodiment one; difference is: adsorbing fiber layer 2 polyvinylidene fluoride pvdf membrane is made; the carrier protein solution of coupling CL is chicken ovalbumin (OVA); stealthy contrast trace 7 rabbit anti-mouse IgG antibody solution is made on cellulose membrane; cellulose rete 4 adopts carboxylated cellulose film; 8-2 is the handle end greenism film covered above absorbent material layer 5, and the stealthy trace band that detects is " ┬ " with stealthy contrast trace band.
For detecting blood sample: extract serum and diluted the testing sample making 1:2 ~ 10 with physiological saline.
Result judgement and method of operating are all with embodiment one.
Embodiment four: test strips structure is substantially identical with embodiment one, and difference is: adsorbing fiber layer 2 is made with polyester film, cellulose rete 4 adopts carboxylated cellulose film, and the stealthy carrier protein solution detecting coupling CL in trace 6 is hemocyanin (KLH).For detecting urine sample, can directly get urine as testing sample.Detect trace band and contrast trace band and be " ┴ ".Method of operating and result decision method are with example one.
Embodiment five: test strips structure is substantially identical with embodiment one, and difference is: stealthy contrast trace 7 goat anti-rabbit igg antibody solution is made on cellulose membrane, and adsorbing fiber layer 2 is made with nylon membrane.Detect trace band and contrast trace band and be " ├ ".Detect sample, result judgement and method of operating with example one.
Embodiment six: substantially identical with embodiment one, difference is: fluorescence antibody fibrage 3 is adsorbed with the polyclonal antibody of anti-CL, detection sample is milk sample.Detect trace band and contrast trace band and be " ┤ ".
Embodiment seven: substantially identical with embodiment one, difference is: fluorescence antibody fibrage 3 is adsorbed with the polyclonal antibody of anti-CL, detection sample is blood sample.
Embodiment eight: substantially identical with embodiment one, difference is: fluorescence antibody fibrage 3 is adsorbed with anti-CL polyclonal antibody, detection sample is urine sample.
Embodiment nine: substantially identical with embodiment one, difference is: in stealthy detection trace 6, coupling CL carrier protein solution is hemocyanin (KLH), detects sample, result judgement and method of operating with embodiment one, does not repeat.
Embodiment ten: substantially identical with embodiment one, difference is: in stealthy detection trace 6, coupling CL carrier protein solution is chicken ovalbumin (OVA), detects sample, result judgement and method of operating with embodiment one, does not repeat.
Embodiment 11: the sensitivity of test strips of the present invention, specific detection
1, the detection of sensitivity: use phosphate buffered solution PBS(PH7.4) or distilled water respectively configuration concentration be 4,2,1,0.5,0.25,0.125,0.0625, the CL standard items of 0ng/mL, loading 80-100uL in test strips of the present invention, react after 5-10 minute, observations under ultraviolet light.
Test strips reacted under ultraviolet light is taken pictures, picture drawing tools is opened, select T line region with the rectangle tool, measure its shading value (L
t), deduct the bias light angle value (L between T line (detection line) and C line (control line)
b), with inhibiting rate L
t-L
bfor ordinate, with the logarithm value of different CL concentration for horizontal ordinate, drawing standard suppresses curve, carries out correlation regression analysis, calculates the IC of this test strips to CL
50and lowest detectable limit.After measured, this test paper to the Regression Equations of CL is: y=-49.952x+131.57, and related coefficient is R
2=0.9929, go out the IC of this test paper to CL according to regression equation calculation
50for 429.50pg/mL, the lowest detection of this test paper is limited to 107.74pg/mL, shows that test paper has higher sensitivity to CL.See Fig. 3.
2, specific detection: using the similar drugs Ractopamine of CL, Cimaterol and daimeton, sulphadiazine, oxolinic acid, penicillin, tetracycline chloromycetin, neomycin, olaquindox as competitor, the concentration configuring above-mentioned mark product is 1mg/mL, with its inhibiting rate of up-conversion fluorescence detection paper, with the IC of this test paper to CL
50with the IC of each competitor
50number percent be its cross reacting rate.
Measurement result sees the following form 1.Can find out, the specificity of this test strips is better, no cross reaction equal to other drug.
| Compound | Half-inhibition concentration IC 50（ng/mL） | Cross reactivity (%) |
| Clenobuterol hydrochloride | 0.43 | 100 |
| Ractopamine | > 1.0×10 3 | < 0.04 |
| Cimaterol | > 1.0×10 3 | < 0.04 |
| Daimeton | > 1.0×10 3 | < 0.04 |
| Sulphadiazine | > 1.0×10 3 | < 0.04 |
| Oxolinic acid | > 1.0×10 3 | < 0.04 |
| Penicillin | > 1.0×10 3 | < 0.04 |
| Tetracycline | > 1.0×10 3 | < 0.04 |
| Chloromycetin | > 1.0×10 3 | < 0.04 |
| Neomycin | > 1.0×10 3 | < 0.04 |
| Olaquindox | > 1.0×10 3 | < 0.04 |
Claims (7)
1. the immuno-chromatographic test paper strip based on the detection Clenbuterol of upper conversion fluorescent nano particle mark, bottom is supporting layer, middle layer is adsorbed layer, protective seam is fixed on adsorbed layer, adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, fluorescence antibody fibrage, cellulose rete and handle end from test lead, it is characterized in that: the stealth be provided with on cellulose rete with the carrier protein solution of coupling CL is printed detects trace, be also provided with the stealth contrast trace printed with goat anti-mouse igg, rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution; Described fluorescence antibody fibrage adopts the glass fibre cotton of absorption fluorescence antibody to make, and fluorescence antibody adopts NaYF
4: the CL monoclonal antibody of Yb:Er nanoparticle label or polyclonal antibody;
Described NaYF
4: Yb:Er nano particle is with NaYF
4for matrix, Yb
3+for sensitizer, the diameter formed by Yb and Er codope is the nano particle of 50-200nm;
Described NaYF
4: the CL monoclonal antibody of Yb:Er nanoparticle label or polyclonal antibody are prepared by following methods, and concrete steps are:
(1) surface siliconization of fluorescent nano particle
By NaYF
4: it is in the ethanolic solution of 10% that Yb:Er fluorescent nano particle is dispersed in mass concentration, under agitation adds 5ml strong aqua, and reaction 10min, then adds 2ml tetraethyl orthosilicate, room temperature reaction 3h; The centrifugal 5min of 6000rpm, disperses with ethanol after washing;
(2) surface amination of fluorescent nano particle
The fluorescent nano particle of surface siliconization is added in the mixed solution of methyl alcohol that volume ratio is 3:5, acetone, disperse through ultrasound wave, add N-(2-the aminoethyl)-3-aminopropyl trimethoxysilane of 5 μ l, 70 DEG C of water-bath 1h, the centrifugal 5min of 6000rpm, the precipitation ethanol obtained is washed, dry;
(3) mark of CL antibody
By the NaYF that surface is modified through amination
4: Yb:Er nano particle PBS buffer solution disperses, add the glutaraldehyde solution of mass concentration 25% wherein, room temperature reaction 3 hours, centrifugally wash away unnecessary glutaraldehyde, then be dispersed in PBS buffer solution by reactant liquor, add monoclonal antibody or the polyclonal antibody of CL, 4 DEG C are reacted 3 hours, through centrifuge washing, namely obtain NaYF
4: the CL monoclonal antibody of Yb:Er nanoparticle label or polyclonal antibody, be placed in PBS buffer solution, 4 DEG C of preservations.
2. immuno-chromatographic test paper strip according to claim 1, is characterized in that: described adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film; Absorbent material layer absorbent filter, the supporting layer toughness material do not absorbed water; Cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film;
the carrier protein of coupling CL is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin.
3. immuno-chromatographic test paper strip according to claim 1, is characterized in that: described stealth detect trace and stealthy contrast trace be arranged in parallel "
︱ ︱" orthoscopic trace, or be " 10 " font arrangement trace, or be " ┬ ┬ " font arrangement trace, or be " ┴ ┴ " font arrangement trace, or be " ├ ├ " font arrangement trace, or be " ┤ ┤ " font arrangement trace.
4. immuno-chromatographic test paper strip according to claim 1; it is characterized in that: on described adsorbing fiber layer, fluorescence antibody fibrage and absorbent material layer, be coated with diaphragm; the diaphragm that adsorbing fiber layer is corresponding with fluorescence antibody fibrage intersection is printed with sample mark line, this mark line deflection adsorbing fiber layer side 0.3-0.7cm.
5. the immuno-chromatographic test paper strip according to any one of claim 1-4, is characterized in that: described NaYF4:Yb:Er nano particle adopts coprecipitation preparation, and concrete grammar is as follows:
Get the Y (NO that concentration is 0.2mol/L respectively
3)
3yb (the NO of 20ml, 0.2mol/L
3)
3er (the NO of 2ml, 0.2mol/L
3)
3the EDTA-Na21ml of 0.5ml, 1mol/L, adds in flask, and 50 DEG C of oil bath stirring reaction 1h, obtain reactant liquor A;
Get the NaF solution of 50ml, 1mol/L, add in polytetrafluoroethylcontainer container, under 50 DEG C of stirred in water bath, reactant liquor A is poured in NaF solution, reaction 1min; The centrifugal 5min of 6000rpm, abandoning supernatant must precipitate; Add water in precipitation, ultrasonic washing 3 times, the centrifugal 5min of 7000rpm; Again by precipitation absolute ethyl alcohol ultrasonic washing 2 times, the centrifugal 5min of 10000rpm, is placed in 80 DEG C of baking oven dried overnight by the solid obtained; Dried solid is put into muffle furnace, in 500 DEG C of calcining 5h, namely obtains NaYF
4: Yb:Er fluorescent nano particle.
6. the immuno-chromatographic test paper strip according to any one of claim 1-4, is characterized in that: described NaYF
4: Yb:Er nano particle adopts hydrothermal synthesis method preparation, and concrete grammar is as follows:
Get the Y (NO that concentration is 0.2mol/L respectively
3)
34ml, Yb (NO
3)
30.5ml, Er (NO
3)
30.1ml, EDTA-Na4ml, mix and fully react, and obtains reactant liquor A; Take NaOH 0.7g, join in the mixed liquor of 8ml oleic acid and 12ml ethanol, mixing, the under agitation slow NaF solution injecting 10mL, 1mol/L in mixed liquor, after filling, continue to be stirred to solution and to be translucent shape; Reactant liquor A is poured in described solution, mixes, ageing 20-30min; Then potpourri is transferred in polytetrafluoroethylcontainer container, 130-150 DEG C of oil bath reaction 12-18h, the product obtained after filtration, washing, dry, namely obtain NaYF
4: Yb:Er fluorescent nano particle.
7. the preparation method of immuno-chromatographic test paper strip described in claim 1, is characterized in that: the method comprises the following steps:
(1) preparation of CL monoclonal antibody or polyclonal antibody;
(2) preparation of fluorescence antibody: first prepare NaYF
4: Yb:Er fluorescent material, described fluorescent material is with NaYF
4for matrix, Yb
3+for sensitizer, formed the nano particle of diameter 50-200nm by Yb and Er codope; Then CL monoclonal antibody or polyclonal antibody are carried out fluorescence labeling;
(3) preparation of adsorbing fiber layer: adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film are made;
(4) preparation of cellulose rete: cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film, with point sample instrument diverse location specking detection trace and contrast trace respectively on cellulose membrane, dries;
(5) assembling of test strips: adsorbing fiber layer, fluorescence antibody fibrage, cellulose rete, absorbent material layer are attached to from right to left successively and scribble on the supporting layer of bonding agent, successively supporting layer, adsorbed layer and protective seam are assembled into test strips;
Described fluorescently-labeled concrete steps are as follows:
(1) surface siliconization of fluorescent nano particle
By NaYF
4: it is in the ethanolic solution of 10% that Yb:Er fluorescent nano particle is dispersed in mass concentration, under agitation adds 5ml strong aqua, and reaction 10min, then adds 2ml tetraethyl orthosilicate, room temperature reaction 3h; The centrifugal 5min of 6000rpm, disperses with ethanol after washing;
(2) surface amination of fluorescent nano particle
The fluorescent nano particle of surface siliconization is added in the mixed solution of methyl alcohol that volume ratio is 3:5, acetone, disperse through ultrasound wave, add N-(2-the aminoethyl)-3-aminopropyl trimethoxysilane of 5 μ l, 70 DEG C of water-bath 1h, the centrifugal 5min of 6000rpm, the precipitation ethanol obtained is washed, dry;
(3) mark of CL antibody
By the NaYF that surface is modified through amination
4: Yb:Er nano particle PBS buffer solution disperses, add the glutaraldehyde solution of mass concentration 25% wherein, room temperature reaction 3 hours, centrifugally wash away unnecessary glutaraldehyde, then be dispersed in PBS buffer solution by reactant liquor, add monoclonal antibody or the polyclonal antibody of CL, 4 DEG C are reacted 3 hours, through centrifuge washing, namely obtain NaYF
4: the CL monoclonal antibody of Yb:Er nanoparticle label or polyclonal antibody, be placed in PBS buffer solution, 4 DEG C of preservations.
?
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