CN105424923A - Color-fluorescence dual-function immunochromatography test strip and preparation method thereof - Google Patents
Color-fluorescence dual-function immunochromatography test strip and preparation method thereof Download PDFInfo
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- FXXACINHVKSMDR-UHFFFAOYSA-N acetyl bromide Chemical compound CC(Br)=O FXXACINHVKSMDR-UHFFFAOYSA-N 0.000 claims description 4
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 3
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 2
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- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- 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 Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the field of biological detection, and particularly relates to a color-fluorescence dual-function immunochromatography test strip and a preparation method thereof. The test strip comprises a base plate (1). A sample addition pad (2), a marker combination pad (3), a detection pad (4) and a water absorption pad (5) are sequentially arranged on one face of the base plate (1), wherein the detection pad (4) is provided with a detection line T (41) and a quality control line C (42), and the water absorption pad (5) provides power for a capillary tube siphoning, the adjacent pads are sequentially connected in an overlapped mode, and a color-fluorescence dual-function microsphere marker probe coats the marker combination pad (3). According to the color-fluorescence dual-function immunochromatography test strip and the preparation method thereof, negative and positive attribute preliminary qualitative judgment can be performed on the detection result through naked eyes, the detection result can also be analyzed through an instrument, and accurate detection data are obtained.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of colour-fluorescent dual-function immuno-chromatographic test paper strip and preparation method thereof.
Background technology
At immunochromatography detection field, there is the detection mode of two kinds of main flows at present: a kind of is qualitative detection immuno-chromatographic test paper strip by naked eyes interpretation, comprise colloidal gold mark test paper, color latex mark test paper etc., its advantage can make the content of determinand in sample judging qualitatively without the need to configuring any checkout equipment, shortcoming is cannot be quantitative, can not provide concentration data accurately, and its detection sensitivity is on the low side, generally can only the determinand of concentrations more than ng/ml rank; Another kind is the fluorescence immune chromatography test paper bar by instrument interpretation, its advantage is by instrument interpretation data, concentration data accurately can be provided, and test strips height 2-3 order of magnitude that detection sensitivity marks than collaurum or color latex, but its shortcoming is the content concn situation that cannot obtain determinand when not having readout instrument completely.
Summary of the invention
Invention broadly provides a kind of colour-fluorescent dual-function immuno-chromatographic test paper strip and preparation method thereof, its testing result both carried out the judgement of yin and yang attribute initial characterization by naked eyes, again by instrumental analysis, obtained and detected data accurately.
A kind of colour-fluorescent dual-function immuno-chromatographic test paper strip, comprise base plate, the one side of base plate is sequentially with application of sample pad, the label pad of interpolation testing sample, is provided with the detecting pad of detection line T line and nature controlling line C line and the adsorptive pads of capillary siphoning power is provided, adjacent each pad overlaps successively, label pad is coated with colour-fluorescent dual-function microballoon label probe.
Preferably, described colour-fluorescent dual-function microballoon label probe contains determinand antibody colour-fluorescent dual-function microballoon label and rabbit, antigen-antibody colour-fluorescent dual-function microballoon the label of mouse or other and determinand antigen-antibody no cross reaction, determinand antibody colour-Fluorescent microsphere marker is combined by the antigen-antibody relevant to determinand or ligand receptor and colour-fluorescent dual-function microballoon and is prepared from, rabbit, antigen-antibody colour-fluorescent dual-function microballoon the label of mouse or other and determinand antigen-antibody no cross reaction is by rabbit, mouse or other antigen-antibodies with determinand antigen-antibody no cross reaction and colour-fluorescent dual-function microballoon are combined and are prepared from.
Preferably, described detection line T line is coated with the antigen-antibody relevant to determinand or ligand receptor, and nature controlling line C line is coated with the antigen-antibody irrelevant with determinand or ligand receptor.
Preferably; the particle diameter of described colour-fluorescent dual-function microballoon is 50-1000nm; the material of colour-fluorescent dual-function microballoon is the one in polystyrene microsphere, poly (methyl methacrylate) micro-sphere, polyvinyl pyridine microspheres, poly-ethyl methyl acrylate microballoon, lipid microsphere and silicon dioxide microsphere, and described colour-fluorescent dual-function microsphere surface is with one or more in carboxyl, amino, aldehyde radical, acetyl bromide, iodoacetyl, mercapto and epoxy radicals or be not with group.
Preferably, the color of described colour-fluorescent dual-function microballoon is the macroscopic dark color of one in redness, blueness, green, purple and black; Fluorescence is the one in Rare Earth Chelate fluorescence, quantum dot fluorescence and common fluorescent; The wavelength difference that the wavelength of color emission and fluorescent material are launched is more than or equal to 30nm.
Preferably, described application of sample pad is the material with filtration, the material of application of sample pad is glass fibre, polyester film, sponge, filter paper or through being added with one or more the phosphate buffer in surfactant, carbohydrate, protide, macromolecule polymer, salt or the glass fibre after Tris-HCl damping fluid dipping pretreatment, polyester film, sponge, filter paper;
Preferably, the material of described label pad be glass fibre, polyester film or through being added with one or more the phosphate buffer in surfactant, carbohydrate, protide, macromolecule polymer, salt or the glass fibre after Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl damping fluid) dipping pretreatment or polyester film;
Preferably, described detecting pad is the porosint with strong hydrophobic property, and the material of detecting pad is nitrocellulose filter or cellulose acetate membrane, and T line and C line are at least one.
A preparation method for colour-fluorescent dual-function immuno-chromatographic test paper strip, comprises the following steps:
(I) preparation of application of sample pad
Glass fibre, polyester film, sponge or filter paper is soaked with the phosphate buffer (PBS damping fluid) containing ethylenediamine tetraacetic acid (EDTA), polysorbas20 (tween-20), polyvinylpyrrolidone (PVP) and bovine serum albumin(BSA) (BSA), then be placed in baking oven to dry, hermetically drying saves backup;
(II) preparation of label pad
Glass fibre or polyester film is soaked with the PBS damping fluid containing trehalose, bovine serum albumin(BSA), tween-20 and PVP, then be placed in baking oven to dry, again colour-fluorescent dual-function microballoon label probe is sprayed on the glass fibre after process oven dry or polyester film, again be placed in baking oven to dry, hermetically drying saves backup;
(III) preparation of detecting pad
The bag quilt of detection line T line: by the antigen-antibody relevant to determinand or ligand receptor and with the PBS buffer solution containing trehalose and lauryl sodium sulfate (SDS), obtained spray coating liquor one, is sprayed on spray coating liquor one and forms detection line T line apart from detecting pad left end 12mm place;
The bag quilt of nature controlling line C line: the phosphate buffer containing trehalose and SDS dissolves by the antigen-antibody had nothing to do with determinand or ligand receptor, obtained spray coating liquor two, is sprayed on apart from detecting pad left end 18mm place formation detection line T line by spray coating liquor two;
By for subsequent use for the detecting pad kept dry sprayed;
(IV) assembling of test strips
On Polyvinylchloride (PVC) base plate successively overlap processed application of sample pad, be sprayed with the colour-label pad of fluorescent dual-function microballoon label probe, the detecting pad being coated with detection line T line and nature controlling line C line and adsorptive pads, cut into the test strips that 4mm is wide after assembling.
Preferably, concrete preparation method is as follows for colour described in step (II)-fluorescent dual-function microballoon label probe:
The preparation of (b) colour-fluorescent dual-function microballoon: add microballoon prepared by methylene chloride or tetrahydrofuran and step (a) in deionized water, obtain microspheres solution, the fluorescent marker and fat-soluble color pigment that are dissolved in methylene chloride or tetrahydrofuran are dropwise added in microspheres solution also to seal and stir, then depressurization remove methylene chloride or tetrahydrofuran, last centrifugal treating, obtain colour-fluorescent dual-function microballoon, for subsequent use;
The preparation of (c) determinand antibody colour-fluorescent dual-function microballoon label: the colour prepared with damping fluid dilution step (b)-fluorescent dual-function microballoon, add carbodiimide and N-hydroxy-succinamide, on shaking table after activation, centrifugal treating, remove supernatant, add damping fluid to redissolve to precipitation, then the antigen-antibody relevant to determinand or ligand receptor is added, coupling on shaking table, then monoethanolamine and bovine serum albumin(BSA) is added, sealing is spent the night, last centrifugal treating, remove supernatant, with damping fluid, precipitation is redissolved again, ultrasonic process, obtained determinand antibody colour-Fluorescent microsphere marker,
(d) rabbit, the preparation of the antigen-antibody colour-Fluorescent microsphere marker of mouse or other and determinand antigen-antibody no cross reaction: dilute colour-fluorescent dual-function microballoon with damping fluid, add carbodiimide and rabbit, the antigen-antibody of mouse or other and determinand antigen-antibody no cross reaction, coupling on shaking table, then monoethanolamine and bovine serum albumin(BSA) is added, sealing is spent the night, last centrifugal treating, remove supernatant, redissolve with damping fluid again, ultrasonic process, obtained rabbit, antigen-antibody colour-the Fluorescent microsphere marker of mouse or other and determinand antigen-antibody no cross reaction,
The preparation of (e) colour-fluorescent dual-function microballoon label probe: determinand antibody colour-Fluorescent microsphere marker and rabbit, mouse or other mix by a certain percentage with the antigen-antibody colour-Fluorescent microsphere marker of determinand antigen-antibody no cross reaction, be sprayed on obtained colour-fluorescent dual-function microballoon label probe on pad.
Adopt above-mentioned colour-fluorescent dual-function immuno-chromatographic test paper strip, the present invention has the following advantages:
Colour prepared by the present invention-fluorescent dual-function immuno-chromatographic test paper strip both can carry out the judgement of yin and yang attribute initial characterization by naked eyes to testing result, when not needing professional detecting instrument, qualitative judgement is made to the content of determinand in sample, detection sensitivity is high, can determinand below qualitative detection ng/ml rank; By instrument, quantitative test is carried out to result again, obtain concentration data accurately, and test strips height 2-3 order of magnitude that detection sensitivity marks than existing collaurum or color latex, accuracy of detection is high, detection demand various under meeting different test item, environment, object, sensitivity requirement situation.
Accompanying drawing explanation
Fig. 1 is the structural representation of colour of the present invention-fluorescent dual-function immuno-chromatographic test paper strip.
Wherein: 1, base plate, 2, application of sample pad, 3, label pad, 4, detecting pad, 41, detection line T line, 42, nature controlling line C line, 5, adsorptive pads.
Embodiment
A kind of colour-fluorescent dual-function immuno-chromatographic test paper strip, comprise base plate 1, the one side of base plate 1 is sequentially with application of sample pad 2, the label pad 3 of interpolation testing sample, is provided with the detecting pad 4 of detection line T line 41 and nature controlling line C line 42 and the adsorptive pads 5 of capillary siphoning power is provided, adjacent each pad overlaps successively, label pad 3 is coated with colour-fluorescent dual-function microballoon label probe.
Colour-fluorescent dual-function microballoon label probe contains determinand antibody colour-fluorescent dual-function microballoon label and rabbit, antigen-antibody colour-fluorescent dual-function microballoon the label of mouse or other and determinand antigen-antibody no cross reaction, determinand antibody colour-fluorescent dual-function microballoon label is combined by the antigen-antibody relevant to determinand or ligand receptor and colour-fluorescent dual-function microballoon and is prepared from, rabbit, mouse or other and determinand antigen-antibody no cross reaction antigen-antibody colour-fluorescent dual-function microballoon label are by rabbit, mouse or other antigen-antibodies with determinand antigen-antibody no cross reaction and colour-fluorescent dual-function microballoon are combined and are prepared from.
Detection line T line 41 is coated with the antigen-antibody relevant to determinand or ligand receptor, and nature controlling line C line 42 is coated with the antigen-antibody irrelevant with determinand or ligand receptor.
Thing to be detected can be biotoxin and metabolin, small molecule chemicals and high molecular weight protein, nucleic acid etc., all can use ELISA test strip of the present invention.For Aflatoxins M1 and chloromycetin in following specific embodiment, schematically illustrate test strips of the present invention and there is colour-fluorescent dual-function.When detecting other materials, with class of operation in embodiment seemingly, do and simply replace.
When described biotoxin is Aflatoxins M1, colour-fluorescent dual-function microballoon label probe contains Aflatoxins M1 antibody colour-fluorescent dual-function microballoon label and rabbit igg colour-Fluorescent microsphere marker.Detection line T line 41 is coated with Aflatoxins M1-bovine serum albumin(BSA) conjugate, and nature controlling line C line 42 is coated with goat-anti rabbit polyclonal antibody.
When described small molecule chemicals is chloromycetin, colour-fluorescent dual-function microballoon label probe contains chloramphenicol antibody colour-fluorescent dual-function microballoon label and chicken IgG colour-Fluorescent microsphere marker.Detection line T line 41 is coated with chloromycetin-bovine serum albumin(BSA) conjugate, and nature controlling line C line 42 is coated with goat-anti chicken polyclonal antibody.
The particle diameter of described colour-fluorescent dual-function microballoon is 50-1000nm; the material of colour-fluorescent dual-function microballoon is the one in polystyrene microsphere, poly (methyl methacrylate) micro-sphere, polyvinyl pyridine microspheres, poly-ethyl methyl acrylate microballoon, lipid microsphere and silicon dioxide microsphere, and described colour-fluorescent dual-function microsphere surface is with one or more in carboxyl, amino, aldehyde radical, acetyl bromide, iodoacetyl, mercapto and epoxy radicals or be not with group.
The color of described colour-fluorescent dual-function microballoon is the macroscopic dark color of one in redness, blueness, green, purple and black; Fluorescence is the one in Rare Earth Chelate fluorescence, quantum dot fluorescence and common fluorescent; The wavelength difference that the wavelength of color emission and fluorescent material are launched is more than or equal to 30nm.
Application of sample pad 2 for having the material of filtration, the material of application of sample pad 2 is glass fibre, polyester film, sponge, filter paper or through being added with one or more the phosphate buffer in surfactant, carbohydrate, protide, macromolecule polymer, salt or the glass fibre after Tris-HCl damping fluid dipping pretreatment, polyester film, sponge, filter paper;
The material of label pad 3 is glass fibre, polyester film or through being added with one or more the phosphate buffer in surfactant, carbohydrate, protide, macromolecule polymer, salt or the glass fibre after Tris-HCl damping fluid dipping pretreatment or polyester film;
Detecting pad 4 is for having the porosint of strong hydrophobic property, and the material of detecting pad 4 is nitrocellulose filter or cellulose acetate membrane, and T line and C line are at least one.
The preparation method of a kind of colour-fluorescent dual-function immuno-chromatographic test paper strip comprises the following steps:
(I) preparation of application of sample pad 2
Soak glass fibre, polyester film, sponge or filter paper with the PBS damping fluid containing EDTA, tween-20, PVP and BSA, be then placed in baking oven and dry, hermetically drying saves backup;
(II) preparation of label pad 3
Glass fibre or polyester film is soaked with the PBS damping fluid containing trehalose, bovine serum albumin(BSA), tween-20 and PVP, then be placed in baking oven to dry, again colour-fluorescent dual-function microballoon label probe is sprayed on the glass fibre after process oven dry or polyester film, again be placed in baking oven to dry, hermetically drying saves backup.
Concrete preparation method is as follows for above-mentioned colour-fluorescent dual-function microballoon label probe:
The preparation of (a) microballoon: to get in polystyrene, polymethylmethacrylate, polyvinyl pyridine, poly-ethyl methyl acrylate, lipid and silicon dioxide a kind of, and to be dissolved in the deionized water for stirring of sodium dodecylsulphonate even, removal aeroseal is carried out to it and heats, add potassium persulfate after depressurization to stir, then stirring liquid is filtered, to the filtrate centrifugal purification collected, obtain microballoon, for subsequent use;
The preparation of (b) colour-fluorescent dual-function microballoon: add microballoon prepared by methylene chloride or tetrahydrofuran and step (a) in deionized water, obtain microspheres solution, the fluorescent marker and fat-soluble color pigment that are dissolved in methylene chloride or tetrahydrofuran are dropwise added in microspheres solution also to seal and stir, then depressurization remove methylene chloride or tetrahydrofuran, last centrifugal treating, obtain colour-fluorescent dual-function microballoon, for subsequent use;
The preparation of (c) determinand antibody colour-fluorescent dual-function microballoon label: the colour prepared with damping fluid dilution step (b)-fluorescent dual-function microballoon, add carbodiimide and N-hydroxy-succinamide, on shaking table after activation, centrifugal treating, remove supernatant, add damping fluid to redissolve to precipitation, then the antigen-antibody relevant to determinand or ligand receptor is added, coupling on shaking table, then monoethanolamine and bovine serum albumin(BSA) is added, sealing is spent the night, last centrifugal treating, remove supernatant, with damping fluid, precipitation is redissolved again, ultrasonic process, obtained determinand antibody colour-fluorescent dual-function microballoon label,
(d) rabbit, the preparation of the antigen-antibody colour-fluorescent dual-function microballoon label of mouse or other and determinand antigen-antibody no cross reaction: dilute colour-fluorescent dual-function microballoon with damping fluid, add carbodiimide and rabbit, the antigen-antibody of mouse or other and determinand antigen-antibody no cross reaction, coupling on shaking table, then monoethanolamine and bovine serum albumin(BSA) is added, sealing is spent the night, last centrifugal treating, remove supernatant, redissolve with damping fluid again, ultrasonic process, obtained rabbit, antigen-antibody colour-fluorescent dual-function microballoon label colour-the Fluorescent microsphere marker of mouse or other and determinand antigen-antibody no cross reaction,
The preparation of (e) colour-fluorescent dual-function microballoon label probe: determinand antibody colour-fluorescent dual-function microballoon label and rabbit, mouse or other mix with the antigen-antibody colour-Fluorescent microsphere marker of determinand antigen-antibody no cross reaction and be sprayed on pad, obtained colour-fluorescent dual-function microballoon label probe.
(III) preparation of detecting pad 4
The bag quilt of detection line T line 41: the PBS buffer solution containing trehalose and SDS by the antigen-antibody relevant to determinand or ligand receptor, obtained spray coating liquor one, is sprayed on spray coating liquor one and forms detection line T line 41 apart from detecting pad 4 left end 12mm place;
The bag quilt of nature controlling line C line 42: the phosphate buffer containing trehalose and SDS dissolves by the antibody of the antigen had nothing to do with determinand or ligand receptor, obtained spray coating liquor two, is sprayed on apart from detecting pad 4 left end 18mm place formation nature controlling line C line 42 by spray coating liquor two;
By for subsequent use for detecting pad 4 kept dry sprayed;
(IV) assembling of test strips
On PVC base plate 1 successively overlap processed application of sample pad 2, be sprayed with the colour-label pad 3 of fluorescent dual-function microballoon label probe, the detecting pad 4 being coated with detection line T line 41 and nature controlling line C line 42 and adsorptive pads 5, cut into the test strips that 4mm is wide after assembling.
Specific embodiment
Embodiment 1
The difunctional immuno-chromatographic test paper strip of Aflatoxins M1 blueness-time-resolved fluorescence
One, the preparation method of test strips
The preparation of (a) microballoon
The acrylic monomers of the styrene monomer and 0.8mmol of getting 8mmol is dissolved in the deionized water of 10ml containing the sodium dodecylsulphonate of 0.3mmol, is added by mixed liquor in round-bottomed flask, stirs by magnetic stir bar.Then with high pure nitrogen, air in round-bottomed flask is eliminated, heated sealed to 70 DEG C, add the potassium persulfate of 0.3ml0.2mmol, after sealing oxygen barrier stirring reaction 8h, be down to room temperature.Then be the Filter paper filtering of 8 μm by reactant liquor aperture, collect filtrate centrifugal purification, centrifugal condition is the 50000g10 DEG C of centrifugal 15min of constant temperature, remove supernatant, redissolve with 5ml deionized water and precipitate, cyclic washing three times, finally add concentration be 0.05% sodium dodecylsulphonate and concentration be that the sodium azide of 0.05% is in 4 DEG C of preservations.The different carboxyl density of microsphere surface can be obtained by regulating the amount adding acrylic acid monomer; By regulating the amount of sodium dodecylsulphonate and potassium persulfate, the microballoon of different-grain diameter can be prepared.
The preparation of the difunctional microballoon of (b) blueness-time-resolved fluorescence
Get the microballoon that completes of above-mentioned preparation that 1ml solid content is 10%, add in 9ml deionized water, then add 3ml methylene chloride, be placed in 37 DEG C of isoperibols, magnetic agitation 30min, obtain pre-service microspheres solution.Get 10mg Europium chelate (Eu: β-NTA:TOPO=1:3:3) and 5mg Disperse Blue 2BLN, be dissolved in 1ml methylene chloride, then above-mentioned dropwise is added in pre-service microspheres solution, sealing, 37 DEG C of temperature constant magnetic stirring 5h.Then open sealing lid, the methylene chloride in solution system is volatilized totally under 37 DEG C of conditions.Proceeded to by solution in centrifuge tube, centrifuge washing three times, centrifugal condition is: the 50000g10 DEG C of centrifugal 15min of constant temperature.Remove supernatant, front twice centrifugal precipitation obtained ethanol of 50% redissolves, the 0.05M borate buffer of precipitation 10mlpH8.0 centrifugal for the last time redissolves, then add concentration be 0.05% sodium dodecylsulphonate and concentration be 0.05% sodium azide 4 DEG C preservation.
The preparation of (c) Aflatoxins M1 antibody blueness-time-resolved fluorescence microballoon label
Get solid content be 1% above-mentioned blueness-time-resolved fluorescence difunctional microballoon 100 μ L to be diluted in concentration be 0.05mol/L, in the 400 μ L borate buffers of pH8.0, add the N-hydroxy-succinamide (NHS) (purchased from Shanghai Jing Chun biochemical technology incorporated company) that carbodiimide (EDC) (purchased from Shanghai Jing Chun biochemical technology incorporated company) that 30 μ L concentration are 10mg/mL and 60 μ L concentration are 10mg/mL, under 50 revs/min of conditions, 15min is activated by being placed on rotary shaker under above-mentioned mixed liquor room temperature condition, then the centrifugal 10min of 40000g, remove supernatant, with 0.05mol/L, the borate buffer of pH8.0 redissolves and precipitates, then 80W ultrasonic process 30S, add 25 μ g Aflatoxins M1 mouse monoclonal antibodies (purchased from Fei Ce bio tech ltd, Shanghai), room temperature to be placed on rotary shaker 50 revs/min of couplings 2 hours, add the monoethanolamine (purchased from Shanghai Jing Chun biochemical technology incorporated company) of 50mmol/L, BSA (purchased from Xi Bao bio tech ltd, Shanghai) the solution 50 μ L of 10% closes and spends the night.The centrifugal 10min of last 40000g, redissolve with the borate buffer of 0.05mol/L, pH8.0 and precipitate, cyclic washing 2 ~ 3 times, then 80W ultrasonic process 30S, be placed in 4 ~ 8 DEG C of refrigerators and save backup.
The preparation of (d) rabbit igg blueness-time-resolved fluorescence microballoon label
Get solid content be 1% above-mentioned blueness-time-resolved fluorescence difunctional microballoon 100 μ L be diluted in 0.05mol/L, in the 400 μ L borate buffers of pH8.0, add the carbodiimide (EDC) (purchased from Shanghai Jing Chun biochemical technology incorporated company) of 30 μ L10mg/mL, add 100 μ g rabbit iggs (purchased from Bo You bio tech ltd, Changsha) again, 50 revs/min of couplings 2 hours will to be placed on rotary shaker under above-mentioned mixed liquor room temperature condition, add the monoethanolamine (purchased from Shanghai Jing Chun biochemical technology incorporated company) of 50mmol/L, BSA (purchased from Xi Bao bio tech ltd, Shanghai) the solution 50 μ L of 10% closes and spends the night.Centrifugal 10 minutes of last 40000g, redissolve with the borate buffer of 0.05mol/LpH8.0 and precipitate, cyclic washing 2 ~ 3 times, then 80W ultrasonic process 30S, be placed in 4 ~ 8 DEG C of refrigerators and save backup.
The preparation of (e) test strips
(I) preparation of application of sample pad 2
With tween-20, the PVP of 0.5% (m/v) of the EDTA containing 10mmol/L, 1% (v/v), the PBS damping fluid (0.2mol/L of the BSA of 0.5% (m/v), pH8.0) glass fibre element film application of sample pad is soaked, then be placed in 37 DEG C of baking oven bakings 2 little of bone dry, be placed in hermetically drying packaging bag and save backup.
(II) preparation of label pad 3
First use the phosphate buffer of the 0.1mol/LpH7.4 of the bovine serum albumin(BSA) of the trehalose containing 2.5% (m/v), 1% (m/v), the tween-20 of 1% (v/v), the PVP of 0.5% (m/v) to soak pad Fusion5 (purchased from GE company), be then placed in 37 DEG C of baking ovens and dry 2 hours.Aflatoxins M1 antibody blue-fluorescent difunctional microballoon label probe is mixed and made into after the phosphate buffer of the Aflatoxins M1 antibody blueness-time-resolved fluorescence microballoon label above-mentioned preparation completed again and rabbit igg blueness-time-resolved fluorescence microballoon label 0.1mol/L, pH7.4 dilutes 500 times and 1200 times respectively, above-mentioned probe gold spraying instrument is sprayed on the Fusion5 after process oven dry, be placed in 37 DEG C of baking ovens to dry after 2 hours, be placed in hermetically drying packaging bag and save backup.
(III) preparation of detecting pad 4
The bag quilt of detection line T line 41
It is 0.025mg/mL that Aflatoxins M1 and bovine serum albumin(BSA) conjugate (purchased from Fei Ce bio tech ltd, Shanghai) are dissolved to final concentration with the phosphate buffer that the concentration of the SDS of the trehalose and 0.05% (v/v) containing 1.5% (m/v) is 0.01mol/L, pH7.4, is sprayed on by above-mentioned mixed liquor Membrane jetter and forms detection line T line 41 apart from nitrocellulose filter left end 12mm place.
The bag quilt of nature controlling line C line 42
The goat anti-rabbit igg monoclonal antibody phosphate buffer that the concentration of the SDS of the trehalose and 0.05% (v/v) containing 1.5% (m/v) is 0.01mol/L, pH7.4 is dissolved to final concentration 1.0mg/mL, above-mentioned mixed liquor Membrane jetter is sprayed on and forms nature controlling line C line 42 apart from nitrocellulose filter left end 18mm place.The nitrocellulose filter sprayed is dried 2 hours under 37 DEG C of conditions, is placed in drying at room temperature environment and saves backup.
(IV) assembling of test strips
As shown in Figure 1, described colour-fluorescent dual-function immuno-chromatographic test paper strip comprises base plate 1, the one side of base plate 1 is sequentially with application of sample pad 2, the label pad 3 of interpolation testing sample, is provided with the detecting pad 4 of detection line T line 41 and nature controlling line C line 42 and provides the adsorptive pads 5 of capillary siphoning power, adjacent each pad overlaps successively and pastes on PVC backing.
The wide test strips of 4mm is cut into after the application of sample pad 2 of the present embodiment process, the label pad 3 being sprayed with Aflatoxins M1 antibody blue-fluorescent difunctional microballoon label probe, the detecting pad 4 being coated with detection line T line 41 and nature controlling line C line 42 and adsorptive pads 5 being assembled, during loading plastics get stuck, then loading is built-in with in the aluminium foil bag of drying agent, drying at room temperature is preserved, and the shelf-life can reach more than 2 years.
During the plastics with well and detection window get stuck if above-mentioned test strips be fixed on, then make test card.
Two, the determination of fluorescent quantitation typical curve
Aflatoxins M1 standard items lactogenesis is diluted to 0ng/mL, 0.025ng/mL, 0.075ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/mL, 8.1ng/mL, the each Concentration Testing of toxin is often planted 10 times according to Standard Operating Procedure, then by measured numerical value input Microdetection food safety management software (micrometering bio tech ltd, Nanjing), the parameters of typical curve can be obtained, then by the Data Enter IC-cards such as the parameter of typical curve, batch number.
Three, testing sample detects
1. detection method
The difunctional immuno-chromatographic test paper strip of Aflatoxins M1 blue-fluorescent prepared is lain against on desktop, gets 100 μ L milk sample to be measured (20-30 DEG C), drop to application of sample pad 2 place of test strips, timing 5 minutes, observe testing result.
2. result judges
1. naked eyes interpretation
When the T line naked eyes of test strip are invisible, when C line naked eyes are visible, illustrate that the concentration of Aflatoxins M1 in milk sample is higher than 0.3ng/ml; When the T line naked eyes of test strip are visible, when C line naked eyes are visible, illustrate that the concentration of Aflatoxins M1 in milk sample is lower than 0.3ng/ml; When the first naked eyes of C are invisible, regardless of T line, whether naked eyes are visible, and it is invalid to be all judged to, and need again detect.
2. Fluorescent reader interpretation
Test strip is inserted in Fluorescent reader, reading can be carried out by read key and automatically calculate, the concentration of Aflatoxins M1 shows by liquid crystal display, also by thermal printer field print that readout instrument carries, also by GPRS and WIFI wireless communication module on instrument, detection data are sent to high in the clouds data management platform in real time, realize the real-time monitoring to testing result.
3. ELISA test strip result accuracy judges
Be add Aflatoxins M1 standard solution in the fresh milk of 0 to detecting Aflatoxins M1 content through high performance liquid chromatography GC-MS, its concentration is made to reach 0ng/mL, 0.025ng/mL, 0.075ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/mL respectively, 8.1ng/mL, detect the accuracy of test strips prepared by the present embodiment respectively.Repeat above test experience 10 times, get the mean value of instrument sentence read result.
Test experience result is as shown in table 1 below.
Table 1 detection experiment result 1
Test result shows: the difunctional test strip of blue-fluorescent microballoon prepared by the present embodiment in use, both can judge that whether the amount of Aflatoxins M1 in milk sample was more than 0.3ng/ml by naked eyes, carry out initial characterization interpretation, learn whether milk sample is qualified milk; Also obtain accurately quantitatively testing result by Fluorescent reader, well meet various detection demand.
Embodiment 2
Chloromycetin redness-rhodamine fluorescent dual-function immuno-chromatographic test paper strip
One, the preparation method of test strips
The synthesis of (a) microballoon
The acrylic monomers getting the styrene monomer 0.8mmol of 8mmol is dissolved in the deionized water of 10ml containing the sodium dodecylsulphonate of 0.3mmol, is added by mixed liquor in round-bottomed flask, stirs by magnetic stir bar.Then with high pure nitrogen, air in round-bottomed flask is eliminated, heated sealed to 70 DEG C, add the potassium persulfate of 0.3ml0.2mmol, after sealing oxygen barrier stirring reaction 8h, be down to room temperature.Then be the Filter paper filtering of 8 μm by reactant liquor aperture, collect filtrate centrifugal purification, centrifugal condition is the 50000g10 DEG C of centrifugal 15min of constant temperature, remove supernatant, redissolve with 5ml deionized water and precipitate, cyclic washing three times, finally add concentration be 0.05% sodium dodecylsulphonate and concentration be that the sodium azide of 0.05% is in 4 DEG C of preservations.The different carboxyl density of microsphere surface can be obtained by regulating the amount adding acrylic acid monomer; By regulating the amount of sodium dodecylsulphonate and potassium persulfate, the microballoon of different-grain diameter can be prepared.
The preparation of (b) redness-rhodamine fluorescent dual-function microballoon
Get the microballoon that completes of above-mentioned preparation that 1ml solid content is 10%, add in 9ml deionized water, then add 3.5ml tetrahydrofuran, be placed in room temperature environment, magnetic agitation 30min, obtain pre-service microspheres solution.Get 3mg rhodamine B and 5mg disperse red SER, be dissolved in 2ml tetrahydrofuran, then add in pre-service microspheres solution by above-mentioned dropwise, under magnetic agitation condition, room temperature keeps 5h.Then open sealing lid, under rotatory vacuum condition, make the tetrahydrofuran volatilization in solution system clean.Proceeded to by solution in centrifuge tube, centrifuge washing three times, centrifugal condition is: the 50000g10 DEG C of centrifugal 15min of constant temperature.Remove supernatant, front twice centrifugal precipitation obtained ethanol of 50% redissolves, the 0.05M borate buffer of precipitation 10mlpH8.0 centrifugal for the last time redissolves, then add concentration be 0.05% sodium dodecylsulphonate and concentration be 0.05% sodium azide 4 DEG C preservation.
The preparation of (c) chloramphenicol antibody redness-rhodamine Fluorescent microsphere marker
Get solid content be 1% above-mentioned redness-rhodamine fluorescent dual-function microballoon 100 μ L to be diluted in concentration be 0.05mol/L, in the 400 μ L borate buffers of pH8.0, add the N-hydroxy-succinamide (NHS) (purchased from Shanghai Jing Chun biochemical technology incorporated company) that carbodiimide (EDC) (purchased from Shanghai Jing Chun biochemical technology incorporated company) that 30 μ L concentration are 10mg/mL and 60 μ L concentration are 10mg/mL, under 50 revs/min of conditions, 15min is activated by being placed on rotary shaker under above-mentioned mixed liquor room temperature condition, then the centrifugal 10min of 40000g, remove supernatant, with 0.05mol/L, the borate buffer of pH8.0 redissolves and precipitates, then 80W ultrasonic process 30S, add 20 μ g chloromycetin mouse monoclonal antibodies (purchased from Fei Ce bio tech ltd, Shanghai), room temperature to be placed on rotary shaker 50 revs/min of couplings 2 hours, add the monoethanolamine (purchased from Shanghai Jing Chun biochemical technology incorporated company) of 50mmol/L, BSA (purchased from Xi Bao bio tech ltd, Shanghai) the solution 50 μ L of 10% closes and spends the night.The centrifugal 10min of last 40000g, redissolve with the borate buffer of 0.05mol/L, pH8.0 and precipitate, cyclic washing 2 ~ 3 times, then 80W ultrasonic process 30S, be placed in 4 ~ 8 DEG C of refrigerators and save backup.
The preparation of (d) rabbit igg redness-rhodamine Fluorescent microsphere marker
Get solid content be 1% above-mentioned redness-rhodamine fluorescent dual-function microballoon 100 μ L be diluted in 0.05mol/L, in the 400 μ L borate buffers of pH8.0, add the carbodiimide (EDC) (purchased from Shanghai Jing Chun biochemical technology incorporated company) of 30 μ L10mg/mL, add 100 μ g rabbit iggs (purchased from Bo You bio tech ltd, Changsha) again, 50 revs/min of couplings 2 hours will to be placed on rotary shaker under above-mentioned mixed liquor room temperature condition, add the monoethanolamine (purchased from Shanghai Jing Chun biochemical technology incorporated company) of 50mmol/L, BSA (purchased from Xi Bao bio tech ltd, Shanghai) the solution 50 μ L of 10% closes and spends the night.Centrifugal 10 minutes of last 40000g, redissolve with the borate buffer of 0.05mol/LpH8.0 and precipitate, cyclic washing 2 ~ 3 times, then 80W ultrasonic process 30S, be placed in 4 ~ 8 DEG C of refrigerators and save backup.
The preparation of (e) test strips
(I) preparation of application of sample pad 2
With tween-20, the PVP of 0.5% (m/v) of the EDTA containing 10mmol/L, 1% (v/v), the PBS damping fluid (0.2mol/L of the BSA of 0.5% (m/v), pH8.0) glass fibre element film application of sample pad is soaked, then be placed in 37 DEG C of baking oven bakings 2 little of bone dry, be placed in hermetically drying packaging bag and save backup.
(II) preparation of label pad 3
First use the phosphate buffer of the 0.1mol/LpH7.4 of the bovine serum albumin(BSA) of the trehalose containing 2.5% (m/v), 1% (m/v), the tween-20 of 1% (v/v), the PVP of 0.5% (m/v) to soak pad Fusion5 (purchased from GE company), be then placed in 37 DEG C of baking ovens and dry 2 hours.Chloramphenicol antibody redness-rhodamine fluorescent dual-function microballoon label probe is mixed into after the phosphate buffer of the chloramphenicol antibody redness-rhodamine Fluorescent microsphere marker above-mentioned preparation completed again and rabbit igg redness-rhodamine Fluorescent microsphere marker 0.1mol/L, pH7.4 dilutes 400 times and 1000 times respectively, above-mentioned probe gold spraying instrument is sprayed on the Fusion5 after process oven dry, be placed in 37 DEG C of baking ovens to dry after 2 hours, be placed in hermetically drying packaging bag and save backup.
(III) preparation of detecting pad 4
The bag quilt of detection line T line 41
It is 0.025mg/mL that chloromycetin and bovine serum albumin(BSA) conjugate (purchased from Fei Ce bio tech ltd, Shanghai) are dissolved to final concentration with the phosphate buffer that the concentration of trehalose and 0.05% (v/v) SDS containing 1.5% (m/v) is 0.01mol/L, pH7.4, is sprayed on by above-mentioned mixed liquor Membrane jetter and forms detection line T line 41 apart from nitrocellulose filter left end 12mm place.
The bag quilt of nature controlling line C line 42
The goat anti-rabbit igg monoclonal antibody phosphate buffer that the concentration of the SDS of the trehalose and 0.05% (v/v) containing 1.5% (m/v) is 0.01mol/L, pH7.4 is dissolved to final concentration 1.0mg/mL, above-mentioned mixed liquor Membrane jetter is sprayed on and forms nature controlling line C line 42 apart from nitrocellulose filter left end 18mm place.The nitrocellulose filter sprayed is dried 2 hours under 37 DEG C of conditions, is placed in drying at room temperature environment and saves backup.
(IV) assembling of test strips
As shown in Figure 1, described colour-fluorescent dual-function immuno-chromatographic test paper strip comprises base plate 1, the one side of base plate 1 is sequentially with application of sample pad 2, the label pad 3 of interpolation testing sample, is provided with the detecting pad 4 of detection line T line 41 and nature controlling line C line 42 and provides the adsorptive pads 5 of capillary siphoning power, adjacent each pad overlaps successively and pastes on PVC backing.
By the application of sample pad 2 of the present embodiment process, be sprayed with the chloramphenicol antibody redness-label pad 3 of rhodamine fluorescent dual-function microballoon label probe, the detecting pad 4 being coated with detection line T line 41 and nature controlling line C line 42 and adsorptive pads 5 and cut into the wide test strips of 4mm after assembling, during loading plastics get stuck, then loading is built-in with in the aluminium foil bag of drying agent, drying at room temperature is preserved, and the shelf-life can reach more than 2 years.
During the plastics with well and detection window get stuck if above-mentioned test strips be fixed on, then make test card.
Two, the determination of fluorescent quantitation typical curve
Chloromycetin standard items lactogenesis is diluted to 0ng/mL, 0.025ng/mL, 0.05ng/mL, 0.1ng/mL, 0.3ng/mL, 0.5ng/mL, 1.0ng/mL, the each Concentration Testing of toxin is often planted 10 times according to Standard Operating Procedure, then by measured numerical value input Microdetection food safety management software (micrometering bio tech ltd, Nanjing), the parameters of typical curve can be obtained, then by the Data Enter IC-cards such as the parameter of typical curve, batch number.
Three, testing sample detects
1. detection method
By the chloromycetin prepared redness-rhodamine fluorescent dual-function immuno-chromatographic test paper strip lies against on desktop, get 100 μ L milk sample to be measured (20-30 DEG C), drop to application of sample pad 2 place of test strips, timing 5 minutes, observe testing result.
2. result judges
1. naked eyes interpretation
When the T line naked eyes of test strip are invisible, when C line naked eyes are visible, illustrate that the concentration of chloromycetin in milk sample is higher than 0.1ng/ml; When the T line naked eyes of test strip are visible, when C line naked eyes are visible, illustrate that the concentration of chloromycetin in milk sample is lower than 0.1ng/ml; When the first naked eyes of C are invisible, regardless of T line, whether naked eyes are visible, and it is invalid to be all judged to, and need again detect.
2. Fluorescent reader interpretation
Test strip is inserted in Fluorescent reader, reading can be carried out by read key and automatically calculate, the concentration of chloromycetin shows by liquid crystal display, also by thermal printer field print that readout instrument carries, also by GPRS and WIFI wireless communication module on instrument, detection data are sent to high in the clouds data management platform in real time, realize the real-time monitoring to testing result.
3. ELISA test strip result accuracy judges
To through high performance liquid chromatography GC-MS detect chloromycetin content be add chloromycetin standard solution in the fresh milk of 0, its concentration is made to reach 0ng/mL, 0.025ng/mL, 0.05ng/mL, 0.1ng/mL, 0.3ng/mL, 0.5ng/mL respectively, 1.0ng/mL, detect the accuracy of test strips prepared by the present embodiment respectively, repeat above test experience 10 times, get the mean value of instrument sentence read result.
Test experience result is as shown in table 2 below.
Table 2 detection experiment result 2
Redness-difunctional test strip of rhodamine fluorescent microsphere prepared by the present embodiment in use, both can judge that whether the amount of chloromycetin in milk sample is more than 0.1ng/ml, carried out initial characterization judgement by naked eyes, learn whether milk sample is qualified milk; Also obtain accurately quantitatively testing result by Fluorescent reader, well meet various detection demand.
To one skilled in the art, according to technical scheme described above and design, other various corresponding change and deformation can be made, and all these change and deformation all should belong within the protection domain of the claims in the present invention.
Claims (8)
1. colour-fluorescent dual-function immuno-chromatographic test paper strip, it is characterized in that: comprise base plate (1), the one side of base plate (1) is sequentially with application of sample pad (2), the label pad (3) of interpolation testing sample, is provided with the detecting pad (4) of detection line T line (41) and nature controlling line C line (42) and the adsorptive pads (5) of capillary siphoning power is provided, adjacent each pad overlaps successively, label pad (3) is coated with colour-fluorescent dual-function microballoon label probe.
2. colour according to claim 1-fluorescent dual-function immuno-chromatographic test paper strip, it is characterized in that: colour-fluorescent dual-function microballoon label probe contains determinand antibody colour-fluorescent dual-function microballoon label and rabbit, IgG colour-fluorescent dual-function microballoon the label of mouse or other and determinand antigen-antibody no cross reaction, determinand antibody colour-Fluorescent microsphere marker is combined by the antigen-antibody relevant to determinand or ligand receptor and colour-fluorescent dual-function microballoon and is prepared from, rabbit, IgG colour-the Fluorescent microsphere marker of mouse or other and determinand antigen-antibody no cross reaction is by rabbit, mouse or other IgG with determinand antigen-antibody no cross reaction and colour-fluorescent dual-function microballoon are combined and are prepared from.
3. colour according to claim 2-fluorescent dual-function immuno-chromatographic test paper strip, it is characterized in that: detection line T line (41) is coated with antibody or the ligand receptor of the antigen relevant to determinand, nature controlling line C line (42) is coated with the antibody of the antigen relevant to determinand or ligand receptor or is coated with antibody or the ligand receptor of the antigen had nothing to do with determinand.
4. colour according to claim 2-fluorescent dual-function immuno-chromatographic test paper strip; it is characterized in that: the particle diameter of described colour-fluorescent dual-function microballoon is 50-1000nm; the material of colour-fluorescent dual-function microballoon is the one in polystyrene microsphere, poly (methyl methacrylate) micro-sphere, polyvinyl pyridine microspheres, poly-ethyl methyl acrylate microballoon, lipid microsphere and silicon dioxide microsphere, and described colour-fluorescent dual-function microsphere surface is with one or more in carboxyl, amino, aldehyde radical, acetyl bromide, iodoacetyl, mercapto and epoxy radicals or be not with group.
5. the colour according to any one of claim 2-4-fluorescent dual-function immuno-chromatographic test paper strip, is characterized in that: the color of described colour-fluorescent dual-function microballoon is the macroscopic dark color of one in redness, blueness, green, purple and black; Fluorescence is the one in Rare Earth Chelate fluorescence, quantum dot fluorescence and common fluorescent; The wavelength difference that the wavelength of color emission and fluorescent material are launched is more than or equal to 30nm.
6. colour according to claim 5-fluorescent dual-function immuno-chromatographic test paper strip, it is characterized in that: application of sample pad (2) for having the material of filtration, the material of application of sample pad (2) is glass fibre, polyester film, sponge, filter paper or through being added with one or more the phosphate buffer in surfactant, carbohydrate, protide, macromolecule polymer, salt or the glass fibre after Tris-HCl damping fluid dipping pretreatment, polyester film, sponge, filter paper;
The material of label pad (3) is glass fibre, polyester film or through being added with one or more the phosphate buffer in surfactant, carbohydrate, protide, macromolecule polymer, salt or the glass fibre after Tris-HCl damping fluid dipping pretreatment or polyester film;
Detecting pad (4) is for having the porosint of strong hydrophobic property, and the material of detecting pad (4) is nitrocellulose filter or cellulose acetate membrane, and detection line T line (41) and nature controlling line C line (42) are at least one.
7. a preparation method for the colour-fluorescent dual-function immuno-chromatographic test paper strip of claim 6, is characterized in that: comprise the following steps:
(I) preparation of application of sample pad (2)
Soak glass fibre, polyester film, sponge or filter paper with the PBS damping fluid containing EDTA, tween-20, PVP and BSA, be then placed in baking oven and dry, hermetically drying saves backup;
(II) preparation of label pad (3)
Glass fibre or polyester film is soaked with the PBS damping fluid containing trehalose, bovine serum albumin(BSA), tween-20 and PVP, then be placed in baking oven to dry, again colour-fluorescent dual-function microballoon label probe is sprayed on the glass fibre after process oven dry or polyester film, again be placed in baking oven to dry, hermetically drying saves backup;
(III) preparation of detecting pad (4)
The bag quilt of detection line T line (41): the PBS buffer solution containing trehalose and SDS by the antigen-antibody relevant to determinand or ligand receptor, obtained spray coating liquor one, is sprayed on spray coating liquor one and forms detection line T line (41) apart from detecting pad (4) left end 12mm place;
The bag quilt of nature controlling line C line (42): the phosphate buffer containing trehalose and SDS dissolves by the antigen-antibody had nothing to do with determinand or ligand receptor, obtained spray coating liquor two, is sprayed on spray coating liquor two and forms nature controlling line C line (42) apart from detecting pad (4) left end 18mm place;
By for subsequent use for detecting pad (4) kept dry sprayed;
(IV) assembling of test strips
PVC base plate (1) overlaps application of sample pad (2), the label pad (3) being sprayed with colour-fluorescent dual-function microballoon label probe, the detecting pad (4) being coated with detection line T line (41) and nature controlling line C line (42) and adsorptive pads (5) successively that processed, after assembling, cuts into the test strips that 4mm is wide.
8. the preparation method of colour according to claim 7-fluorescent dual-function immuno-chromatographic test paper strip, is characterized in that: concrete preparation method is as follows for colour described in step (II)-fluorescent dual-function microballoon label probe:
The preparation of (a) microballoon: adopt microemulsion legal system to be polystyrene microsphere, poly (methyl methacrylate) micro-sphere, polyvinyl pyridine microspheres, the one of gathering in ethyl methyl acrylate microballoon, lipid microsphere and silicon dioxide microsphere of 50-1000nm for particle diameter, described microsphere surface is with one or more in carboxyl, amino, aldehyde radical, acetyl bromide, iodoacetyl, mercapto and epoxy radicals or be not with group;
The preparation of (b) colour-fluorescent dual-function microballoon: add microballoon prepared by methylene chloride or tetrahydrofuran and step (a) in deionized water, obtain microspheres solution, the fluorescent marker and fat-soluble color pigment that are dissolved in methylene chloride or tetrahydrofuran are dropwise added in microspheres solution also to seal and stir, then depressurization remove methylene chloride or tetrahydrofuran, last centrifugal treating, obtain colour-fluorescent dual-function microballoon, for subsequent use;
The preparation of (c) determinand antibody colour-fluorescent dual-function microballoon label: the colour prepared with damping fluid dilution step (b)-fluorescent dual-function microballoon, add carbodiimide and N-hydroxy-succinamide, on shaking table after activation, centrifugal treating, remove supernatant, add damping fluid to redissolve to precipitation, then the antigen-antibody relevant to determinand or ligand receptor is added, coupling on shaking table, then monoethanolamine and bovine serum albumin(BSA) is added, sealing is spent the night, last centrifugal treating, remove supernatant, with damping fluid, precipitation is redissolved again, ultrasonic process, obtained determinand antibody colour-Fluorescent microsphere marker,
(d) rabbit, the preparation of the antigen-antibody colour-Fluorescent microsphere marker of mouse or other and determinand antigen-antibody no cross reaction: dilute colour-fluorescent dual-function microballoon with damping fluid, add carbodiimide and rabbit, the antigen-antibody of mouse or other and determinand antigen-antibody no cross reaction, coupling on shaking table, then monoethanolamine and bovine serum albumin(BSA) is added, sealing is spent the night, last centrifugal treating, remove supernatant, redissolve with damping fluid again, ultrasonic process, obtained rabbit, antigen-antibody colour-the Fluorescent microsphere marker of mouse or other and determinand antigen-antibody no cross reaction,
The preparation of (e) colour-fluorescent dual-function microballoon label probe: determinand antibody colour-Fluorescent microsphere marker and rabbit, mouse or other mix by a certain percentage with the antigen-antibody colour-Fluorescent microsphere marker of determinand antigen-antibody no cross reaction, be sprayed on obtained colour-fluorescent dual-function microballoon label probe on pad.
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