CN105424923B - Difunctional immuno-chromatographic test paper strip of color fluorescence and preparation method thereof - Google Patents
Difunctional immuno-chromatographic test paper strip of color fluorescence and preparation method thereof Download PDFInfo
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- CN105424923B CN105424923B CN201510576098.XA CN201510576098A CN105424923B CN 105424923 B CN105424923 B CN 105424923B CN 201510576098 A CN201510576098 A CN 201510576098A CN 105424923 B CN105424923 B CN 105424923B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention belongs to field of biological detection, more particularly to difunctional immuno-chromatographic test paper strip of a kind of color fluorescence and preparation method thereof, including bottom plate (1), sample-adding pad (2), label pad (3), the detecting pad (4) provided with detection line T lines (41) and nature controlling line C line (42) and the adsorptive pads (5) that capillary siphoning power is provided of addition testing sample are sequentially with the one side of bottom plate (1), adjacent each pad is overlapped successively, and the difunctional microballoon label probe of color fluorescence is coated with label pad (3).Difunctional immuno-chromatographic test paper strip of color fluorescence of the present invention and preparation method thereof, its testing result both can carry out yin and yang attribute initial characterization judgement by naked eyes, can obtain accurately detection data by Instrumental Analysis again.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of colour-fluorescent dual-function immuno-chromatographic test paper strip and its
Preparation method.
Background technology
In immunochromatography detection field, there is the detection mode of two kinds of main flows at present：A kind of is by the qualitative of naked eyes interpretation
Immuno-chromatographic test paper strip, including colloidal gold mark test paper, color latex mark test paper etc. are detected, its advantage is without configuration
Any detection device can be made to the content of determinand in sample qualitatively to be judged, shortcoming is no standard measure, it is impossible to is provided accurate
True concentration data, and its detection sensitivity is relatively low, typically can only determinand of the concentrations more than ng/ml ranks；It is another
Kind is by the fluorescence immune chromatography test paper bar of instrument interpretation, and its advantage is by instrument interpretation data, it is possible to provide accurately dense
Degrees of data, and detection sensitivity is than the high 2-3 order of magnitude of test strips that collaurum or color latex mark, but its shortcoming be
There is no the content concn situation that can not obtain determinand in the case of readout instrument completely.
The content of the invention
Invention broadly provides a kind of colour-fluorescent dual-function immuno-chromatographic test paper strip and preparation method thereof, and it is detected
As a result both yin and yang attribute initial characterization judgement can be carried out by naked eyes, accurately detection data can be obtained by Instrumental Analysis again.
A kind of colour-fluorescent dual-function immuno-chromatographic test paper strip, including bottom plate, addition is sequentially with the one side of bottom plate and is treated
Test sample product are loaded pad, label pad, the detecting pad provided with detection line T lines and nature controlling line C line and offer capillary siphoning
The adsorptive pads of power, adjacent each pad are overlapped successively, and chromatic colour-fluorescent dual-function microballoon mark physical prospecting is coated on label pad
Pin.
Preferably, the colour-fluorescent dual-function microballoon label probe contains determinand antibody colour-fluorescent dual-function
Microballoon label and rabbit, mouse or other are micro- with antigen-antibody colour-fluorescent dual-function of determinand antigen-antibody no cross reaction
Ball label, determinand antibody colour-Fluorescent microsphere marker is by the antigen-antibody or ligand receptor related to determinand and coloured silk
Color-fluorescent dual-function microballoon is combined and is prepared, rabbit, mouse or other antigen-antibodies with determinand antigen-antibody no cross reaction
Colour-fluorescent dual-function microballoon label by rabbit, mouse or other antigen-antibodies with determinand antigen-antibody no cross reaction and
Colour-fluorescent dual-function microballoon is combined and is prepared.
Preferably, the detection line T lines are coated with the antigen-antibody or ligand receptor related to determinand, nature controlling line C line
It is coated with the antigen-antibody or ligand receptor unrelated with determinand.
Preferably, the particle diameter of the colour-fluorescent dual-function microballoon is 50-1000nm, colour-fluorescent dual-function microballoon
Material be polystyrene microsphere, poly (methyl methacrylate) micro-sphere, polyvinyl pyridine microspheres, poly- ethyl methyl acrylate microballoon,
One kind in lipid microsphere and silicon dioxide microsphere, the colour-fluorescent dual-function microsphere surface with carboxyl, amino, aldehyde radical,
In acetyl bromide, iodoacetyl, mercapto and epoxy radicals one or more or without group.
Preferably, the color of the colour-fluorescent dual-function microballoon is one in red, blueness, green, purple and black
The macroscopic dark color of kind；Fluorescence is one kind in Rare Earth Chelate fluorescence, quantum dot fluorescence and common fluorescent；Color emission
Wavelength and the wavelength difference of fluorescent material transmitting are more than or equal to 30nm.
Preferably, it is described sample-adding pad be the material with filtration, be loaded pad material be glass fibre, polyester film,
Sponge, filter paper are one or more in surfactant, carbohydrate, protide, macromolecule polymer, salt by being added with
Glass fibre, polyester film, sponge, filter paper after phosphate buffer or Tris-HCl buffer solution dipping pretreatments；
Preferably, the material of the label pad be glass fibre, polyester film or by added with surfactant,
One or more phosphate buffers or trishydroxymethylaminomethane hydrochloric acid in carbohydrate, protide, macromolecule polymer, salt
Glass fibre or polyester film after salt (Tris-HCl buffer solutions) dipping pretreatment；
Preferably, the detecting pad is the porous material with strong hydrophobic property, and the material of detecting pad is nitrocellulose
Film or cellulose acetate film, T lines and C lines are at least one.
A kind of preparation method of colour-fluorescent dual-function immuno-chromatographic test paper strip, comprises the following steps：
(I) preparation of sample-adding pad
With containing ethylenediamine tetra-acetic acid (EDTA), polysorbas20 (tween-20), polyvinylpyrrolidone (PVP) and cow's serum
Phosphate buffer (PBS) immersion glass fibre, polyester film, sponge or the filter paper of albumin (BSA), are subsequently placed in baking oven
Middle drying, hermetically drying save backup；
(II) preparation of label pad
With PBS immersion glass fibre or polyester containing trehalose, bovine serum albumin(BSA), tween-20 and PVP
Film, it is subsequently placed in baking oven and dries, then colour-fluorescent dual-function microballoon label probe is sprayed at the glass after processing drying
On fiber or polyester film, it is again placed in drying in baking oven, hermetically drying saves backup；
(III) preparation of detecting pad
The coating of detection line T lines：The antigen-antibody or ligand receptor related to determinand are contained into trehalose and ten with using
The PBS dissolving of sodium dialkyl sulfate (SDS), is made spray coating liquor one, spray coating liquor one is sprayed on away from detecting pad left end
Detection line T lines are formed at 12mm；
The coating of nature controlling line C line：By the antigen-antibody or ligand receptor unrelated with determinand with containing trehalose and SDS
Phosphate buffer is dissolved, and spray coating liquor two is made, and spray coating liquor two is sprayed on away from forming detection line T lines at detecting pad left end 18mm；
The detecting pad kept dry sprayed is standby；
(IV) assembling of test strips
Overlap treated sample-adding pad successively on polyvinyl chloride (PVC) bottom plate, be sprayed with colour-fluorescent dual-function microballoon mark
Remember the label pad of physical prospecting pin, be coated with detection line T lines and the detecting pad and adsorptive pads of nature controlling line C line, assembling finishes
The wide test strips of 4mm are cut into afterwards.
Preferably, colour described in step (II)-specific preparation method of fluorescent dual-function microballoon label probe is as follows：
(b) preparation of colour-fluorescent dual-function microballoon：Dichloromethane or tetrahydrofuran and step are added in deionized water
(a) microballoon completed is prepared, microspheres solution is obtained, by the fluorescent marker for being dissolved in dichloromethane or tetrahydrofuran and fat-soluble colour
Pigment is added dropwise in microspheres solution and sealed and stirs, and then depressurization and removes dichloromethane or tetrahydrofuran, most
Centrifugal treating afterwards, colour-fluorescent dual-function microballoon is obtained, it is standby；
(c) preparation of determinand antibody colour-fluorescent dual-function microballoon label：Prepared with buffer solution dilution step (b)
The colour of completion-fluorescent dual-function microballoon, carbodiimide and n-hydroxysuccinimide are added, after being activated on shaking table, centrifugation
Processing, remove supernatant, add buffer solution and precipitation is redissolved, then add the antigen-antibody related to determinand or part by
Body, in being coupled on shaking table, monoethanolamine and bovine serum albumin(BSA) are then added, sealing overnight, last centrifugal treating, removes supernatant
Liquid, then precipitation is redissolved with buffer solution, it is ultrasonically treated, determinand antibody colour-Fluorescent microsphere marker is made；
(d) rabbit, mouse or other antigen-antibody colour-Fluorescent microsphere markers with determinand antigen-antibody no cross reaction
Preparation：Colour-fluorescent dual-function microballoon is diluted with buffer solution, adds carbodiimide and rabbit, mouse or other and determinand antigen
The antigen-antibody of antibody no cross reaction, in being coupled on shaking table, monoethanolamine and bovine serum albumin(BSA) then being added, sealing is stayed overnight,
Last centrifugal treating, remove supernatant, then redissolved with buffer solution, be ultrasonically treated, be made rabbit, mouse or other resist with determinand antigen
Antigen-antibody colour-Fluorescent microsphere marker of body no cross reaction；
(e) preparation of colour-fluorescent dual-function microballoon label probe：Determinand antibody colour-Fluorescent microsphere marker
Antigen-antibody colour-Fluorescent microsphere marker with rabbit, mouse or other and determinand antigen-antibody no cross reaction is by certain ratio
Example mixing, is sprayed on pad and colour-fluorescent dual-function microballoon label probe is made.
Using above-mentioned colour-fluorescent dual-function immuno-chromatographic test paper strip, the present invention has advantages below：
Colour prepared by the present invention-fluorescent dual-function immuno-chromatographic test paper strip can both be carried out by naked eyes to testing result
Yin and yang attribute initial characterization judges, when not needing professional detecting instrument, qualitative judgement is made to the content of determinand in sample, examines
High sensitivity is surveyed, can be with the determinand below qualitative detection ng/ml ranks；Quantitative analysis can be carried out to result by instrument again,
Accurate concentration data is obtained, and detection sensitivity is higher 2-3 than the test strips that existing collaurum or color latex mark individual
The order of magnitude, accuracy of detection are high, and meeting various detection in the case of different detection projects, environment, purpose, sensitivity requirement needs
Ask.
Brief description of the drawings
Fig. 1 is the structural representation of colour-fluorescent dual-function immuno-chromatographic test paper strip of the present invention.
Wherein：1st, bottom plate, 2, sample-adding pad, 3, label pad, 4, detecting pad, 41, detection line T lines, 42, nature controlling line C
Line, 5, adsorptive pads.
Embodiment
A kind of colour-fluorescent dual-function immuno-chromatographic test paper strip, including bottom plate 1, addition is sequentially with the one side of bottom plate 1
Sample-adding pad 2, label pad 3, the detecting pad 4 provided with detection line T lines 41 and nature controlling line C line 42 and the offer hair of testing sample
The adsorptive pads 5 of tubule siphon power, adjacent each pad are overlapped successively, and it is micro- that chromatic colour-fluorescent dual-function is coated on label pad 3
Ball label probe.
Colour-fluorescent dual-function microballoon label probe contains determinand antibody colour-fluorescent dual-function microballoon label
With rabbit, mouse or other antigen-antibody colour-fluorescent dual-function microballoon labels with determinand antigen-antibody no cross reaction, treat
Thing antibody colour-fluorescent dual-function microballoon label is surveyed by the antigen-antibody or ligand receptor related to determinand and colour-glimmering
The difunctional microballoon of light is combined and is prepared, rabbit, mouse or other are colored-glimmering with determinand antigen-antibody no cross reaction antigen-antibody
The difunctional microballoon label of light is by rabbit, mouse or other antigen-antibodies and colour with determinand antigen-antibody no cross reaction-glimmering
The difunctional microballoon of light, which combines, to be prepared.
Detection line T lines 41 are coated with the antigen-antibody or ligand receptor related to determinand, nature controlling line C line 42 be coated with
Determinand unrelated antigen-antibody or ligand receptor.
Thing to be detected can be biotoxin and its metabolin, small molecule chemicals and high molecular weight protein, nucleic acid etc.,
The ELISA test strip of the present invention can be used.In specific examples below by taking Aflatoxins M1 and chloramphenicol as an example, schematically say
Bright test strips of the invention have colour-fluorescent dual-function.It is similar with being operated in embodiment when detecting other materials, do simple
Replacement.
When the biotoxin is Aflatoxins M1, colour-fluorescent dual-function microballoon label probe contains aspergillus flavus
Toxin M1 antibody colour-fluorescent dual-function microballoon label and rabbit igg colour-Fluorescent microsphere marker.Detection line T lines 41 are coated with
There is Aflatoxins M1-bovine serum albumin(BSA) conjugate, nature controlling line C line 42 is coated with goat-anti rabbit polyclonal antibody.
When the small molecule chemicals are chloramphenicol, colour-fluorescent dual-function microballoon label probe contains chloramphenicol
Antibody colour-fluorescent dual-function microballoon label and chicken IgG colours-Fluorescent microsphere marker.It is mould that detection line T lines 41 are coated with chlorine
Element-bovine serum albumin(BSA) conjugate, nature controlling line C line 42 are coated with goat-anti chicken polyclonal antibody.
The particle diameter of the colour-fluorescent dual-function microballoon is 50-1000nm, and the material of colour-fluorescent dual-function microballoon is
Polystyrene microsphere, poly (methyl methacrylate) micro-sphere, polyvinyl pyridine microspheres, poly- ethyl methyl acrylate microballoon, lipid are micro-
One kind in ball and silicon dioxide microsphere, the colour-fluorescent dual-function microsphere surface is with carboxyl, amino, aldehyde radical, acetyl bromide
In base, iodoacetyl, mercapto and epoxy radicals one or more or without group.
The color of the colour-fluorescent dual-function microballoon is a kind of naked eyes in red, blueness, green, purple and black
Visible dark color；Fluorescence is one kind in Rare Earth Chelate fluorescence, quantum dot fluorescence and common fluorescent；The wavelength of color emission and
The wavelength difference of fluorescent material transmitting is more than or equal to 30nm.
Sample-adding pad 2 is the material with filtration, and the material of sample-adding pad 2 is glass fibre, polyester film, sponge, filter paper
Or by added with one or more phosphoric acid buffers in surfactant, carbohydrate, protide, macromolecule polymer, salt
Glass fibre, polyester film, sponge, filter paper after liquid or Tris-HCl buffer solution dipping pretreatments；
The material of label pad 3 is glass fibre, polyester film or passed through added with surfactant, carbohydrate, albumen
After one or more phosphate buffers or Tris-HCl buffer solution dipping pretreatments in class, macromolecule polymer, salt
Glass fibre or polyester film；
Detecting pad 4 is the porous material with strong hydrophobic property, and the material of detecting pad 4 is fine for nitrocellulose filter or acetic acid
Plain film is tieed up, T lines and C lines are at least one.
A kind of preparation method of colour-fluorescent dual-function immuno-chromatographic test paper strip comprises the following steps：
(I) it is loaded the preparation of pad 2
With PBS immersion glass fibre, polyester film, sponge or filter containing EDTA, tween-20, PVP and BSA
Paper, it is subsequently placed in baking oven and dries, hermetically drying saves backup；
(II) preparation of label pad 3
With PBS immersion glass fibre or polyester containing trehalose, bovine serum albumin(BSA), tween-20 and PVP
Film, it is subsequently placed in baking oven and dries, then colour-fluorescent dual-function microballoon label probe is sprayed at the glass after processing drying
On fiber or polyester film, it is again placed in drying in baking oven, hermetically drying saves backup.
Above-mentioned colour-specific preparation method of fluorescent dual-function microballoon label probe is as follows：
(a) preparation of microballoon：Take polystyrene, polymethyl methacrylate, polyvinyl pyridine, poly- ethylacrylic acid first
A kind of be dissolved in the deionized water of dodecyl sodium sulfate in ester, lipid and silica is stirred, and it is removed
Aeroseal is simultaneously heated, and potassium peroxydisulfate stirring is added after depressurization, and then to stirring liquid filtering, the filtrate centrifugation to collection is pure
Change, obtain microballoon, it is standby；
(b) preparation of colour-fluorescent dual-function microballoon：Dichloromethane or tetrahydrofuran and step are added in deionized water
(a) microballoon completed is prepared, microspheres solution is obtained, by the fluorescent marker for being dissolved in dichloromethane or tetrahydrofuran and fat-soluble colour
Pigment is added dropwise in microspheres solution and sealed and stirs, and then depressurization and removes dichloromethane or tetrahydrofuran, most
Centrifugal treating afterwards, colour-fluorescent dual-function microballoon is obtained, it is standby；
(c) preparation of determinand antibody colour-fluorescent dual-function microballoon label：Prepared with buffer solution dilution step (b)
The colour of completion-fluorescent dual-function microballoon, carbodiimide and n-hydroxysuccinimide are added, after being activated on shaking table, centrifugation
Processing, remove supernatant, add buffer solution and precipitation is redissolved, then add the antigen-antibody related to determinand or part by
Body, in being coupled on shaking table, monoethanolamine and bovine serum albumin(BSA) are then added, sealing overnight, last centrifugal treating, removes supernatant
Liquid, then precipitation is redissolved with buffer solution, it is ultrasonically treated, determinand antibody colour-fluorescent dual-function microballoon label is made；
(d) rabbit, mouse or other antigen-antibody colour-fluorescent dual-function microballoons with determinand antigen-antibody no cross reaction
The preparation of label：Dilute colour-fluorescent dual-function microballoon with buffer solution, add carbodiimide and rabbit, mouse or other with it is to be measured
The antigen-antibody of thing antigen-antibody no cross reaction, in being coupled on shaking table, then add monoethanolamine and bovine serum albumin(BSA), sealing
Overnight, last centrifugal treating, supernatant is removed, then is redissolved with buffer solution, is ultrasonically treated, rabbit, mouse or other and determinand is made
The antigen-antibody colour of antigen-antibody no cross reaction-fluorescent dual-function microballoon label colour-Fluorescent microsphere marker；
(e) preparation of colour-fluorescent dual-function microballoon label probe：Determinand antibody colour-fluorescent dual-function microballoon
Antigen-antibody colour-Fluorescent microsphere marker of label and rabbit, mouse or other and determinand antigen-antibody no cross reaction is mixed
Merging is sprayed on pad, and colour-fluorescent dual-function microballoon label probe is made.
(III) preparation of detecting pad 4
The coating of detection line T lines 41：The antigen-antibody or ligand receptor related to determinand are used and contain trehalose and SDS
PBS dissolving, spray coating liquor one is made, spray coating liquor one is sprayed on away from forming detection line T lines at the left end 12mm of detecting pad 4
41；
The coating of nature controlling line C line 42：The antibody of the antigen unrelated with determinand or ligand receptor are used containing trehalose and
SDS phosphate buffer dissolving, is made spray coating liquor two, spray coating liquor two is sprayed on away from forming Quality Control at the left end 18mm of detecting pad 4
Line C lines 42；
The kept dry of detecting pad 4 sprayed is standby；
(IV) assembling of test strips
Overlap treated sample-adding pad 2 successively on PVC bottom plates 1, be sprayed with colour-fluorescent dual-function microballoon label probe
Label pad 3, be coated with detection line T lines 41 and the detecting pad 4 and adsorptive pads 5 of nature controlling line C line 42, after assembling
Cut into the wide test strips of 4mm.
Specific embodiment
Embodiment 1
Aflatoxins M1 blueness-difunctional the immuno-chromatographic test paper strip of time-resolved fluorescence
First, the preparation method of test strips
(a) preparation of microballoon
8mmol styrene monomer and 0.8mmol acrylic monomers is taken to be dissolved in dodecyls of the 10ml containing 0.3mmol
In the deionized water of sodium sulfonate, mixed liquor is added in round-bottomed flask, stirred with magnetic stir bar.Then high pure nitrogen is used
Air in round-bottomed flask is eliminated, heated sealed adds 0.3ml 0.2mmol potassium peroxydisulfate, sealing oxygen barrier stirring to 70 DEG C
After reacting 8h, room temperature is down to.Then the filter paper that reaction solution aperture is 8 μm is filtered, collects filtrate centrifugal purification, centrifugal condition
15min is centrifuged for 10 DEG C of constant temperature of 50000g, supernatant is removed, is redissolved and precipitated with 5ml deionized waters, repeatedly washing three times, finally
The dodecyl sodium sulfate that concentration is 0.05% and the sodium azide that concentration is 0.05% are added in 4 DEG C of preservations.Added by regulation
The amount for entering the monomer of acrylic acid can obtain the different carboxyl density of microsphere surface；By adjusting dodecyl sodium sulfate and over cure
The amount of sour potassium, you can prepare the microballoon of different-grain diameter.
(b) preparation of the difunctional microballoon of blueness-time-resolved fluorescence
The microballoon for taking the above-mentioned preparation that 1ml solid contents are 10% to complete, adds in 9ml deionized waters, adds 3ml dichloros
Methane, it is placed in 37 DEG C of isoperibols, magnetic agitation 30min, obtains pretreatment microspheres solution.Take 10mg Europium chelates (Eu:β-
NTA:TOPO=1：3：3) with 5mg Disperse Blue 2BLNs, it is dissolved in 1ml dichloromethane, then above-mentioned solution is added dropwise pre-
Handle in microspheres solution, sealing, 37 DEG C of temperature constant magnetic stirring 5h.Then sealing lid is opened, makes the dichloromethane in solution system
Alkane volatilizees totally under the conditions of 37 DEG C.Solution is transferred in centrifuge tube, three times, centrifugal condition is centrifuge washing：50000g 10℃
Constant temperature centrifuges 15min.Supernatant is removed, the preceding precipitation centrifuged twice is redissolved with 50% ethanol, the precipitation centrifuged for the last time
Redissolved with 10ml pH8.0 0.05M borate buffers, add dodecyl sodium sulfate that concentration is 0.05% and concentration is
0.05% 4 DEG C of preservations of sodium azide.
(c) preparation of Aflatoxins M1 antibody blueness-time-resolved fluorescence microballoon label
The difunctional μ L of microballoon 100 of above-mentioned blueness-time-resolved fluorescence for taking solid content to be 1% are diluted in concentration and are
In 0.05mol/L, pH8.0 400 μ L borate buffers, add the carbodiimide (EDC) that 30 μ L concentration are 10mg/mL and (be purchased from
Shanghai Jing Chun biochemical technologies limited company) and 60 μ L concentration be 10mg/mL n-hydroxysuccinimide (NHS) (be purchased from
Shanghai Jing Chun biochemical technologies limited company), it will be placed under above-mentioned mixed liquor room temperature condition on rotary shaker at 50 revs/min
Under the conditions of activate 15min, then 40000g centrifuge 10min, remove supernatant, with 0.05mol/L, pH8.0 borate buffer
Redissolve and precipitate, then 80W is ultrasonically treated 30S, and adding 25 μ g Aflatoxins M1 mouse monoclonal antibodies, (flying survey biotechnology purchased from Shanghai has
Limit company), room temperature is placed on rotary shaker 50 revs/min and is coupled 2 hours, and the monoethanolamine for adding 50mmol/L is (brilliant purchased from Shanghai
Pure biochemical technology limited company), the 10% μ L of BSA (being purchased from Shanghai Xi Bao bio tech ltd) solution 50 closed
Night.Last 40000g centrifugations 10min, precipitation is redissolved with 0.05mol/L, pH8.0 borate buffer, is washed 2~3 times repeatedly,
Then 80W is ultrasonically treated 30S, is placed in 4~8 DEG C of refrigerators and saves backup.
(d) preparation of rabbit igg blueness-time-resolved fluorescence microballoon label
Take solid content be 1% the difunctional μ L of microballoon 100 of above-mentioned blueness-time-resolved fluorescence be diluted in 0.05mol/L,
In pH8.0 400 μ L borate buffers, 30 μ L10mg/mL carbodiimide (EDC) is added (purchased from the brilliant pure biochemical technology in Shanghai
Limited company), 100 μ g rabbit iggs (being purchased from Changsha Bo You bio tech ltd) are added, by above-mentioned mixed liquor room temperature
Under the conditions of be placed on rotary shaker 50 revs/min and be coupled 2 hours, add 50mmol/L monoethanolamine (purchased from the brilliant pure biochemistry in Shanghai
Science and Technology Co., Ltd.), 10% μ L of BSA (being purchased from Shanghai Xi Bao bio tech ltd) solution 50 closing overnight.Most
40000g is centrifuged 10 minutes afterwards, redissolves precipitation with 0.05mol/L pH8.0 borate buffer, washing 2~3 times repeatedly, then
80W is ultrasonically treated 30S, is placed in 4~8 DEG C of refrigerators and saves backup.
(e) preparation of test strips
(I) it is loaded the preparation of pad 2
With the EDTA containing 10mmol/L, 1% (v/v) tween-20,0.5% (m/v) PVP, 0.5% (m/v)
BSA PBS (0.2mol/L, pH8.0) immersion glass fibre element film sample-adding pad, is subsequently placed in 37 DEG C of baking ovens and toasts 2
Hour to being completely dried, is placed in hermetically drying packaging bag and saved backup.
(II) preparation of label pad 3
Use first containing 2.5% (m/v) trehalose, 1% (m/v) bovine serum albumin(BSA), 1% (v/v) tween-
20th, 0.5% (m/v) PVP 0.1mol/L pH7.4 phosphate buffer immersion pad Fusion5 (being purchased from GE companies),
It is subsequently placed in 37 DEG C of baking ovens and dries 2 hours.Aflatoxins M1 antibody blueness-time resolution that above-mentioned preparation is completed again is glimmering
The phosphate buffer point of light microballoon label and rabbit igg blueness-time-resolved fluorescence microballoon label 0.1mol/L, pH7.4
The difunctional microballoon label probe of Aflatoxins M1 antibody blue-fluorescent Xi Shi not be mixed and made into after 500 times and 1200 times, will
Above-mentioned probe is sprayed on the Fusion5 after processing is dried with gold spraying instrument, is placed in 37 DEG C of baking ovens after drying 2 hours, is placed in sealing
Saved backup in dry packing bag.
(III) preparation of detecting pad 4
The coating of detection line T lines 41
By Aflatoxins M1 and bovine serum albumin(BSA) conjugate (being purchased from Shanghai Fei Ce bio tech ltd) with containing
There is the phosphate buffer that 1.5% (m/v) trehalose and 0.05% (v/v) SDS concentration are 0.01mol/L, pH7.4 to dissolve
To final concentration of 0.025mg/mL, above-mentioned mixed liquor is sprayed on away from forming inspection at nitrocellulose filter left end 12mm with Membrane jetter
Survey line T lines 41.
The coating of nature controlling line C line 42
Goat anti-rabbit igg monoclonal antibody is used to the concentration containing 1.5% (m/v) trehalose and 0.05% (v/v) SDS
Final concentration 1.0mg/mL is dissolved to for 0.01mol/L, pH7.4 phosphate buffer, above-mentioned mixed liquor is sprayed on Membrane jetter
Away from forming nature controlling line C line 42 at nitrocellulose filter left end 18mm.The nitrocellulose filter sprayed is dried under the conditions of 37 DEG C
It is dry 2 hours, it is placed in drying at room temperature environment and saves backup.
(IV) assembling of test strips
As shown in figure 1, the colour-fluorescent dual-function immuno-chromatographic test paper strip includes bottom plate 1, it is suitable in the one side of bottom plate 1
Sequence is provided with the sample-adding pad 2, label pad 3, the detection provided with detection line T lines 41 and nature controlling line C line 42 of addition testing sample
Pad 4 and the adsorptive pads 5 for providing capillary siphoning power, adjacent each pad are overlapped and pasted successively on PVC backings.
By the treated sample-adding pad 2 of the present embodiment, it is sprayed with the difunctional microballoon mark of Aflatoxins M1 antibody blue-fluorescent
The label pad 3 of physical prospecting pin, the detecting pad 4 for being coated with detection line T lines 41 and nature controlling line C line 42 and adsorptive pads 5 assemble
After cut into the wide test strips of 4mm, be fitted into during plastics get stuck, be then charged into the aluminium foil bag for being built-in with drier, room temperature
Kept dry, the shelf-life was up to more than 2 years.
If above-mentioned test strips are fixed on during the plastics with well and detection window get stuck, detection card is made.
2nd, the determination of fluorescent quantitation standard curve
Aflatoxins M1 standard items are diluted to 0ng/mL, 0.025ng/mL, 0.075ng/mL, 0.3ng/ with lactogenesis
ML, 0.9ng/mL, 2.7ng/mL, 8.1ng/mL, according to each Concentration Testing of the every kind of toxin of Standard Operating Procedure 10 times, then will
In measured numerical value input Microdetection food safety managements software (Nanjing micrometering bio tech ltd), i.e.,
The parameters of standard curve can be obtained, then by the Data Enter IC-card such as the parameter of standard curve, batch number.
3rd, testing sample detects
1. detection method
The difunctional immuno-chromatographic test paper strip of Aflatoxins M1 blue-fluorescent for preparing completion is lain against on desktop, taken
100 μ L milk samples to be measured (20-30 DEG C), are added dropwise at the sample-adding pad 2 of test strips, timing 5 minutes, observe testing result.
2. result judgement
1. naked eyes interpretation
When the T lines naked eyes of test strip are invisible, when C lines naked eyes are visible, illustrate the dense of Aflatoxins M1 in milk sample
Degree is higher than 0.3ng/ml；When test strip T lines naked eyes it is visible, C lines naked eyes it is visible when, illustrate Aflatoxins M1 in milk sample
Concentration be less than 0.3ng/ml；As C, first naked eyes are invisible, no matter whether naked eyes are visible for T lines, it is invalid to be judged to, and need to detect again.
2. Fluorescent reader interpretation
Test strip is inserted in Fluorescent reader, reading and automatic calculating, aspergillus flavus poison can be carried out by read key
Plain M1 concentration can be shown by liquid crystal display, also can also pass through instrument by the thermal printer field print that readout instrument carries
On device GPRS and WIFI wireless communication modules by testing number factually when be sent to high in the clouds data management platform, realize to testing result
Real-time monitoring.
3. the accurate sex determination of ELISA test strip result
Aspergillus flavus is added to being detected through high performance liquid chromatography GC-MS in the fresh milk that Aflatoxins M1 content is 0
Toxin M1 standard solutions, make its concentration respectively reach 0ng/mL, 0.025ng/mL, 0.075ng/mL, 0.3ng/mL, 0.9ng/
ML, 2.7ng/mL, 8.1ng/mL, the accuracy that the present embodiment prepares the test strips completed are detected respectively.Detected more than repeating real
Test 10 times, take the average value of instrument sentence read result.
Test experience result is as shown in table 1 below.
Table 1 detects result of the test 1
Test result shows：The present embodiment prepares the difunctional test strip of blue-fluorescent microballoon completed when in use,
Both it can visually judge that the amount of Aflatoxins M1 in milk sample whether more than 0.3ng/ml, carried out initial characterization interpretation, learns milk sample
Whether it is qualified milk；Also it can be obtained by Fluorescent reader and accurately quantify testing result, meet various inspection well
Survey demand.
Embodiment 2
Chloramphenicol red-rhodamine fluorescent dual-function immuno-chromatographic test paper strip
First, the preparation method of test strips
(a) synthesis of microballoon
8mmol styrene monomer 0.8mmol acrylic monomers is taken to be dissolved in dodecyl sulphurs of the 10ml containing 0.3mmol
In the deionized water of sour sodium, mixed liquor is added in round-bottomed flask, stirred with magnetic stir bar.Then will with high pure nitrogen
Air eliminates in round-bottomed flask, and heated sealed adds 0.3ml 0.2mmol potassium peroxydisulfate to 70 DEG C, and sealing oxygen barrier stirring is anti-
After answering 8h, room temperature is down to.Then the filter paper that reaction solution aperture is 8 μm is filtered, collects filtrate centrifugal purification, centrifugal condition is
10 DEG C of constant temperature of 50000g centrifuge 15min, remove supernatant, are redissolved and precipitated with 5ml deionized waters, wash three times, finally add repeatedly
Enter the dodecyl sodium sulfate that concentration is 0.05% and the sodium azide that concentration is 0.05% in 4 DEG C of preservations.Added by adjusting
The amount of the monomer of acrylic acid can obtain the different carboxyl density of microsphere surface；By adjusting dodecyl sodium sulfate and persulfuric acid
The amount of potassium, you can prepare the microballoon of different-grain diameter.
(b) preparation of red-rhodamine fluorescent dual-function microballoon
The microballoon for taking the above-mentioned preparation that 1ml solid contents are 10% to complete, adds in 9ml deionized waters, adds 3.5ml tetra-
Hydrogen furans, is placed in room temperature environment, magnetic agitation 30min, obtains pretreatment microspheres solution.Take 3mg rhodamine Bs and 5mg disperse reds
SER, it is dissolved in 2ml tetrahydrofurans, then above-mentioned solution is added dropwise in pretreatment microspheres solution, room under the conditions of magnetic agitation
Temperature keeps 5h.Then sealing lid is opened, the tetrahydrofuran in solution system is volatilized under the conditions of rotatory vacuum clean.By solution
It is transferred in centrifuge tube, three times, centrifugal condition is centrifuge washing：10 DEG C of constant temperature centrifugation 15min of 50000g.Remove supernatant, it is preceding twice from
The precipitation of gains in depth of comprehension is redissolved with 50% ethanol, the precipitation 10ml pH8.0 centrifuged for the last time 0.05M borate buffers
Redissolve, add the 4 DEG C of preservations of the dodecyl sodium sulfate that concentration is 0.05% and the sodium azide that concentration is 0.05%.
(c) preparation of chloramphenicol antibody red-rhodamine Fluorescent microsphere marker
It is 0.05mol/ to take above-mentioned red-μ L of rhodamine fluorescent dual-function microballoon 100 that solid content is 1% to be diluted in concentration
L, in pH8.0 400 μ L borate buffers, it is (pure purchased from Shanghai crystalline substance to add the carbodiimide (EDC) that 30 μ L concentration are 10mg/mL
Biochemical technology limited company) and 60 μ L concentration be 10mg/mL n-hydroxysuccinimide (NHS) (purchased from Shanghai crystalline substance it is pure
Biochemical technology limited company), it will be placed under above-mentioned mixed liquor room temperature condition on rotary shaker under the conditions of 50 revs/min living
Change 15min, then 40000g centrifuges 10min, removes supernatant, redissolves precipitation with 0.05mol/L, pH8.0 borate buffer,
Then 80W is ultrasonically treated 30S, adds 20 μ g chloramphenicol mouse monoclonal antibodies (being purchased from Shanghai Fei Ce bio tech ltd), room temperature is put
It is coupled 2 hours in 50 revs/min on rotary shaker, adds 50mmol/L monoethanolamine (purchased from the brilliant pure biochemical technology share in Shanghai
Co., Ltd), 10% μ L of BSA (being purchased from Shanghai Xi Bao bio tech ltd) solution 50 closing overnight.Last 40000g
10min is centrifuged, redissolves precipitation with 0.05mol/L, pH8.0 borate buffer, is washed repeatedly 2~3 times, then at 80W ultrasounds
30S is managed, is placed in 4~8 DEG C of refrigerators and saves backup.
(d) preparation of rabbit igg red-rhodamine Fluorescent microsphere marker
Take solid content be 1% above-mentioned red-μ L of rhodamine fluorescent dual-function microballoon 100 be diluted in 0.05mol/L,
In pH8.0 400 μ L borate buffers, 30 μ L 10mg/mL carbodiimide (EDC) is added (purchased from the brilliant pure biochemical technology in Shanghai
Limited company), 100 μ g rabbit iggs (being purchased from Changsha Bo You bio tech ltd) are added, by above-mentioned mixed liquor room temperature
Under the conditions of be placed on rotary shaker 50 revs/min and be coupled 2 hours, add 50mmol/L monoethanolamine (purchased from the brilliant pure biochemistry in Shanghai
Science and Technology Co., Ltd.), 10% μ L of BSA (being purchased from Shanghai Xi Bao bio tech ltd) solution 50 closing overnight.Most
40000g is centrifuged 10 minutes afterwards, redissolves precipitation with 0.05mol/L pH8.0 borate buffer, washing 2~3 times repeatedly, then
80W is ultrasonically treated 30S, is placed in 4~8 DEG C of refrigerators and saves backup.
(e) preparation of test strips
(I) it is loaded the preparation of pad 2
With the EDTA containing 10mmol/L, 1% (v/v) tween-20,0.5% (m/v) PVP, 0.5% (m/v)
BSA PBS (0.2mol/L, pH8.0) immersion glass fibre element film sample-adding pad, is subsequently placed in 37 DEG C of baking ovens and toasts 2
Hour to being completely dried, is placed in hermetically drying packaging bag and saved backup.
(II) preparation of label pad 3
Use first containing 2.5% (m/v) trehalose, 1% (m/v) bovine serum albumin(BSA), 1% (v/v) tween-
20th, 0.5% (m/v) PVP 0.1mol/L pH7.4 phosphate buffer immersion pad Fusion5 (being purchased from GE companies),
It is subsequently placed in 37 DEG C of baking ovens and dries 2 hours.The chloramphenicol antibody red that above-mentioned preparation is completed again-rhodamine fluorescent microsphere mark
Note thing and rabbit igg red-rhodamine Fluorescent microsphere marker dilute 400 times respectively with 0.1mol/L, pH7.4 phosphate buffer
With 1000 times after be mixed into chloramphenicol antibody red-rhodamine fluorescent dual-function microballoon label probe, by above-mentioned probe spray
Jin Yi is sprayed on the Fusion5 after processing drying, is placed in 37 DEG C of baking ovens after drying 2 hours, is placed in hermetically drying packaging bag
Save backup.
(III) preparation of detecting pad 4
The coating of detection line T lines 41
By chloramphenicol and bovine serum albumin(BSA) conjugate (being purchased from Shanghai Fei Ce bio tech ltd) with containing 1.5%
(m/v) trehalose and 0.05% (v/v) SDS concentration is dissolved to final concentration for 0.01mol/L, pH7.4 phosphate buffer
For 0.025mg/mL, above-mentioned mixed liquor is sprayed on away from forming detection line T lines at nitrocellulose filter left end 12mm with Membrane jetter
41。
The coating of nature controlling line C line 42
Goat anti-rabbit igg monoclonal antibody is used to the concentration containing 1.5% (m/v) trehalose and 0.05% (v/v) SDS
Final concentration 1.0mg/mL is dissolved to for 0.01mol/L, pH7.4 phosphate buffer, above-mentioned mixed liquor is sprayed on Membrane jetter
Away from forming nature controlling line C line 42 at nitrocellulose filter left end 18mm.The nitrocellulose filter sprayed is dried under the conditions of 37 DEG C
It is dry 2 hours, it is placed in drying at room temperature environment and saves backup.
(IV) assembling of test strips
As shown in figure 1, the colour-fluorescent dual-function immuno-chromatographic test paper strip includes bottom plate 1, it is suitable in the one side of bottom plate 1
Sequence is provided with the sample-adding pad 2, label pad 3, the detection provided with detection line T lines 41 and nature controlling line C line 42 of addition testing sample
Pad 4 and the adsorptive pads 5 for providing capillary siphoning power, adjacent each pad are overlapped and pasted successively on PVC backings.
By the treated sample-adding pad 2 of the present embodiment, it is sprayed with chloramphenicol antibody red-rhodamine fluorescent dual-function microballoon mark
The label pad 3 of physical prospecting pin, the detecting pad 4 for being coated with detection line T lines 41 and nature controlling line C line 42 and adsorptive pads 5 assemble
After cut into the wide test strips of 4mm, be fitted into during plastics get stuck, be then charged into the aluminium foil bag for being built-in with drier, room temperature
Kept dry, the shelf-life was up to more than 2 years.
If above-mentioned test strips are fixed on during the plastics with well and detection window get stuck, detection card is made.
2nd, the determination of fluorescent quantitation standard curve
Chloramphenicol standard items are diluted to 0ng/mL, 0.025ng/mL, 0.05ng/mL, 0.1ng/mL, 0.3ng/ with lactogenesis
ML, 0.5ng/mL, 1.0ng/mL, then will be measured according to each Concentration Testing of the every kind of toxin of Standard Operating Procedure 10 times
In numerical value input Microdetection food safety managements software (Nanjing micrometering bio tech ltd), you can marked
The parameters of directrix curve, then by the Data Enter IC-card such as the parameter of standard curve, batch number.
3rd, testing sample detects
1. detection method
Chloramphenicol red-rhodamine fluorescent dual-function the immuno-chromatographic test paper strip for preparing completion is lain against on desktop, taken
100 μ L milk samples to be measured (20-30 DEG C), are added dropwise at the sample-adding pad 2 of test strips, timing 5 minutes, observe testing result.
2. result judgement
1. naked eyes interpretation
When test strip T lines naked eyes it is invisible, C lines naked eyes it is visible when, illustrate that the concentration of chloramphenicol in milk sample is higher than
0.1ng/ml；When test strip T lines naked eyes it is visible, C lines naked eyes it is visible when, illustrate that the concentration of chloramphenicol in milk sample is less than
0.1ng/ml；As C, first naked eyes are invisible, no matter whether naked eyes are visible for T lines, it is invalid to be judged to, and need to detect again.
2. Fluorescent reader interpretation
Test strip is inserted in Fluorescent reader, reading and automatic calculating can be carried out by read key, chloramphenicol
Concentration can be shown by liquid crystal display, also can be by the thermal printer field print that readout instrument carries, can also be by instrument
GPRS and WIFI wireless communication modules by testing number factually when be sent to high in the clouds data management platform, realize the reality to testing result
When monitor.
3. the accurate sex determination of ELISA test strip result
Chloramphenicol standard items are added into the fresh milk for being 0 through high performance liquid chromatography GC-MS detection chloromycetin content
Solution, its concentration is set to respectively reach 0ng/mL, 0.025ng/mL, 0.05ng/mL, 0.1ng/mL, 0.3ng/mL, 0.5ng/mL,
1.0ng/mL, the accuracy that the present embodiment prepares the test strips completed is detected respectively, repeat above test experience 10 times, take instrument
The average value of sentence read result.
Test experience result is as shown in table 2 below.
Table 2 detects result of the test 2
The present embodiment prepares the red-difunctional test strip of rhodamine fluorescent microsphere completed when in use, both can meat
Eye judges that the amount of chloramphenicol in milk sample whether more than 0.1ng/ml, carries out initial characterization judgement, learns whether milk sample is qualified ox
Milk；Also it can be obtained by Fluorescent reader and accurately quantify testing result, meet various detection demand well.
It will be apparent to those skilled in the art that technical scheme that can be as described above and design, make other various
Corresponding change and deformation, and all these changes and deformation should all belong to the protection domain of the claims in the present invention
Within.
Claims (6)
- A kind of 1. colour-fluorescent dual-function immuno-chromatographic test paper strip, it is characterised in that：Including bottom plate（1）, bottom plate（1）One side On be sequentially with addition testing sample sample-adding pad（2）, label pad（3）, provided with detection line T lines（41）With nature controlling line C Line（42）Detecting pad（4）With the adsorptive pads for providing capillary siphoning power（5）, adjacent each pad overlaps successively, and label combines Pad（3）Upper coating chromatic colour-fluorescent dual-function microballoon label probe；The colour-fluorescent dual-function microballoon label probe Antibody containing determinand or acceptor colour-fluorescent dual-function microballoon label and rabbit or mouse and antigen no cross reaction to be detected IgG colours-fluorescent dual-function microballoon label, antibody or acceptor colour-Fluorescent microsphere marker of determinand are by determinand Antibody or acceptor and colour-fluorescent dual-function microballoon combine and be prepared, rabbit or mouse and antigen no cross reaction to be detected IgG colours-Fluorescent microsphere marker is micro- by rabbit or mouse and the IgG and colour-fluorescent dual-function of antigen no cross reaction to be detected Chou is closed and is prepared；The preparation method of the colour-fluorescent dual-function microballoon is：Microemulsion method is used to prepare particle diameter as the poly- of 50-1000nm Phenylethylene micro ball, poly (methyl methacrylate) micro-sphere, polyvinyl pyridine microspheres, poly- ethyl methyl acrylate microballoon, lipid microsphere With one kind in silicon dioxide microsphere, the microsphere surface carries carboxyl, adds dichloromethane or tetrahydrochysene furan in deionized water The microballoon completed is muttered and prepared, microspheres solution is obtained, by the fluorescent marker for being dissolved in dichloromethane or tetrahydrofuran and fat-soluble coloured silk Color pigment is added dropwise in microspheres solution and sealed and stirs, and then depressurization and removes dichloromethane or tetrahydrofuran, Last centrifugal treating, obtain colour-fluorescent dual-function microballoon.
- 2. colour according to claim 1-fluorescent dual-function immuno-chromatographic test paper strip, it is characterised in that：Detection line T lines （41）It is coated with antigen or part to be detected, nature controlling line C line（42）It is coated with goat anti-rabbit igg or sheep anti-mouse igg.
- 3. colour according to claim 1 or 2-fluorescent dual-function immuno-chromatographic test paper strip, it is characterised in that：The coloured silk The color of color-fluorescent dual-function microballoon is a kind of macroscopic dark color in red, blueness, green, purple and black；Fluorescence It is one kind in Rare Earth Chelate fluorescence, quantum dot fluorescence；The wavelength difference of wavelength and the fluorescent material transmitting of color emission is more than Equal to 30nm.
- 4. colour according to claim 3-fluorescent dual-function immuno-chromatographic test paper strip, it is characterised in that：Sample-adding pad（2）For Material with filtration, sample-adding pad（2）Material be by more added with surfactant, carbohydrate, protide, macromolecule Glass fibre, the polyester of one or more phosphate buffers or Tris-HCl buffer solution dipping pretreatments in polymers, salt Film, sponge, filter paper；Label pad（3）Material be by added with surfactant, carbohydrate, protide, macromolecule polymer, salt In one or more phosphate buffers or Tris-HCl buffer solution dipping pretreatments glass fibre or polyester film；Detecting pad（4）For the porous material with strong hydrophobic property, detecting pad（4）Material it is fine for nitrocellulose filter or acetic acid Tie up plain film, detection line T lines（41）And nature controlling line C line（42）It is at least one.
- A kind of 5. preparation method of the colour of claim 4-fluorescent dual-function immuno-chromatographic test paper strip, it is characterised in that：Including Following steps：（Ⅰ）Sample-adding pad（2）PreparationGlass fibre, polyester film, sponge or filter paper are soaked with the PBS containing EDTA, tween-20, PVP and BSA, so After be placed in baking oven and dry, hermetically drying saves backup；（Ⅱ）Label pad（3）PreparationGlass fibre or polyester film are soaked with the PBS containing trehalose, bovine serum albumin(BSA), tween-20 and PVP, so After be placed in baking oven and dry, then colour-fluorescent dual-function microballoon label probe is sprayed at the glass fibre after processing drying Or on polyester film, it is again placed in drying in baking oven, hermetically drying saves backup；（Ⅲ）Detecting pad（4）PreparationDetection line T lines（41）Coating：By antigen to be detected or part, the PBS containing trehalose and SDS dissolves, system Spray coating liquor one is obtained, spray coating liquor one is sprayed on away from detecting pad（4）Detection line T lines are formed at left end 12mm（41）；Nature controlling line C line（42）Coating：Goat anti-rabbit igg or sheep anti-mouse igg is molten with the phosphate buffer containing trehalose and SDS Solution, spray coating liquor two is made, spray coating liquor two is sprayed on away from detecting pad（4）Nature controlling line C line is formed at left end 18mm（42）；The detecting pad that will have been sprayed（4）Kept dry is standby；（Ⅳ）The assembling of test stripsIn PVC bottom plates（1）On overlap treated sample-adding pad successively（2）, be sprayed with colour-fluorescent dual-function microballoon label probe Label pad（3）, be coated with detection line T lines（41）And nature controlling line C line（42）Detecting pad（4）And adsorptive pads（5）, The wide test strips of 4 mm are cut into after assembling.
- 6. the preparation method of colour according to claim 5-fluorescent dual-function immuno-chromatographic test paper strip, it is characterised in that： Step（Ⅱ）Described in colour-specific preparation method of fluorescent dual-function microballoon label probe it is as follows：（a）The preparation of microballoon：Microemulsion method is used to prepare polystyrene microsphere, polymethyl of the particle diameter for 50-1000nm One in sour methyl esters microballoon, polyvinyl pyridine microspheres, poly- ethyl methyl acrylate microballoon, lipid microsphere and silicon dioxide microsphere Kind, the microsphere surface carries carboxyl；（b）The preparation of colour-fluorescent dual-function microballoon：Dichloromethane or tetrahydrofuran and step are added in deionized water（a） The microballoon completed is prepared, microspheres solution is obtained, by the fluorescent marker for being dissolved in dichloromethane or tetrahydrofuran and fat-soluble colored face Material is added dropwise in microspheres solution and sealed and stirs, and then depressurization and removes dichloromethane or tetrahydrofuran, finally Centrifugal treating, colour-fluorescent dual-function microballoon is obtained, it is standby；（c）The preparation of antibody or the acceptor colour of determinand-fluorescent dual-function microballoon label：With buffer solution dilution step（b） Colour-fluorescent dual-function the microballoon completed is prepared, adds carbodiimide and n-hydroxysuccinimide, after being activated on shaking table, Centrifugal treating, supernatant is removed, add buffer solution and precipitation is redissolved, then add the antibody or acceptor related to determinand, in It is coupled on shaking table, then adds monoethanolamine and bovine serum albumin(BSA), sealing overnight, last centrifugal treating, removes supernatant, then use Buffer solution redissolves to precipitation, is ultrasonically treated, and the antibody or acceptor colour-Fluorescent microsphere marker of determinand is made；（d）Rabbit or mouse and the preparation of IgG colours-Fluorescent microsphere marker of antigen no cross reaction to be detected：It is dilute with buffer solution Colour-fluorescent dual-function microballoon is released, carbodiimide and rabbit or mouse and the antibody of antigen no cross reaction to be detected are added, in shaking table Upper coupling, then adds monoethanolamine and bovine serum albumin(BSA), and overnight, last centrifugal treating removes supernatant for sealing, then with buffering Liquid redissolves, and is ultrasonically treated, and rabbit or mouse and IgG colours-Fluorescent microsphere marker of antigen no cross reaction to be detected is made；（e）The preparation of colour-fluorescent dual-function microballoon label probe：Antibody or acceptor colour-fluorescent microsphere mark of determinand Note thing mixes produce by a certain percentage with rabbit or mouse with IgG colours-Fluorescent microsphere marker of antigen no cross reaction to be detected.
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