CN109061142A - Detect the Nano silver grain delustring immuno-chromatographic test paper strip of Aflatoxins M1 - Google Patents

Detect the Nano silver grain delustring immuno-chromatographic test paper strip of Aflatoxins M1 Download PDF

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Publication number
CN109061142A
CN109061142A CN201810895940.XA CN201810895940A CN109061142A CN 109061142 A CN109061142 A CN 109061142A CN 201810895940 A CN201810895940 A CN 201810895940A CN 109061142 A CN109061142 A CN 109061142A
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bsa
silver
detection
solution
test strips
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熊勇华
江湖
郭亮
李响敏
赖卫华
聂丽娟
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Nanchang University
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Nanchang University
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Abstract

The present invention is to detect the Nano silver grain delustring immuno-chromatographic test paper strip of Aflatoxins M1, belong to analysis detection field, it discloses the immuno-chromatographic test paper strip of detection small-molecule substance: the fluorescent material for modifying bovine serum albumin(BSA) is mixed with detection antigen and anti-immunoglobulin G antibody respectively, it is sprayed on test strips specific region and the Nano silver grain (silver-colored delustring probe) of monoclonal antibody specific is marked to be sprayed on test strips bonding pad as detection line and nature controlling line.When determinand is not present in sample solution, antigen binding is detected in silver-colored delustring probe and detection line, absorb the exciting light of fluorescent material or emits light and makes detection line unstressed configuration, and nature controlling line has fluorescence；When there are when determinand for sample solution, preferentially in conjunction with determinand, the silver-colored delustring number of probes being incorporated in detection line at this time is reduced silver-colored delustring probe, and is incorporated in the increase of the silver-colored delustring number of probes on nature controlling line, test strips detection line fluorescence is caused to increase, and nature controlling line fluorescence declines.The present invention is sensitiveer compared with the line interpretation mode that disappears of Traditional immunochromatographic test strips detection small-molecule substance.

Description

Detect the Nano silver grain delustring immuno-chromatographic test paper strip of Aflatoxins M1
Technical field
The invention belongs to immune analysis detection technique field, it is related to detecting the Nano silver grain delustring of Aflatoxins M1 Immuno-chromatographic test paper strip.
Background technique
Immunochromatography technique has many advantages, such as that speed is fast, anti-matrix interference ability is strong, easy to operate, is on-site quick screening Prefered method and the fields innovation research such as food safety, medical and health, environmental protection hot spot.In recent years, based on immune The colloidal gold strip of chromatographic technique is widely applied, however traditional colloidal gold immunochromatographimethod method is because of detection sensitivity It is not high, detect certain micro or trace analysis object such as food in pollute in terms of there are still larger defects.Cause This, improves the detection sensitivity of immune chromatography method and prolongs its scope of application for opening up and have very important significance.Fluorescent marker Object has higher signal strength, test strips detection sensitivity can be improved compared with traditional colloid gold label object.
Small molecule determinand is detected using test strips, usually using competitive mechanism, i.e. artificial antigen in detection line It competes the antibody of fluorescent material label jointly with determinand, signal strength and determinand content is detected inversely, when to be measured When object content is in reduced levels, stronger detection signal is to the minor change of determinand content and insensitive, therefore sensitivity It is relatively low.Building fluorescence reduction mode can make object concentration to be checked directly proportional to fluorescence intensity on competition immuno-chromatographic test paper strip, From scratch, detection sensitivity and accuracy can be improved in object i.e. to be checked fluorescence signal in the presence of trace.Compared to conventional glue Body gold particle, Nano silver grain have higher Molar Extinction ability, therefore have stronger fluorescence cut down ability, so as into One step improves test strips detection sensitivity.In addition, the extinction spectra of Nano silver grain increases with particle diameter and is increased, silver nanoparticle Particle diameter from 10nm to 100nm, from 400nm to 500nm, widen by corresponding surface plasmon resonance (SPR) absorption peak Colloidal gold fluorescence " be quenched " immuno-chromatographic test paper strip fluorescent material range of choice (gold nanoparticle SPR absorption peak 500nm with On).
Summary of the invention
Nano silver grain delustring immuno-chromatographic test paper strip of the invention absorbs the excitation of fluorescent material based on Nano silver grain Light emits light and its fluorescence signal is caused to weaken, and is used for fast qualitative and quantitative detection small-molecule substance.Nano silver grain disappears The structure composition of light immuno-chromatographic test paper strip is as shown in attached drawing 1, including filter paper, sample pad, bonding pad, NC film and blotting paper etc. five Part forms, and wherein filter paper, sample pad and bonding pad stacking are assembled in NC film one end, and absorbent paper layer is stacked in the NC film other end, and five Part is integrally attached on sticky paper jam.Fluorescent material-BSA is mixed with small-molecule substance to be measured detection antigen and is fixed on test strips Detection line, fluorescent material-BSA are mixed with IgG Fc fragments specific identification antibody (secondary antibody) and are fixed on test strips control line (matter Control line), Nano silver grain and small-molecule substance specific antibody complex (silver-colored delustring probe) are fixed on test strips bonding pad, Substance solution to be checked is filtered by filter paper, enters bonding pad through sample pad, small molecule to be measured is absorbing water in conjunction with silver-colored delustring probe Under paper capillarity, solution to be checked flows in test strips, proceeds on detection line, not the silver in conjunction with small molecule to be measured Detection antigen binding on delustring probe and detection line, remaining silver-colored delustring probe proceed on nature controlling line and two resistive connections It closes.Ten minutes later, test strips read the fluorescence values that detection line and nature controlling line are read in instrument in fluorescence to sample-adding ten to two.Due to silver The exciting light or transmitting light of the absorption fluorescent material of nanoparticle specificity, lead to the fluorescent value and silver particles quantity in detection line It is inversely proportional, and the concentration direct proportionality with small-molecule substance to be checked, when being free of small-molecule substance to be checked in sample, detection There is no fluorescence signal on line.A kind of Nano silver grain delustring immune chromatography test paper detecting small-molecule substance of the present invention Item, the preparation method comprises the following steps:
(1) fluorescent material and bovine serum albumin(BSA) (abbreviation BSA) pass through Covalent bonding together between carboxyl and amino
Selected fluorescent material is fluorescent microsphere, quantum dot microsphere, quantum dot or fluorescent dye；Fluorescent material need to be passed through first Cross surface amino groups or carboxyl modified, then on BSA carboxyl or amino it is sub- by 1- (3- dimethylamino-propyl) -3- ethyl carbon two Amine hydrochlorate (abbreviation EDC) activates covalent bond, obtains fluorescent material-BSA；The most suitable excitation wavelength and emission maximum of fluorescent material Both wavelength at least one is in 400nm between 500nm；
(2) silver-colored delustring probe is prepared:
1) using seed, gradually growth method synthesizes Nano silver grain, measures ultraviolet absorption peak to the silver particles of each step, when Peak value stops growing when reaching with the maximum excitation wavelength or consistent launch wavelength of selected pairing fluorescent material；Concrete scheme Are as follows: sodium citrate (abbreviation SC) concentration a) silver-colored seed preparation: is adjusted in 100mL system to 5mM, tannic acid (abbreviation TA) 0.1mM, heating are vigorously stirred, and are boiled at the beginning, and 1mL 25mM AgNO is added immediately3, solution is immediately turned to glassy yellow, supplies System volume obtains the seed silver solution of seed silver size about 15nm to 100mL；B) silver particles are gradually grown: in 100mL system 16.5mL water is added in middle removal 19.5mL reaction solution, and temperature is set to 90 DEG C, is added 0.5mL SC (25mM), 1.5mL TA (2.5mM), 1mL AgNO3(25mM) supplies system volume to 100mL, stirs 30min；Repeat " 19.5mL reaction solution is removed, 16.5mL water, 0.5mL SC (25mM), 1.5mL TA (2.5mM) and 1mL AgNO is added3(25mM), the system volume of supplying is extremely 100mL, 90 DEG C of stirring 30min " this processes, UV scanning monitors the situation of change of Nano silver grain SPR absorption peak, until obtaining It is identical as the maximum excitation wavelength or launch wavelength for matching fluorescent material to obtain SPR absorption peak, obtains Nano silver grain；
2) Nano silver grain is in conjunction with small molecule monoclonal antibody to be checked: taking 1mL silver nano-particle solution, (population is about 1010To 1012), 5000 go to 10000 turns of centrifugation 10min, abandon supernatant；Precipitating is resuspended in 1mL containing small molecular antibody (IgG to be checked The 0.1M pH 7.0-7.5 boric acid solution of 1 μ g of content to 80 μ g)；2h is stirred on 20 DEG C of magnetic stirring apparatus, 5000 go to 10000 Turn centrifugation 10min, abandons supernatant；Precipitating is resuspended in 0.1M boric acid solution of the pH 7.0 to 7.5 containing 0.1% (w/v) BSA, 5min Afterwards, 5,000 10000 turns of centrifugation 10min are gone to, precipitating is resuspended in the 0.1M that 0.25mL pH 7.0 to 7.5 contains 0.1% (w/v) BSA In boric acid solution, silver-colored delustring probe is obtained, 4 DEG C save backup；
(3) small molecule determinand and BSA detection antigen (small molecule-BSA) preparation: are passed through into Covalent bonding together；
The group of non-antibody recognition site on first activated micromolecular determinand is then logical with amino on BSA molecule or carboxyl It crosses Covalent bonding together and obtains small molecule determinand-BSA；This method is identical as the preparation method of small molecule artificial antigen；
(4) assembling of Nano silver grain delustring immuno-chromatographic test paper strip:
Test strips include the five part groups such as filter paper, sample pad, bonding pad, nitrocellulose filter (abbreviation NC film) and blotting paper At；Wherein filter paper, sample pad and bonding pad stacking are assembled in NC film one end, and absorbent paper layer is stacked in the NC film other end, and five parts are whole Body is fixed on sticky paper jam；Silver-colored delustring probe is wherein carried on bonding pad, the fixed number of probes of each test strips bonding pad is 106To 108It is a；The detection antigen and fluorescent material-BSA of small molecule to be checked are fixed in detection line, are consolidated in each test strips detection line Determine small molecule detection 0.1 μ g of antigen to 0.5 μ g；Anti-immunoglobulin G (abbreviation IgG) antibody i.e. secondary antibody and glimmering is fixed on nature controlling line Stimulative substance-BSA fixes anti-igg antibody 10ng to 100ng on each test strips nature controlling line；It is fixed in detection line and nature controlling line glimmering The amount of stimulative substance-BSA is with fluorescent material Mass Calculation for 1ng to 50ng；Matter is sprayed at detection line 4.0mm to 6.0mm Control line；It is cut into width 3.8mm every, is fitted into test strips cartridge, cartridge is identical as conventional colloidal gold quick measuring card box, inside has Test strips fixed card slot, in the filter paper position of test strips, there are wells, and there are inspections for detection line and Quality Control line position on Nc film Survey window；The loading together with desiccant of test strips detection card is protected from light bag and is sealed for use.
Further, refinement application of the aforementioned test strips in specific field:
The invention also discloses detect aflatoxin M1Nano silver grain delustring immuno-chromatographic test paper strip, determinand Matter is aflatoxin M1(abbreviation AFM1), fluorescent material is fluorescein isothiocynate microballoon；
The preparation method is as follows:
(1) amino surface fluorescein isothiocynate microballoon is in conjunction with BSA:
In 25mL phosphate buffer (abbreviation PB, 0.01M, pH 6.0), 20mg amino surface isosulfocyanic acid fluorescence is added Plain microballoon, 5mg BSA, 200 μ g EDC after 45min is stirred at room temperature, adds 200 μ g EDC, 25mg BSA, 1h are stirred at room temperature, 8000 turns of centrifugation 10min, precipitating are redissolved in 2mL water, obtain fluorescent microsphere-BSA, 4 DEG C save backup；
(2) silver-colored delustring probe synthesis:
1) synthesis of Nano silver grain includes that seed is generated and gradually grown.A) it silver-colored seed preparation: is adjusted in 100mL system Whole sodium citrate (SC) concentration to 5mM, tannic acid (TA) 0.1mM, heating is vigorously stirred, boils at the beginning, and 1mL is added immediately AgNO3(25mM), solution is immediately turned to glassy yellow, supplies system volume to 100mL, obtains the seed of seed silver size about 15nm Silver-colored solution；B) silver particles are gradually grown: removing 19.5mL reaction solution in 100mL system, 16.5mL water is added, temperature is set to It 90 DEG C, is added 0.5mL SC (25mM), 1.5mL TA (2.5mM), 1mL AgNO3(25mM), supplies system volume to 100mL, Stir 30min.Repeat " remove 19.5mL reaction solution, be added 16.5mL water, 0.5mL SC (25mM), 1.5mL TA (2.5mM) and 1mL AgNO3(25mM), supplies system volume to 100mL, 90 DEG C stirring 30min " this process 16 times, obtain SPR absorption peak It is consistent with fluorescein isothiocynate microballoon maximum excitation light for the Nano silver grain of 494nm；
2) Nano silver grain and aspergillus flavus resisting toxin M1Monoclonal antibody combines: taking 1mL silver nano-particle solution (population About 6 × 1010It is a), 5000 turns of centrifugation 10min abandon supernatant；Precipitating is resuspended in 1mL containing AFM1The 0.1M boric acid solution of 20 μ g of monoclonal antibody (NaOH tune pH to 7.4)；2h is stirred on 20 DEG C of magnetic stirring apparatus, is centrifuged 10min, abandons supernatant；PH 7.4 is resuspended in containing 0.1% (w/v) in the 0.1M boric acid solution of BSA, after 5min, 5000 turns of centrifugation 10min, precipitating is resuspended in 0.25mL pH 7.4 and contains The 0.1M boric acid solution of 0.1% (w/v) BSA, obtains silver-colored delustring probe solution, and 4 DEG C save backup；
(3) AFM is synthesized1Detect antigen:
By 1mg AFM1, 0.5mg succinic anhydride and 2mL pyridine mixing, 120 DEG C are heated to reflux 3h, with 200 after being dried with nitrogen μ L dimethylformamide (abbreviation DMF) dissolution, is added 1mg N, N'- dicyclohexylcarbodiimide (abbreviation DCC) and 1mg N- hydroxyl Base succinimide (abbreviation NHS), is stirred at room temperature 4h, by 10000 turns of centrifugation 10min of mixed liquor, takes supernatant that 2mL is added dropwise In the 0.13M sodium bicarbonate solution of the BSA containing 10mg.Room temperature is slowly stirred overnight, is transferred to (the letter of 4 DEG C of phosphate buffered salines Claim PBS, 0.01M, pH 7.4) in dialyse 72h.
(4) assembling of Nano silver grain delustring epidemic disease chromatograph test strip:
Test strips include five parts such as filter paper, sample pad, bonding pad, NC film and blotting paper composition；Wherein, sample pad is used 50mM pH 7.4 phosphate buffer solution (comprising 1% BSA, 0.5% polysorbas20 and 0.05% Sodium azide) infiltration, And in 60 DEG C of dry 2h；PBS (pH 7.5) buffer solution (including 0.2% polysorbas20 and 2% sucrose) leaching of bonding pad 0.01M Profit, in 60 DEG C of dry 4h, silver-colored delustring probe solution is sprayed on bonding pad after diluting 10 times with the concentration of 3 μ L/cm；It will test anti- Former (1.2mg/mL) is sprayed at the detection line of NC film, the anti-mouse IgG (0.2mg/ of donkey after mixing with fluorescent microsphere-BSA (100 μ g/mL) ML it is sprayed on the nature controlling line of NC film after) being mixed with fluorescent microsphere-BSA (100 μ g/mL), it is 0.75 μ that each wire spraying amount, which is arranged, NC film after spraying is placed in 37 DEG C of vacuum oven dry 6h by L/cm；The commercialized filter paper and blotting paper without Specially treated directly uses；Filter paper, sample pad and bonding pad stacking are assembled in NC film one end, and it is another that absorbent paper layer is stacked in NC film End, five parts are integrally attached on sticky paper jam；Nature controlling line is sprayed at detection line 4.0mm to 6.0mm；It is cut into width 3.8mm every is fitted into test strips cartridge, and cartridge is identical as conventional colloidal gold quick measuring card box, inside there is test strips fixed card slot, In the filter paper position of test strips, there are wells, and there are detection windows for detection line and Quality Control line position on Nc film；Test strips inspection The loading together with desiccant of survey card is protected from light bag and is sealed for use.
The invention also discloses the Nano silver grain delustring immuno-chromatographic test paper strip of salbutamol, to be measured in detection pig urine Substance is salbutamol, and fluorescent material is the carboxyl surface quantum point microballoon of maximum emission wavelength 620nm；The preparation method is as follows:
(1) the carboxyl surface quantum point microballoon of maximum emission wavelength 620nm is in conjunction with BSA:
In 25mL PB (0.01M, pH 6.0), 10mg carboxyl surface quantum point microballoon is added, 200 μ g EDC are stirred at room temperature After 20min, add 5mL BSA aqueous solution (0.5%, w/v), be stirred at room temperature 1h, 8000 turns of centrifugation 10min, precipitating redissolve in In 2mL water, quantum dot microsphere-BSA is obtained, 4 DEG C save backup；
(2) silver-colored delustring probe synthesis:
1) synthesis of Nano silver grain includes that seed is generated and gradually grown.A) it silver-colored seed preparation: is adjusted in 100mL system Whole sodium citrate (SC) concentration to 5mM, tannic acid (TA) 0.1mM, heating is vigorously stirred, boils at the beginning, and 1mL is added immediately AgNO3(25mM), solution is immediately turned to glassy yellow, supplies system volume to 100mL, obtains the seed of seed silver size about 15nm Silver-colored solution；B) silver particles are gradually grown: removing 19.5mL reaction solution in 100mL system, 16.5mL water is added, temperature is set to It 90 DEG C, is added 0.5mL SC (25mM), 1.5mL TA (2.5mM), 1mL AgNO3(25mM), supplies system volume to 100mL, Stir 30min.Repeat " remove 19.5mL reaction solution, be added 16.5mL water, 0.5mL SC (25mM), 1.5mL TA (2.5mM) and 1mL AgNO3(25mM), supplies system volume to 100mL, 90 DEG C stirring 30min " this process 11 times, obtain SPR absorption peak It is consistent with quantum dot microsphere exciting light for the Nano silver grain of 450nm；
2) Nano silver grain is in conjunction with anti-salbutamol monoclonal antibody: taking 1mL silver nano-particle solution, (population is about 1.3×1011It is a), 7000 turns of centrifugation 10min abandon supernatant；The 0.1M boric acid that precipitating is resuspended in 5 μ g of 1mL monoclonal antibody containing salbutamol is molten Liquid (NaOH tune pH to 7.2)；2h is stirred at room temperature, is centrifuged 10min, abandons supernatant；It is resuspended in the 0.1M pH containing 0.1% (w/v) BSA In the 7.2 0.1M boric acid solutions containing 0.1% (w/v) BSA, after 5min, 7000 turns of centrifugation 10min, precipitating is resuspended in 0.25mL pH 7.2 contain the 0.1M boric acid solution of 0.1% (w/v) BSA, and 4 DEG C save backup；
(3) synthesis salbutamol detects antigen:
1mg salbutamol is dissolved in 0.5mL dehydrated alcohol, adds 1mg succinic anhydride, 2h is stirred at room temperature, with 200 after being dried with nitrogen μ L DMF dissolution, is added 1mg DCC and 1mg NHS, 4h is stirred at room temperature, supernatant is added dropwise 10000 turns of centrifugation 10min In the 0.13M sodium bicarbonate solution of 2mL BSA containing 10mg.Room temperature is slowly stirred overnight, is transferred to 4 DEG C of PBS (0.01M, pH 7.4) Middle dialysis 72h.
(4) assembling of Nano silver grain delustring immuno-chromatographic test paper strip:
Test strips include five parts such as filter paper, sample pad, bonding pad, NC film and blotting paper composition；Wherein, sample pad is used 50mM pH 7.4 phosphate buffer solution (comprising 1% BSA, 0.4% polysorbas20 and 0.03% Sodium azide) infiltration, The dry 10h of dehumidification at room temperature；PBS (pH7.4) buffer solution (including 0.2% polysorbas20 and 2% sucrose) of bonding pad 0.01M Infiltration, the dry 12h of dehumidification, silver-colored delustring probe solution are sprayed on bonding pad after diluting 5 times with the concentration of 3.5 μ L/cm at room temperature； It will test the detection line that NC film is sprayed at after antigen (1.0mg/mL) is mixed with quantum dot microsphere-BSA (80 μ g/mL), the anti-mouse of donkey IgG (0.25mg/mL) is sprayed on the nature controlling line of NC film after mixing with quantum dot microsphere-BSA (80 μ g/mL), and each line spray is arranged Painting amount is 0.77 μ L/cm, and the NC film after spraying is placed in 37 DEG C of vacuum oven dry 6h；Commercialized filter paper and suction Water paper is not specially treated, directly uses；Filter paper, sample pad and bonding pad stacking are assembled in NC film one end, and absorbent paper layer is stacked in The NC film other end, five parts are integrally attached on sticky paper jam；Nature controlling line is sprayed at detection line 4.0mm to 6.0mm；Cutting It at width 3.8mm every, is fitted into test strips cartridge, cartridge is identical as conventional colloidal gold quick measuring card box, inside has test strips solid Determine card slot, there are wells in the filter paper position of test strips, and there are detection windows for detection line and Quality Control line position on Nc film；Examination The loading together with desiccant of paper slip detection card is protected from light bag and is sealed for use.
The invention also discloses, the Nano silver grain delustring immuno-chromatographic test paper strip of Enrofloxacin in chicken is detected, it is to be measured Substance is Enrofloxacin, and fluorescent material is 7- amino -4- methylcoumarin-BSA (abbreviation AMCA-BSA)；The preparation method is as follows:
(1) silver-colored delustring probe synthesis:
1) synthesis of Nano silver grain includes that seed is generated and gradually grown.A) it silver-colored seed preparation: is adjusted in 100mL system Whole sodium citrate (SC) concentration to 5mM, tannic acid (TA) 0.1mM, heating is vigorously stirred, boils at the beginning, and 1mL is added immediately AgNO3(25mM), solution is immediately turned to glassy yellow, supplies system volume to 100mL, obtains the seed of seed silver size about 15nm Silver-colored solution；B) silver particles are gradually grown: removing 19.5mL reaction solution in 100mL system, 16.5mL water is added, temperature is set to It 90 DEG C, is added 0.5mL SC (25mM), 1.5mL TA (2.5mM), 1mL AgNO3(25mM), supplies system volume to 100mL, Stir 30min.Repeat " remove 19.5mL reaction solution, be added 16.5mL water, 0.5mL SC (25mM), 1.5mL TA (2.5mM) and 1mL AgNO3(25mM), supplies system volume to 100mL, 90 DEG C stirring 30min " this process 9 times, obtaining SPR absorption peak is The Nano silver grain of 442nm, it is consistent with AMCA transmitting light；
2) Nano silver grain is in conjunction with anti-enrofloxacin monoclonal antibody: taking 1mL silver nano-particle solution, (population is about 1.6×1011It is a), 7500 turns of centrifugation 10min abandon supernatant；It is molten that precipitating is resuspended in 0.1M boric acid of the 1mL containing husky 6.5 μ g of Enrofloxacin Liquid (NaOH tune pH to 7.5)；2h is stirred at room temperature, is centrifuged 10min, abandons supernatant；It is resuspended in the 0.1M pH containing 0.1% (w/v) BSA In the 7.5 0.1M boric acid solutions containing 0.1% (w/v) BSA, after 5min, 7500 turns of centrifugation 10min, precipitating is resuspended in 0.25mL pH 7.5 contain the 0.1M boric acid solution of 0.1% (w/v) BSA, and 4 DEG C save backup；
(2) synthesis Enrofloxacin detects antigen:
1mg Enrofloxacin, 1mg NHS and 1mg EDC are weighed, is dissolved in 0.2mL DMF, 10 μ L triethylamines, room is added Temperature stirring 4h, is added dropwise in the 0.13M sodium bicarbonate solution of 2mL BSA containing 10mg, and room temperature is slowly stirred overnight, is transferred to 4 DEG C Dialyse 72h in PBS (0.01M, pH 7.4).
(3) assembling of Nano silver grain delustring immuno-chromatographic test paper strip:
Test strips include five parts such as filter paper, sample pad, bonding pad, NC film and blotting paper composition；Wherein, sample pad is used 50mM pH 7.4 phosphate buffer solution (comprising 1% BSA, 0.4% polysorbas20 and 0.03% Sodium azide) infiltration, The dry 10h of dehumidification at room temperature；Bonding pad (includes 0.2% polysorbas20 and 2% sugarcane with PBS (pH 7.4) buffer solution of 0.01M Sugar) infiltration, the dry 12h of dehumidification at room temperature, silver-colored delustring probe solution with the concentration of 4 μ L/cm is sprayed on bonding pad after diluting 5 times On；It will test the detection line that NC film is sprayed at after antigen (1.75mg/mL) is mixed with AMCA-BSA (60 μ g/mL), the anti-mouse two of donkey Anti- (0.25mg/mL) is sprayed on the nature controlling line of NC film after mixing with AMCA-BSA (60 μ g/mL), and each wire spraying amount, which is arranged, is NC film after spraying is placed in 37 DEG C of vacuum oven dry 6h by 0.75 μ L/cm；Commercialized filter paper and blotting paper without Specially treated directly uses；Filter paper, sample pad and bonding pad stacking are assembled in NC film one end, and it is another that absorbent paper layer is stacked in NC film End, five parts are integrally attached on sticky paper jam；Nature controlling line is sprayed at detection line 4.0mm to 6.0mm；It is cut into width 3.8mm every is fitted into test strips cartridge, and cartridge is identical as conventional colloidal gold quick measuring card box, inside there is test strips fixed card slot, In the filter paper position of test strips, there are wells, and there are detection windows for detection line and Quality Control line position on Nc film；Test strips inspection The loading together with desiccant of survey card is protected from light bag and is sealed for use.
The invention also discloses detect the Nano silver grain delustring immune chromatography test paper of melamine in milk and milk powder Item, test substance are melamine, and fluorescent material is the carboxyl surface quantum point of maximum emission wavelength 590nm；Preparation method is such as Under:
(1) the carboxyl surface quantum point of maximum emission wavelength 590nm is in conjunction with BSA:
In 20mL PB (0.01M, pH 6.0), 1mg carboxyl surface quantum point is added, 20min is stirred at room temperature in 200 μ g EDC Afterwards, 20mL BSA aqueous solution (1%, w/v) is added, 1h is stirred at room temperature, is slowly added to dilute hydrochloric acid tune pH to 5.0, then 19000 turns It is centrifuged 20min, precipitating is redissolved in 2mL water,
Quantum dot-BSA is obtained, 4 DEG C save backup；
(2) silver-colored delustring probe synthesis:
1) synthesis of Nano silver grain includes that seed is generated and gradually grown.A) it silver-colored seed preparation: is adjusted in 100mL system Whole sodium citrate (SC) concentration to 5mM, tannic acid (TA) 0.1mM, heating is vigorously stirred, boils at the beginning, and 1mL is added immediately AgNO3(25mM), solution is immediately turned to glassy yellow, supplies system volume to 100mL, obtains the seed of seed silver size about 15nm Silver-colored solution；B) silver particles are gradually grown: removing 19.5mL reaction solution in 100mL system, 16.5mL water is added, temperature is set to It 90 DEG C, is added 0.5mL SC (25mM), 1.5mL TA (2.5mM), 1mL AgNO3(25mM), supplies system volume to 100mL, Stir 30min.Repeat " remove 19.5mL reaction solution, be added 16.5mL water, 0.5mL SC (25mM), 1.5mL TA (2.5mM) and 1mL AgNO3(25mM), supplies system volume to 100mL, 90 DEG C stirring 30min " this process 11 times, obtain SPR absorption peak It is consistent with -590 exciting light of quantum dot for the Nano silver grain of 450nm；
2) Nano silver grain is in conjunction with melamine monoclonal antibody: taking 1mL silver nano-particle solution (population about 1.3 ×1011It is a), 7000 turns of centrifugation 10min abandon supernatant；Precipitating is resuspended in the 0.1M boric acid solution of 5 μ g of 1mL monoclonal antibody containing melamine (NaOH tune pH to 7.5)；2h is stirred at room temperature, is centrifuged 10min, abandons supernatant；It is resuspended in the 0.1M pH containing 0.1% (w/v) BSA In the 7.5 0.1M boric acid solutions containing 0.1% (w/v) BSA, after 5min, 7000 turns of centrifugation 10min, precipitating is resuspended in 0.25mL pH 7.5 contain the 0.1M boric acid solution of 0.1% (w/v) BSA, and 4 DEG C save backup；
(3) production of melamine detects antigen:
1mg melamine is dissolved in 0.2mL pyridine solution, and succinic anhydride 1mg is added, 4h is stirred at room temperature.It is dried with nitrogen, obtains To white product be dissolved in 200 μ L DMF, be added 1mg NHS, 1mg DCC, 4h is stirred at room temperature.By 10000 turns of mixed liquor It is centrifuged 10min, supernatant is taken to be added dropwise in the 0.13M sodium bicarbonate solution of 2mL BSA containing 10mg.Room temperature was slowly stirred Night is transferred in 4 DEG C of PBS (0.01M, pH 7.4) 72h that dialyses.
(4) assembling of Nano silver grain delustring immuno-chromatographic test paper strip:
Test strips include five parts such as filter paper, sample pad, bonding pad, NC film and blotting paper composition；Wherein, sample pad is used 50mM pH 7.4 phosphate buffer solution (comprising 1% BSA, 0.4% polysorbas20 and 0.03% Sodium azide) infiltration, The dry 10h of dehumidification at room temperature；PBS (pH7.4) buffer solution (including 0.2% polysorbas20 and 2% sucrose) of bonding pad 0.01M Infiltration, the dry 12h of dehumidification, silver-colored delustring probe solution are sprayed on bonding pad after diluting 5 times with the concentration of 5 μ L/cm at room temperature；It will Detection antigen (1.6mg/mL) is sprayed at the detection line of NC film, the anti-mouse secondary antibody of donkey after mixing with quantum dot-BSA (80 μ g/mL) (0.2mg/mL) is sprayed on the nature controlling line of NC film after mixing with quantum dot-BSA (80 μ g/mL), and each wire spraying amount is arranged and is NC film after spraying is placed in 37 DEG C of vacuum oven dry 6h by 0.85 μ L/cm；Commercialized filter paper and blotting paper without Specially treated directly uses；Filter paper, sample pad and bonding pad stacking are assembled in NC film one end, and it is another that absorbent paper layer is stacked in NC film End, five parts are integrally attached on sticky paper jam；Nature controlling line is sprayed at detection line 4.0mm to 6.0mm；It is cut into width 3.8mm every is fitted into test strips cartridge, and cartridge is identical as conventional colloidal gold quick measuring card box, inside there is test strips fixed card slot, In the filter paper position of test strips, there are wells, and there are detection windows for detection line and Quality Control line position on Nc film；Test strips inspection The loading together with desiccant of survey card is protected from light bag and is sealed for use.
The structure composition of test strips is as shown in attached drawing 1.
The use of Nano silver grain delustring test strips: after substance solution to be checked is added on the well of test strips cartridge, Under blotting paper capillarity, solution to be checked flows in test strips, filters out larger impurity through filter paper first, flows through sample Pad, into bonding pad, substance to be checked proceeds on detection line, is not associated with the silver of substance to be checked in conjunction with silver-colored delustring probe Antigen binding is detected in probe and detection line, remaining silver-colored probe proceeds on nature controlling line and two anti-bindings.It is loaded 10min To 20min, test strips cartridge is put into fluorescence and reads the fluorescence values for reading detection line and nature controlling line in instrument.Due to silver particles spy The exciting light or transmitting light of anisotropic absorption fluorescent material, cause fluorescent value and Nano silver grain quantity in detection line to be inversely proportional Example, and the concentration direct proportionality with substance to be checked.When not containing small-molecule substance to be checked in sample, without glimmering in detection line Optical signal.It needs to draw standard curve when quantitative detection.
When determinand is not present in sample solution, silver-colored delustring probe under capillary forces, is creeped on nitrocellulose filter Detection line and detection antigen binding are reached, a small amount of remaining silver-colored delustring probe proceeds on nature controlling line and two anti-bindings, by In the photoextinction of Nano silver grain, test strips detection line does not have fluorescence, and nature controlling line has fluorescence；When there are to be measured for sample solution When object, first in conjunction with determinand, the silver-colored delustring probe in conjunction with detection line is reduced silver-colored delustring probe at this time, and is incorporated in Quality Control Silver-colored delustring number of probes on line increases, and test strips detection line fluorescence is caused to increase, and nature controlling line fluorescence declines.
The concentration direct proportionality of the method for the present invention fluorescence intensity and small-molecule substance to be checked, in small-molecule substance trace In the presence of, from scratch, the line interpretation mode that disappears compared with Traditional immunochromatographic test strips detection small-molecule substance is sensitiveer for fluorescence.
In Nano silver grain delustring immuno-chromatographic test paper strip detection line of the present invention fluorescent value be incorporated in silver nanoparticle above Number of particles is inversely proportional, directly proportional to small molecule content to be measured, when not containing target small-molecule substance in solution to be checked, detection line Upper no fluorescence.
The novelty of the present invention is propose the new Nano silver grain delustring immuno-chromatographic test paper strip of one kind to detect small point The method of sub- substance, this method is using the higher Nano silver grain of molar extinction coefficient as delustring probe, specific absorption The exciting light or transmitting light of fluorescent material, are turned using the competition immunochromatography backward read mode that fluorescence reduction mode will test small molecule It is changed to positive reading mode, and then improves detection sensitivity.
Technical solution of the present invention has the advantages that
(1) technical solution of the present invention high sensitivity will be silver-colored since the molar extinction coefficient of Nano silver grain is higher than colloidal gold Detection sensitivity can be improved in positive reading mode of the nanoparticle as delustring probe application in small molecule competition immunochromatography.
(2) present invention has widened the range of choice that the fluorescent material of test strips " is quenched " in fluorescence, and partial size 10nm is to 100nm's The SPR absorption peak of Nano silver grain is in 400nm between 500nm, can matching maximum excitation wavelength or launch wavelength range exists 400nm is to the fluorescent material between 500nm, and the SPR absorption peak of colloidal gold is in 500nm or more.
Detailed description of the invention
Fig. 1 is Nano silver grain delustring immuno-chromatographic test paper strip structure chart.The bottom is sticky paper jam, and in-between position is viscous Nc film is pasted, Nc film sprays small molecule detection antigen and fluorescent material-BSA to be measured in detection line position in advance, in Quality Control line position Spray anti-igg antibody (secondary antibody) and fluorescent material-BSA.Bonding pad, which is successively pasted, on the sticky paper jam on the Nc film left side (is coated with silver Delustring probe), sample pad and filter paper, bonding pad right side edge and Nc film left side edge stacking 1 to 2mm, sample pad is layered in knot Close pad above, left side, which is staggered, 1 to 2mm to be pasted on paper jam, and filter paper is layered in sample pad, and same left side, which is staggered, 1 to 2mm pastes On paper jam.Blotting paper, blotting paper left side edge and Nc film right side edge stacking 1 to 2mm are pasted on the right of Nc film.It is to be measured when detection Sample solution enters Nc film under blotting paper capillarity, flows from left to right through filter paper, sample pad, bonding pad.
Fig. 2 is Nano silver grain delustring immuno-chromatographic test paper strip sample detection schematic diagram.When detecting positive sample, silver disappears Light probe is not incorporated in detection line, and fluorescent material generates transmitting light when exciting light irradiates；When detecting negative sample, silver-colored delustring Probe is incorporated in detection line, and specificity absorbs the exciting light or transmitting light of fluorescent material, unstressed configuration signal when exciting light irradiates.
Specific embodiment
In order to which the present invention is more clearly understood, with reference to embodiments, the present invention will be described in further detail.It answers Work as understanding, described herein specific examples are only used to explain the present invention, is not intended to limit the present invention.
All mouse IgG class monoclonal antibodies involved in embodiment: aspergillus flavus resisting toxin M1Monoclonal antibody resists Enrofloxacin monoclonal antibody, anti-melamine monoclonal antibody and anti-salbutamol monoclonal antibody, fluorescent material: amino table Face fluorescein isothiocynate microballoon and 7- amino -4- methylcoumarin and BSA compound (AMCA-BSA) etc. are Sino-German by Wuxi Bai Er Bioisystech Co., Ltd provides；Carboxyl modified quantum dot involved in this experiment and carboxyl modified quantum dot microsphere are by beauty Ocean-nano company of state provides；All small molecule standard substances involved in this experiment are purchased from Sigma company.
The manufacturing process of the normal equation of all quantitative detection small-molecule substances involved in embodiment is equal are as follows: at 20 20 parts of gradient mark-on samples are made, through sample in the small molecule standard substance to be measured for mixing various concentration in negative sample matrix respectively It respectively takes 70 μ L sample liquids that test strips well is added after product pre-treatment, reads detection line fluorescent value and nature controlling line fluorescence after a certain period of time The two ratio is set as ordinate by value, and the logarithm of mark-on sample small molecular material concentration is set as abscissa, and it is bent to draw standard Line removes both ends and deviates linear biggish point, guarantees coefficient R2The standard side of quantitative detection is arrived not less than 0.985 Journey and the range of linearity.
Compound breviary name and its Chinese name:
SC/ sodium citrate, TA/ tannic acid, PB/ phosphate buffer, PBS/ phosphate buffered saline, BSA/ cow's serum Albumin, EDC/1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride, dimethylformamide/DMF, N, bis- ring of N'- Hexyl carbodiimide/DCC, IgG/ immunoglobulin G, AMCA/7- amino -4- methylcoumarin.
Embodiment 1
Milk is detected based on the fluorescence subtractive immunization chromatograph test strip between Nano silver grain and fluorescein isothiocynate microballoon Middle aflatoxin M1
(1) amino surface fluorescein isothiocynate microballoon is in conjunction with BSA:
In 25mL phosphate buffer (abbreviation PB, 0.01M, pH 6.0), 20mg amino surface isosulfocyanic acid fluorescence is added Plain microballoon, 5mg BSA, 200 μ g EDC after 45min is stirred at room temperature, adds 200 μ g EDC, 25mg BSA, 1h are stirred at room temperature, 8000 turns of centrifugation 10min, precipitating are redissolved in 2mL water, obtain fluorescent microsphere-BSA, 4 DEG C save backup；
(2) silver-colored delustring probe synthesis:
1) synthesis of Nano silver grain includes that seed is generated and gradually grown.A) it silver-colored seed preparation: is adjusted in 100mL system Whole sodium citrate (SC) concentration to 5mM, tannic acid (TA) 0.1mM, heating is vigorously stirred, boils at the beginning, and 1mL is added immediately AgNO3(25mM), solution is immediately turned to glassy yellow, supplies system volume to 100mL, obtains the seed of seed silver size about 15nm Silver-colored solution；B) silver particles are gradually grown: removing 19.5mL reaction solution in 100mL system, 16.5mL water is added, temperature is set to It 90 DEG C, is added 0.5mL SC (25mM), 1.5mL TA (2.5mM), 1mL AgNO3(25mM), supplies system volume to 100mL, Stir 30min.Repeat " remove 19.5mL reaction solution, be added 16.5mL water, 0.5mL SC (25mM), 1.5mL TA (2.5mM) and 1mL AgNO3(25mM), supplies system volume to 100mL, 90 DEG C stirring 30min " this process 16 times, obtain SPR absorption peak It is consistent with fluorescein isothiocynate microballoon maximum excitation light for the Nano silver grain of 494nm；
2) Nano silver grain and aspergillus flavus resisting toxin M1Monoclonal antibody combines: taking 1mL silver nano-particle solution (population About 6 × 1010It is a), 5000 turns of centrifugation 10min abandon supernatant；Precipitating is resuspended in 1mL containing AFM1The 0.1M boric acid solution of 20 μ g of monoclonal antibody (NaOH tune pH to 7.4)；2h is stirred on 20 DEG C of magnetic stirring apparatus, is centrifuged 10min, abandons supernatant；PH 7.4 is resuspended in containing 0.1% (w/v) in the 0.1M boric acid solution of BSA, after 5min, 5000 turns of centrifugation 10min, precipitating is resuspended in 0.25mL pH 7.4 and contains The 0.1M boric acid solution of 0.1% (w/v) BSA, obtains silver-colored delustring probe solution, and 4 DEG C save backup；
(3) AFM is synthesized1Detect antigen:
By 1mg AFM1, 0.5mg succinic anhydride and 2mL pyridine mixing, 120 DEG C are heated to reflux 3h, with 200 after being dried with nitrogen μ L dimethylformamide (abbreviation DMF) dissolution, is added 1mg N, N'- dicyclohexylcarbodiimide (abbreviation DCC) and 1mg N- hydroxyl Base succinimide (abbreviation NHS), is stirred at room temperature 4h, by 10000 turns of centrifugation 10min of mixed liquor, takes supernatant that 2mL is added dropwise In the 0.13M sodium bicarbonate solution of the BSA containing 10mg.Room temperature is slowly stirred overnight, is transferred to (the letter of 4 DEG C of phosphate buffered salines Claim PBS, 0.01M, pH 7.4) in dialyse 72h.
(4) assembling of Nano silver grain delustring epidemic disease chromatograph test strip: test strips include filter paper, sample pad, bonding pad, NC film It is formed with five parts such as blotting papers；Wherein, sample pad with the phosphate buffer solution of 50mM pH 7.4 (comprising 1% BSA, 0.5% polysorbas20 and 0.05% Sodium azide) infiltration, and in 60 DEG C of dry 2h；The PBS (pH 7.5) of bonding pad 0.01M Buffer solution (including 0.2% polysorbas20 and 2% sucrose) infiltration, in 60 DEG C of dry 4h, after silver-colored delustring probe solution dilutes 10 times It is sprayed on bonding pad with the concentration of 3 μ L/cm；It will test antigen (1.2mg/mL) to mix with fluorescent microsphere-BSA (100 μ g/mL) It is sprayed at the detection line of NC film afterwards, the anti-mouse IgG (0.2mg/mL) of donkey is sprayed at after mixing with fluorescent microsphere-BSA (100 μ g/mL) On the nature controlling line of NC film, it is 0.75 μ L/cm that each wire spraying amount, which is arranged, and the NC film after spraying is placed in 37 DEG C of vacuum oven Middle dry 6h；The commercialized filter paper and blotting paper is not specially treated, directly uses；Filter paper, sample pad with combine bed course Stacked group is mounted in NC film one end, and absorbent paper layer is stacked in the NC film other end, and five parts are integrally attached on sticky paper jam；Apart from detection line Nature controlling line is sprayed at 4.0mm to 6.0mm；It is cut into width 3.8mm every, is fitted into test strips cartridge, cartridge and conventional glue Body gold quick measuring card box is identical, inside there is test strips fixed card slot, and in the filter paper position of test strips, there are wells, detects on Nc film There are detection windows for line and Quality Control line position；The loading together with desiccant of test strips detection card is protected from light bag and is sealed for use.(1) Amino surface fluorescein isothiocynate microballoon is in conjunction with BSA
(5) sample pre-treatments and detection:
1mL whole milk rotates 2min with micro centrifuge, removes upper-layer fat, takes out 70 μ L from lower layer and test strips are added Well.Skim milk is not required to centrifugation step, directly takes out 70 μ L and test strips well is added, fluorescence reading is put into after 20min In instrument, excitation wavelength 494nm, Detection wavelength 517nm read fluorescent value on detection line and nature controlling line, bring normal equation into: y =0.20ln (x)+0.71, wherein y is the ratio of fluorescent value on detection line and nature controlling line, and x is aflatoxin M in milk1 Concentration, unit ng/mL.Aflatoxin M in this method quantitative detection milk1The normal equation range of linearity be 0.4ng/mL To 10ng/mL, i.e., aflatoxin M when y value range is between 0.52 to 1.171Content meets this equation relationship.We are legal Property detection milk in aflatoxin M1Detection limit be 0.2ng/mL, i.e. aflatoxin M in milk1Concentration is greater than 0.2ng/ When mL, occurs fluorescence signal in detection line.
Embodiment 2
Based in the fluorescence subtractive immunization chromatograph test strip detection pig urine between Nano silver grain and quantum dot microsphere -620 Salbutamol
(1) the carboxyl surface quantum point microballoon of maximum emission wavelength 620nm is in conjunction with BSA:
In 25mL PB (0.01M, pH 6.0), 10mg carboxyl surface quantum point microballoon is added, 200 μ g EDC are stirred at room temperature After 20min, add 5mL BSA aqueous solution (0.5%, w/v), be stirred at room temperature 1h, 8000 turns of centrifugation 10min, precipitating redissolve in In 2mL water, quantum dot microsphere-BSA is obtained, 4 DEG C save backup；
(2) silver-colored delustring probe synthesis:
1) synthesis of Nano silver grain includes that seed is generated and gradually grown.A) it silver-colored seed preparation: is adjusted in 100mL system Whole sodium citrate (SC) concentration to 5mM, tannic acid (TA) 0.1mM, heating is vigorously stirred, boils at the beginning, and 1mL is added immediately AgNO3(25mM), solution is immediately turned to glassy yellow, supplies system volume to 100mL, obtains the seed of seed silver size about 15nm Silver-colored solution；B) silver particles are gradually grown: removing 19.5mL reaction solution in 100mL system, 16.5mL water is added, temperature is set to It 90 DEG C, is added 0.5mL SC (25mM), 1.5mL TA (2.5mM), 1mL AgNO3(25mM), supplies system volume to 100mL, Stir 30min.Repeat " remove 19.5mL reaction solution, be added 16.5mL water, 0.5mL SC (25mM), 1.5mL TA (2.5mM) and 1mL AgNO3(25mM), supplies system volume to 100mL, 90 DEG C stirring 30min " this process 11 times, obtain SPR absorption peak It is consistent with quantum dot microsphere exciting light for the Nano silver grain of 450nm；
2) Nano silver grain is in conjunction with anti-salbutamol monoclonal antibody: taking 1mL silver nano-particle solution, (population is about 1.3×1011It is a), 7000 turns of centrifugation 10min abandon supernatant；The 0.1M boric acid that precipitating is resuspended in 5 μ g of 1mL monoclonal antibody containing salbutamol is molten Liquid (NaOH tune pH to 7.2)；2h is stirred at room temperature, is centrifuged 10min, abandons supernatant；It is resuspended in the 0.1M pH containing 0.1% (w/v) BSA In the 7.2 0.1M boric acid solutions containing 0.1% (w/v) BSA, after 5min, 7000 turns of centrifugation 10min, precipitating is resuspended in 0.25mL pH 7.2 contain the 0.1M boric acid solution of 0.1% (w/v) BSA, and 4 DEG C save backup；
(3) synthesis salbutamol detects antigen:
1mg salbutamol is dissolved in 0.5mL dehydrated alcohol, adds 1mg succinic anhydride, 2h is stirred at room temperature, with 200 after being dried with nitrogen μ L DMF dissolution, is added 1mg DCC and 1mg NHS, 4h is stirred at room temperature, supernatant is added dropwise 10000 turns of centrifugation 10min In the 0.13M sodium bicarbonate solution of 2mL BSA containing 10mg.Room temperature is slowly stirred overnight, is transferred to 4 DEG C of PBS (0.01M, pH 7.4) Middle dialysis 72h.
(4) assembling of Nano silver grain delustring immuno-chromatographic test paper strip:
Test strips include five parts such as filter paper, sample pad, bonding pad, NC film and blotting paper composition；Wherein, sample pad is used 50mM pH 7.4 phosphate buffer solution (comprising 1% BSA, 0.4% polysorbas20 and 0.03% Sodium azide) infiltration, The dry 10h of dehumidification at room temperature；PBS (pH7.4) buffer solution (including 0.2% polysorbas20 and 2% sucrose) of bonding pad 0.01M Infiltration, the dry 12h of dehumidification, silver-colored delustring probe solution are sprayed on bonding pad after diluting 5 times with the concentration of 3.5 μ L/cm at room temperature； It will test the detection line that NC film is sprayed at after antigen (1.0mg/mL) is mixed with quantum dot microsphere-BSA (80 μ g/mL), the anti-mouse of donkey IgG (0.25mg/mL) is sprayed on the nature controlling line of NC film after mixing with quantum dot microsphere-BSA (80 μ g/mL), and each line spray is arranged Painting amount is 0.77 μ L/cm, and the NC film after spraying is placed in 37 DEG C of vacuum oven dry 6h；Commercialized filter paper and suction Water paper is not specially treated, directly uses；Filter paper, sample pad and bonding pad stacking are assembled in NC film one end, and absorbent paper layer is stacked in The NC film other end, five parts are integrally attached on sticky paper jam；Nature controlling line is sprayed at detection line 4.0mm to 6.0mm；Cutting It at width 3.8mm every, is fitted into test strips cartridge, cartridge is identical as conventional colloidal gold quick measuring card box, inside has test strips solid Determine card slot, there are wells in the filter paper position of test strips, and there are detection windows for detection line and Quality Control line position on Nc film；Examination The loading together with desiccant of paper slip detection card is protected from light bag and is sealed for use.
(5) sample pre-treatments and detection:
The transparent pig urine samples of visual sense do not need pre-treatment, directly take 70 μ L that test strips detection is added.The pig of state muddiness Urine samples rotate 2min through micro centrifuge, take 70 μ L of supernatant that test strips well is added, are put into test strips fluorescence after 15min It reads in instrument, excitation wavelength 450nm, Detection wavelength 620nm, reads fluorescent value on detection line and nature controlling line, bring standard side into Journey: y=0.324ln (x)+0.976, wherein y is the ratio of fluorescent value on detection line and nature controlling line, and x is husky butylamine in pig urine Determining alcohol, unit ng/mL.This method quantitative detection pig urine in salbutamol the normal equation range of linearity be 0.2ng/mL extremely 5ng/mL, i.e., salbutamol content meets this equation relationship when y value range is between 0.45 to 1.72.This method qualitative detection pig The detection limit of salbutamol is 0.1ng/mL in urine, i.e., when salbutamol concentration is greater than 0.1ng/mL in pig urine, goes out in detection line Existing fluorescence signal.
Embodiment 3
Based on the Enrofloxacin in the fluorescence subtractive immunization chromatograph test strip detection chicken between Nano silver grain and AMCA
(1) silver-colored delustring probe synthesis:
1) synthesis of Nano silver grain includes that seed is generated and gradually grown.A) it silver-colored seed preparation: is adjusted in 100mL system Whole sodium citrate (SC) concentration to 5mM, tannic acid (TA) 0.1mM, heating is vigorously stirred, boils at the beginning, and 1mL is added immediately AgNO3(25mM), solution is immediately turned to glassy yellow, supplies system volume to 100mL, obtains the seed of seed silver size about 15nm Silver-colored solution；B) silver particles are gradually grown: removing 19.5mL reaction solution in 100mL system, 16.5mL water is added, temperature is set to It 90 DEG C, is added 0.5mL SC (25mM), 1.5mL TA (2.5mM), 1mL AgNO3(25mM), supplies system volume to 100mL, Stir 30min.Repeat " remove 19.5mL reaction solution, be added 16.5mL water, 0.5mL SC (25mM), 1.5mL TA (2.5mM) and 1mL AgNO3(25mM), supplies system volume to 100mL, 90 DEG C stirring 30min " this process 9 times, obtaining SPR absorption peak is The Nano silver grain of 442nm, it is consistent with AMCA transmitting light；
2) Nano silver grain is in conjunction with anti-enrofloxacin monoclonal antibody: taking 1mL silver nano-particle solution, (population is about 1.6×1011It is a), 7500 turns of centrifugation 10min abandon supernatant；It is molten that precipitating is resuspended in 0.1M boric acid of the 1mL containing husky 6.5 μ g of Enrofloxacin Liquid (NaOH tune pH to 7.5)；2h is stirred at room temperature, is centrifuged 10min, abandons supernatant；It is resuspended in the 0.1M pH containing 0.1% (w/v) BSA In the 7.5 0.1M boric acid solutions containing 0.1% (w/v) BSA, after 5min, 7500 turns of centrifugation 10min, precipitating is resuspended in 0.25mL pH 7.5 contain the 0.1M boric acid solution of 0.1% (w/v) BSA, and 4 DEG C save backup；
(2) synthesis Enrofloxacin detects antigen:
1mg Enrofloxacin, 1mg NHS and 1mg EDC are weighed, is dissolved in 0.2mL DMF, 10 μ L triethylamines, room is added Temperature stirring 4h, is added dropwise in the 0.13M sodium bicarbonate solution of 2mL BSA containing 10mg, and room temperature is slowly stirred overnight, is transferred to 4 DEG C Dialyse 72h in PBS (0.01M, pH 7.4).
(3) assembling of Nano silver grain delustring immuno-chromatographic test paper strip:
Test strips include five parts such as filter paper, sample pad, bonding pad, NC film and blotting paper composition；Wherein, sample pad is used 50mM pH 7.4 phosphate buffer solution (comprising 1% BSA, 0.4% polysorbas20 and 0.03% Sodium azide) infiltration, The dry 10h of dehumidification at room temperature；PBS (pH7.4) buffer solution (including 0.2% polysorbas20 and 2% sucrose) of bonding pad 0.01M Infiltration, the dry 12h of dehumidification, silver-colored delustring probe solution are sprayed on bonding pad after diluting 5 times with the concentration of 4 μ L/cm at room temperature；It will Detection antigen (1.75mg/mL) is sprayed at the detection line of NC film, the anti-mouse secondary antibody of donkey after mixing with AMCA-BSA (60 μ g/mL) (0.25mg/mL) is sprayed on the nature controlling line of NC film after mixing with AMCA-BSA (60 μ g/mL), and each wire spraying amount is arranged and is NC film after spraying is placed in 37 DEG C of vacuum oven dry 6h by 0.75 μ L/cm；Commercialized filter paper and blotting paper without Specially treated directly uses；Filter paper, sample pad and bonding pad stacking are assembled in NC film one end, and it is another that absorbent paper layer is stacked in NC film End, five parts are integrally attached on sticky paper jam；Nature controlling line is sprayed at detection line 4.0mm to 6.0mm；It is cut into width 3.8mm every is fitted into test strips cartridge, and cartridge is identical as conventional colloidal gold quick measuring card box, inside there is test strips fixed card slot, In the filter paper position of test strips, there are wells, and there are detection windows for detection line and Quality Control line position on Nc film；Test strips inspection The loading together with desiccant of survey card is protected from light bag and is sealed for use.
(4) sample pre-treatments and detection:
1g chicken meat sample is taken, 80 DEG C of homogenization 5min are added 3mL 0.01M PB (pH7.0, NaCI containing 0.6M), whirlpool Whirlpool concussion mixes 3min, 4000 turns of centrifugation 5min, recycles supernatant, and residue extracts once again.Merge total 6mL extracting solution twice, It takes 70 μ L that test strips well is added, test strips fluorescence is put into after 15min and is read in instrument, excitation wavelength 350nm, Detection wavelength 442nm reads fluorescent value on detection line and nature controlling line, brings normal equation into: y=0.342ln (x)+0.397, wherein y is detection The ratio of fluorescent value on line and nature controlling line, x are determining enrofloxacin content in chicken, and unit is μ g/kg.This method quantitative detection chicken The normal equation range of linearity of Enrofloxacin is 0.5 μ g/kg to 18 μ g/kg in meat, i.e., when y value range is between 0.16 to 1.38 Determining enrofloxacin content meets this equation relationship in chicken.The detection limit of Enrofloxacin is 0.25 μ in this method qualitative detection chicken When Enrofloxacin concentration is greater than 0.25 μ g/kg that is, in chicken, there is fluorescence signal in detection line in g/kg.
Embodiment 4
Based in the fluorescence subtractive immunization chromatograph test strip detection milk powder and milk between Nano silver grain and quantum dot -590 Melamine
(1) the carboxyl surface quantum point of maximum emission wavelength 590nm is in conjunction with BSA:
In 20mL PB (0.01M, pH 6.0), 1mg carboxyl surface quantum point is added, 20min is stirred at room temperature in 200 μ g EDC Afterwards, 20mL BSA aqueous solution (1%, w/v) is added, 1h is stirred at room temperature, is slowly added to dilute hydrochloric acid tune pH to 5.0, then 19000 turns It is centrifuged 20min, precipitating is redissolved in 2mL water, obtains quantum dot-BSA, 4 DEG C save backup；
(2) silver-colored delustring probe synthesis:
1) synthesis of Nano silver grain includes that seed is generated and gradually grown.A) it silver-colored seed preparation: is adjusted in 100mL system Whole sodium citrate (SC) concentration to 5mM, tannic acid (TA) 0.1mM, heating is vigorously stirred, boils at the beginning, and 1mL is added immediately AgNO3(25mM), solution is immediately turned to glassy yellow, supplies system volume to 100mL, obtains the seed of seed silver size about 15nm Silver-colored solution；B) silver particles are gradually grown: removing 19.5mL reaction solution in 100mL system, 16.5mL water is added, temperature is set to It 90 DEG C, is added 0.5mL SC (25mM), 1.5mL TA (2.5mM), 1mL AgNO3(25mM), supplies system volume to 100mL, Stir 30min.Repeat " remove 19.5mL reaction solution, be added 16.5mL water, 0.5mL SC (25mM), 1.5mL TA (2.5mM) and 1mL AgNO3(25mM), supplies system volume to 100mL, 90 DEG C stirring 30min " this process 11 times, obtain SPR absorption peak It is consistent with -590 exciting light of quantum dot for the Nano silver grain of 450nm；
2) Nano silver grain is in conjunction with melamine monoclonal antibody: taking 1mL silver nano-particle solution (population about 1.3 ×1011It is a), 7000 turns of centrifugation 10min abandon supernatant；Precipitating is resuspended in the 0.1M boric acid solution of 5 μ g of 1mL monoclonal antibody containing melamine (NaOH tune pH to 7.5)；2h is stirred at room temperature, is centrifuged 10min, abandons supernatant；It is resuspended in the 0.1M pH containing 0.1% (w/v) BSA In the 7.5 0.1M boric acid solutions containing 0.1% (w/v) BSA, after 5min, 7000 turns of centrifugation 10min, precipitating is resuspended in 0.25mL pH 7.5 contain the 0.1M boric acid solution of 0.1% (w/v) BSA, and 4 DEG C save backup；
(3) production of melamine detects antigen:
1mg melamine is dissolved in 0.2mL pyridine solution, and succinic anhydride 1mg is added, 4h is stirred at room temperature.It is dried with nitrogen, obtains To white product be dissolved in 200 μ L DMF, be added 1mg NHS, 1mg DCC, 4h is stirred at room temperature.By 10000 turns of mixed liquor It is centrifuged 10min, supernatant is taken to be added dropwise in the 0.13M sodium bicarbonate solution of 2mL BSA containing 10mg.Room temperature was slowly stirred Night is transferred in 4 DEG C of PBS (0.01M, pH 7.4) 72h that dialyses.
(4) assembling of Nano silver grain delustring immuno-chromatographic test paper strip:
Test strips include five parts such as filter paper, sample pad, bonding pad, NC film and blotting paper composition；Wherein, sample pad is used 50mM pH 7.4 phosphate buffer solution (comprising 1% BSA, 0.4% polysorbas20 and 0.03% Sodium azide) infiltration, The dry 10h of dehumidification at room temperature；PBS (pH7.4) buffer solution (including 0.2% polysorbas20 and 2% sucrose) of bonding pad 0.01M Infiltration, the dry 12h of dehumidification, silver-colored delustring probe solution are sprayed on bonding pad after diluting 5 times with the concentration of 5 μ L/cm at room temperature；It will Detection antigen (1.6mg/mL) is sprayed at the detection line of NC film, the anti-mouse secondary antibody of donkey after mixing with quantum dot-BSA (80 μ g/mL) (0.2mg/mL) is sprayed on the nature controlling line of NC film after mixing with quantum dot-BSA (80 μ g/mL), and each wire spraying amount is arranged and is NC film after spraying is placed in 37 DEG C of vacuum oven dry 6h by 0.85 μ L/cm；Commercialized filter paper and blotting paper without Specially treated directly uses；Filter paper, sample pad and bonding pad stacking are assembled in NC film one end, and it is another that absorbent paper layer is stacked in NC film End, five parts are integrally attached on sticky paper jam；Nature controlling line is sprayed at detection line 4.0mm to 6.0mm；It is cut into width 3.8mm every is fitted into test strips cartridge, and cartridge is identical as conventional colloidal gold quick measuring card box, inside there is test strips fixed card slot, In the filter paper position of test strips, there are wells, and there are detection windows for detection line and Quality Control line position on Nc film；Test strips inspection The loading together with desiccant of survey card is protected from light bag and is sealed for use.
(4) sample pre-treatments and detection:
Full-cream and incomplete skimmed milk power: taking 1g milk powder, and 8mL ultrapure water is added, and whirlpool shakes 2min, takes out 1mL with micro- Type centrifuge is centrifuged 2min, removes upper-layer fat.
Skimmed milk power: taking 1g milk powder, and 8mL ultrapure water is added, and whirlpool shakes 2min.
Plain chocolate: 1mL is taken out with micro centrifuge and is centrifuged 2min, removes upper-layer fat.
Skim milk is not required to pre-treatment.
Above-mentioned sample takes out 70 μ L respectively and test strips well is added, and test strips fluorescence is put into after 15min and is read in instrument, is swashed Emission wavelength 450nm, Detection wavelength 590nm read fluorescent value on detection line and nature controlling line, bring normal equation into respectively.
Milk powder and skimmed milk power: y=0.304ln (x)+0.363, wherein y is the ratio of fluorescent value on detection line and nature controlling line Value, x are the melamine concentration in milk powder, and unit is μ g/kg.The standard side of melamine in this method quantitative detection milk powder The journey range of linearity is 1.6 μ g/kg to 40 μ g/kg, i.e., testing melamine content in milk powder accords with when y value range is between 0.5 to 1.48 Close this equation relationship.The detection limit of melamine is 0.8 μ g/kg, i.e. melamine in milk powder in this method qualitative detection milk powder When concentration is greater than 0.8 μ g/kg, there is fluorescence signal in detection line.
Plain chocolate and skim milk: y=0.304ln (x)+0.997, wherein y is fluorescent value on detection line and nature controlling line Ratio, x are the melamine concentration in milk, and unit is μ g/L.The standard of melamine in this method quantitative detection milk The equation range of linearity is 0.2 μ g/L to 5 μ g/L, i.e., content of melamine meets in milk when y value range is between 0.5 to 1.48 This equation relationship.The detection limit of melamine is 0.1 μ g/L, i.e. melamine concentration in milk in this method qualitative detection milk When greater than 0.1 μ g/L, there is fluorescence signal in detection line.

Claims (1)

1. detecting aflatoxin M1Nano silver grain delustring immuno-chromatographic test paper strip, it is characterised in that the preparation method is as follows:
Test substance is aflatoxin M1Abbreviation AFM1, fluorescent material is fluorescein isothiocynate microballoon；
(1) amino surface fluorescein isothiocynate microballoon is in conjunction with BSA:
In 6.0 phosphate buffer of 25mL 0.01MpH, 20mg amino surface fluorescein isothiocynate microballoon, 5mg is added BSA, 200 μ g EDC after 45min is stirred at room temperature, add 200 μ g EDC, 25mg BSA, and 1h, 8000 turns of centrifugations are stirred at room temperature 10min, precipitating are redissolved in 2mL water, obtain fluorescent microsphere-BSA, 4 DEG C save backup；
(2) silver-colored delustring probe synthesis:
1) synthesis of Nano silver grain includes that seed is generated and gradually grown, a) silver-colored seed preparation: adjusting lemon in 100mL system Lemon acid sodium SC concentration to 5mM, tannic acid TA 0.1mM, heating is vigorously stirred, boils at the beginning, and 1mL is added immediately 25mMAgNO3, solution is immediately turned to glassy yellow, supplies system volume to 100mL, obtains the seed silver of seed silver size about 15nm Solution；B) silver particles are gradually grown: removing 19.5mL reaction solution in 100mL system, 16.5mL water is added, temperature is set to 90 DEG C, 0.5mL 25mM SC, 1.5mL 2.5mM TA, 1mL 25mMAgNO is added3, system volume is supplied to 100mL, stirring 30min repeats " to remove 19.5mL reaction solution, 16.5mL water, 0.5mL 25mM SC, 1.5mL 2.5mM TA and 1mL is added 25mMAgNO3, supply system volume to 100mL, 90 DEG C stirring 30min " this process 16 times, acquisition SPR absorption peak is 494nm Nano silver grain, it is consistent with fluorescein isothiocynate microballoon maximum excitation light；
2) Nano silver grain and aspergillus flavus resisting toxin M1Monoclonal antibody combines: taking 1mL silver nano-particle solution, 5000 turns of centrifugations 10min abandons supernatant；Precipitating is resuspended in 1mL containing AFM1The pH of 20 μ g of monoclonal antibody is adjusted to 7.4 0.1M boric acid solution；20 DEG C of magnetic force 2h is stirred on blender, is centrifuged 10min, abandons supernatant；It is resuspended in 0.1M boric acid solution of the pH 7.4 containing 0.1% (w/v) BSA, After 5min, 5000 turns of centrifugation 10min, precipitating is resuspended in the 0.1M boric acid solution that 0.25mL pH 7.4 contains 0.1% (w/v) BSA, Silver-colored delustring probe solution is obtained, 4 DEG C save backup；
(3) AFM is synthesized1Detect antigen:
By 1mgAFM1, 0.5mg succinic anhydride and 2mL pyridine mixing, 120 DEG C are heated to reflux 3h, with 200 μ L diformazans after being dried with nitrogen The dissolution of base formamide, is added 1mg N, N'- dicyclohexylcarbodiimide and 1mg n-hydroxysuccinimide, 4h is stirred at room temperature, By 10000 turns of centrifugation 10min of mixed liquor, supernatant is taken to be added dropwise in the 0.13M sodium bicarbonate solution of 2mL BSA containing 10mg, Room temperature is slowly stirred overnight, is transferred to 4 DEG C of 0.01M, dialyse 72h in 7.4 phosphate buffered saline of pH；
(4) assembling of Nano silver grain delustring epidemic disease chromatograph test strip:
Test strips include filter paper, sample pad, bonding pad, NC film and five part of blotting paper composition；Wherein, sample pad is with including 1% The phosphate buffer solution of the 50mM pH 7.4 of BSA, 0.5% polysorbas20 and 0.05% Sodium azide infiltrates, and in 60 DEG C of dryings 2h；Bonding pad is infiltrated with comprising the PBS buffer solution of 0.2% polysorbas20 and the 0.01M pH 7.5 of 2% sucrose, in 60 DEG C of dryings 4h, silver-colored delustring probe solution are sprayed on bonding pad after diluting 10 times with the concentration of 3 μ L/cm；By 1.2mg/mL detection antigen with The detection line of NC film, the anti-mouse IgG of 0.2mg/mL donkey and 100 μ g/mL fluorescence are sprayed at after 100 μ g/mL fluorescent microsphere-BSA mixing It is sprayed on the nature controlling line of NC film after microballoon-BSA mixing, it is 0.75 μ L/cm that each wire spraying amount, which is arranged, by the NC film after spraying Dry 6h is placed in 37 DEG C of vacuum oven；The commercialized filter paper and blotting paper is not specially treated, directly uses；Filter Paper, sample pad and bonding pad stacking are assembled in NC film one end, and absorbent paper layer is stacked in the NC film other end, and five parts are integrally attached to glue On property paper jam；Nature controlling line is sprayed at detection line 4.0mm to 6.0mm；It is cut into width 3.8mm every, is packed into test strips card In box, cartridge is identical as conventional colloidal gold quick measuring card box, inside there is test strips fixed card slot, the filter paper position of test strips there are Well, there are detection windows for detection line and Quality Control line position on NC film；Test strips detection card is packed into together with desiccant to be kept away Light bag is sealed for use.
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